Osteoblasts are considered as a major factor contributing to bone development and mineralization,however,few studies have been done to establish and evaluate the primary cultured tibial osteoblast model of broiler chi...Osteoblasts are considered as a major factor contributing to bone development and mineralization,however,few studies have been done to establish and evaluate the primary cultured tibial osteoblast model of broiler chicks.Therefore,in the present study,two experiments were conducted to establish and evaluate the primary cultured tibial osteoblast model of broiler chicks.In experiment 1,osteoblasts were isolated from the tibia of one-day-old Arbor Acre male broiler chicks using the explant method and identified through the cell morphology,alkaline phosphatase(ALP)and alizarin red staining.Experiment 2 was carried out to evaluate the vitality and mineralization of primary cultured tibial osteoblasts of broilers on days 4,8,12,16,20,24,28 and 32 after incubation,respectively.The results from experiment 1 demonstrated that primary cultured tibial osteoblasts of broilers showed a spindle-shaped,triangular or polygonal morphology.More than 95%of the cells were stained blue-black after ALP staining,and mineralized nodules were formed after 4 days of continuous incubation.In experiment 2,lactate dehydrogenase(LDH)activity stayed at a relatively stabilized level although incubation time affected(P=0.0012)it during the whole culture period.Additionally,incubation time affected(P≤0.0001)the number and proportion of the area of mineralized nodules.They increased linearly and quadratically(P<0.04)with the increase of incubation time,and remained at a stabilized level from 24 to 32 days of incubation.The estimates of the optimal incubation time were 17 and 26 days based on the best fitted broken-line or quadratic models(P<0.0001)of the number and proportion of the area of mineralized nodules,respectively.These results indicate that the primary cultured tibial osteoblast model of broilers has been established successfully by the explant method,and it showed typical osteoblast morphology and characteristics of ALP activity and mineralization,and could maintain a relatively stabilized vitality from 4 to 32 days of incubation;and the optimal incubation time of primary tibial osteoblasts was 17 to 26 days.Therefore,it could be used to further study the underlying mechanisms of bone development and mineralization of broiler chicks.展开更多
BACKGROUND: Choosing proper donor cells is one of keys in experimental and clinical studies on cell replacement therapy (CRT) for treating Parkinson disease (PD). Embryonic mesencephalic precursor cells (MPCs) can sta...BACKGROUND: Choosing proper donor cells is one of keys in experimental and clinical studies on cell replacement therapy (CRT) for treating Parkinson disease (PD). Embryonic mesencephalic precursor cells (MPCs) can stably differentiate into dopaminergic neuron after in vitro proliferated culture. As compared with embryonic stem cell and neural stem cell strains, cell composition of embryonic MPCs after primary culture is also the most close to that of embryonic mesencephalic ventral cell suspension without proliferated culture. Successful experience accumulated in the latter suggests that primary cultured embryonic MPCs might be the most potential donor cells in clinical application with CRT for treating PD so far. OBJECTIVE: To investigate the feasibility of primary cultured embryonic precursor cells cultured primarily as donor cells in CRT for treating PD in rats. DESIGN: A randomized and controlled trial taking SD rats as experimental animals. SETTING: Department of Neurosurgery, Huashan Hospital Affiliated to Fudan University. MATERIALS: This experiment was carried out at the Institute of Neuroscience, Shanghai Institute for Biological Science, Chinese Academy of Sciences from July 2003 to June 2004. Totally 26 female SD rats, with body mass of 200 to 220 g, were provided by Shanghai Experimental Animal Center of Chinese Academy of Sciences. METHODS: Stereotaxic injection of 6-hydroxydopamine into the medial forebrain bundle were perfored to develop PD model rat. Among 26 SD rats, 20 rats achieved a more than 5 turns/min in apomorphine induced rotation test, reaching the standard of PD model rats. Immunohistochemical detection was performed on 1 out of 20 model rats after execution, and the other 19 rats were randomly divided into control group (n=5), sham transplantation group (n=5)and cell grafted group (n=9). Primary cultured E12 MPC cell suspension (1.2×1011 L-1)were used as donor cells. 4 μL primary cultured E12 MPC cell suspension prepared freshly was injected into the lesioned corpus striatum of rats in cell grafted group, and 4 μL D-Hank’s solution was injected in sham transplantation group in the same way. There was no injection in control group. Apomorphine-induced rotation rate of PD rats were recorded respectively in cell grafted group and sham transplantation group pre-operation (initial value) and at postoperative 2, 4, 6 and 16 weeks. Apomorphine-induced rotation rate of PD rats was recorded in control group at postoperative 2 months (initial value) and following 2,4,6 and 16 weeks. To determine TH antigen with immunohistological ABC method (DAB developing) at 6 months post-transplantation to investigate the differentiation and survival of donor cells in the host body. MAIN OUTCOME MEASURES: Apomorphine-induced rotation behavior before and after transplantation and the survival and differentiation of implanted cells in the host body at 6 months post-transplantation. RESULTS: Among 19 model rats, one rat died after transplantation respectively in the cell grafted group and sham transplantation group; finally 17 model rats entered the stage of result analysis. Relative apomorphine-induced rotation rate was significantly decreased in the cell grafted group as compared with that before transplantation, with significant difference (P < 0.01,P < 0.05);the mean value of relative apomorphine-induced rotation rate was significantly decreased at postoperative 16 weeks in cell grafted group as compared with that of corresponding relative rotation rate in control group, also with significant difference (P < 0.05).Immunohistological results showed that donor cells could differentiate into large and multi-polar dopaminergic neurons in the host body. CONCLUSION: Primary cultured embryonic MPCs can be used as the donor cells in CRT for treating PD.展开更多
Objective The successful establishment of a tumor cell bank is based on the premise that the target cells can be cultured by a legitimate approach.In this experiment,we used primary culture to select and detect breast...Objective The successful establishment of a tumor cell bank is based on the premise that the target cells can be cultured by a legitimate approach.In this experiment,we used primary culture to select and detect breast cancer cells in vitro,which can provide experimental ideas and methods for the establishment of a living tumor tissue cell bank.Methods Fifty-two specimens were collected over a two-year period from people with breast cancer who needed surgical treatment in our hospital.Cells were isolated and used to establish successful cell culture.Cell activity and cell purity were measured before liquid nitrogen cryopreservation.Results(1)At the initial culture stage,cells grew with adherence.Cell multiplication could be seen after the cell medium was exchanged three times.Cell viability was above 86%,while the viability of the target cells was above 75%,as detected by hematoxylin and eosin(HE)staining.(2)The number of breast cancer cells decreased,while the number of fibroblasts increased after five rounds of passage.(3)The success rate was 73.08%,which did not include polluted cells and those that were not successfully cryopreserved.Conclusion(1)breast cancer cells could be selected from primary culture in vitro through an appropriate method.(2)Exchange of the cell medium and further cell passage improved cell multiplication.(3)The experimental results could be monitored using trypan blue and HE staining.(4)The success of breast cancer cell culture in vitro could be used as a reference for other cell culture,so as to establish a tumor tissue cell bank.展开更多
The aim of the study was to investigate whether phosphorus(P) transporters, type IIb sodium-dependent phosphate cotransporter(NaP-IIb) and inorganic phosphate transporter 2(PiT2), were directly involved in P absorptio...The aim of the study was to investigate whether phosphorus(P) transporters, type IIb sodium-dependent phosphate cotransporter(NaP-IIb) and inorganic phosphate transporter 2(PiT2), were directly involved in P absorption across primary cultured duodenal epithelial cell monolayers of chick embryos. The siRNAs against NaP-IIb or PiT2 were designed, synthesized and transfected into primary cultured duodenal epithelial cells of chick embryos. Then, the inhibitory efficiency of siRNAs against NaP-IIb or PiT2 was analyzed, and the most efficacious siRNAs were selected to be used for subsequent P absorption experiments. Briefly, primary cultured duodenal epithelial cells of chick embryos were transfected with either NaP-IIb or PiT2 siRNAs and grown in confluent monolayers on transwell plates. The untransfected or transfected cell monolayers were then incubated in an uptake medium containing 0 or 0.25 mmol L^(–1) of P as KH_(2) PO_(4) to measure the P absorption across duodenal epithelial cell monolayers. The results showed that among the siRNAs designed, si-1372 and si-890 were demonstrated to be the most effective in inhibiting the NaPIIb and PiT2 expressions, respectively. Supplemental P increased(P=0.065) the protein abundance of PiT2 and enhanced(P<0.0001) P absorption in primary cultured duodenal epithelial cell of chick embryos. Furthermore, NaPIIb silencing decreased(P=0.07) P absorption across duodenal epithelial cell monolayers, while PiT2 silencing had no effect(P=0.345). It is concluded that the NaP-IIb, but not PiT2, might be directly involved in the P absorption of chick duodenal epithelial cells.展开更多
OBJECTIVE To establish a method for primary cultured and iden-tified Sprague-Dawley(SD) rats cerebral cortical astrocytes. METHODS Cerebral co-rtex of SD neonatal rats within 24 h was taken with stereo microscope and ...OBJECTIVE To establish a method for primary cultured and iden-tified Sprague-Dawley(SD) rats cerebral cortical astrocytes. METHODS Cerebral co-rtex of SD neonatal rats within 24 h was taken with stereo microscope and was cut topieces(1 mm^3),digested by Accutase and 0.1% DNAase(37℃,15 min),anddispersed cell suspension was made by mechanical method and filtered. The fibroblast cells and microglia were removed through differential adhesion and sha-ke. Passaged cells were identified by immunofluorescent with anti-Glial fibrillaryacidic protein(GFAP) antibody. RESULTS The astrocytes of rats cerebral cortex were cultured in this method,which had a large number of cells,good activity,high purity,abundant and elongated cell processes,and were interwoven into a network,showing a typical and good growth state. The third generation of the cel s comprised >95% astrocytes. CONCLUSION This simple and reliable cultivation method of astrocyte from rat cerebral tissue is established with high purity,and in a good growth condition.展开更多
Objective:To establish a simple and efficient culture method of primary rabbit brain microvascular endothelial cells,provide important carriers and tool cells for the research of related cerebrovascular diseases.Metho...Objective:To establish a simple and efficient culture method of primary rabbit brain microvascular endothelial cells,provide important carriers and tool cells for the research of related cerebrovascular diseases.Methods:The cerebral cortexes of rabbits were collected aseptic and inoculated after cutting,passing through cell sieve,bovine serum albumin density gradient centrifugation,typeⅡcollagenase digestion,finally inoculated and cultured.The cultured cells were identified by cell morphological observation and angiogenesis experiment.Results:Under the inverted microscope,the cells were short fusiform or polygonal,and grew in clusters and adhere to the wall.After the cells were densely fused,they would be in a typical monolayer flat,“pebbled"mosaic arrangement.Tube formation test had the ability to form tubes structure.Conclusion:This method can successfully separate and cultivate primary rabbit brain microvascular endothelial cells.展开更多
The sweet taste receptors comprised of T1r2 and T1r3, sense glucose concentrations in the gastrointestine. While hyperglycemia was reported to decrease the T1R2 and T1R3 tanscript levels in healthy subjects, no change...The sweet taste receptors comprised of T1r2 and T1r3, sense glucose concentrations in the gastrointestine. While hyperglycemia was reported to decrease the T1R2 and T1R3 tanscript levels in healthy subjects, no change was observed in type 2 diabetes patients. We investigated which glucose level and nutrients affect those transcript levels in MIN 6 and primary cultured taste buds cells using quantitative Reverse Trancription Polymerase Chain Reaction. High glucose diminished T1r2 transcript levels in MIN 6 and primary cultured taste buds cells. Resveratrol and its analogue augmented transcript levels of T1r1 and T1r2 above normal levels in MIN 6 cells in the medium with 25 mM glucose. Adenine, but not guanine, augmented T1r2 transcript levels of MIN 6 cells in the medium with 25 mM glucose. These results imply that nutrients in meals could affect sweet taste sensitivity by modulating T1r2 transcript levels in response to blood glucose levels.展开更多
Objective: To find out an optimal condition for isola-tion and primary culture of hepatocytes.Method: Rat hepatocytes were isolated by a two-stepcollagenase perfusion, and cultured in hepatozyme-SFM. The reduction of ...Objective: To find out an optimal condition for isola-tion and primary culture of hepatocytes.Method: Rat hepatocytes were isolated by a two-stepcollagenase perfusion, and cultured in hepatozyme-SFM. The reduction of MTT to formazan salt was ex-amined. Supernatant medium was collected for analy-sis of alanine aminotransferase(ALT) and ureagene-sis.Results: The two-step collagenase perfusion yielded39±12×10~6 cells/g liver tissue with a viability of88%±2%. Fine morphology and stable urea synthesisfor one week could be achieved when hepatocytes werecultured in Hepatozyme-SFM.Conclusion: High yield of hepatocytes can be isolatedwith two-step collagenese perfusion. Hepatozyme-SFM is suitable for sustained growth of hepatocytes.展开更多
The effects of bis(7) tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate induced cell injury were investigated in primary cerebral cortical neurons of rats. Exposure of cultured neurons (12 d...The effects of bis(7) tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate induced cell injury were investigated in primary cerebral cortical neurons of rats. Exposure of cultured neurons (12 days after plating) to 0.5 mmol/L glutamate for 30 min resulted in significant cell damage. Pretreatment with bis(7) tacrine (0.03 1.0 μmol/L) reduced the glutamate induced neurotoxicity in a concentration dependent manner and the maximal response was seen at 1 μmol/L with approximately 30% protection. A receptor binding assay showed that bis(7) tacrine can completely displace MK 801 binding to rat cortical membrane with an IC 50 of 0.57 μmol/L. These findings suggest that bis(7) tacrine can directly interact with N methyl D aspartate receptor channel complex, which may contribute to the inhibitor’s protective effects against glutamate induced excitotoxicity. Thus, it is possible that anti glutamate/anti AChE synergism is responsible for potentially better Alzheimer’s therapy of bis(7) tacrine relative to tacrine.展开更多
The primary routes of potential human exposure to N-nitrosodiethylamine (NDEA) are ingestion, inhalation, and dermal contact. Air, diet and smoking contribute to potential human exposure at levels of a few μg of NDEA...The primary routes of potential human exposure to N-nitrosodiethylamine (NDEA) are ingestion, inhalation, and dermal contact. Air, diet and smoking contribute to potential human exposure at levels of a few μg of NDEA/day. Potential exposure depends on the ability of the nitrosamines to migrate from the product into the body. The first step in the metabolic degradation of NDEA by cytochrome oxidase (CYPs) enzymes is the introduction of a hydroxyl group and in human esophage and liver CYP2A3 and CYP2E1 participate on this metabolism. Measuring cytotoxicity in female rat primary hepatocytes cultures, were used to understand the CYP induction and metaboli-zation correlated with low NDEA concentrations. We observed that NDEA at different concentrations in the absence of CYPs inducers, was able to induce CYP2B1, CYP2B2, CYP2E1, CYP3A1 and CYP4A3. A positive NDEA synergistic effect on the levels of mRNA, was observed in the presence of pyrazole (300 μM) for CYP2B1 and CYP2B2 and for pregnenolone 16- carbonitrile (0.15 μM) for CYP2E1. Negative NDEA synergistic effects were observed for ethanol (0.3%) for CYP3A1, pyrazol (300 μM) for CYP2A1 and CYP2E1, and phenobarbital (1 mM) for CYP2A1. These facts are extremally important once that these metabolites can be directly related to the primary DNA lesions. We consider that studies to elucidate the biological factors that determine the shape of the dose-response curve are crucial for low-dose extrapolations of risk.展开更多
Satellite glial cells surround neurons within dorsal root ganglia. Previous studies have focused on single-cell suspensions of cultured neurons derived from rat dorsal root ganglia. At present, the primary culture met...Satellite glial cells surround neurons within dorsal root ganglia. Previous studies have focused on single-cell suspensions of cultured neurons derived from rat dorsal root ganglia. At present, the primary culture method for satellite glial cells derived from rat dorsal root ganglia requires no digestion skill. Hence, the aim of the present study was to establish a novel primary culture method for satellite glial cells derived from dorsal root ganglia. Neonatal rat spine was collected and an incision made to expose the transverse protrusion and remove dorsal root ganglia. Dorsal root ganglia were freed from nerve fibers, connective tissue, and capsule membranes, then rinsed and transferred to 6-well plates, and cultured in a humidified 5% CO_2 incubator at 37°C. After 3 days in culture, some cells had migrated from dorsal root ganglia. After subculture, cells were identified by immunofluorescence labeling for three satellite glial cell-specific markers: glutamine synthetase, glial fibrillary acidic protein, and S100β. Cultured cells expressed glutamine synthetase, glial fibrillary acidic protein, and S100β, suggesting they are satellite glial cells with a purity of > 95%. Thus, we have successfully established a novel primary culture method for obtaining high-purity satellite glial cells from rat dorsal root ganglia without digestion.展开更多
Craniopharynigoma samples were collected from 36 patients. Out of the 36 samples, 29 achieved successful sub-culturing, with a success rate of 80.6%. Immunohistochemistry staining showed that cytokeratin-7 was positiv...Craniopharynigoma samples were collected from 36 patients. Out of the 36 samples, 29 achieved successful sub-culturing, with a success rate of 80.6%. Immunohistochemistry staining showed that cytokeratin-7 was positively expressed in the cytomembrane and cytoplasm of craniopharyngioma cells at 6-8 passages, confirming that all cultured cells were squamous epithelial cells. The doubling time of craniopharyngioma cells was 3 days, as confirmed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In this study, craniopharyngioma cells cultured in vitro were established; however, establishment of immortalized craniopharyngioma cell lines requires further research.展开更多
Objective To investigate the protective effects of quercetin on cadmium-induced cytotoxicity in primary cultures of rat proximal tubular(rPT)cells.Methods Primary cultures of rPT cells undergoing exponential growth we...Objective To investigate the protective effects of quercetin on cadmium-induced cytotoxicity in primary cultures of rat proximal tubular(rPT)cells.Methods Primary cultures of rPT cells undergoing exponential growth were incubated with 1.0 μg/mL quercetin and/or cadmium(2.5,5.0 μmol/L),in a serum-free medium at 37 ℃ at different time intervals.Commercial kits were used and flow cytometric analyses were performed on rPT cell cultures to assay apoptosis and oxidative stress.Results Exposure of rPT cells to cadmium acetate(2.5,5.0 μmol/L)induced a decrease in cell viability,caused an increase in apoptotic rate and apoptotic morphological changes.Simultaneously,elevation of intracellular reactive oxygen species,malondialdehyde and calcium levels,depletion of mitochondrial membrane potential and intracellular glutathione,and inhibition of Na +,K +-ATPase,Ca 2+-ATPase,glutathione peroxidase(GSH-Px),catalase(CAT),and superoxide dismutase(SOD)activities were revealed during the cadmium exposure of rPT cells.However,simultaneous supplementation with 1 μg/mL quercetin protected rPT cells against cadmium-induced cytotoxicity through inhibiting apoptosis,attenuating lipid peroxidation,renewing mitochondrial function and elevating the intracellular antioxidants(non-enzymatic and enzymic)levels.Conclusion The present study has suggested that quercetin,as a widely distributed dietary antioxidant,contributes potentially to prevent cadmium-induced cytotoxicity in rPT cells.展开更多
Objective To investigate the development and characterizations of the hepatocytes isolated from fetal ovine and to determine the effect of hypoxia on their growth and metabolism.Methods Fresh hepatocytes were isolated...Objective To investigate the development and characterizations of the hepatocytes isolated from fetal ovine and to determine the effect of hypoxia on their growth and metabolism.Methods Fresh hepatocytes were isolated from the liver of fetal ovine at late gestation, cultured in specific media, and exposed to normoxia(21% O2) or hypoxia(2% O2).The cellular characteristics and population purity were identified by immunocytochemistry and flow cytometry(FCM).The effects of hypoxia on cell cycle and apoptosis of the hepatocytes were evaluated by FCM, whereas the cellular ultrastructure changes were examined with a transmission electron microscope.Results The cell purity of hepatocytes was over 95%.Under hypoxia exposure, the hepatocytes showed a gradual increase in proportion at the S phase and in proliferative index, followed with a compatible increase in apoptosis and progressively decreased cell viability.Additionally, the organelles of the hepatocytes demonstrated dramatic changes, including swelling of mitochondria, disorder in cristae arrangement, expansion of endoplasmic reticulum, and a large number of circular lipid droplets emerging in the cytoplasm.Conclusion Fetal ovine hepatocytes could be primarily cultured in a short-term culture system with a high purity of over 95% and with their preserved original characteristics.Hypoxia could induce changes in ultrastructural and inhibit the proliferation of cultured fetal ovine hepatocytes through apoptotic mechanisms.展开更多
To establish a better method of primary culture for alveolar epithelial type Ⅱ cells (AEC Ⅱ) and to study its bionomics, alveolar epithelial type Ⅱ cells were isolated by digestion with tryp- sin and collagenase, w...To establish a better method of primary culture for alveolar epithelial type Ⅱ cells (AEC Ⅱ) and to study its bionomics, alveolar epithelial type Ⅱ cells were isolated by digestion with tryp- sin and collagenase, which were then purified by plated into culture flask coated with rat immu- noglobulin G. The purified AEC Ⅱ were identified by alkaline phosphatase staining, electron mi- croscopy, immunocytochemical staining of pulmonary surfactant protein A (SPA). The SPA expres- sion and transfection characteristics were compared with those of A549 cell line. The results showed that AEC Ⅱ could be isolated by digestion with trysin and collagenase and purified by adhesive pu- rification by using IgG, with a yield of about 2–3×107, and a purity of about 75%–84 %. Cells could be quickly identified with AKP staining. AECⅡ were different from A549 cell line in terms of SPA expression and transfection characteristics. It is concluded that adhesive purification with IgG can improve the purity of AECⅡ, and AKP staining is simple in cell identification. AECⅡ can not be completely replaced by A549 cells in some studies because the differences between them, such as SPA expression.展开更多
BACKGROUND: Evidence illustrates that androgen has a neuroprotective role.However,whether androgen also has the protective effect on hippocampal neurons during free radical mediated injury remains unclear.OBJECTIVE: T...BACKGROUND: Evidence illustrates that androgen has a neuroprotective role.However,whether androgen also has the protective effect on hippocampal neurons during free radical mediated injury remains unclear.OBJECTIVE: To investigate the neuroprotective effect of androgen on hippocampal neurons during free radical damage.DESIGN,TIME AND SETTING: A controlled in vitro experiment was performed at the Department of Human Anatomy,Cell Culture Lab,and Neuroendocrinology Lab,Basic Medical School,Hebei Medical University from February to June 2009.MATERIALS: Testosterone was provided by Tianjin Jinyao Amino Acid Company,China.METHODS: Primary cultured neurons from 24 Sprague Dawley rats were randomly assigned into four groups: control,H2O2,testosterone,and testosterone (pre-added) plus H2O2 groups.MAIN OUTCOME MEASURES: The positive cell ratio of microtubule associated protein-II and neuron specific enolase was determined by immunocytochemistry.Neuronal morphology was observed by hematoxylin-eosin staining and Nissl staining.Cell vitality and viability were determined using an inverted phase contrast microscope.The content of nitric oxide synthase,malondialdehyde,and superoxide dismutase were measured with a spectrophotometer.RESULTS: As compared with the control group,cell vitality and viability,and superoxide dismutase level were significantly decreased in the H2O2 group (P < 0.05),while nitric oxide synthase and malondialdehyde levels were significantly increased (P < 0.05).Neuronal vitality and viability as well as superoxide dismutase level in the testosterone plus H2O2 group were significantly greater than in the H2O2 group (P < 0.05),and nitric oxide synthase and malondialdehyde levels were significantly less than in the H2O2 group (P < 0.05).CONCLUSION: Androgen partially reversed H2O2-induced neuronal damage and protected neurons.展开更多
Integrins mediate cell adhesion to the extracellular matrix (ECM). In particular, integrin alphavbeta3 recognizes the RGD motif as a ligand-binding site on various extracellular molecules of the extracellular matrix. ...Integrins mediate cell adhesion to the extracellular matrix (ECM). In particular, integrin alphavbeta3 recognizes the RGD motif as a ligand-binding site on various extracellular molecules of the extracellular matrix. Integrin aphavbeta3 has been associated with high malignant potential in breast cancer cells, and has signalized the onset of widespread metastasis. In recent years, several antagonists of integrin alphavbeta3, including snake venom disintegrins, have been used as potential anti-cancer agents. In the present work, the effect of contortrostatin, a disintegrin isolated from the venom of the snake Agkistrodon contortrix, was studied on primary cultures of human breast cancer cell. Scanning and transmission electron microscopy were employed in order to examine alterations in cell morphology and fluorescent microscopy and visualize changes in distribution of integrin alphavbeta3 and talin. Fluorescent localization of caspase 8 was made in order to visualize any sign of proapoptotosis and western immunoblotting of integrin, talin and annexin was undertaken in order to identify changes. The results suggest that the snake venom contortrostatin seriously affects cell morphology, adhesion and mobility and induces breast cancer cells to apoptosis.展开更多
The aim of this study was to investigate the cytotoxicity of modified nonequilibrium plasma with chlorhexidine digluconate(CHX) on human gingival fibroblasts(HGFs), and to evaluate the biosecurity of modified nonequil...The aim of this study was to investigate the cytotoxicity of modified nonequilibrium plasma with chlorhexidine digluconate(CHX) on human gingival fibroblasts(HGFs), and to evaluate the biosecurity of modified nonequilibrium plasma with 2% CHX as a new method of root canal treatment. Tissue samples taken from human gingiva were primarily cultured and passaged. Cells from passages 3–7 were used. HGFs were treated by modified nonequilibrium plasma with 2% CHX for 0 min(control group), 30 s, 1 min, 1.5 min, 3 min, 5 min, and 10 min, respectively, and then they were incubated for 0, 24, and 48 h. After that, cell counting kit-8(CCK-8) assay was applied to analyze the cytotoxicity of modified nonequilibrium plasma with 2% CHX on HGFs. There was no significant difference between the 0 h group treated with the modified nonequilibrium plasma for 1 min and the control group(P>0.05). However, there were significant differences between all the other treated groups and the control group(P<0.05). When treated for 1.5 min or shorter, the cell viability was obviously increased; while treated for 3 min or longer, it was obviously reduced. Moreover, when successively cultured for 0, 24, and 48 h, cell viability was decreased at first and then increased in the 3-min-treated and 5-min-treated groups. The modified nonequilibrium plasma with 2% CHX was of no influence on cell viability in 1.5 min treatment, and it could be safely used on root canal treatment.展开更多
BACKGROUND: It is the key point for vascular endothelialgrowth factor (VEGF121) gene related therapy as to how totransfect and express the gene safely, effectively and repeat-edly. This study was designed to investiga...BACKGROUND: It is the key point for vascular endothelialgrowth factor (VEGF121) gene related therapy as to how totransfect and express the gene safely, effectively and repeat-edly. This study was designed to investigate the VEGF121transfection and expression in primary cultured rat hepato-cyte.METHODS: After construction of vector internal ribosomeentry site-enhanced yellow fluorescent protein (pIRES-EY-FP)/VEGF121, the transfection and expression of the exoge-nous VEGF121 gene in primary cultured rat hepatocyteswere observed through RT-PCR, Western blot and fluores-cent microscopy.RESULTS: pIRES-EYFP/VEGF121 plasmid was construct-ed and transfected successfully into primary cultured rathepatocytes, the transfection and expression of gene in pri-mary cultured rat hepatocytes were examined by RT-PCRand Western blot, and yellow-green fluorescence was ob-served through a fluorescent microscope.CONCLUSION: The successful transfection and expressionof plasmid pIRES-EYFP/VEGF121 in primary cultured rathepatocytes provides a foundation for hepatocyte transplan-tation and gene therapy after modification of hepatocytesby the gene.展开更多
基金supported by the Key Program of the National Natural Science Foundation of China(31630073)the Initiation Funds of Yangzhou University for Distinguished Scientists,Chinathe Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences(ASTIP-IAS09)。
文摘Osteoblasts are considered as a major factor contributing to bone development and mineralization,however,few studies have been done to establish and evaluate the primary cultured tibial osteoblast model of broiler chicks.Therefore,in the present study,two experiments were conducted to establish and evaluate the primary cultured tibial osteoblast model of broiler chicks.In experiment 1,osteoblasts were isolated from the tibia of one-day-old Arbor Acre male broiler chicks using the explant method and identified through the cell morphology,alkaline phosphatase(ALP)and alizarin red staining.Experiment 2 was carried out to evaluate the vitality and mineralization of primary cultured tibial osteoblasts of broilers on days 4,8,12,16,20,24,28 and 32 after incubation,respectively.The results from experiment 1 demonstrated that primary cultured tibial osteoblasts of broilers showed a spindle-shaped,triangular or polygonal morphology.More than 95%of the cells were stained blue-black after ALP staining,and mineralized nodules were formed after 4 days of continuous incubation.In experiment 2,lactate dehydrogenase(LDH)activity stayed at a relatively stabilized level although incubation time affected(P=0.0012)it during the whole culture period.Additionally,incubation time affected(P≤0.0001)the number and proportion of the area of mineralized nodules.They increased linearly and quadratically(P<0.04)with the increase of incubation time,and remained at a stabilized level from 24 to 32 days of incubation.The estimates of the optimal incubation time were 17 and 26 days based on the best fitted broken-line or quadratic models(P<0.0001)of the number and proportion of the area of mineralized nodules,respectively.These results indicate that the primary cultured tibial osteoblast model of broilers has been established successfully by the explant method,and it showed typical osteoblast morphology and characteristics of ALP activity and mineralization,and could maintain a relatively stabilized vitality from 4 to 32 days of incubation;and the optimal incubation time of primary tibial osteoblasts was 17 to 26 days.Therefore,it could be used to further study the underlying mechanisms of bone development and mineralization of broiler chicks.
文摘BACKGROUND: Choosing proper donor cells is one of keys in experimental and clinical studies on cell replacement therapy (CRT) for treating Parkinson disease (PD). Embryonic mesencephalic precursor cells (MPCs) can stably differentiate into dopaminergic neuron after in vitro proliferated culture. As compared with embryonic stem cell and neural stem cell strains, cell composition of embryonic MPCs after primary culture is also the most close to that of embryonic mesencephalic ventral cell suspension without proliferated culture. Successful experience accumulated in the latter suggests that primary cultured embryonic MPCs might be the most potential donor cells in clinical application with CRT for treating PD so far. OBJECTIVE: To investigate the feasibility of primary cultured embryonic precursor cells cultured primarily as donor cells in CRT for treating PD in rats. DESIGN: A randomized and controlled trial taking SD rats as experimental animals. SETTING: Department of Neurosurgery, Huashan Hospital Affiliated to Fudan University. MATERIALS: This experiment was carried out at the Institute of Neuroscience, Shanghai Institute for Biological Science, Chinese Academy of Sciences from July 2003 to June 2004. Totally 26 female SD rats, with body mass of 200 to 220 g, were provided by Shanghai Experimental Animal Center of Chinese Academy of Sciences. METHODS: Stereotaxic injection of 6-hydroxydopamine into the medial forebrain bundle were perfored to develop PD model rat. Among 26 SD rats, 20 rats achieved a more than 5 turns/min in apomorphine induced rotation test, reaching the standard of PD model rats. Immunohistochemical detection was performed on 1 out of 20 model rats after execution, and the other 19 rats were randomly divided into control group (n=5), sham transplantation group (n=5)and cell grafted group (n=9). Primary cultured E12 MPC cell suspension (1.2×1011 L-1)were used as donor cells. 4 μL primary cultured E12 MPC cell suspension prepared freshly was injected into the lesioned corpus striatum of rats in cell grafted group, and 4 μL D-Hank’s solution was injected in sham transplantation group in the same way. There was no injection in control group. Apomorphine-induced rotation rate of PD rats were recorded respectively in cell grafted group and sham transplantation group pre-operation (initial value) and at postoperative 2, 4, 6 and 16 weeks. Apomorphine-induced rotation rate of PD rats was recorded in control group at postoperative 2 months (initial value) and following 2,4,6 and 16 weeks. To determine TH antigen with immunohistological ABC method (DAB developing) at 6 months post-transplantation to investigate the differentiation and survival of donor cells in the host body. MAIN OUTCOME MEASURES: Apomorphine-induced rotation behavior before and after transplantation and the survival and differentiation of implanted cells in the host body at 6 months post-transplantation. RESULTS: Among 19 model rats, one rat died after transplantation respectively in the cell grafted group and sham transplantation group; finally 17 model rats entered the stage of result analysis. Relative apomorphine-induced rotation rate was significantly decreased in the cell grafted group as compared with that before transplantation, with significant difference (P < 0.01,P < 0.05);the mean value of relative apomorphine-induced rotation rate was significantly decreased at postoperative 16 weeks in cell grafted group as compared with that of corresponding relative rotation rate in control group, also with significant difference (P < 0.05).Immunohistological results showed that donor cells could differentiate into large and multi-polar dopaminergic neurons in the host body. CONCLUSION: Primary cultured embryonic MPCs can be used as the donor cells in CRT for treating PD.
基金a grant from the Science Foundation of Inner Mongolia Autonomous Region People’s Hospital(No.2016015).
文摘Objective The successful establishment of a tumor cell bank is based on the premise that the target cells can be cultured by a legitimate approach.In this experiment,we used primary culture to select and detect breast cancer cells in vitro,which can provide experimental ideas and methods for the establishment of a living tumor tissue cell bank.Methods Fifty-two specimens were collected over a two-year period from people with breast cancer who needed surgical treatment in our hospital.Cells were isolated and used to establish successful cell culture.Cell activity and cell purity were measured before liquid nitrogen cryopreservation.Results(1)At the initial culture stage,cells grew with adherence.Cell multiplication could be seen after the cell medium was exchanged three times.Cell viability was above 86%,while the viability of the target cells was above 75%,as detected by hematoxylin and eosin(HE)staining.(2)The number of breast cancer cells decreased,while the number of fibroblasts increased after five rounds of passage.(3)The success rate was 73.08%,which did not include polluted cells and those that were not successfully cryopreserved.Conclusion(1)breast cancer cells could be selected from primary culture in vitro through an appropriate method.(2)Exchange of the cell medium and further cell passage improved cell multiplication.(3)The experimental results could be monitored using trypan blue and HE staining.(4)The success of breast cancer cell culture in vitro could be used as a reference for other cell culture,so as to establish a tumor tissue cell bank.
基金supported by the Key Program of the National Natural Science Foundation of China(31630073)the National Natural Science Foundation of China(31472116)+2 种基金the National Key R&D Program of China(2017YFD0502200)the China Agriculture Research System of MOF and MARA(CARS-41)the Agricultural Science and Technology Innovation Program(ASTIP-IAS09)。
文摘The aim of the study was to investigate whether phosphorus(P) transporters, type IIb sodium-dependent phosphate cotransporter(NaP-IIb) and inorganic phosphate transporter 2(PiT2), were directly involved in P absorption across primary cultured duodenal epithelial cell monolayers of chick embryos. The siRNAs against NaP-IIb or PiT2 were designed, synthesized and transfected into primary cultured duodenal epithelial cells of chick embryos. Then, the inhibitory efficiency of siRNAs against NaP-IIb or PiT2 was analyzed, and the most efficacious siRNAs were selected to be used for subsequent P absorption experiments. Briefly, primary cultured duodenal epithelial cells of chick embryos were transfected with either NaP-IIb or PiT2 siRNAs and grown in confluent monolayers on transwell plates. The untransfected or transfected cell monolayers were then incubated in an uptake medium containing 0 or 0.25 mmol L^(–1) of P as KH_(2) PO_(4) to measure the P absorption across duodenal epithelial cell monolayers. The results showed that among the siRNAs designed, si-1372 and si-890 were demonstrated to be the most effective in inhibiting the NaPIIb and PiT2 expressions, respectively. Supplemental P increased(P=0.065) the protein abundance of PiT2 and enhanced(P<0.0001) P absorption in primary cultured duodenal epithelial cell of chick embryos. Furthermore, NaPIIb silencing decreased(P=0.07) P absorption across duodenal epithelial cell monolayers, while PiT2 silencing had no effect(P=0.345). It is concluded that the NaP-IIb, but not PiT2, might be directly involved in the P absorption of chick duodenal epithelial cells.
文摘OBJECTIVE To establish a method for primary cultured and iden-tified Sprague-Dawley(SD) rats cerebral cortical astrocytes. METHODS Cerebral co-rtex of SD neonatal rats within 24 h was taken with stereo microscope and was cut topieces(1 mm^3),digested by Accutase and 0.1% DNAase(37℃,15 min),anddispersed cell suspension was made by mechanical method and filtered. The fibroblast cells and microglia were removed through differential adhesion and sha-ke. Passaged cells were identified by immunofluorescent with anti-Glial fibrillaryacidic protein(GFAP) antibody. RESULTS The astrocytes of rats cerebral cortex were cultured in this method,which had a large number of cells,good activity,high purity,abundant and elongated cell processes,and were interwoven into a network,showing a typical and good growth state. The third generation of the cel s comprised >95% astrocytes. CONCLUSION This simple and reliable cultivation method of astrocyte from rat cerebral tissue is established with high purity,and in a good growth condition.
基金This study was supported by the National Natural Science Foundation of China(81072714)the Natural Science Foundation of Fujian Province,China(No.2017J01545)。
文摘Objective:To establish a simple and efficient culture method of primary rabbit brain microvascular endothelial cells,provide important carriers and tool cells for the research of related cerebrovascular diseases.Methods:The cerebral cortexes of rabbits were collected aseptic and inoculated after cutting,passing through cell sieve,bovine serum albumin density gradient centrifugation,typeⅡcollagenase digestion,finally inoculated and cultured.The cultured cells were identified by cell morphological observation and angiogenesis experiment.Results:Under the inverted microscope,the cells were short fusiform or polygonal,and grew in clusters and adhere to the wall.After the cells were densely fused,they would be in a typical monolayer flat,“pebbled"mosaic arrangement.Tube formation test had the ability to form tubes structure.Conclusion:This method can successfully separate and cultivate primary rabbit brain microvascular endothelial cells.
文摘The sweet taste receptors comprised of T1r2 and T1r3, sense glucose concentrations in the gastrointestine. While hyperglycemia was reported to decrease the T1R2 and T1R3 tanscript levels in healthy subjects, no change was observed in type 2 diabetes patients. We investigated which glucose level and nutrients affect those transcript levels in MIN 6 and primary cultured taste buds cells using quantitative Reverse Trancription Polymerase Chain Reaction. High glucose diminished T1r2 transcript levels in MIN 6 and primary cultured taste buds cells. Resveratrol and its analogue augmented transcript levels of T1r1 and T1r2 above normal levels in MIN 6 cells in the medium with 25 mM glucose. Adenine, but not guanine, augmented T1r2 transcript levels of MIN 6 cells in the medium with 25 mM glucose. These results imply that nutrients in meals could affect sweet taste sensitivity by modulating T1r2 transcript levels in response to blood glucose levels.
文摘Objective: To find out an optimal condition for isola-tion and primary culture of hepatocytes.Method: Rat hepatocytes were isolated by a two-stepcollagenase perfusion, and cultured in hepatozyme-SFM. The reduction of MTT to formazan salt was ex-amined. Supernatant medium was collected for analy-sis of alanine aminotransferase(ALT) and ureagene-sis.Results: The two-step collagenase perfusion yielded39±12×10~6 cells/g liver tissue with a viability of88%±2%. Fine morphology and stable urea synthesisfor one week could be achieved when hepatocytes werecultured in Hepatozyme-SFM.Conclusion: High yield of hepatocytes can be isolatedwith two-step collagenese perfusion. Hepatozyme-SFM is suitable for sustained growth of hepatocytes.
文摘The effects of bis(7) tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate induced cell injury were investigated in primary cerebral cortical neurons of rats. Exposure of cultured neurons (12 days after plating) to 0.5 mmol/L glutamate for 30 min resulted in significant cell damage. Pretreatment with bis(7) tacrine (0.03 1.0 μmol/L) reduced the glutamate induced neurotoxicity in a concentration dependent manner and the maximal response was seen at 1 μmol/L with approximately 30% protection. A receptor binding assay showed that bis(7) tacrine can completely displace MK 801 binding to rat cortical membrane with an IC 50 of 0.57 μmol/L. These findings suggest that bis(7) tacrine can directly interact with N methyl D aspartate receptor channel complex, which may contribute to the inhibitor’s protective effects against glutamate induced excitotoxicity. Thus, it is possible that anti glutamate/anti AChE synergism is responsible for potentially better Alzheimer’s therapy of bis(7) tacrine relative to tacrine.
文摘The primary routes of potential human exposure to N-nitrosodiethylamine (NDEA) are ingestion, inhalation, and dermal contact. Air, diet and smoking contribute to potential human exposure at levels of a few μg of NDEA/day. Potential exposure depends on the ability of the nitrosamines to migrate from the product into the body. The first step in the metabolic degradation of NDEA by cytochrome oxidase (CYPs) enzymes is the introduction of a hydroxyl group and in human esophage and liver CYP2A3 and CYP2E1 participate on this metabolism. Measuring cytotoxicity in female rat primary hepatocytes cultures, were used to understand the CYP induction and metaboli-zation correlated with low NDEA concentrations. We observed that NDEA at different concentrations in the absence of CYPs inducers, was able to induce CYP2B1, CYP2B2, CYP2E1, CYP3A1 and CYP4A3. A positive NDEA synergistic effect on the levels of mRNA, was observed in the presence of pyrazole (300 μM) for CYP2B1 and CYP2B2 and for pregnenolone 16- carbonitrile (0.15 μM) for CYP2E1. Negative NDEA synergistic effects were observed for ethanol (0.3%) for CYP3A1, pyrazol (300 μM) for CYP2A1 and CYP2E1, and phenobarbital (1 mM) for CYP2A1. These facts are extremally important once that these metabolites can be directly related to the primary DNA lesions. We consider that studies to elucidate the biological factors that determine the shape of the dose-response curve are crucial for low-dose extrapolations of risk.
基金supported by the National Natural Science Foundation of China,No.31560295(to LYL)the Priority Union Foundation of Yunnan Department of Science and Technology and Kunming Medical University of China,No.2015FB098(to JHG)+1 种基金the Project of Major Scientific and Technological Achievements Cultivation of Kunming Medical University of China,No.CGPY201802(to LYL)the Health Science and Technology Plan Projects of Yunnan Province of China,No.2014NS202(to JHG)
文摘Satellite glial cells surround neurons within dorsal root ganglia. Previous studies have focused on single-cell suspensions of cultured neurons derived from rat dorsal root ganglia. At present, the primary culture method for satellite glial cells derived from rat dorsal root ganglia requires no digestion skill. Hence, the aim of the present study was to establish a novel primary culture method for satellite glial cells derived from dorsal root ganglia. Neonatal rat spine was collected and an incision made to expose the transverse protrusion and remove dorsal root ganglia. Dorsal root ganglia were freed from nerve fibers, connective tissue, and capsule membranes, then rinsed and transferred to 6-well plates, and cultured in a humidified 5% CO_2 incubator at 37°C. After 3 days in culture, some cells had migrated from dorsal root ganglia. After subculture, cells were identified by immunofluorescence labeling for three satellite glial cell-specific markers: glutamine synthetase, glial fibrillary acidic protein, and S100β. Cultured cells expressed glutamine synthetase, glial fibrillary acidic protein, and S100β, suggesting they are satellite glial cells with a purity of > 95%. Thus, we have successfully established a novel primary culture method for obtaining high-purity satellite glial cells from rat dorsal root ganglia without digestion.
基金supported by the National Natural Science Foundation of China, No. 30872646 and 30973082
文摘Craniopharynigoma samples were collected from 36 patients. Out of the 36 samples, 29 achieved successful sub-culturing, with a success rate of 80.6%. Immunohistochemistry staining showed that cytokeratin-7 was positively expressed in the cytomembrane and cytoplasm of craniopharyngioma cells at 6-8 passages, confirming that all cultured cells were squamous epithelial cells. The doubling time of craniopharyngioma cells was 3 days, as confirmed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In this study, craniopharyngioma cells cultured in vitro were established; however, establishment of immortalized craniopharyngioma cell lines requires further research.
基金supported by the National Nature Science Foundation of China (No. 31101870)Shandong Provincial Natural Science Foundation of China (No.ZR2010CQ014)
文摘Objective To investigate the protective effects of quercetin on cadmium-induced cytotoxicity in primary cultures of rat proximal tubular(rPT)cells.Methods Primary cultures of rPT cells undergoing exponential growth were incubated with 1.0 μg/mL quercetin and/or cadmium(2.5,5.0 μmol/L),in a serum-free medium at 37 ℃ at different time intervals.Commercial kits were used and flow cytometric analyses were performed on rPT cell cultures to assay apoptosis and oxidative stress.Results Exposure of rPT cells to cadmium acetate(2.5,5.0 μmol/L)induced a decrease in cell viability,caused an increase in apoptotic rate and apoptotic morphological changes.Simultaneously,elevation of intracellular reactive oxygen species,malondialdehyde and calcium levels,depletion of mitochondrial membrane potential and intracellular glutathione,and inhibition of Na +,K +-ATPase,Ca 2+-ATPase,glutathione peroxidase(GSH-Px),catalase(CAT),and superoxide dismutase(SOD)activities were revealed during the cadmium exposure of rPT cells.However,simultaneous supplementation with 1 μg/mL quercetin protected rPT cells against cadmium-induced cytotoxicity through inhibiting apoptosis,attenuating lipid peroxidation,renewing mitochondrial function and elevating the intracellular antioxidants(non-enzymatic and enzymic)levels.Conclusion The present study has suggested that quercetin,as a widely distributed dietary antioxidant,contributes potentially to prevent cadmium-induced cytotoxicity in rPT cells.
基金funded by National Natural Science Foundation [81370719 and 81671535]Jiangsu Science Foundation [BE2015642]+3 种基金Jiangsu Key Discipline of Human Assisted Reproduction Medicine Foundation [FXK201749]Jiangsu Provincial Medical Talent of the Project of Invigorating Healthcare through Science,Technology and Education [ZDRCA2016044]and Chinese Medical Association Clinical Medicine Research Reproductive Medicine [17020270696]The Priority Academic Program Development of the Jiangsu Higher Education Institutes(PAPD)
文摘Objective To investigate the development and characterizations of the hepatocytes isolated from fetal ovine and to determine the effect of hypoxia on their growth and metabolism.Methods Fresh hepatocytes were isolated from the liver of fetal ovine at late gestation, cultured in specific media, and exposed to normoxia(21% O2) or hypoxia(2% O2).The cellular characteristics and population purity were identified by immunocytochemistry and flow cytometry(FCM).The effects of hypoxia on cell cycle and apoptosis of the hepatocytes were evaluated by FCM, whereas the cellular ultrastructure changes were examined with a transmission electron microscope.Results The cell purity of hepatocytes was over 95%.Under hypoxia exposure, the hepatocytes showed a gradual increase in proportion at the S phase and in proliferative index, followed with a compatible increase in apoptosis and progressively decreased cell viability.Additionally, the organelles of the hepatocytes demonstrated dramatic changes, including swelling of mitochondria, disorder in cristae arrangement, expansion of endoplasmic reticulum, and a large number of circular lipid droplets emerging in the cytoplasm.Conclusion Fetal ovine hepatocytes could be primarily cultured in a short-term culture system with a high purity of over 95% and with their preserved original characteristics.Hypoxia could induce changes in ultrastructural and inhibit the proliferation of cultured fetal ovine hepatocytes through apoptotic mechanisms.
基金the National Natural Sciences Foundation of China (No. 30500224 and No. 30400193).
文摘To establish a better method of primary culture for alveolar epithelial type Ⅱ cells (AEC Ⅱ) and to study its bionomics, alveolar epithelial type Ⅱ cells were isolated by digestion with tryp- sin and collagenase, which were then purified by plated into culture flask coated with rat immu- noglobulin G. The purified AEC Ⅱ were identified by alkaline phosphatase staining, electron mi- croscopy, immunocytochemical staining of pulmonary surfactant protein A (SPA). The SPA expres- sion and transfection characteristics were compared with those of A549 cell line. The results showed that AEC Ⅱ could be isolated by digestion with trysin and collagenase and purified by adhesive pu- rification by using IgG, with a yield of about 2–3×107, and a purity of about 75%–84 %. Cells could be quickly identified with AKP staining. AECⅡ were different from A549 cell line in terms of SPA expression and transfection characteristics. It is concluded that adhesive purification with IgG can improve the purity of AECⅡ, and AKP staining is simple in cell identification. AECⅡ can not be completely replaced by A549 cells in some studies because the differences between them, such as SPA expression.
文摘BACKGROUND: Evidence illustrates that androgen has a neuroprotective role.However,whether androgen also has the protective effect on hippocampal neurons during free radical mediated injury remains unclear.OBJECTIVE: To investigate the neuroprotective effect of androgen on hippocampal neurons during free radical damage.DESIGN,TIME AND SETTING: A controlled in vitro experiment was performed at the Department of Human Anatomy,Cell Culture Lab,and Neuroendocrinology Lab,Basic Medical School,Hebei Medical University from February to June 2009.MATERIALS: Testosterone was provided by Tianjin Jinyao Amino Acid Company,China.METHODS: Primary cultured neurons from 24 Sprague Dawley rats were randomly assigned into four groups: control,H2O2,testosterone,and testosterone (pre-added) plus H2O2 groups.MAIN OUTCOME MEASURES: The positive cell ratio of microtubule associated protein-II and neuron specific enolase was determined by immunocytochemistry.Neuronal morphology was observed by hematoxylin-eosin staining and Nissl staining.Cell vitality and viability were determined using an inverted phase contrast microscope.The content of nitric oxide synthase,malondialdehyde,and superoxide dismutase were measured with a spectrophotometer.RESULTS: As compared with the control group,cell vitality and viability,and superoxide dismutase level were significantly decreased in the H2O2 group (P < 0.05),while nitric oxide synthase and malondialdehyde levels were significantly increased (P < 0.05).Neuronal vitality and viability as well as superoxide dismutase level in the testosterone plus H2O2 group were significantly greater than in the H2O2 group (P < 0.05),and nitric oxide synthase and malondialdehyde levels were significantly less than in the H2O2 group (P < 0.05).CONCLUSION: Androgen partially reversed H2O2-induced neuronal damage and protected neurons.
文摘Integrins mediate cell adhesion to the extracellular matrix (ECM). In particular, integrin alphavbeta3 recognizes the RGD motif as a ligand-binding site on various extracellular molecules of the extracellular matrix. Integrin aphavbeta3 has been associated with high malignant potential in breast cancer cells, and has signalized the onset of widespread metastasis. In recent years, several antagonists of integrin alphavbeta3, including snake venom disintegrins, have been used as potential anti-cancer agents. In the present work, the effect of contortrostatin, a disintegrin isolated from the venom of the snake Agkistrodon contortrix, was studied on primary cultures of human breast cancer cell. Scanning and transmission electron microscopy were employed in order to examine alterations in cell morphology and fluorescent microscopy and visualize changes in distribution of integrin alphavbeta3 and talin. Fluorescent localization of caspase 8 was made in order to visualize any sign of proapoptotosis and western immunoblotting of integrin, talin and annexin was undertaken in order to identify changes. The results suggest that the snake venom contortrostatin seriously affects cell morphology, adhesion and mobility and induces breast cancer cells to apoptosis.
基金supported by grants from the National Natural Science Foundation of China(No.81271189)the Hubei Provincial Science and Technology Support Program of China(No.2015BCE058)
文摘The aim of this study was to investigate the cytotoxicity of modified nonequilibrium plasma with chlorhexidine digluconate(CHX) on human gingival fibroblasts(HGFs), and to evaluate the biosecurity of modified nonequilibrium plasma with 2% CHX as a new method of root canal treatment. Tissue samples taken from human gingiva were primarily cultured and passaged. Cells from passages 3–7 were used. HGFs were treated by modified nonequilibrium plasma with 2% CHX for 0 min(control group), 30 s, 1 min, 1.5 min, 3 min, 5 min, and 10 min, respectively, and then they were incubated for 0, 24, and 48 h. After that, cell counting kit-8(CCK-8) assay was applied to analyze the cytotoxicity of modified nonequilibrium plasma with 2% CHX on HGFs. There was no significant difference between the 0 h group treated with the modified nonequilibrium plasma for 1 min and the control group(P>0.05). However, there were significant differences between all the other treated groups and the control group(P<0.05). When treated for 1.5 min or shorter, the cell viability was obviously increased; while treated for 3 min or longer, it was obviously reduced. Moreover, when successively cultured for 0, 24, and 48 h, cell viability was decreased at first and then increased in the 3-min-treated and 5-min-treated groups. The modified nonequilibrium plasma with 2% CHX was of no influence on cell viability in 1.5 min treatment, and it could be safely used on root canal treatment.
文摘BACKGROUND: It is the key point for vascular endothelialgrowth factor (VEGF121) gene related therapy as to how totransfect and express the gene safely, effectively and repeat-edly. This study was designed to investigate the VEGF121transfection and expression in primary cultured rat hepato-cyte.METHODS: After construction of vector internal ribosomeentry site-enhanced yellow fluorescent protein (pIRES-EY-FP)/VEGF121, the transfection and expression of the exoge-nous VEGF121 gene in primary cultured rat hepatocyteswere observed through RT-PCR, Western blot and fluores-cent microscopy.RESULTS: pIRES-EYFP/VEGF121 plasmid was construct-ed and transfected successfully into primary cultured rathepatocytes, the transfection and expression of gene in pri-mary cultured rat hepatocytes were examined by RT-PCRand Western blot, and yellow-green fluorescence was ob-served through a fluorescent microscope.CONCLUSION: The successful transfection and expressionof plasmid pIRES-EYFP/VEGF121 in primary cultured rathepatocytes provides a foundation for hepatocyte transplan-tation and gene therapy after modification of hepatocytesby the gene.
