Objective To investigate the effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocyte L-02 cells. Methods Lipid peroxide (LPO) level, reduced glutathione (GSH) content, DNA damage...Objective To investigate the effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocyte L-02 cells. Methods Lipid peroxide (LPO) level, reduced glutathione (GSH) content, DNA damage, apoptosis, and cell cycle analysis were measured after in vitro cultured L-02 cells were exposed to sodium fluoride at different doses (40 μg/mL, 80 μg/mL, and 160 μg/mL) for 24 hours. Results Fluoride caused an increase of LPO levels and a decrease of GSH content in L-02 cells. There appeared to be an obvious dose-effect relationship between the fluoride concentration and the observed changes. Fluoride also caused DNA damage and apoptosis and increased the cell number in S phase of cell cycle in the cells tested. There was a statistically significant difference in DNA damage and apoptosis when comparing the high dose of fluoride treated cells with the low dose of fluoride treated cells. Conclusion Fluoride can cause lipid peroxidation, DNA damage, and apoptosis in the L-02 cell experimental model and there is a significant positive correlation between fluoride concentration and these pathological changes.展开更多
BACKGROUND:Total saponins of Panax ginseng(TSPG) exhibits neuroprotection against Parkinson's disease in the substantia nigra. OBJECTIVE:To investigate the effects of TSPG on human embryonic neural stem cells(NSCs...BACKGROUND:Total saponins of Panax ginseng(TSPG) exhibits neuroprotection against Parkinson's disease in the substantia nigra. OBJECTIVE:To investigate the effects of TSPG on human embryonic neural stem cells(NSCs) proliferation and differentiation into dopaminergic neurons using in vitro studies,and to observe NSC differentiation in a mouse model of Parkinson's disease,as well as behavioral changes before and after transplantation. DESIGN,TIME AND SETTING:In vitro neural cell biology trial and in vivo randomized,controlled animal trial were performed at the Institute of Basic Medical Sciences,Chongqing Medical University between September 2004 and December 2007. MATERIALS:TSPG(purity>95%) was isolated,extracted,and identified by Chongqing Academy of Chinese Materia Medica.Recombinant human basic fibroblast growth factor(bFGF) and recombinant human epidermal growth factor(EGF) were purchased from PeproTech,USA.A total of 25 C57/BL6J mice,aged 18-20 weeks were included.Twenty were used to establish a Parkinson's disease model with i.p.injection of MPTP(1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine) and TSPG alone or combined with interleukin-1(IL-1)-treated NSCs prior to transplantation into the corpus striatum.The remaining five mice were pretreated for 3 days with TSPG prior to MPTP injection,serving as the TSPG prevention group. METHODS:Primary NSCs were isolated,cultured and purified from embryonic cerebral cortex. Immunocytochemistry was employed to detect specific antigen expression in the NSCs.In vitro experiment:(1) to induce proliferation,NSCs were treated with TSPG,EGF+bFGF,or TSPG+EGF+bFGF,respectively;(2) to induce dopaminergic neuronal differentiation,NSCs were treated with TSPG,IL-1,or TSPG+IL-1,respectively. MAIN OUTCOME MEASURES:In vitro experiment:the effects of TSPG on NSCs proliferation were evaluated with flow cytometry and MTT assay.Tyrosine hydroxylase expression was determined by immunocytochemistry assay to observe effects of TSPG on dopaminergic neuronal differentiation.In vivo experiment:differentiation of grafted NSCs in the mouse brain was determined by immunohistochemical staining.Behavioral changes were evaluated by spontaneous activity frequency,memory function,and score of paralysis agitans. RESULTS:(1) NSCs were cultured and passaged for more than three passages. Immunocytochemistry revealed positive nestin staining,as well as neurofilament protein and glial fibrillary acidic protein.(2) TSPG significantly increased NSC proliferation,in particular when combined with EGF and bFGF,which was twice as effective as FGF or bFGF alone.TSPG also induced dopaminergic differentiation in NSCs,in particular when TSPG was added together with IL-1,resulting in an effect five times greater than that of IL-1 alone.(3) At day 30 following transplantation,most NSCs in the TSPG prevention group differentiated into dopaminergic neurons,and the scores of paralysis agitans,spontaneous activity,and memory function were significantly increased compared with TSPG alone or TSPG+IL-1 groups(P<0.05). CONCLUSION:TSPG stimulated NSC proliferation,in particular when combined with FGF and bFGF.TSPG significantly induced dopaminergic neuronal differentiation of NSCs,and the effect was greater when combined with IL-1.In addition,TSPG greatly improved behavior in the Parkinson's disease mouse model following NSC transplantation.Following NSC transplantation, TSPG pretreatment exhibited superior efficacy over either TSPG alone or TSPG in combination with IL-1,in terms of behavioral improvements in the Parkinson's disease mouse model.展开更多
BACKGROUND:Numerous current studies have suggested that human telomerase reverse transcriptase(hTERT) gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses;however,the mechan...BACKGROUND:Numerous current studies have suggested that human telomerase reverse transcriptase(hTERT) gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses;however,the mechanism of action remains unknown. OBJECTIVE:To evaluate the neuroprotective effects and possible mechanism of action of hTERT gene transfection in human embryonic cortical neurons treated with beta-amyloid fragment 25-35 (Aβ_(25-35)). DESIGN,TIME AND SETTING:The randomized,controlled and molecular biological studies were performed at the Department of Anatomy and Brain Research,Zhongshan School of Medicine,Sun Yat-sen University,China,from September 2005 to June 2008. MATERIALS:AdEasy-1 Expression System was gifted by Professor Guoquan Gao from Sun Yat-Sen University,China.Human cortical neurons were derived from 12-20 week old aborted fetuses,obtained from the Guangzhou Maternal and Child Health Hospital,China.Mouse anti-Cdk5 and mouse anti-p16 monoclonal antibodies(Lab Vision,USA),and mouse anti-hTERT monoclonal antibody(Epitomics,USA),were used in this study. METHODS:(1) Recombinant adenovirus vectors,encoding hTERT(Ad-hTERT) and green fluorescent protein(Ad-GFP),were constructed using the AdEasy-1 Expression System.Human embryonic cortical neurons in the Ad-hTERT group were transfected with Ad-hTERT for 1-21 days. Likewise,human embryonic cortical neurons in the Ad-GFP group were transfected with Ad-GFP for 1-21 days.Human embryonic cortical neurons in the control group were cultured as normal.(2) Human embryonic cortical neurons in the Ad-hTERT group were treated with 10μmol/L Aβ_(25-35) for 24 hours.Normal human embryonic cortical neurons treated with 10μmol/LAβ_(25-35) for 24 hours served as a model group.Human embryonic cortical neurons in the Ad-GFP and control groups were not treated with Aβ_(25-35). MAIN OUTCOME MEASURES:Expression of hTERT in human embryonic cortical neurons was evaluated by immunocytochemical staining and Western blot assay.Telomerase activity was measured using a PcR-based telomeric repeat amplification protocol(TRAP) ELISA kit.Neural activity in human embryonic cortical neurons was examined by MTT assay;apoptosis was measured using TUNEL assay;and Cdk5 and p16 protein expressions were measured by Westem blot. RESULTS:Expression of hTERT protein was significantly increased and peaked at day 3 post-transfection in the Ad-hTERT group.No hTERT expression was detected in the Ad-GFP and control groups.Telomerase activity was significantly greater in the Ad-hTERT group compared with the Ad-GFP and control groups(P<0.01).Compared with the control group,cell activity was significantly decreased(P<0.05),and cell apoptotic rate,Cdk5 and p16 expression were significantly increased(P<0.01) in the model group.Compared with the model group,cell activity was increased in the Ad-hTERT group,and peaked at day 3 post-transfection(P<0.05). Neuroprotective effects also peaked at day 3 post-transfection;and the apoptotic rate,Cdk5 and p16 expression significantly decreased(P<0.01). CONCLUSION:Expression of hTERT in human embryonic cortical neurons can relieve Aβ_(25-35)-induced neuronal apoptosis.The possible mechanism by which hTERT produces these neuroprotective effects may be associated with inhibition of Cdk5 and p16 expression.展开更多
Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection ...Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV.RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time-and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.展开更多
Neural cells cultures from human embryo brain of 9° - 11°W gestational age have been used to study ERα (Estrogens Receptor α) and to perform toxicity test for Mitomycin C and Methotrexate. Histochemical co...Neural cells cultures from human embryo brain of 9° - 11°W gestational age have been used to study ERα (Estrogens Receptor α) and to perform toxicity test for Mitomycin C and Methotrexate. Histochemical confirmation of cellular neuronal phenotype was based on histochemical evidence of NSE (Neuron Specific Enolase).The detection of ERα in neuronal cells was performed with a rabbit Monoclonal Antibody. ERα was absent both on neurons grown in vitro and on tissue brain specimens. This finding is apparently in contrast with the positive immunoreactivity of ERα and ERβ reported by other Authors on foetal and adult CNS (Central Nervous System). The absence of nuclear ERα on neurons in culture and in brain tissue specimens in our experiment is not in contrast with the relevant physiologic role of estrogens on nervous central system, but it could be correlated to the embryonic period of life and could represent a protection of male brain from an undue estrogens imprinting. The mitomycin C, alkylation agent, has shown in our experiment a major neurotoxic and cytostatic power in comparison with methotrexate. Our conclusion is that human embryo neuronal culture in vitro is a powerful instrument for physiology and human therapy for cancer and neurodegenerative diseases.展开更多
BACKGROUND: Cell culture in vitro trials have demonstrated that neurotrophin-3 (NT-3) can enhance the survival of sensory neurons and sympathetic neurons, and can also support embryo-derived motor neurons. This effect...BACKGROUND: Cell culture in vitro trials have demonstrated that neurotrophin-3 (NT-3) can enhance the survival of sensory neurons and sympathetic neurons, and can also support embryo-derived motor neurons. This effect is dependent on nerve growth factor on the surface of cells. Understanding the role of NT-3 and its receptor in the early development of human embryonic brains will help to investigate the correlation between early survival of nerve cells and the microenvironment of neural regeneration. OBJECTIVE: To observe the proliferation of cerebral neurons in the development of human embryonic brain, and to investigate the location, expression and distribution of NT-3 and its receptor TrkC during human brain development. DESIGN, TIME AND SETTING: An observation study on cells was performed in the Department of Human Anatomy, Histology and Embryology, Chengdu Medical College in September 2007. MATERIALS: Fifteen specimens of fresh human embryo, aged 6 weeks, were used in this study. METHODS: The proliferation of cerebral neurons was detected using proliferating cell nuclear antigen, and the immunocytochemistry ABC technique was applied to observe the location, expression and distribution of NT-3 and its receptor TrkC in the brain of the human embryo. MAIN OUTCOME MEASURES: Location, expression and distribution of NT-3 and its receptor in the brain of the human embryo. RESULTS: In the early period (aged 6 weeks) of human embryonic development, proliferating cell nuclear antigen-positive reactive substances were mainly observed in the nucleus of the forebrain ventricular zone and subventricular zone, and the intensity was stronger in the subventricular zone than the forebrain ventricle. NT-3 positive reactive substance was mainly distributed in the cytoblastema of the forebrain neuroepithelial layer and nerve cell process, while TrkC was mainly distributed in the cell membrane of the forebrain ventricular zone and subventricular zone. During embryonic development, NT-3 and TrkC showed a positive immune reaction to a greater or lesser extent in ependymal epithelium. CONCLUSION: During early human embryonic development, cerebral nerve cells proliferate in the ventricular zone and subventricular zone, and NT-3 is expressed in the neural axon. The results show that the highly expressed NT-3 could promote the proliferation of neural axons and maintain the neuron body’s survival.展开更多
Neural tube development comprises neural induction,neural epithelial cell proliferation,and apoptosis,as well as migration of nerve cells.Too much or too little apoptosis leads to abnormal nervous system development.T...Neural tube development comprises neural induction,neural epithelial cell proliferation,and apoptosis,as well as migration of nerve cells.Too much or too little apoptosis leads to abnormal nervous system development.The present study analyzed expression and distribution of apoptotic-related factors,including Fas,FasL,and caspase-3,during human embryonic neural tube development.Experimental results showed that increased caspase-3 expression promoted neural apoptosis via a mitochondrial-mediated intrinsic pathway at 4 weeks during early human embryonic neural tube development.Subsequently,Fas and FasL expression increased during embryonic development.The results suggest that neural cells influence neural apoptosis through synergistic effects of extrinsic pathways.Therefore,neural apoptosis during the early period of neural tube development in the human embryo might be regulated by the death receptor induced apoptotic extrinsic pathways.展开更多
Early human embryogenesis is a very sophisticated process due to its unique gene regulatory network.Autophagy has been suggested to play an important role in mediating the development of early embryonic cells in mamma...Early human embryogenesis is a very sophisticated process due to its unique gene regulatory network.Autophagy has been suggested to play an important role in mediating the development of early embryonic cells in mammals.However,evidence showing how autophagy regulates early human embryogenesis remains to be further explored.In this study,we systematically investigated the human transcriptome and methylome patterns of autophagy-related(ATG)genes in early embryonic cells at single-cell resolution.We analyzed the transcriptomic data of 365 cells and methylome data of 265 cells.The results showed that most ATG genes remained epigenetically active and were expressed stably throughout early embryogenesis,whereas the dynamics varied among different developmental stages.This evidence indicated that the autophagy pathway was constitutively activated and exerted a fundamental role in early human embryo development.Our work,for the first time,comprehensively reveals the features of autophagy during early human embryo development.展开更多
Summary: An early embryo co-culture system with human decidual stromal cells was established to study its effect on early embryonic cleavage and growth in vitro. Three hundred and eight 2-cell mouse embryos were co-cu...Summary: An early embryo co-culture system with human decidual stromal cells was established to study its effect on early embryonic cleavage and growth in vitro. Three hundred and eight 2-cell mouse embryos were co-cultured with human decidual stromal cell monolayer in MEM+0. 4 % bovine serum albumin (BSA) and 163 embryos cultured in MEM+15 % FCS alone as control. Among the mouse 2-cell embryos co-cultured with human decidual stromal cells, 72.73 % developed to the morula stage and 67.21 % cavitated to blastocysts with 59. 74 % hatching, as compared with 61. 34 % to morula stage, 48. 47 % to blastocysts and none hatching in the controls, respectively. Co-cultured embryos cleaved slightly faster than controls and showed no or less fragmentation than those in the control. These results suggested that human decidual stromal cells can support early embryonic development and yield a reasonable number of embryos with good quality up to blastocyst stage.展开更多
The precise determination of zona pellucida (ZP) hardness is largely unknown due to the lack of appropriate measuring and modelling methods. In this study, we have used experimental and theoretical models to describe ...The precise determination of zona pellucida (ZP) hardness is largely unknown due to the lack of appropriate measuring and modelling methods. In this study, we have used experimental and theoretical models to describe the mechanical behavior of a single oocyte cell to improve the assisted reproductive technology (ART) outcomes by assessing oocyte/embryo quality. This paper presents the development of: i) a microinjection model to estimate the force of ZP penetration, ii) a micropipette aspiration model to determine the corresponding hardness, and iii) an experimental procedure to generate the required data for these two models. Our results show that the estimated penetration force provides a performance target for the penetration process during intracytoplasmic sperm injection (ICSI), while the estimated corresponding hardness serves as an indicator of the extent of deformation sustained by the oocyte prior to penetration. Evaluation of these results shows that a routine assessment of ZP hardness under microinjection would allow for the identification of certain oocyte pools for which further manipulation is recommended in order to improve injection, hatching and finally ART outcomes.展开更多
Objective To investigate the sensitivity of the human sperm motility assay for detecting endotoxin in culture medium Materials & Methods Motile sperm were separated and exposed to different concentrations of endot...Objective To investigate the sensitivity of the human sperm motility assay for detecting endotoxin in culture medium Materials & Methods Motile sperm were separated and exposed to different concentrations of endotoxin (0.5 ng/mL, 1 ng/mL, 10 ng/mL, 1 000 ng/mL, 10 000 ng/mL, and 50 000 ng/mL), and sperm motility was determined after incubation. Effects of endotoxin on sperm motility in media without albumin were also examined. In addition, at the same concentrations of endotoxin (0.5 ng/mL, 1 ng/mL, and 10 ng/mL), the sensitivity of the human sperm motility assay was compared to those of 1-cell and 2-cell mouse embryo bioassays.Results At levels of 0.5 ng/mL～1 000 ng/mL endotoxin in media with 2 mg/mL albumin, sperm did not show significant change in motility during 24 h of incubation when compared with the control (P>0.05). However, the sperm motility was significantly inhibited at endotoxin dosages of 10 000 and 50 000 ng/mL. In the absence of albumin supplementation, at endotoxin levels of 50 000 ng/mL, and 1 000 ng/mL, there was a marked decrease in sperm motility compared with the control after 2 h or 8 h of incubation, respectively (P<0.01). In media containing 0.5 ng/mL and 1 ng/mL endotoxin, 1-cell and 2-cell mouse embryos had significantly reduced developmental rates in all developmental stages, and at the level of 10 ng/mL, the development of the embryos was arrested.Conclusion The human sperm motility assay could detect high levels of endotoxin in culture medium but its sensitivity to endotoxin would be inferior to that of the 1-cell or 2-cell mouse embryo bioassay. In the absence of albumin supplementation, the sensitivity of the sperm motility assay could be improved.展开更多
Objective To determine whether the presence of bacterial endotoxin in the commercial culture media utilized for human in vitro fertilization (IVF), and evaluate the difference in detecting endotoxin in culture medium ...Objective To determine whether the presence of bacterial endotoxin in the commercial culture media utilized for human in vitro fertilization (IVF), and evaluate the difference in detecting endotoxin in culture medium between the human sperm motility assay and the 2-cell mouse embryo assay. Methods Thirty-six batches of culture media commonly used in IVF laboratories from 3 manufacturers were determined for the presence of endotoxin before using the medium for the assisted reproductive programs (group A). After being used, 25 specimens among above media were also tested (group B). The chromogenic limulus amoebocyte lysate (LAL) test was used for quantification the content of endotoxin. In addition, the human sperm motility assay was compared with the 2-cell mouse embryo assay to evaluate the difference in detecting endotoxin in culture medium. Results Endotoxin was not detected in group A. However, 2 samples were positive in group B. Sperm did not show significant change in motility in group A during 24 h of incubation when compared with the control (P>0.05). However, in group A the 2-cell embryo development to blastocyst was suppressed in 3 batches of media. Conclusions Regular screening of each batch of culture medium should be performed if possible although there was no evidence of endotoxin contamination in commercially prepared pre-tested media. Culture environment should be stringently controlled in case the medium is polluted. The sensitivity of the sperm motility assay was lower than that of the mouse embryo assay for detecting low levels of endotoxin or toxic compounds in the medium.展开更多
In this study, we cloned human KGF (hKGF) genes using RT-PCR techniques and developed a eukaryotic expression plasmid vector capable of directing the expression of functional hKGF. Monolayer culture of human embryo lu...In this study, we cloned human KGF (hKGF) genes using RT-PCR techniques and developed a eukaryotic expression plasmid vector capable of directing the expression of functional hKGF. Monolayer culture of human embryo lung fibro-blast (HLF) was used for isolation of total RNA. Then the total RNA was purified and reverse- transcribed into cDNA using an oligo (dT) primer. A full PCR fragment for hKGF was generated and cloned. Restriction digestion and nucleo-tide sequence analysis validated the complete hKGF transcription. The hKGF cDNA fragment was inserted into pEGFP-C2 vector by means of recombinant DNA technology and verified by restriction analysis and sequencing. We have constructed pEGFP-C2-hKGF encoding the green fluorescent protein (GFP). Furthermore, hKGF had the effect on AEC II proliferation. These results suggest that the potential appli-cation of a hKGF plasmid of gene expression should be useful for sustained AEC proliferation, and its in vivo efficacy needs to be validated. Keywords:展开更多
基金The work was supported by grants from the National Nature Science Foundation of China (No. 30271155) China national key basic research and development program (No. 2022CB512908).
文摘Objective To investigate the effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocyte L-02 cells. Methods Lipid peroxide (LPO) level, reduced glutathione (GSH) content, DNA damage, apoptosis, and cell cycle analysis were measured after in vitro cultured L-02 cells were exposed to sodium fluoride at different doses (40 μg/mL, 80 μg/mL, and 160 μg/mL) for 24 hours. Results Fluoride caused an increase of LPO levels and a decrease of GSH content in L-02 cells. There appeared to be an obvious dose-effect relationship between the fluoride concentration and the observed changes. Fluoride also caused DNA damage and apoptosis and increased the cell number in S phase of cell cycle in the cells tested. There was a statistically significant difference in DNA damage and apoptosis when comparing the high dose of fluoride treated cells with the low dose of fluoride treated cells. Conclusion Fluoride can cause lipid peroxidation, DNA damage, and apoptosis in the L-02 cell experimental model and there is a significant positive correlation between fluoride concentration and these pathological changes.
文摘BACKGROUND:Total saponins of Panax ginseng(TSPG) exhibits neuroprotection against Parkinson's disease in the substantia nigra. OBJECTIVE:To investigate the effects of TSPG on human embryonic neural stem cells(NSCs) proliferation and differentiation into dopaminergic neurons using in vitro studies,and to observe NSC differentiation in a mouse model of Parkinson's disease,as well as behavioral changes before and after transplantation. DESIGN,TIME AND SETTING:In vitro neural cell biology trial and in vivo randomized,controlled animal trial were performed at the Institute of Basic Medical Sciences,Chongqing Medical University between September 2004 and December 2007. MATERIALS:TSPG(purity>95%) was isolated,extracted,and identified by Chongqing Academy of Chinese Materia Medica.Recombinant human basic fibroblast growth factor(bFGF) and recombinant human epidermal growth factor(EGF) were purchased from PeproTech,USA.A total of 25 C57/BL6J mice,aged 18-20 weeks were included.Twenty were used to establish a Parkinson's disease model with i.p.injection of MPTP(1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine) and TSPG alone or combined with interleukin-1(IL-1)-treated NSCs prior to transplantation into the corpus striatum.The remaining five mice were pretreated for 3 days with TSPG prior to MPTP injection,serving as the TSPG prevention group. METHODS:Primary NSCs were isolated,cultured and purified from embryonic cerebral cortex. Immunocytochemistry was employed to detect specific antigen expression in the NSCs.In vitro experiment:(1) to induce proliferation,NSCs were treated with TSPG,EGF+bFGF,or TSPG+EGF+bFGF,respectively;(2) to induce dopaminergic neuronal differentiation,NSCs were treated with TSPG,IL-1,or TSPG+IL-1,respectively. MAIN OUTCOME MEASURES:In vitro experiment:the effects of TSPG on NSCs proliferation were evaluated with flow cytometry and MTT assay.Tyrosine hydroxylase expression was determined by immunocytochemistry assay to observe effects of TSPG on dopaminergic neuronal differentiation.In vivo experiment:differentiation of grafted NSCs in the mouse brain was determined by immunohistochemical staining.Behavioral changes were evaluated by spontaneous activity frequency,memory function,and score of paralysis agitans. RESULTS:(1) NSCs were cultured and passaged for more than three passages. Immunocytochemistry revealed positive nestin staining,as well as neurofilament protein and glial fibrillary acidic protein.(2) TSPG significantly increased NSC proliferation,in particular when combined with EGF and bFGF,which was twice as effective as FGF or bFGF alone.TSPG also induced dopaminergic differentiation in NSCs,in particular when TSPG was added together with IL-1,resulting in an effect five times greater than that of IL-1 alone.(3) At day 30 following transplantation,most NSCs in the TSPG prevention group differentiated into dopaminergic neurons,and the scores of paralysis agitans,spontaneous activity,and memory function were significantly increased compared with TSPG alone or TSPG+IL-1 groups(P<0.05). CONCLUSION:TSPG stimulated NSC proliferation,in particular when combined with FGF and bFGF.TSPG significantly induced dopaminergic neuronal differentiation of NSCs,and the effect was greater when combined with IL-1.In addition,TSPG greatly improved behavior in the Parkinson's disease mouse model following NSC transplantation.Following NSC transplantation, TSPG pretreatment exhibited superior efficacy over either TSPG alone or TSPG in combination with IL-1,in terms of behavioral improvements in the Parkinson's disease mouse model.
基金the National Key Basic Research Program of China,No. 2006cb500700the National Natural Science Foundation of China,No.30470904the Natural Science and Technology Foundation of Guangdong Province,No. 04009356, 2008B030301320
文摘BACKGROUND:Numerous current studies have suggested that human telomerase reverse transcriptase(hTERT) gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses;however,the mechanism of action remains unknown. OBJECTIVE:To evaluate the neuroprotective effects and possible mechanism of action of hTERT gene transfection in human embryonic cortical neurons treated with beta-amyloid fragment 25-35 (Aβ_(25-35)). DESIGN,TIME AND SETTING:The randomized,controlled and molecular biological studies were performed at the Department of Anatomy and Brain Research,Zhongshan School of Medicine,Sun Yat-sen University,China,from September 2005 to June 2008. MATERIALS:AdEasy-1 Expression System was gifted by Professor Guoquan Gao from Sun Yat-Sen University,China.Human cortical neurons were derived from 12-20 week old aborted fetuses,obtained from the Guangzhou Maternal and Child Health Hospital,China.Mouse anti-Cdk5 and mouse anti-p16 monoclonal antibodies(Lab Vision,USA),and mouse anti-hTERT monoclonal antibody(Epitomics,USA),were used in this study. METHODS:(1) Recombinant adenovirus vectors,encoding hTERT(Ad-hTERT) and green fluorescent protein(Ad-GFP),were constructed using the AdEasy-1 Expression System.Human embryonic cortical neurons in the Ad-hTERT group were transfected with Ad-hTERT for 1-21 days. Likewise,human embryonic cortical neurons in the Ad-GFP group were transfected with Ad-GFP for 1-21 days.Human embryonic cortical neurons in the control group were cultured as normal.(2) Human embryonic cortical neurons in the Ad-hTERT group were treated with 10μmol/L Aβ_(25-35) for 24 hours.Normal human embryonic cortical neurons treated with 10μmol/LAβ_(25-35) for 24 hours served as a model group.Human embryonic cortical neurons in the Ad-GFP and control groups were not treated with Aβ_(25-35). MAIN OUTCOME MEASURES:Expression of hTERT in human embryonic cortical neurons was evaluated by immunocytochemical staining and Western blot assay.Telomerase activity was measured using a PcR-based telomeric repeat amplification protocol(TRAP) ELISA kit.Neural activity in human embryonic cortical neurons was examined by MTT assay;apoptosis was measured using TUNEL assay;and Cdk5 and p16 protein expressions were measured by Westem blot. RESULTS:Expression of hTERT protein was significantly increased and peaked at day 3 post-transfection in the Ad-hTERT group.No hTERT expression was detected in the Ad-GFP and control groups.Telomerase activity was significantly greater in the Ad-hTERT group compared with the Ad-GFP and control groups(P<0.01).Compared with the control group,cell activity was significantly decreased(P<0.05),and cell apoptotic rate,Cdk5 and p16 expression were significantly increased(P<0.01) in the model group.Compared with the model group,cell activity was increased in the Ad-hTERT group,and peaked at day 3 post-transfection(P<0.05). Neuroprotective effects also peaked at day 3 post-transfection;and the apoptotic rate,Cdk5 and p16 expression significantly decreased(P<0.01). CONCLUSION:Expression of hTERT in human embryonic cortical neurons can relieve Aβ_(25-35)-induced neuronal apoptosis.The possible mechanism by which hTERT produces these neuroprotective effects may be associated with inhibition of Cdk5 and p16 expression.
文摘Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV.RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time-and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.
文摘Neural cells cultures from human embryo brain of 9° - 11°W gestational age have been used to study ERα (Estrogens Receptor α) and to perform toxicity test for Mitomycin C and Methotrexate. Histochemical confirmation of cellular neuronal phenotype was based on histochemical evidence of NSE (Neuron Specific Enolase).The detection of ERα in neuronal cells was performed with a rabbit Monoclonal Antibody. ERα was absent both on neurons grown in vitro and on tissue brain specimens. This finding is apparently in contrast with the positive immunoreactivity of ERα and ERβ reported by other Authors on foetal and adult CNS (Central Nervous System). The absence of nuclear ERα on neurons in culture and in brain tissue specimens in our experiment is not in contrast with the relevant physiologic role of estrogens on nervous central system, but it could be correlated to the embryonic period of life and could represent a protection of male brain from an undue estrogens imprinting. The mitomycin C, alkylation agent, has shown in our experiment a major neurotoxic and cytostatic power in comparison with methotrexate. Our conclusion is that human embryo neuronal culture in vitro is a powerful instrument for physiology and human therapy for cancer and neurodegenerative diseases.
文摘BACKGROUND: Cell culture in vitro trials have demonstrated that neurotrophin-3 (NT-3) can enhance the survival of sensory neurons and sympathetic neurons, and can also support embryo-derived motor neurons. This effect is dependent on nerve growth factor on the surface of cells. Understanding the role of NT-3 and its receptor in the early development of human embryonic brains will help to investigate the correlation between early survival of nerve cells and the microenvironment of neural regeneration. OBJECTIVE: To observe the proliferation of cerebral neurons in the development of human embryonic brain, and to investigate the location, expression and distribution of NT-3 and its receptor TrkC during human brain development. DESIGN, TIME AND SETTING: An observation study on cells was performed in the Department of Human Anatomy, Histology and Embryology, Chengdu Medical College in September 2007. MATERIALS: Fifteen specimens of fresh human embryo, aged 6 weeks, were used in this study. METHODS: The proliferation of cerebral neurons was detected using proliferating cell nuclear antigen, and the immunocytochemistry ABC technique was applied to observe the location, expression and distribution of NT-3 and its receptor TrkC in the brain of the human embryo. MAIN OUTCOME MEASURES: Location, expression and distribution of NT-3 and its receptor in the brain of the human embryo. RESULTS: In the early period (aged 6 weeks) of human embryonic development, proliferating cell nuclear antigen-positive reactive substances were mainly observed in the nucleus of the forebrain ventricular zone and subventricular zone, and the intensity was stronger in the subventricular zone than the forebrain ventricle. NT-3 positive reactive substance was mainly distributed in the cytoblastema of the forebrain neuroepithelial layer and nerve cell process, while TrkC was mainly distributed in the cell membrane of the forebrain ventricular zone and subventricular zone. During embryonic development, NT-3 and TrkC showed a positive immune reaction to a greater or lesser extent in ependymal epithelium. CONCLUSION: During early human embryonic development, cerebral nerve cells proliferate in the ventricular zone and subventricular zone, and NT-3 is expressed in the neural axon. The results show that the highly expressed NT-3 could promote the proliferation of neural axons and maintain the neuron body’s survival.
文摘Neural tube development comprises neural induction,neural epithelial cell proliferation,and apoptosis,as well as migration of nerve cells.Too much or too little apoptosis leads to abnormal nervous system development.The present study analyzed expression and distribution of apoptotic-related factors,including Fas,FasL,and caspase-3,during human embryonic neural tube development.Experimental results showed that increased caspase-3 expression promoted neural apoptosis via a mitochondrial-mediated intrinsic pathway at 4 weeks during early human embryonic neural tube development.Subsequently,Fas and FasL expression increased during embryonic development.The results suggest that neural cells influence neural apoptosis through synergistic effects of extrinsic pathways.Therefore,neural apoptosis during the early period of neural tube development in the human embryo might be regulated by the death receptor induced apoptotic extrinsic pathways.
基金supported by the National Key Research and Development Program(2019YFA0801400,2017YFA01038012018YFC1004000)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDA16020703)。
文摘Early human embryogenesis is a very sophisticated process due to its unique gene regulatory network.Autophagy has been suggested to play an important role in mediating the development of early embryonic cells in mammals.However,evidence showing how autophagy regulates early human embryogenesis remains to be further explored.In this study,we systematically investigated the human transcriptome and methylome patterns of autophagy-related(ATG)genes in early embryonic cells at single-cell resolution.We analyzed the transcriptomic data of 365 cells and methylome data of 265 cells.The results showed that most ATG genes remained epigenetically active and were expressed stably throughout early embryogenesis,whereas the dynamics varied among different developmental stages.This evidence indicated that the autophagy pathway was constitutively activated and exerted a fundamental role in early human embryo development.Our work,for the first time,comprehensively reveals the features of autophagy during early human embryo development.
基金We are grateful to Dr. Qi Zhou for helpful suggestions. This work was supported by National Key R＆D Program of China （2017YFC1001901 and 2017YFC1001600）, the Science and Technology Planning Project of Guangdong Province （2015B020228002）, the Guangzhou Science and Technology Project （201707010085） and the National Natural Science Foundation of China （Grant No. 81771579）.
文摘Summary: An early embryo co-culture system with human decidual stromal cells was established to study its effect on early embryonic cleavage and growth in vitro. Three hundred and eight 2-cell mouse embryos were co-cultured with human decidual stromal cell monolayer in MEM+0. 4 % bovine serum albumin (BSA) and 163 embryos cultured in MEM+15 % FCS alone as control. Among the mouse 2-cell embryos co-cultured with human decidual stromal cells, 72.73 % developed to the morula stage and 67.21 % cavitated to blastocysts with 59. 74 % hatching, as compared with 61. 34 % to morula stage, 48. 47 % to blastocysts and none hatching in the controls, respectively. Co-cultured embryos cleaved slightly faster than controls and showed no or less fragmentation than those in the control. These results suggested that human decidual stromal cells can support early embryonic development and yield a reasonable number of embryos with good quality up to blastocyst stage.
文摘The precise determination of zona pellucida (ZP) hardness is largely unknown due to the lack of appropriate measuring and modelling methods. In this study, we have used experimental and theoretical models to describe the mechanical behavior of a single oocyte cell to improve the assisted reproductive technology (ART) outcomes by assessing oocyte/embryo quality. This paper presents the development of: i) a microinjection model to estimate the force of ZP penetration, ii) a micropipette aspiration model to determine the corresponding hardness, and iii) an experimental procedure to generate the required data for these two models. Our results show that the estimated penetration force provides a performance target for the penetration process during intracytoplasmic sperm injection (ICSI), while the estimated corresponding hardness serves as an indicator of the extent of deformation sustained by the oocyte prior to penetration. Evaluation of these results shows that a routine assessment of ZP hardness under microinjection would allow for the identification of certain oocyte pools for which further manipulation is recommended in order to improve injection, hatching and finally ART outcomes.
基金This study was supported by the Science&Technology Commission of Guangdong Province,P.R.China
文摘Objective To investigate the sensitivity of the human sperm motility assay for detecting endotoxin in culture medium Materials & Methods Motile sperm were separated and exposed to different concentrations of endotoxin (0.5 ng/mL, 1 ng/mL, 10 ng/mL, 1 000 ng/mL, 10 000 ng/mL, and 50 000 ng/mL), and sperm motility was determined after incubation. Effects of endotoxin on sperm motility in media without albumin were also examined. In addition, at the same concentrations of endotoxin (0.5 ng/mL, 1 ng/mL, and 10 ng/mL), the sensitivity of the human sperm motility assay was compared to those of 1-cell and 2-cell mouse embryo bioassays.Results At levels of 0.5 ng/mL～1 000 ng/mL endotoxin in media with 2 mg/mL albumin, sperm did not show significant change in motility during 24 h of incubation when compared with the control (P>0.05). However, the sperm motility was significantly inhibited at endotoxin dosages of 10 000 and 50 000 ng/mL. In the absence of albumin supplementation, at endotoxin levels of 50 000 ng/mL, and 1 000 ng/mL, there was a marked decrease in sperm motility compared with the control after 2 h or 8 h of incubation, respectively (P<0.01). In media containing 0.5 ng/mL and 1 ng/mL endotoxin, 1-cell and 2-cell mouse embryos had significantly reduced developmental rates in all developmental stages, and at the level of 10 ng/mL, the development of the embryos was arrested.Conclusion The human sperm motility assay could detect high levels of endotoxin in culture medium but its sensitivity to endotoxin would be inferior to that of the 1-cell or 2-cell mouse embryo bioassay. In the absence of albumin supplementation, the sensitivity of the sperm motility assay could be improved.
基金This is a part of the project (No. 010399) supported by Natural Science Foundation of Guangdong Province,China
文摘Objective To determine whether the presence of bacterial endotoxin in the commercial culture media utilized for human in vitro fertilization (IVF), and evaluate the difference in detecting endotoxin in culture medium between the human sperm motility assay and the 2-cell mouse embryo assay. Methods Thirty-six batches of culture media commonly used in IVF laboratories from 3 manufacturers were determined for the presence of endotoxin before using the medium for the assisted reproductive programs (group A). After being used, 25 specimens among above media were also tested (group B). The chromogenic limulus amoebocyte lysate (LAL) test was used for quantification the content of endotoxin. In addition, the human sperm motility assay was compared with the 2-cell mouse embryo assay to evaluate the difference in detecting endotoxin in culture medium. Results Endotoxin was not detected in group A. However, 2 samples were positive in group B. Sperm did not show significant change in motility in group A during 24 h of incubation when compared with the control (P>0.05). However, in group A the 2-cell embryo development to blastocyst was suppressed in 3 batches of media. Conclusions Regular screening of each batch of culture medium should be performed if possible although there was no evidence of endotoxin contamination in commercially prepared pre-tested media. Culture environment should be stringently controlled in case the medium is polluted. The sensitivity of the sperm motility assay was lower than that of the mouse embryo assay for detecting low levels of endotoxin or toxic compounds in the medium.
文摘In this study, we cloned human KGF (hKGF) genes using RT-PCR techniques and developed a eukaryotic expression plasmid vector capable of directing the expression of functional hKGF. Monolayer culture of human embryo lung fibro-blast (HLF) was used for isolation of total RNA. Then the total RNA was purified and reverse- transcribed into cDNA using an oligo (dT) primer. A full PCR fragment for hKGF was generated and cloned. Restriction digestion and nucleo-tide sequence analysis validated the complete hKGF transcription. The hKGF cDNA fragment was inserted into pEGFP-C2 vector by means of recombinant DNA technology and verified by restriction analysis and sequencing. We have constructed pEGFP-C2-hKGF encoding the green fluorescent protein (GFP). Furthermore, hKGF had the effect on AEC II proliferation. These results suggest that the potential appli-cation of a hKGF plasmid of gene expression should be useful for sustained AEC proliferation, and its in vivo efficacy needs to be validated. Keywords:
