To explore the effect of NF κB on bcl x gene transcription in extended drug resistance leukemia cell line HL 60/E6, drug resistant subline HL 60/E6 was derived by intermittently exposing HL 60 cells to 6 ng/ml epirub...To explore the effect of NF κB on bcl x gene transcription in extended drug resistance leukemia cell line HL 60/E6, drug resistant subline HL 60/E6 was derived by intermittently exposing HL 60 cells to 6 ng/ml epirubicin. Indirect immunofluorescence was used to demonstrate the location of NF κB RelA in HL 60/E6 cells. FCM analysis and RT PCR were used to detect the efficiency of liposome mediated ODN transfection and the change of bcl x L mRNA levels after 5 μmol/L phosphorothioate (PS) derivatized antisense (AS) oligodeoxynucleotide (ODN) directed to RelA was transferred into HL 60/E6 cells. The results showed that RelA remained persistently active and located at the nuclei of HL 60/E6 cells,but in the cytoplasm of HL 60 cells, the efficiency of liposome mediated ODN transfection was significantly higher than that of null ODN ( P <0.01 in 4 h, 6 h, 12 h, 24 h). Exposure of HL 60/E6 cells to 5 μmol/L AS PS ODN directed to RelA led to a maximal 40 % decline of bcl x L mRNA levels within 8 h. The inhibition rate of bcl x L mRNA was (15±1.79) %, (28±2.34) %, (40±3.47) %, (20±1.54) % in 4 h, 6 h, 8 h, 15 h, respectively, but it was less than 15 % in control group. It was concluded that NF κB was involved in regulating bcl x transcription. It was suggested that NF κB was an important factor for drug resistance in leukemia cells.展开更多
AIM:To study the inhibition of nuclear factor kappa-B p65(NF-κB p65)antisense oligodeoxynucleotide(ASODN)on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2(TGF-β2...AIM:To study the inhibition of nuclear factor kappa-B p65(NF-κB p65)antisense oligodeoxynucleotide(ASODN)on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2(TGF-β2)in vitro.·METHODS:NF-κBp65ASODNand NF-κB p65 missense oligodeoxynucleotide(MSODN)were designed and synthesized.Human lens epithelial cell line(HLE B-3)cells were prepared for study and divided into 7 groups.Control group was HLE B-3 cells cultured in vitro in dulbecco's modified eagle medium(DMEM).T1,T2,and T3 group were HLE B-3 cells cultured in vitro in DMEM with 10 ng/m L TGF-β2 for 6h,12h,24h respectively.A+T group was HLE B-3 cells cultured with 10 ng/m L TGF-β2for 24h after transfected by NF-κB p65 ASODN for 24h.M+T group was HLE B-3 cells cultured with 10 ng/m L TGF-β2 for 24h after transfected by NF-κB p65 MSODN for 24h.The negative control group was HLE B-3 cells cultured with 10 ng/m L TGF-β2 for 24h after cultured with transfer agent(Hi Per Fect)for 24h.Cell morphology was observed at different time points using an inverted microscope.The expression of NF-κB p65 m RNA was detected with reverse transcription-polymerase chain reaction(RT-PCR),and the expression ofα-smooth muscle actin(α-SMA)protein was assayed with ELISA.·RESULTS:With the TGF-β2 stimulation prolongation,the expression of NF-κB p65 m RNA and a-SMA protein increased in T1,T2,T3 groups compared with the control group,and the difference was statistically significant(P<0.05).NF-κB p65 ASODN lowered the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2.NF-κB p65 MSODN and Hi Per Fect did not lower the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2.The difference between control group and A+T group was not statistically significant(P>0.05),but the difference among A+T group and other groups was statistically significant(P<0.05).·CONCLUSION:NF-κB p65 ASODN could lower the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2,and antagonized TGF-β2-induced transdifferentiation of HLE B-3 in vitro.NF-κB p65ASODN could be used as a new biological therapeutic target of posterior capsular opacification.展开更多
To assess the role of follicle stimulating hormone receptor(FSHR) gene expression in regulating expression of FSHR protein in the plasma membrane, the effects of a porcine FSHR cDNA antisense oligodeoxynucleotide (ODN...To assess the role of follicle stimulating hormone receptor(FSHR) gene expression in regulating expression of FSHR protein in the plasma membrane, the effects of a porcine FSHR cDNA antisense oligodeoxynucleotide (ODN) on FSHR mRNA levels and 125 I FSH binding were determined in Chinese hamster ovary cells expression recombinant porcine FSHR (pFSHR CHO cells). An 18 mer phosphorothioate endcapped antisense ODN that corresponded to the region surrounding the translation initiation codon of the porcine FSHR cDNA was synthesized. An 18 mer nonsense sequence of identical nucleotide composition, which had little homology to known DNA sequences, was synthesized for use as a control. pFSHR CHO cells were cultured in 24 well plates (10 5 cells/well) in the absence or presence of 1 20 μmol/L antisense or nonsense ODN for 24 h and then assayed for porcine FSHR mRNA, using quantitative reverse transcription and competitive polymerase chain reaction, and for 125 I FSH binding activity. Treatment with 10 μmol/L antisense ODN caused a paradoxical increase in porcine FSHR mRNA from 0.89±0.06 to 1.64±0.08 ng/mg total RNA ( P <0.05). Transfection with lipofectamine and 0.33 μmol/L antisense ODN caused an increase in porcine mRNA from 0.95±0.08 to 1.53±0.07 ng/mg total RAN. This was probably due to upregulation of mRNA synthesis resulting from inhibition of porcine FSHR protein translation. The nonsense ODN had no effect on porcine FSHR mRNA. Antisense, but not nonsense, ODN (10 μmol/L) inhibited membrane binding of 125 I FSH by 13.6± 0.8 % ( P <0.05) in 24 h. Treatment of cells with antisense ODN (10 μmol/L) for 48 h resulted in a 76±1.5 % ( P <0.05) inhibition of 125 I FSH binding. In contrast, transfection with lipofectamine and 0.33 μmol/L antisense ODN at 0 h caused a 76.1±1.3 % ( P <0.05) reduction in binding within 24 h. Binding had returned to 52.3±2.3 % ( P < 0.05) of normal by 48 h. These results indicate that an antisense ODN corresponding to the region of the translation start site of the porcine FSHR cDNA is an effective specific inhibitor of porcine FSHR synthesis and that inhibition of receptor synthesis causes a decrease in functional membrane bound FSHR.展开更多
INTRODUCTIONEpidermal-growth-factor receptor(EGFR) is apolypeptide with 1186 amino acids,which binds toEGF family growth factors.Two major naturalligands in the family interact with EGFR:one isEGF,the other is transfo...INTRODUCTIONEpidermal-growth-factor receptor(EGFR) is apolypeptide with 1186 amino acids,which binds toEGF family growth factors.Two major naturalligands in the family interact with EGFR:one isEGF,the other is transforming growth factor-α(TGF-α).When EGF or TGF-α,binds to EGFR,tyrosine kinase activity is induced which in turntriggers a series of events regulating the cellgrowth.The importance of EGFR in growthregulating pathways was confirmed by the fact展开更多
Objective: To explore the probability of vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotides as a developing new therapeutic strategy for glioma. Methods: VEGF protein expression was detected by ...Objective: To explore the probability of vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotides as a developing new therapeutic strategy for glioma. Methods: VEGF protein expression was detected by S-P immunohistochemical technique. Tumor cell apoptosis was observed by TUNEL method. Results: Compared with control, VEGF protein expression was inhibited by antisense oligodeoxynucleotides in vitro. And the inhibitory effects increased with the increasing concentration. VEGF positive rate was 82.10% in control group, while in 2.5, 5, 10 mmol/L AODN groups, they were 70.00%, 57.85%, 53.20% respectively. No inhibition effect was found in the cell lines treated with missense and sense oligodeoxynucleotides. In vivo, antisense oligodeoxy-nucleotides therapy also inhibited VEGF protein expression and induced the increase of apoptotic tumor cells. However, it has no effect on tumor cell proliferation. Conclusion: It is hopeful that VEGF antisense oligodeoxynucleotides may be a new gene therapy method to glioma through its antiangiogenesis effect by inhibition of VEGF.展开更多
To study the effect of c myc antisense oligodeoxynucleotides (ODNs) on proliferation of pulmonary vascular pericytes (PC) induced by hypoxia, cell culture, dot hybridization using probe of digoxigenin 11 dUTP labeled ...To study the effect of c myc antisense oligodeoxynucleotides (ODNs) on proliferation of pulmonary vascular pericytes (PC) induced by hypoxia, cell culture, dot hybridization using probe of digoxigenin 11 dUTP labeled cDNA, 3H thymidine incorporation, immunocytochemical technique and image analysis methods were used to observe the effect of c myc antisense ODNs on expression of c myc gene and proliferating cell nuclear antigen (PCNA), and 3H thymidine incorporation of PC induced by hypoxia. The results showed that hypoxia could significantly enhance the expression of c myc and PCNA ( P <0.01), and elevate 3H thymidine incorporation of PC ( P <0.01), but antisense ODNs could significantly inhibit the expression of c myc and PCNA ( P <0.05), and 3H thymidine incorporation of PC ( P <0.01). It was suggested that hypoxia could promote the proliferation of PC by up regulating the expression of c myc gene, but c myc antisense ODNs could inhibit hypoxia induced proliferation of PC by downregulating the expression of c myc gene.展开更多
Extracellular matrix (ECM) degradation is an essential step that allows tumor cells to penetrate a tissue barrier and become metastatic. Heparanase (HPSE) is an endoglycosidase that specifically degrades heparin sulfa...Extracellular matrix (ECM) degradation is an essential step that allows tumor cells to penetrate a tissue barrier and become metastatic. Heparanase (HPSE) is an endoglycosidase that specifically degrades heparin sulfate proteoglycans (HSPG), a chief component of ECM. HPSE is not expressed in normal epithelial cells but can be detected in a variety of human carcinomas including pancreatic cancer. In the present study, human pancreatic cancer cell line Panc-1 was transfected with HPSE antisense oligodeoxynucleotide (AS-ODN) in vitro, then the inhibitory effect of AS-ODN on HPSE gene expression and invasive ability of Panc-1 cells in vitro was examined. The HPSE mRNA and protein expression of Panc-1 cells transfected with AS-ODN was significantly inhibited. However, there were no marked inhibitory effects in Panc-1 cells treated with nonsense oligodeoxynucleotide (NS-ODN). Moreover, a modified Boyden chamber assay demonstrated that transfection with HPSE AS-ODN significantly inhibited invasive potential of Panc-1 cells in vitro after AS-ODN transfection. This suggests that HPSE AS-ODN may contribute to the inhibition of HPSE mRNA and protein expression, and results in a decrease of the invasive ability of Panc-1 in vitro.展开更多
To study the effect of tTG fully phosphorothioated antisense oligodeoxynucleotides (tTG-ASDON) on tTG expression in cultured bovine trabecular meshwork cells (BTMCs) in vitro and explore a new treatment alternative fo...To study the effect of tTG fully phosphorothioated antisense oligodeoxynucleotides (tTG-ASDON) on tTG expression in cultured bovine trabecular meshwork cells (BTMCs) in vitro and explore a new treatment alternative for primary open angle glaucoma (POAG), the ASDON1 and ASDON2 complementary to the protein codogram region of tTG were designed, synthesized and phosphorothioated according to the secondary structure of tTG. The ASDON1 and ASDON2 were embedded in Lipofectamine and transfected into BTMCs. The untreated group served as negative controls. The expression of tTG in the mRNA and protein level were measured by semi-quantitative RT-PCR and immunohistochemical technique-Supervision method respectively. Our results showed that both the mRNA and the protein of tTG with tTG-ASDON1 and tTG-ASDON2 were significantly decreased as compared with that of the controls (P<0.05). On the other hand, no significant difference was found between the ASDON1 group and the ASDON2 group. It is concluded that the expression of tTG mRNA and protein in cultured BTMC are down-regulated by tTG- ASDON. As a result, tTG-ASDON may be used for the treatment of POAG through the inhibitory effect on the expression of tTG.展开更多
Objective: Aurora A kinase representing a family of evolutionarily conserved mitotic serine/threonine kinases has been found elevated in human lung adenocarcinoma cell line A549. It is suggested that the overexpressio...Objective: Aurora A kinase representing a family of evolutionarily conserved mitotic serine/threonine kinases has been found elevated in human lung adenocarcinoma cell line A549. It is suggested that the overexpression of Aurora A con- tributes to the carcinogenesis, chromosomal instability (CIN), and de-differentiation of lung cancers. To address its possibility as a therapeutic target for lung cancer, we employed the antisense oligodeoxynucleotide (ASODN) technique to inhibit Aurora A expression and investigate its effects on tumor growth and cell cycle of A549, as well as the chemosensitivity to paclitaxel. Methods: Aurora A ASODN was synthesized and transfected into A549 cells by lipofectAMINE 2000. Aurora A mRNA and protein expression were examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respec- tively. Cell cycle distribution was observed by flow cytometer. MTT assay was used to evaluate cell inhibition ratio before and after transfection. Results: The proliferation of the A549 cells was inhibited by Aurora A ASODN dose and time dependently. It was also observed that the IC50 of A549 cells after 48 hours’ treatment of ASODN was about 300 nmol/L and under such circumstances, the Aurora A mRNA and protein expression significantly decreased (P < 0.05), along with the induction of ac- cumulation of cells in S phase and the G2-M transition. Furthermore, cell inhibition ratio of the combination of Aurora A ASODN and paclitaxel was higher significantly than paclitaxel (P < 0.05) or Aurora A ASODN alone (P < 0.05). Conclusion: Inhibition of Aurora A expression can result in the suppression of cell growth and chemosensitizing activity to paclitaxel in human lung cancer cell line A549.展开更多
AIM: To study the distribution and stability of antisense oligodeoxynucleotide (ASODN) in Walker-256 cells and their distribution in liver, lung and kidney tissues after being infused alone or mixed with lipiodol via ...AIM: To study the distribution and stability of antisense oligodeoxynucleotide (ASODN) in Walker-256 cells and their distribution in liver, lung and kidney tissues after being infused alone or mixed with lipiodol via hepatic artery in a rat liver tumor model.METHODS: 5'-Isothiocyananate (FITC)-labeled vascular endothelial growth factor (VEGF) ASODN was added into Walker-256 cell culture media. Its distribution in cells was observed by fluorescence microscope at different time points. Walker-256 carcinosarcoma was transplanted into Wistar rat liver to establish a liver cancer model. 5'-FITC-labeled VEGF ASODN mixed with (mixed group, n = 6) or without (TAI group, n = 6)ultra-fluid lipiodol was administrated via hepatic artery.Frozen samples of liver, lung and kidney tissue were taken from rats after 1, 3 and 6 d, respectively. The distribution of ASODN was observed under fluorescent microscope.RESULTS: ASODN could enter cytoplasm within 2 h and nuclei within 6 h. Accumulation of ASODN reached the peak point in nuclei at 12 h, and then disappeared gradually. No fluorescence could be seen in cells at 48 h. In vivo experiment, on d 1 and 3 the fluorescence staining in liver was stronger in mixed group than in TAI group and more fluorescence could be detected in lung and kidney in TAI group than in mixed group. On d 6, no fluorescence could be detected in TAI group, but faint fluorescence could be seen in mixed group. ASODN could be seen in cancer cells and normal hepatic cells. In mixed group, ASODN was mainly distributed in liver tumor tissues.CONCLUSION: ASODN can transfect Walker-256 cells.ASODN mixed with lipiodol infusion via hepatic artery can be used in the treatment of HCC.展开更多
Purpose: To investiagate the effect of antisense oligonucleotides (ODN) to Nogo-A mRNA expression in oligodendrocytes and to establish the base for further research of repair of optic nerve injury.Method: (1)Oligodend...Purpose: To investiagate the effect of antisense oligonucleotides (ODN) to Nogo-A mRNA expression in oligodendrocytes and to establish the base for further research of repair of optic nerve injury.Method: (1)Oligodendrocytes were obtained by inoculating the optic nerve of newborn (2 days)rats and were identified with galactocerebroside(GC) antibody immunocytochemical stain. (2) In order to observe the effects of antisense ODN on cultured cells,we set up five groups, including the groups of three concentration of antisense Nogo-A ODN (2μM、5μM、l0μM),a group with the random sequence added to the medium and the control group. Reverse transcription-polymerase chain reaction(RT-PCR) was adopted to study the effects of ODN on the expression of Nogo-A in oligodendrocytes.Results: (1)Three days after inoculation, a few of round or fusiform shape cells migrated from optic nerve tissue; About 11 days later, the coverlips were completely covered by the cells;The cells identified with GC antibody immunocytochemical stain were positive cells.(2)The result of RT-PCR study showed that antisense Nogo-A ODN could significantly and specifically inhibit the expression of Nogo-A after 24 hours ( P＜ 0.01). Random sequence has no effect on Nogo-A expresson.Conclusion:Antisense Nogo-A ODN can effectively and specifically inhibit the expression of Nogo-A.展开更多
Objective and Methods: Excessive accumulation of collagen typeⅠ and type Ⅲ causes the formation of keloids and hypertrophic scars. To understand the mechanism by which antisense oligodeoxynucleotide (Oligo) acts on ...Objective and Methods: Excessive accumulation of collagen typeⅠ and type Ⅲ causes the formation of keloids and hypertrophic scars. To understand the mechanism by which antisense oligodeoxynucleotide (Oligo) acts on in vitro transcrption α1 (I) collagen gene, isotopes (α-32pGTP) was incorporated into 2 SP6 in vitro transcription systems. Results and Conclu- sion: Oligo 2 (at the transcription start region) could effectively inhibit in vitro transcription of pGEM3-Col13 and the control (random oligodeoxynucleotides) showed no inhibition. However, oligo 1 (at the transcription start region) obviously inhibited the in vitro transcription of pGEM3-Col14, while Oligo 2, which targeted at the down stream region (about 200 bp) of the promoter showed no significant inhibition effect.展开更多
Hepatocellular carcinoma (HCC) is one of the most common cancer-related causes of death worldwide. The epidermal growth factor receptor (EGFR) is highly expressed in many human tumors and provides a new target for ant...Hepatocellular carcinoma (HCC) is one of the most common cancer-related causes of death worldwide. The epidermal growth factor receptor (EGFR) is highly expressed in many human tumors and provides a new target for anticancer drug development. The aim of the present study was to explore the effect of EGFR antisense oligodeoxynucleotide on human HCC. METHODS:SMMC-7721 cells in culture were treated with 10 μmol/L antisense-odn for 24, 48, 72 hours respectively and MTT assay was adopted to determine the proliferation of tumor cells in vitro. About 2 ×106 SMMC-7721 cells with or without pretreatment(30 μmol/L oligodeoxynucleotide) were inoculated into subcutaneous flap of 21 nude mice, of which 7 were treated with EGFR antisense-odn, 7 with scrambled oligodeoxynucleotide (scrambled-odn), and 7 not treated in vivo. RESULTS:In vitro, after 24, 48, 72 hours the inhibitory rate of proliferation of SMMC-7721 cells treated with EGFR antisense-odn was 8%, 32%, and 34% respectively. In vivo after 8 weeks, no palpable tumor was found in 1/7 mice receiving cells pretreated with antisense-odn, whereas 7/7 untreated mice and 6/7 mice treated with scrambled-odn developed palpable tumors. Tumor growth in antisense-odn treated mice was significantly inhibited in comparison with that of those untreated (P【0.01) or treated with scram-bled-odn (P【0.05). CONCLUSIONS:Antisense oligodeoxynucleotide acts as a specific growth inhibitor on SMMC-7721 in a sequence specific and time-dependent manner. EGFR antisense-odn can significantly inhibit the proliferation of human hepatoma cell in vitro as well as in vivo, indicating that EGFR may play an important role in the development of hepatoma and will be a new target for its treatment.展开更多
This paper reviews the significant study on SRS leukemia virus (SRSV) in recent years in China. A series of results about the proteins, nucleic acids and function, CDNA libraries, molecular cloning of SRSV, and inhibi...This paper reviews the significant study on SRS leukemia virus (SRSV) in recent years in China. A series of results about the proteins, nucleic acids and function, CDNA libraries, molecular cloning of SRSV, and inhibitory effect of antisense oligodeoxynucleotides on SRSV are reported. It has proved that SRSV is useful to study the vival etiology and pathogenesis of human leukemial.展开更多
Objective: To investigate whether the Bc1-2 antisense oligonucleotide(ASODN) may enhance radiation-induced apoptosis in Raji cell line. Methods: Cell surviving fraction was determined using the trypan blue dye exclusi...Objective: To investigate whether the Bc1-2 antisense oligonucleotide(ASODN) may enhance radiation-induced apoptosis in Raji cell line. Methods: Cell surviving fraction was determined using the trypan blue dye exclusion assay. The expression level of bc1-2 protein was assayed by immunofluorescence using fluoresce isothiocyanate label. Apoptosis was detected by Giemsa staining and flow cytomertric cell cycle analysis. Results: It was found that Bc1-2 ASODN combined with radiation had significantly reduced the number of viable cells (P<0.05). There was no difference on cell survival between mismatch Bc1-2 oligodeoxynucleotide/radiation combination and radiation-treated cells alone. Bc1-2 ASODN combined with radiation could significantly inhibit expression of Bc1-2 protein in Raji cells (P<0.05). Cells treated with Bc1-2 ASODN combined with radiation at 72 h displayed classic apoptotic changes. Apoptosis rates of Raji cells treated with Bc1-2 oligodeoxynucleotide/radiation combination and radiation-treated cells alone, respectively. Conclusion: Bc1-2 antisense oligonucleotide can enhance radiation-induced apoptosis in Raji cell line.展开更多
AIM: To investigate the combined effects of K-ras antisense oligodeoxynucleotide (K-ras ASODN) specif ic to GTT point mutation at codon 12 and type Ⅰ insulin-like growth factor receptor (IGF-IR) antisense oligodeoxyn...AIM: To investigate the combined effects of K-ras antisense oligodeoxynucleotide (K-ras ASODN) specif ic to GTT point mutation at codon 12 and type Ⅰ insulin-like growth factor receptor (IGF-IR) antisense oligodeoxynucleotide (IGF-IR ASODN) on proliferation and apoptosis of human pancreatic cancer Patu8988 cells in vitro and in vivo. METHODS: K-ras gene point mutation and its style at codon 12 of human pancreatic cancer cell line Patu8988 were detected by using polymerase chain reaction with special sequence primers (PCR-SSP) and sequence analysis. According to the mutation style, K-ras mutation ASODN specifi c to K-ras point mutation at codon 12 was designed and composed. After K-ras ASODN and IGF-IR ASODN treated on Patu8988 cells respectively or cooperatively, the proliferation and morphological change of Patu8988 cells were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony forming assay andtransmission electron microscopy; the expression of K-ras and IGF-IR mRNA and protein in the treated cells was measured by reverse-transcript polymerase chain reaction (RT-PCR) and flow cytometry respectively; apoptosis was determined by flow cytometry. The combined antitumor activity of K-ras ASODN and IGF-IR ASODN was evaluated in BALB/c nude mice bearing human pancreatic cancer inoculated with Patu8988 cells. RESULTS: The results of PCR-SSP and sequence analysis showed that the human pancreatic cancer cell line Patu8988 had point mutation at codon 12, and the mutation style was GGT→GTT. 2-32 μg/mL K-ras ASODN and 2-32 μg/mL IGF-IR ASODN could inhibit Patu8988 cells' growth, induce apoptosis and decrease the expression of K-ras and IGF-IR mRNA and protein alone. However, there was much more effective inhibition of growth and induction of apoptosis by their combination than by each one alone. In tumor bearing mice, the combination of K-ras ASODN and IGF-IR ASODN showed a signif icant inhibitory effect on the growth of transplanted pancreatic cancer, resulting in a statistically signif icant difference compared with each alone. CONCLUSION: It has been found that K-ras ASODN combined with IGF-IR ASODN could cooperatively inhibit the growth of Patu8988 cells, and induce their apoptosis via reinforcing specific down regulation of K-ras and IGF-IR mRNA and protein expression.展开更多
Objective To study the inhibitory effects of caspase-3 mRNA antisense oligodeoxynucleotides (ASODN) on expressions of caspase-3 and it’s mRNA in γ-radiation induced apoptotic HL-60 cells, and screen the effective AS...Objective To study the inhibitory effects of caspase-3 mRNA antisense oligodeoxynucleotides (ASODN) on expressions of caspase-3 and it’s mRNA in γ-radiation induced apoptotic HL-60 cells, and screen the effective ASODN. Methods ASODN-1 and ASODN-2 targeting 5′-noncoding region and initial translation region of caspase-3 mRNA were respectively designed, synthesized and introduced into HL-60 cells by means of liposome-mediated transfection followed by 10Gy γ-radiation exposures. TUNEL assay was conducted to investigate the morphologic change and apoptotic percentage of HL-60 cells 18 h later. Immunocytochemical staining and one step RT-PCR were respectively performed to detect the expressions of caspase-3 and it’s mRNA. Mismatched oligodeoxynucleotide (MODN) transfected and un-transfected HL-60 cells were taken as control. Results TUNEL assay found that the apoptotic percentages in ASODN-1 and ASODN-2 groups were significantly reduced compared with the control groups (P<0.01) when the final concentration of both ASODNs was ≥3μmol/L. Immunocytochemistry showed that caspase-3 positive cell percentages were reduced but the average gray values increased significantly compared with the control groups (P<0.01). RT-PCR showed expressions of caspase-3 mRNA was decreased after ASODN transfection. Furthermore, ASODN-1 proved more effective in inhibiting HL-60 cell apoptosis than ASODN-2 (P<0.01). Conclusion Caspase-3 mRNA ASODNs can prevent HL-60 cells from apoptosis induced by γ-radiation and reduce expression of caspase-3 and its mRNA. These effects are dose dependent in a certain range.展开更多
Objective:To investigate the effect of MUC2 antisense oligodeoxynucleotide(ASODN)on cell proliferation,adhesion and proteolytic enzyme in human gastric carcinoma cell line(SGC7901).Methods:Phosphorothioate MUC2 ASODN ...Objective:To investigate the effect of MUC2 antisense oligodeoxynucleotide(ASODN)on cell proliferation,adhesion and proteolytic enzyme in human gastric carcinoma cell line(SGC7901).Methods:Phosphorothioate MUC2 ASODN was synthesized and packaged by lipofectin,and then transfected to SGC7901 cells.The expression of MUC2 mRNA and protein after transfection was detected by reverse transcription-polymerase chain reaction(RT-PCR)and immunohistochemical method respectively,and the effect of MUC2 ASODN on cell proliferation,adhesion and proteolytic enzyme was determined by flow cytometry(FCM),MTT method,Rose Bengal and immunohistochemical method.Results:Compared with the blank control group,ASODN efficiently downregulated the expression of MUC2 mRNA and protein in SGC7901 cells 48h after transfection(P<0.01).Various concentrations of ASODN could significantly inhibit the growth of SGC7901,and the inhibition peaked at the 48th hour after transfection(P<0.05).The apoptosis rate of the experimental group was about 4.38%,and the percentage of S-phase cells rose while G0/G1-phase cells fell because most of them were blocked at S-phase.In addition,cells treated with MUC2 ASODN showed lower adhesion ability with matrix and endothelial cells than control cells in vitro(P<0.01).By immunohistochemical method,the upregulation of E-cadherin proteins and the downregulation of MMP2 and cathepsinD proteins were also observed(P<0.05).Conclusion:MUC2 ASODN could efficiently inhibit SGC7901 cell proliferation,reduce cell adhesion ability and downregulate the expression levels of proteolytic enzyme in vitro.展开更多
文摘To explore the effect of NF κB on bcl x gene transcription in extended drug resistance leukemia cell line HL 60/E6, drug resistant subline HL 60/E6 was derived by intermittently exposing HL 60 cells to 6 ng/ml epirubicin. Indirect immunofluorescence was used to demonstrate the location of NF κB RelA in HL 60/E6 cells. FCM analysis and RT PCR were used to detect the efficiency of liposome mediated ODN transfection and the change of bcl x L mRNA levels after 5 μmol/L phosphorothioate (PS) derivatized antisense (AS) oligodeoxynucleotide (ODN) directed to RelA was transferred into HL 60/E6 cells. The results showed that RelA remained persistently active and located at the nuclei of HL 60/E6 cells,but in the cytoplasm of HL 60 cells, the efficiency of liposome mediated ODN transfection was significantly higher than that of null ODN ( P <0.01 in 4 h, 6 h, 12 h, 24 h). Exposure of HL 60/E6 cells to 5 μmol/L AS PS ODN directed to RelA led to a maximal 40 % decline of bcl x L mRNA levels within 8 h. The inhibition rate of bcl x L mRNA was (15±1.79) %, (28±2.34) %, (40±3.47) %, (20±1.54) % in 4 h, 6 h, 8 h, 15 h, respectively, but it was less than 15 % in control group. It was concluded that NF κB was involved in regulating bcl x transcription. It was suggested that NF κB was an important factor for drug resistance in leukemia cells.
基金Supported by the Outstanding Young Medical Personnel of Qingdao City
文摘AIM:To study the inhibition of nuclear factor kappa-B p65(NF-κB p65)antisense oligodeoxynucleotide(ASODN)on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2(TGF-β2)in vitro.·METHODS:NF-κBp65ASODNand NF-κB p65 missense oligodeoxynucleotide(MSODN)were designed and synthesized.Human lens epithelial cell line(HLE B-3)cells were prepared for study and divided into 7 groups.Control group was HLE B-3 cells cultured in vitro in dulbecco's modified eagle medium(DMEM).T1,T2,and T3 group were HLE B-3 cells cultured in vitro in DMEM with 10 ng/m L TGF-β2 for 6h,12h,24h respectively.A+T group was HLE B-3 cells cultured with 10 ng/m L TGF-β2for 24h after transfected by NF-κB p65 ASODN for 24h.M+T group was HLE B-3 cells cultured with 10 ng/m L TGF-β2 for 24h after transfected by NF-κB p65 MSODN for 24h.The negative control group was HLE B-3 cells cultured with 10 ng/m L TGF-β2 for 24h after cultured with transfer agent(Hi Per Fect)for 24h.Cell morphology was observed at different time points using an inverted microscope.The expression of NF-κB p65 m RNA was detected with reverse transcription-polymerase chain reaction(RT-PCR),and the expression ofα-smooth muscle actin(α-SMA)protein was assayed with ELISA.·RESULTS:With the TGF-β2 stimulation prolongation,the expression of NF-κB p65 m RNA and a-SMA protein increased in T1,T2,T3 groups compared with the control group,and the difference was statistically significant(P<0.05).NF-κB p65 ASODN lowered the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2.NF-κB p65 MSODN and Hi Per Fect did not lower the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2.The difference between control group and A+T group was not statistically significant(P>0.05),but the difference among A+T group and other groups was statistically significant(P<0.05).·CONCLUSION:NF-κB p65 ASODN could lower the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2,and antagonized TGF-β2-induced transdifferentiation of HLE B-3 in vitro.NF-κB p65ASODN could be used as a new biological therapeutic target of posterior capsular opacification.
文摘To assess the role of follicle stimulating hormone receptor(FSHR) gene expression in regulating expression of FSHR protein in the plasma membrane, the effects of a porcine FSHR cDNA antisense oligodeoxynucleotide (ODN) on FSHR mRNA levels and 125 I FSH binding were determined in Chinese hamster ovary cells expression recombinant porcine FSHR (pFSHR CHO cells). An 18 mer phosphorothioate endcapped antisense ODN that corresponded to the region surrounding the translation initiation codon of the porcine FSHR cDNA was synthesized. An 18 mer nonsense sequence of identical nucleotide composition, which had little homology to known DNA sequences, was synthesized for use as a control. pFSHR CHO cells were cultured in 24 well plates (10 5 cells/well) in the absence or presence of 1 20 μmol/L antisense or nonsense ODN for 24 h and then assayed for porcine FSHR mRNA, using quantitative reverse transcription and competitive polymerase chain reaction, and for 125 I FSH binding activity. Treatment with 10 μmol/L antisense ODN caused a paradoxical increase in porcine FSHR mRNA from 0.89±0.06 to 1.64±0.08 ng/mg total RNA ( P <0.05). Transfection with lipofectamine and 0.33 μmol/L antisense ODN caused an increase in porcine mRNA from 0.95±0.08 to 1.53±0.07 ng/mg total RAN. This was probably due to upregulation of mRNA synthesis resulting from inhibition of porcine FSHR protein translation. The nonsense ODN had no effect on porcine FSHR mRNA. Antisense, but not nonsense, ODN (10 μmol/L) inhibited membrane binding of 125 I FSH by 13.6± 0.8 % ( P <0.05) in 24 h. Treatment of cells with antisense ODN (10 μmol/L) for 48 h resulted in a 76±1.5 % ( P <0.05) inhibition of 125 I FSH binding. In contrast, transfection with lipofectamine and 0.33 μmol/L antisense ODN at 0 h caused a 76.1±1.3 % ( P <0.05) reduction in binding within 24 h. Binding had returned to 52.3±2.3 % ( P < 0.05) of normal by 48 h. These results indicate that an antisense ODN corresponding to the region of the translation start site of the porcine FSHR cDNA is an effective specific inhibitor of porcine FSHR synthesis and that inhibition of receptor synthesis causes a decrease in functional membrane bound FSHR.
文摘INTRODUCTIONEpidermal-growth-factor receptor(EGFR) is apolypeptide with 1186 amino acids,which binds toEGF family growth factors.Two major naturalligands in the family interact with EGFR:one isEGF,the other is transforming growth factor-α(TGF-α).When EGF or TGF-α,binds to EGFR,tyrosine kinase activity is induced which in turntriggers a series of events regulating the cellgrowth.The importance of EGFR in growthregulating pathways was confirmed by the fact
文摘Objective: To explore the probability of vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotides as a developing new therapeutic strategy for glioma. Methods: VEGF protein expression was detected by S-P immunohistochemical technique. Tumor cell apoptosis was observed by TUNEL method. Results: Compared with control, VEGF protein expression was inhibited by antisense oligodeoxynucleotides in vitro. And the inhibitory effects increased with the increasing concentration. VEGF positive rate was 82.10% in control group, while in 2.5, 5, 10 mmol/L AODN groups, they were 70.00%, 57.85%, 53.20% respectively. No inhibition effect was found in the cell lines treated with missense and sense oligodeoxynucleotides. In vivo, antisense oligodeoxy-nucleotides therapy also inhibited VEGF protein expression and induced the increase of apoptotic tumor cells. However, it has no effect on tumor cell proliferation. Conclusion: It is hopeful that VEGF antisense oligodeoxynucleotides may be a new gene therapy method to glioma through its antiangiogenesis effect by inhibition of VEGF.
基金This project was supported by a grant from the National Natural Sciences Foundation of China(No.395 70 2 89) .
文摘To study the effect of c myc antisense oligodeoxynucleotides (ODNs) on proliferation of pulmonary vascular pericytes (PC) induced by hypoxia, cell culture, dot hybridization using probe of digoxigenin 11 dUTP labeled cDNA, 3H thymidine incorporation, immunocytochemical technique and image analysis methods were used to observe the effect of c myc antisense ODNs on expression of c myc gene and proliferating cell nuclear antigen (PCNA), and 3H thymidine incorporation of PC induced by hypoxia. The results showed that hypoxia could significantly enhance the expression of c myc and PCNA ( P <0.01), and elevate 3H thymidine incorporation of PC ( P <0.01), but antisense ODNs could significantly inhibit the expression of c myc and PCNA ( P <0.05), and 3H thymidine incorporation of PC ( P <0.01). It was suggested that hypoxia could promote the proliferation of PC by up regulating the expression of c myc gene, but c myc antisense ODNs could inhibit hypoxia induced proliferation of PC by downregulating the expression of c myc gene.
文摘Extracellular matrix (ECM) degradation is an essential step that allows tumor cells to penetrate a tissue barrier and become metastatic. Heparanase (HPSE) is an endoglycosidase that specifically degrades heparin sulfate proteoglycans (HSPG), a chief component of ECM. HPSE is not expressed in normal epithelial cells but can be detected in a variety of human carcinomas including pancreatic cancer. In the present study, human pancreatic cancer cell line Panc-1 was transfected with HPSE antisense oligodeoxynucleotide (AS-ODN) in vitro, then the inhibitory effect of AS-ODN on HPSE gene expression and invasive ability of Panc-1 cells in vitro was examined. The HPSE mRNA and protein expression of Panc-1 cells transfected with AS-ODN was significantly inhibited. However, there were no marked inhibitory effects in Panc-1 cells treated with nonsense oligodeoxynucleotide (NS-ODN). Moreover, a modified Boyden chamber assay demonstrated that transfection with HPSE AS-ODN significantly inhibited invasive potential of Panc-1 cells in vitro after AS-ODN transfection. This suggests that HPSE AS-ODN may contribute to the inhibition of HPSE mRNA and protein expression, and results in a decrease of the invasive ability of Panc-1 in vitro.
文摘To study the effect of tTG fully phosphorothioated antisense oligodeoxynucleotides (tTG-ASDON) on tTG expression in cultured bovine trabecular meshwork cells (BTMCs) in vitro and explore a new treatment alternative for primary open angle glaucoma (POAG), the ASDON1 and ASDON2 complementary to the protein codogram region of tTG were designed, synthesized and phosphorothioated according to the secondary structure of tTG. The ASDON1 and ASDON2 were embedded in Lipofectamine and transfected into BTMCs. The untreated group served as negative controls. The expression of tTG in the mRNA and protein level were measured by semi-quantitative RT-PCR and immunohistochemical technique-Supervision method respectively. Our results showed that both the mRNA and the protein of tTG with tTG-ASDON1 and tTG-ASDON2 were significantly decreased as compared with that of the controls (P<0.05). On the other hand, no significant difference was found between the ASDON1 group and the ASDON2 group. It is concluded that the expression of tTG mRNA and protein in cultured BTMC are down-regulated by tTG- ASDON. As a result, tTG-ASDON may be used for the treatment of POAG through the inhibitory effect on the expression of tTG.
基金Hubei Provincial Science and Technology Key Program Foundation (No. 2004AA304B08)
文摘Objective: Aurora A kinase representing a family of evolutionarily conserved mitotic serine/threonine kinases has been found elevated in human lung adenocarcinoma cell line A549. It is suggested that the overexpression of Aurora A con- tributes to the carcinogenesis, chromosomal instability (CIN), and de-differentiation of lung cancers. To address its possibility as a therapeutic target for lung cancer, we employed the antisense oligodeoxynucleotide (ASODN) technique to inhibit Aurora A expression and investigate its effects on tumor growth and cell cycle of A549, as well as the chemosensitivity to paclitaxel. Methods: Aurora A ASODN was synthesized and transfected into A549 cells by lipofectAMINE 2000. Aurora A mRNA and protein expression were examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respec- tively. Cell cycle distribution was observed by flow cytometer. MTT assay was used to evaluate cell inhibition ratio before and after transfection. Results: The proliferation of the A549 cells was inhibited by Aurora A ASODN dose and time dependently. It was also observed that the IC50 of A549 cells after 48 hours’ treatment of ASODN was about 300 nmol/L and under such circumstances, the Aurora A mRNA and protein expression significantly decreased (P < 0.05), along with the induction of ac- cumulation of cells in S phase and the G2-M transition. Furthermore, cell inhibition ratio of the combination of Aurora A ASODN and paclitaxel was higher significantly than paclitaxel (P < 0.05) or Aurora A ASODN alone (P < 0.05). Conclusion: Inhibition of Aurora A expression can result in the suppression of cell growth and chemosensitizing activity to paclitaxel in human lung cancer cell line A549.
文摘AIM: To study the distribution and stability of antisense oligodeoxynucleotide (ASODN) in Walker-256 cells and their distribution in liver, lung and kidney tissues after being infused alone or mixed with lipiodol via hepatic artery in a rat liver tumor model.METHODS: 5'-Isothiocyananate (FITC)-labeled vascular endothelial growth factor (VEGF) ASODN was added into Walker-256 cell culture media. Its distribution in cells was observed by fluorescence microscope at different time points. Walker-256 carcinosarcoma was transplanted into Wistar rat liver to establish a liver cancer model. 5'-FITC-labeled VEGF ASODN mixed with (mixed group, n = 6) or without (TAI group, n = 6)ultra-fluid lipiodol was administrated via hepatic artery.Frozen samples of liver, lung and kidney tissue were taken from rats after 1, 3 and 6 d, respectively. The distribution of ASODN was observed under fluorescent microscope.RESULTS: ASODN could enter cytoplasm within 2 h and nuclei within 6 h. Accumulation of ASODN reached the peak point in nuclei at 12 h, and then disappeared gradually. No fluorescence could be seen in cells at 48 h. In vivo experiment, on d 1 and 3 the fluorescence staining in liver was stronger in mixed group than in TAI group and more fluorescence could be detected in lung and kidney in TAI group than in mixed group. On d 6, no fluorescence could be detected in TAI group, but faint fluorescence could be seen in mixed group. ASODN could be seen in cancer cells and normal hepatic cells. In mixed group, ASODN was mainly distributed in liver tumor tissues.CONCLUSION: ASODN can transfect Walker-256 cells.ASODN mixed with lipiodol infusion via hepatic artery can be used in the treatment of HCC.
基金This work was supported by the Natural Science National Foundation of China (grant number 30270458)the Application Basic Research Foundation of Chongqing Scientific Committee (grant number 41A1152C)the Fifteenth Medicine and Health Scientific Researc
文摘Purpose: To investiagate the effect of antisense oligonucleotides (ODN) to Nogo-A mRNA expression in oligodendrocytes and to establish the base for further research of repair of optic nerve injury.Method: (1)Oligodendrocytes were obtained by inoculating the optic nerve of newborn (2 days)rats and were identified with galactocerebroside(GC) antibody immunocytochemical stain. (2) In order to observe the effects of antisense ODN on cultured cells,we set up five groups, including the groups of three concentration of antisense Nogo-A ODN (2μM、5μM、l0μM),a group with the random sequence added to the medium and the control group. Reverse transcription-polymerase chain reaction(RT-PCR) was adopted to study the effects of ODN on the expression of Nogo-A in oligodendrocytes.Results: (1)Three days after inoculation, a few of round or fusiform shape cells migrated from optic nerve tissue; About 11 days later, the coverlips were completely covered by the cells;The cells identified with GC antibody immunocytochemical stain were positive cells.(2)The result of RT-PCR study showed that antisense Nogo-A ODN could significantly and specifically inhibit the expression of Nogo-A after 24 hours ( P＜ 0.01). Random sequence has no effect on Nogo-A expresson.Conclusion:Antisense Nogo-A ODN can effectively and specifically inhibit the expression of Nogo-A.
文摘Objective and Methods: Excessive accumulation of collagen typeⅠ and type Ⅲ causes the formation of keloids and hypertrophic scars. To understand the mechanism by which antisense oligodeoxynucleotide (Oligo) acts on in vitro transcrption α1 (I) collagen gene, isotopes (α-32pGTP) was incorporated into 2 SP6 in vitro transcription systems. Results and Conclu- sion: Oligo 2 (at the transcription start region) could effectively inhibit in vitro transcription of pGEM3-Col13 and the control (random oligodeoxynucleotides) showed no inhibition. However, oligo 1 (at the transcription start region) obviously inhibited the in vitro transcription of pGEM3-Col14, while Oligo 2, which targeted at the down stream region (about 200 bp) of the promoter showed no significant inhibition effect.
文摘Hepatocellular carcinoma (HCC) is one of the most common cancer-related causes of death worldwide. The epidermal growth factor receptor (EGFR) is highly expressed in many human tumors and provides a new target for anticancer drug development. The aim of the present study was to explore the effect of EGFR antisense oligodeoxynucleotide on human HCC. METHODS:SMMC-7721 cells in culture were treated with 10 μmol/L antisense-odn for 24, 48, 72 hours respectively and MTT assay was adopted to determine the proliferation of tumor cells in vitro. About 2 ×106 SMMC-7721 cells with or without pretreatment(30 μmol/L oligodeoxynucleotide) were inoculated into subcutaneous flap of 21 nude mice, of which 7 were treated with EGFR antisense-odn, 7 with scrambled oligodeoxynucleotide (scrambled-odn), and 7 not treated in vivo. RESULTS:In vitro, after 24, 48, 72 hours the inhibitory rate of proliferation of SMMC-7721 cells treated with EGFR antisense-odn was 8%, 32%, and 34% respectively. In vivo after 8 weeks, no palpable tumor was found in 1/7 mice receiving cells pretreated with antisense-odn, whereas 7/7 untreated mice and 6/7 mice treated with scrambled-odn developed palpable tumors. Tumor growth in antisense-odn treated mice was significantly inhibited in comparison with that of those untreated (P【0.01) or treated with scram-bled-odn (P【0.05). CONCLUSIONS:Antisense oligodeoxynucleotide acts as a specific growth inhibitor on SMMC-7721 in a sequence specific and time-dependent manner. EGFR antisense-odn can significantly inhibit the proliferation of human hepatoma cell in vitro as well as in vivo, indicating that EGFR may play an important role in the development of hepatoma and will be a new target for its treatment.
文摘This paper reviews the significant study on SRS leukemia virus (SRSV) in recent years in China. A series of results about the proteins, nucleic acids and function, CDNA libraries, molecular cloning of SRSV, and inhibitory effect of antisense oligodeoxynucleotides on SRSV are reported. It has proved that SRSV is useful to study the vival etiology and pathogenesis of human leukemial.
基金this work was supported by the grants from The Natural Science Program Foundation of Gaungdong Province(No.021195) and The Guangzhou City Key Foundation of Science and Technology Program (No.2001-Z- 037-01).
文摘Objective: To investigate whether the Bc1-2 antisense oligonucleotide(ASODN) may enhance radiation-induced apoptosis in Raji cell line. Methods: Cell surviving fraction was determined using the trypan blue dye exclusion assay. The expression level of bc1-2 protein was assayed by immunofluorescence using fluoresce isothiocyanate label. Apoptosis was detected by Giemsa staining and flow cytomertric cell cycle analysis. Results: It was found that Bc1-2 ASODN combined with radiation had significantly reduced the number of viable cells (P<0.05). There was no difference on cell survival between mismatch Bc1-2 oligodeoxynucleotide/radiation combination and radiation-treated cells alone. Bc1-2 ASODN combined with radiation could significantly inhibit expression of Bc1-2 protein in Raji cells (P<0.05). Cells treated with Bc1-2 ASODN combined with radiation at 72 h displayed classic apoptotic changes. Apoptosis rates of Raji cells treated with Bc1-2 oligodeoxynucleotide/radiation combination and radiation-treated cells alone, respectively. Conclusion: Bc1-2 antisense oligonucleotide can enhance radiation-induced apoptosis in Raji cell line.
基金Social development foundation of Suzhou, China, No. SZD0614Young teacher foundation of Soochow UniversityFoundation of health department of Jiangsu Province, China, No. Z200622
文摘AIM: To investigate the combined effects of K-ras antisense oligodeoxynucleotide (K-ras ASODN) specif ic to GTT point mutation at codon 12 and type Ⅰ insulin-like growth factor receptor (IGF-IR) antisense oligodeoxynucleotide (IGF-IR ASODN) on proliferation and apoptosis of human pancreatic cancer Patu8988 cells in vitro and in vivo. METHODS: K-ras gene point mutation and its style at codon 12 of human pancreatic cancer cell line Patu8988 were detected by using polymerase chain reaction with special sequence primers (PCR-SSP) and sequence analysis. According to the mutation style, K-ras mutation ASODN specifi c to K-ras point mutation at codon 12 was designed and composed. After K-ras ASODN and IGF-IR ASODN treated on Patu8988 cells respectively or cooperatively, the proliferation and morphological change of Patu8988 cells were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony forming assay andtransmission electron microscopy; the expression of K-ras and IGF-IR mRNA and protein in the treated cells was measured by reverse-transcript polymerase chain reaction (RT-PCR) and flow cytometry respectively; apoptosis was determined by flow cytometry. The combined antitumor activity of K-ras ASODN and IGF-IR ASODN was evaluated in BALB/c nude mice bearing human pancreatic cancer inoculated with Patu8988 cells. RESULTS: The results of PCR-SSP and sequence analysis showed that the human pancreatic cancer cell line Patu8988 had point mutation at codon 12, and the mutation style was GGT→GTT. 2-32 μg/mL K-ras ASODN and 2-32 μg/mL IGF-IR ASODN could inhibit Patu8988 cells' growth, induce apoptosis and decrease the expression of K-ras and IGF-IR mRNA and protein alone. However, there was much more effective inhibition of growth and induction of apoptosis by their combination than by each one alone. In tumor bearing mice, the combination of K-ras ASODN and IGF-IR ASODN showed a signif icant inhibitory effect on the growth of transplanted pancreatic cancer, resulting in a statistically signif icant difference compared with each alone. CONCLUSION: It has been found that K-ras ASODN combined with IGF-IR ASODN could cooperatively inhibit the growth of Patu8988 cells, and induce their apoptosis via reinforcing specific down regulation of K-ras and IGF-IR mRNA and protein expression.
文摘Objective To study the inhibitory effects of caspase-3 mRNA antisense oligodeoxynucleotides (ASODN) on expressions of caspase-3 and it’s mRNA in γ-radiation induced apoptotic HL-60 cells, and screen the effective ASODN. Methods ASODN-1 and ASODN-2 targeting 5′-noncoding region and initial translation region of caspase-3 mRNA were respectively designed, synthesized and introduced into HL-60 cells by means of liposome-mediated transfection followed by 10Gy γ-radiation exposures. TUNEL assay was conducted to investigate the morphologic change and apoptotic percentage of HL-60 cells 18 h later. Immunocytochemical staining and one step RT-PCR were respectively performed to detect the expressions of caspase-3 and it’s mRNA. Mismatched oligodeoxynucleotide (MODN) transfected and un-transfected HL-60 cells were taken as control. Results TUNEL assay found that the apoptotic percentages in ASODN-1 and ASODN-2 groups were significantly reduced compared with the control groups (P<0.01) when the final concentration of both ASODNs was ≥3μmol/L. Immunocytochemistry showed that caspase-3 positive cell percentages were reduced but the average gray values increased significantly compared with the control groups (P<0.01). RT-PCR showed expressions of caspase-3 mRNA was decreased after ASODN transfection. Furthermore, ASODN-1 proved more effective in inhibiting HL-60 cell apoptosis than ASODN-2 (P<0.01). Conclusion Caspase-3 mRNA ASODNs can prevent HL-60 cells from apoptosis induced by γ-radiation and reduce expression of caspase-3 and its mRNA. These effects are dose dependent in a certain range.
基金This work was supported by the Science Research Foundation of the Health Bureau of Chongqing Municipality(No.2000-48)
文摘Objective:To investigate the effect of MUC2 antisense oligodeoxynucleotide(ASODN)on cell proliferation,adhesion and proteolytic enzyme in human gastric carcinoma cell line(SGC7901).Methods:Phosphorothioate MUC2 ASODN was synthesized and packaged by lipofectin,and then transfected to SGC7901 cells.The expression of MUC2 mRNA and protein after transfection was detected by reverse transcription-polymerase chain reaction(RT-PCR)and immunohistochemical method respectively,and the effect of MUC2 ASODN on cell proliferation,adhesion and proteolytic enzyme was determined by flow cytometry(FCM),MTT method,Rose Bengal and immunohistochemical method.Results:Compared with the blank control group,ASODN efficiently downregulated the expression of MUC2 mRNA and protein in SGC7901 cells 48h after transfection(P<0.01).Various concentrations of ASODN could significantly inhibit the growth of SGC7901,and the inhibition peaked at the 48th hour after transfection(P<0.05).The apoptosis rate of the experimental group was about 4.38%,and the percentage of S-phase cells rose while G0/G1-phase cells fell because most of them were blocked at S-phase.In addition,cells treated with MUC2 ASODN showed lower adhesion ability with matrix and endothelial cells than control cells in vitro(P<0.01).By immunohistochemical method,the upregulation of E-cadherin proteins and the downregulation of MMP2 and cathepsinD proteins were also observed(P<0.05).Conclusion:MUC2 ASODN could efficiently inhibit SGC7901 cell proliferation,reduce cell adhesion ability and downregulate the expression levels of proteolytic enzyme in vitro.
