构建了含抗人端粒酶逆转录酶单链抗体基因ScFv-hTERT的植物表达载体p1304-SH,通过农杆菌介导的叶盘法转化烟草.转基因烟草植株叶片总DNA的PCR,Southern b lot检测结果表明,ScFv-hTERT基因已整合进了转基因烟草植株基因组中;RT-PCR,SDS-P...构建了含抗人端粒酶逆转录酶单链抗体基因ScFv-hTERT的植物表达载体p1304-SH,通过农杆菌介导的叶盘法转化烟草.转基因烟草植株叶片总DNA的PCR,Southern b lot检测结果表明,ScFv-hTERT基因已整合进了转基因烟草植株基因组中;RT-PCR,SDS-PAGE分析证实目的基因已在烟草叶片中成功表达,竞争性ELISA的结果表明,表达的重组抗体与其抗原有良好的结合活性.展开更多
Nasopharyngeal carcinoma （NPC） is a common cancer in Southern China and Southeast Asia. The disease is a poorly differentiated carcinoma without effective cure, and the mechanism underlying its development remains l...Nasopharyngeal carcinoma （NPC） is a common cancer in Southern China and Southeast Asia. The disease is a poorly differentiated carcinoma without effective cure, and the mechanism underlying its development remains largely unknown. Of several factors identified in NPC aetiology in recent years, Epstein-Barr virus （EBV） infection has emerged to be most important. In almost all NPC cells, EBV uses several intracellular mechanisms to cause oncogenic evolution of the infected cells. One such mechanism by which EBV infection induces cellular immortalization is believed to be through the activation of telomerase, an enzyme that is normally repressed but becomes activated during cancer development. Studies show that greater than 85% of primary NPC display high telomerase activity by mechanisms involving EBV infection, consistent with the notion that EBV is commonly involved in inducing cell immortalization. More recently, different EBV proteins have been shown to activate or inhibit the human telomerase reverse transcriptase gene, by modulating intracellular signalling pathways. These findings suggest a new model with a number of challenges towards our understanding, molecular targeting and therapeutic intervention in NPC.展开更多
AIM:To investigate the effect of the demethylating reagent 5-aza-2'-deoxycitidine(DAC) on telomerase activity in hepatocellular carcinoma(HCC) cell lines,SMMC-7721 and HepG2.METHODS:The related gene expression in ...AIM:To investigate the effect of the demethylating reagent 5-aza-2'-deoxycitidine(DAC) on telomerase activity in hepatocellular carcinoma(HCC) cell lines,SMMC-7721 and HepG2.METHODS:The related gene expression in cell lines was examined by real-time reverse transcription-polymerase chain reaction and Western blotting analysis.The telomerase activity was examined by telomeric repeat amplification protocol-enzyme-linked immunosorbent assay and DNA methylation was determined by methylation-specific polymerase chain reaction.RESULTS:The telomerase activity was significantly reduced in both cell lines treated with DAC,accompanied by downregulation of telomerase reverse transcriptase(hTERT).We also observed the effect of DAC on the methylation status of hTERT promoter and the expression of regulatory genes,such as c-myc,p15,p16,p21,E2F1,and WT1.The methylation status of hTERT promoter could be reversed in SMMC-7721 by DAC,but not in HepG2 cells.However,p16 expression could be reactivated by demethylation of its promoter,and c-Myc expression was repressed in both cell lines.Moreover,DAC could enhance the sensitivity to the chemotherapeutic agents,such as cisplatin,by induction of apoptosis of HCC cells.CONCLUSION:The DAC exerts its anti-tumor effects in HCC cells by inhibiting the telomerase activity.展开更多
Objective: To investigate the influence of anti-keratin autoantibodies (AK auto Abs) on telom-erase activity of squamous cell carcinoma cultured in vitro and the mechanisms of the inhibitory effects of AK auto Abs on ...Objective: To investigate the influence of anti-keratin autoantibodies (AK auto Abs) on telom-erase activity of squamous cell carcinoma cultured in vitro and the mechanisms of the inhibitory effects of AK auto Abs on squamous cell carcinoma. Methods: Influence of AK auto Abs on the proliferation of Tca cells was observed by MTT colorimetry. Telomerase activity of cultured Tca cells and human keratinocytes was determined by telomeric repeat amplication protocol-ELISA (TRAP-ELISA) and polyacrylamide gel elec-trophoresis (PAGE). After being treated with AK auto Abs for 36 h at a concentration of 4, 8, 16 mg/L respectively, the changes of telomerase activity of Tea cells were also detected by TRAP-ELISA and PAGE. Results: MTT colorimetric determination showed that the capacity of proliferation of Tca cells correlated negatively with the concentration of AK auto Abs (r= -0. 74, P<0. 01). TRAP-ELISA and PAGE showed that telomerase activity of Tca cells increased significantly compared to that of cultured human keratinocytes (t = 3. 5396, P<0. 01). AK auto Abs at a concentrations of 4, 8, 16 mg/L had significant dose-dependent inhibitory effects on telomerase activity of Tca cells (r= - 0. 8358, P<0. 01). Conclusion: AK auto Abs have a significant dose-dependent inhibitory effect on the proliferation of cultured Tea cells. AK auto Abs inhibit telomerase activity of cultured Tca cells with dose-dependent pattern. It suggests that decrease of telomerase activity may play an important role in the inhibitory effects of AK auto Abs on squamous cell carcinoma.展开更多
文摘构建了含抗人端粒酶逆转录酶单链抗体基因ScFv-hTERT的植物表达载体p1304-SH,通过农杆菌介导的叶盘法转化烟草.转基因烟草植株叶片总DNA的PCR,Southern b lot检测结果表明,ScFv-hTERT基因已整合进了转基因烟草植株基因组中;RT-PCR,SDS-PAGE分析证实目的基因已在烟草叶片中成功表达,竞争性ELISA的结果表明,表达的重组抗体与其抗原有良好的结合活性.
文摘Nasopharyngeal carcinoma （NPC） is a common cancer in Southern China and Southeast Asia. The disease is a poorly differentiated carcinoma without effective cure, and the mechanism underlying its development remains largely unknown. Of several factors identified in NPC aetiology in recent years, Epstein-Barr virus （EBV） infection has emerged to be most important. In almost all NPC cells, EBV uses several intracellular mechanisms to cause oncogenic evolution of the infected cells. One such mechanism by which EBV infection induces cellular immortalization is believed to be through the activation of telomerase, an enzyme that is normally repressed but becomes activated during cancer development. Studies show that greater than 85% of primary NPC display high telomerase activity by mechanisms involving EBV infection, consistent with the notion that EBV is commonly involved in inducing cell immortalization. More recently, different EBV proteins have been shown to activate or inhibit the human telomerase reverse transcriptase gene, by modulating intracellular signalling pathways. These findings suggest a new model with a number of challenges towards our understanding, molecular targeting and therapeutic intervention in NPC.
基金Supported by The National Natural Science Foundation of China,No.30901722,30973433,81000970,81030041,31171321 and 81101622
文摘AIM:To investigate the effect of the demethylating reagent 5-aza-2'-deoxycitidine(DAC) on telomerase activity in hepatocellular carcinoma(HCC) cell lines,SMMC-7721 and HepG2.METHODS:The related gene expression in cell lines was examined by real-time reverse transcription-polymerase chain reaction and Western blotting analysis.The telomerase activity was examined by telomeric repeat amplification protocol-enzyme-linked immunosorbent assay and DNA methylation was determined by methylation-specific polymerase chain reaction.RESULTS:The telomerase activity was significantly reduced in both cell lines treated with DAC,accompanied by downregulation of telomerase reverse transcriptase(hTERT).We also observed the effect of DAC on the methylation status of hTERT promoter and the expression of regulatory genes,such as c-myc,p15,p16,p21,E2F1,and WT1.The methylation status of hTERT promoter could be reversed in SMMC-7721 by DAC,but not in HepG2 cells.However,p16 expression could be reactivated by demethylation of its promoter,and c-Myc expression was repressed in both cell lines.Moreover,DAC could enhance the sensitivity to the chemotherapeutic agents,such as cisplatin,by induction of apoptosis of HCC cells.CONCLUSION:The DAC exerts its anti-tumor effects in HCC cells by inhibiting the telomerase activity.
文摘Objective: To investigate the influence of anti-keratin autoantibodies (AK auto Abs) on telom-erase activity of squamous cell carcinoma cultured in vitro and the mechanisms of the inhibitory effects of AK auto Abs on squamous cell carcinoma. Methods: Influence of AK auto Abs on the proliferation of Tca cells was observed by MTT colorimetry. Telomerase activity of cultured Tca cells and human keratinocytes was determined by telomeric repeat amplication protocol-ELISA (TRAP-ELISA) and polyacrylamide gel elec-trophoresis (PAGE). After being treated with AK auto Abs for 36 h at a concentration of 4, 8, 16 mg/L respectively, the changes of telomerase activity of Tea cells were also detected by TRAP-ELISA and PAGE. Results: MTT colorimetric determination showed that the capacity of proliferation of Tca cells correlated negatively with the concentration of AK auto Abs (r= -0. 74, P<0. 01). TRAP-ELISA and PAGE showed that telomerase activity of Tca cells increased significantly compared to that of cultured human keratinocytes (t = 3. 5396, P<0. 01). AK auto Abs at a concentrations of 4, 8, 16 mg/L had significant dose-dependent inhibitory effects on telomerase activity of Tca cells (r= - 0. 8358, P<0. 01). Conclusion: AK auto Abs have a significant dose-dependent inhibitory effect on the proliferation of cultured Tea cells. AK auto Abs inhibit telomerase activity of cultured Tca cells with dose-dependent pattern. It suggests that decrease of telomerase activity may play an important role in the inhibitory effects of AK auto Abs on squamous cell carcinoma.
