论文部分内容阅读
目的建立稳定转染bcl-2的鳞状细胞癌(鳞癌)细胞系,研究人皮肤鳞癌细胞凋亡的相关通路。方法应用脂质体介导的基因转染法.将含有bcl-2 cDNA的逆转录病毒表达载体pIRESneo质粒导入皮肤鳞癌细胞系SCL-1细胞中.并以空质粒作为对照。经过G-418筛选后获得转入目的基因的细胞克隆,细胞扩大培养后用细胞免疫荧光染色法、RT-PCR和免疫蛋白印迹检测bcl-2的表达情况。然后用C_2-ceramide和抗Fas单克隆抗体诱导细胞株凋亡,利用ELISA法检测可溶性DNA-组蛋白复合物,分析其凋亡的差异。结果转染了pIRESneo-bcl-2的SCL-1细胞bcl-2蛋白和mRNA表达阳性,而转染了pIRESneo空载体的SCL-1细胞为阴性。不同浓度的C_2-ceramide对SCL-1/bcl-2凋亡的诱导差异均无统计学意义,而对SCL-1/vector诱导的凋亡差异均有统计学意义。抗Fas单克隆抗体对SCL-1/bcl-2和SCL-1/vector诱导的凋亡.差异均有统计学意义,P<0.01。结论用基因转染法成功地建立了稳定高表达bcl-2蛋白的SCL-1细胞系,C_2-ceramide诱导的SCL-1凋亡基本可以被bcl-2阻滞,而Fas所诱导的调亡仅部分被阻滞,即SCL-1细胞系在被阻断了线粒体途径后,还可以经由死亡受体通路进行凋亡。
Objective To establish a cell line of squamous cell carcinoma (squamous cell carcinoma) stably transfected with bcl-2 and to study the related pathways of apoptosis in human squamous cell carcinoma. Methods The plasmid pIRESneo containing the bcl-2 cDNA was introduced into the SCL-1 cell line by liposome-mediated gene transfection method, and the empty plasmid was used as the control. Cell clones transfected into the target gene were screened by G-418. The cells were expanded and cultured to detect the expression of bcl-2 by immunofluorescence staining, RT-PCR and Western blotting. C 2 -ceramide and anti-Fas monoclonal antibody were used to induce cell apoptosis. The soluble DNA-histone complex was detected by ELISA and the difference of apoptosis was analyzed. Results The expression of bcl-2 protein and mRNA in SCL-1 cells transfected with pIRESneo-bcl-2 was positive, while the SCL-1 cells transfected with pIRESneo empty vector were negative. There was no significant difference in the induction of SCL-1 / bcl-2 apoptosis between C_2-ceramide and SCL-1 / vector at different concentrations. The apoptosis of SCL-1 / bcl-2 and SCL-1 / vector induced by anti-Fas monoclonal antibody was significantly different (P <0.01). Conclusion The SCL-1 cell line stably expressing bcl-2 protein was established successfully by gene transfection. The apoptosis of SCL-1 induced by C 2 -ceramide was blocked by bcl-2 and the apoptosis induced by Fas Only partially blocked, that is, after being blocked by the mitochondrial pathway, the SCL-1 cell line can also undergo apoptosis via the death receptor pathway.