论文部分内容阅读
目的 进行促凋亡治疗喉癌实验 ,构建和鉴定携带反义HSP70 (heatshockprotein 70 )基因的真核表达载体。方法 将HSP70cDNA反向克隆入 pcDNA 3.1,构建CMV启动子控制的真核表达载体 pcDNA AHSP70 ,用酶切鉴定结果 ;应用该载体转染人喉表皮样癌细胞Hep 2 ,G4 18筛选阳性克隆 ;westernblot和免疫组化检测转染前后瘤细胞的HSP70的表达。检测转染前后瘤细胞的生物学性状。结果 获得 pcDNA AHSP70真核表达载体 ,HSP70反义RNA阻断了Hep 2细胞的HSP70的表达 ,经westernblotting和免疫组化证实 ,实验组瘤细胞不能表达或低表达HSP70 ,而对照和空载体组高表达HSP70。转染反义HSP70的真核表达载体的Hep 2细胞比空载体组和对照组的细胞生长缓慢 ,细胞周期可见实验组出现亚二倍凋亡峰 ,而空载体组没有。结论 本研究成功构建了反义HSP70真核表达载体并在人喉癌细胞得以表达。
Objective To investigate the apoptosis-promoting treatment of laryngeal cancer and construct and identify the eukaryotic expression vector carrying heat shock protein 70 (HSP70) gene. Methods The HSP70 cDNA was reverse cloned into pcDNA 3.1. The eukaryotic expression vector pcDNA AHSP70 controlled by the CMV promoter was constructed and identified by restriction enzyme digestion. The vector was transfected into human laryngeal squamous carcinoma cell line Hep 2 and G4 18 to screen positive clones. Westernblot Immunohistochemistry was used to detect the expression of HSP70 in tumor cells before and after transfection. The biological characteristics of tumor cells before and after transfection were detected. Results The pcDNA AHSP70 eukaryotic expression vector was obtained. HSP70 antisense RNA blocked the expression of HSP70 in Hep 2 cells. Western blotting and immunohistochemistry confirmed that the tumor cells in the experimental group could not express HSP70, Expressed HSP70. Hep 2 cells transfected with the eukaryotic expression vector of antisense HSP70 grew slowly compared with the empty vector group and the control group. The sub-doubling apoptotic peak appeared in the experimental group in the cell cycle, but not in the empty vector group. Conclusion This study successfully constructed antisense HSP70 eukaryotic expression vector and expressed in human laryngeal carcinoma cells.