论文部分内容阅读
目的 :构建前列腺特异性抗原启动子 (PSAP)调控的促凋亡基因caspase 7表达载体并观察其对前列腺癌细胞的促凋亡作用 .方法 :提取基因组DNA ,通过PCR法获得前列腺特异性抗原启动子 (PSAP)基因 ,利用基因重组方法以PSAP替换掉 pCDNA3真核表达载体中的 pCMV启动子 ,构建PSAP pCDNA3载体 .测序正确后 ,将PSAP分别替换绿色荧光蛋白载体pIRES2 EGFP和克隆入带有效应型caspase 7基因的绿色荧光蛋白载体 pIRES2 EGFP Csp7中的pCMV启动子片段 ,分别获得由PSAP启动子调控的绿色荧光蛋白载体和带有效应型Caspase 7基因的真核表达载体PSAP pIRES2 EGFP Csp7.利用脂质体转染法将该载体分别转染至PC 3m(前列腺癌 )、Hep2 (喉癌 )及MC3T3 (正常成纤维 )细胞中 ,通过荧光显微镜观察转染细胞绿色荧光蛋白表达及细胞形态变化 ,免疫组化检测细胞中效应型caspase 7蛋白的表达状况 ,细胞计数观察转染不同表达载体后各类细胞生长变化 .结果 :经酶切鉴定及测序结果证实所构建载体正确 .转染PSAP pIRES2 EGFP Csp7载体后 ,①在PC 3m细胞中可观察到绿色荧光蛋白的表达 ,并可检测到效应型Caspase 7蛋白表达 ,而在Hep2及MC3T3细胞中未观测到绿色荧光 ,②荧光显微镜下可见前列腺癌PC 3m细胞生长受到抑制并出现凋亡细胞形态 ,细胞
OBJECTIVE: To construct a proapoptotic gene caspase 7 expression vector regulated by prostate specific antigen promoter (PSAP) and observe its proapoptotic effect on prostate cancer cells.Methods: Genomic DNA was extracted and prostatic specific antigen was obtained by PCR (PSAP) gene was constructed and pCMV promoter in pCDNA3 eukaryotic expression vector was replaced by PSAP to construct PSAP pCDNA3 vector.After sequenced correctly, PSAP was replaced by green fluorescent protein vector pIRES2 EGFP and cloned into pEGFP- Type green fluorescent protein carrier pIRES2 EGFP Csp7 promoter of the green fluorescent protein vector pSPV promoter were obtained PSAP promoter regulated by the green fluorescent protein vector and the effector Caspase 7 gene eukaryotic expression vector PSAP pIRES2 EGFP Csp7 using The vector was transfected into PC 3m (prostate cancer), Hep2 (laryngeal cancer) and MC3T3 (normal fibroblasts) by liposome transfection method. The expression of green fluorescent protein (GFP) Immunohistochemistry was used to detect the expression of the effector caspase 7 protein in the cells. The cell counts were observed by transfection with different expression The changes of cell growth were observed.Results: The constructed vector was confirmed by restriction enzyme digestion and sequencing.The expression of green fluorescent protein (GFP) was observed in PC 3m cells after transfection with PSAP pIRES2 EGFP Csp7 vector To the expression of effector type Caspase 7, while no green fluorescence was observed in Hep2 and MC3T3 cells. (2) Prostate cancer PC 3m cells were inhibited by fluorescence microscope and apoptotic cell morphology was observed. Cells