pEGFP-C1-反义生存素重组质粒的构建和转染  

Construction and transfection of the recombinant plasmid pEGFP-C1-antisense survivin

作　　者：邹冰[1] 涂水平[1] 谭继宏[1] 马天乐[1] 钟捷[1] 乔敏敏[1] 付华[1] 江石湖[1] 

机构地区：[1]第二医科大学附属瑞金医院消化内科,上海200025

出　　处：《中华消化杂志》2002年第12期711-714,共4页Chinese Journal of Digestion

基　　金：国家自然科学基金 ( 30 10 0 2 2 1);上海市科委项目( 0 14 1190 18)

摘　　要：目的　构建pEGFP C1 反义生存素 (survivin)重组质粒 ,并转染入人MKN 45低分化胃腺癌细胞株。观察转染前后生存素基因的表达及对凋亡的影响 ,从基因水平探讨反义生存素核酸治疗胃癌的分子生物学机制 ,为今后利用反义基因治疗肿瘤提供实验和理论依据。方法　应用基因重组技术构建 pEGFP C1 反义生存素重组质粒 ,通过脂质体转染法转染人MKN 45胃腺癌细胞株 ,以流式细胞仪方法检测细胞凋亡 ,以RT PCR技术检测质粒转染前后生存素基因mRNA的表达。结果　pEGFP C1 反义生存素重组质粒转染MKN 45低分化胃腺癌细胞株后细胞凋亡明显增加 ,G2 /M期细胞减少 ,同时生存素基因在mRNA水平被抑制。结论　反义生存素核酸能抑制细胞增殖 ,减少生存素基因的mRNA表达而促进胃癌细胞凋亡目的　构建pEGFP C1 反义生存素 (survivin)重组质粒 ,并转染入人MKN 45低分化胃腺癌细胞株。观察转染前后生存素基因的表达及对凋亡的影响 ,从基因水平探讨反义生存素核酸治疗胃癌的分子生物学机制 ,为今后利用反义基因治疗肿瘤提供实验和理论依据。方法　应用基因重组技术构建 pEGFP C1 反义生存素重组质粒 ,通过脂质体转染法转染人MKN 45胃腺癌细胞株 ,以流式细胞仪方法检测细胞凋亡 ,以RTObjective To construct recombinant plasmid pEGFP C1 antisense survivin and than transfect it to MKN 45 gastric cancer cells for studying the expression of survivin mRNA and its effects on apoptosis of cancer cells. Methods Recombinant pEGFP C1 antisense survivin plasmid was cloned and transfected to MKN 45 gastric cancer cells by lipofectamine. Apoptosis of gastric cancer cells was assayed by flow cytometry. The levels of survivin mRNA before and after transfection were determined by reverse transcription polymerase chain reaction analysis. Results After recombinant pEGFP C1 antisense survivin plasmid was transfected, the apoptosis of MKN 45 cells was increase, the cell number in G 2/M phase was decreased, and the level of survivin mRNA was inhibited. Conclusions pEGFP C1 antisense suvivin can promote the apoptosis of MKN 45 gastric cancer cells, that might attribute to the inhibition on cell proliferation and expression of survivin mRNA.

关 键 词：基因重组 胃癌 细胞凋亡 PEGFP-C1 反义生存素 重组质粒 构建 

分 类 号：R346[医药卫生—基础医学]