肿瘤相关抗原基因OVA66特异的RNA干扰对肿瘤细胞生物学特性的影响  

The effects of specific interference RNA of the tumor antigen-associated gene on biological charactristics of tumor cells

作　　者：李美星[1] 王颖[1] 王树军[1] 张惠珍[1] 周光炎[1] 尤强[1] 葛海良[1] 

机构地区：[1]上海交通大学医学院上海市免疫学研究所,上海200025

出　　处：《现代免疫学》2005年第6期450-455,共6页Current Immunology

基　　金：国家自然科学基金资助项目(30371598);上海市高校科技发展基金资助项目(03BX20)

摘　　要：构建OVA66基因的特异小干扰RNA表达载体,转染HeLa细胞,分析对该基因表达和肿瘤生物学特性的影响。以RT-PCR、Westernblot方法检测OVA66基因在多种肿瘤细胞系的表达谱。利用计算机辅助设计和合成针对OVA66的特异小干扰RNA的DNA片段,定向克隆于特定的干扰载体(pSUPER)。利用脂质体法将重组载体转染于OVA66高表达细胞系HeLa,筛选得到shRNA-OVA66稳定表达细胞。采用Real-timePCR检测目的基因的表达;FACS、Westernblot方法分析目的蛋白的表达。MTT、3H-TdR掺入等方法检测干扰OVA66基因表达后对HeLa细胞增殖和凋亡的影响。双酶切鉴定得到针对OVA66基因特异的shRNA表达载体(pSUPER-shRNA-OVA66),并经序列分析证实。经检测pSUPER-shRNA-OVA66稳定表达的HeLa细胞(shRNA-OVA66)中目的基因mRNA水平降低;OVA66蛋白表达受到抑制,表明设计的靶片段对目的基因有显著的抑制效果。并显示shRNA-OVA66细胞DNA合成下降;细胞周期分析表明阻断OVA66基因表达可抑制shRNA-OVA66细胞增殖能力,并引发细胞凋亡。OVA66特异的shRNA表达载体构建成功,阻断OVA66基因表达可抑制细胞增殖,促使细胞凋亡,为OVA66蛋白肿瘤生物学功能与肿瘤发生、发展的研究奠定基础。The expression vector for the sequence-specific interference RNA（RNAi） of the tumor-associated gene OVA66 was constructed, tranfected to HeLa cells, and the influence on the expression of this gene and the biological characteristics of tumors were analyzed. In this study, the expression of OVA66 mRNA and the protein contents in some human tumor cell lines were detected by RT-PCR and Western blotting, and the DNA fragment of OVA66-speciflc small RNAi was obtained with the computer aided design and chemical synthesis and then was directionally cloned into the expression vector pSUPER to form recombinant vector pSUPER-shRNA-OVA66. Then, the constructed recombinant vector was transfected to HeLa cells with liposome in which the OVA66 gene was highly expressed. By selection with puromycin, the stable expression cells of shRNA-OVA66 （shRNA-OVA66 cells） was obtained. The interfering effect on the expression of shRNA-OVA66 on OVA66 gene was detected by real-time PCR and the OVA66 protein expression was assayed by Western blotting and FACS. Meanwhile, the proliferation and apoptosis of HeLa cells after interference of the OVA66 gene expression were analyzed by MTT, 3H-TdR incorporation and FACS, and the pSUPER-shRNA-OVA66 was confirmed through double enzymatic digestion and DNA sequence analysis. The expression of OVA66 at mRNA and protein levels were specifically inhibited in the stable expression cells trasfected with pSUPER-shRNA-OVA66, indicating that the designed oligotargeted to OVA66 had significantly inhibitory effect on OVA66 gene expression. It was shown that the DNA synthesis was reduced in shRNA-OVA66 cells as demonstrated by ^3H-TdR incorporation. The cell cycle analysis indicated that the blockage of OVA66 gene expression could induce the inhibition of the proliferative capacity and apoptosis of the shRNA-OVA66 cell. It is concluded that the OVA66-specific shRNA expression vector has been successfully constructed and identified to have the abilities to inhibit cell proliferation and to induce cell ap

关 键 词：OVA66 肿瘤相关基因 干扰shRNA 凋亡 增殖 

分 类 号：R730.231[医药卫生—肿瘤] Q52[医药卫生—临床医学]