靶向于SW1990细胞K-ras exon 1反义RNA腺病毒的构建  

Construction of adenovirus-mediated anti-sense RNA targeting K-ras exon 1 in SW1990 cell line

作　　者：吕纯业[1] 邵成浩[1] 胡先贵[1] 

机构地区：[1]第二军医大学长海医院普通外科,上海200433

出　　处：《第二军医大学学报》2007年第5期500-503,共4页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(30200275).~~

摘　　要：目的:构建靶向于SW1990细胞K-ras exon 1序列的反义RNA腺病毒,观察其对SW1990细胞的增殖和凋亡的影响。方法:将K-ras exon 1 cDNA克隆至穿梭质粒pShuttle-CMV,筛选出反向插入的克隆,与腺病毒骨架质粒pAdEasy-1共转染大肠杆菌BJ5183,同源重组产生腺病毒重组质粒。腺病毒重组质粒转染293细胞进行包装,产生重组腺病毒。腺病毒载体Ad-LacZ转染SW1990细胞,X-gal染色观察感染效率。重组腺病毒感染体外培养的SW1990细胞,MTT法检测SW1990细胞增殖,AnnexinV/PI流式细胞术检测SW1990细胞凋亡。结果:所构建的重组腺病毒PCR扩增出282 bp的目的基因片段;制备出了高滴度重组病毒,CsCl_2密度梯度纯化前后病毒滴度分别为7.6×10~8pfu/ml和5.0×10^(10)pfu/ml,当MOI=100时对SW1990的感染效率接近100%;重组腺病毒体外转染能抑制SW1990细胞增殖(P<0.05),转染后4～5 d,抑制效应达到最高峰,抑制率约为40.5%,也能促进SW1990细胞凋亡,转染后72 h,凋亡率为(22.54±5.38)%(P<0.01)。结论:成功构建了靶向于SW1990细胞K-ras exon 1序列的反义RNA腺病毒,为进一步探讨胰腺癌以K-ras为靶点的反义基因治疗奠定了基础。Objective：To construct an adenovirus mediated anti-sense RNA targeting K ras exon 1 of SW1990 cell line and observe its effect on cell proliferation and apoptosis after transferred into SW1990 cell line. Methods： K ras exon 1 cDNA was cloned into shuttle vector pShuttle CMV and the resultant plasmid was confirmed by enzyme digestion and PCR. Clones with inverted insertion were selected and co transferred into E. coli BJ5183 with an adenoviral backbone plasmid pAdEasy 1 to produce recombinant plasmid by homologous recombination. Recombinants were then selected and transfected into 293 cell line to produce recombinant adenovirus. Recombinant adenovirus production was confirmed by PCR analysis and was amplified and purified; the virus titer was determined. Ad-LacZ was used to infect SW1990 cells and the infection efficiency was observed by X-gal staining. SW1990 cells was infected with the recombinant adenovirus and their proliferation and apoptosis were determined by MTT and annexinV/PI FCM assay. Results： A 282 bp target gene fragment was acquired by PCR; the titer of recombinant adenovirus was 7.6 × 10^8 pfu/m/ before purification by CsCl, gradient centrifugation and 5.0 × 10^10 pfu/ml after CsCl2 gradient centrifugation. When the recombinant adenovirus was at 100 MOI, the infection efficiency of SW1990 cells nearly reached 100K. The transfection of recombinant adenovirus significantly inhibited SW1990 cell proliferation （P〈0.05）, with a maximal inhibitory rate of 40.5% 4-5 days after infection; it also significantly increased SW1990 cell apoptosis, with the apoptotic rate being （22.54±5.38） % 72 hours after infection. Conclusion： We have successfully constructed an anti-sense RNA adenovirus vector targeting K ras exon 1 of SW1990 celt line. paving a way for the anti sense K ras gene therapy of pancreatic carcinoma.

关 键 词：基因 ras RNA 反义 腺病毒 胰腺肿瘤 

分 类 号：R730.5[医药卫生—肿瘤]