TD—IκBα病毒包装及其对肾癌细胞株ACHN增殖和侵袭的影响  

TD-IκBα lentivirus packaging and its effect on proliferation and invasion of clear cell renal cell car-cinoma cell line ACHN

作　　者：李新涛[1] 马鑫[1] 逄海港 高宇[1] 范阳[1] 张旭[1] 

机构地区：[1]解放军总医院泌尿外科肾脏疾病国家重点实验室,北京100853

出　　处：《中华实验外科杂志》2014年第6期1184-1186,共3页Chinese Journal of Experimental Surgery

基　　金：国家重点基础研究发展计划（973计划）资助项目（2013CB530803）;卫生行业科研专项项目泌尿系统重大疾病的防治研究项目（201002010）

摘　　要：目的构建pLVX-TD—IκBα—IRES—ZsGreenl慢病毒表达载体及包装病毒，观察稳定转染TD-IκBα对肾癌细胞株ACHN增殖和侵袭的影响。方法从pCFG5-IEGZ—TD—IκBα质粒上获得的目的基因片段克隆入pLVX-IRES—ZsGreenl载体，并进行测序。将慢病毒载体与包装质粒delta8．91和p-VSVG以4：3：2比例共转染293T细胞，收集病毒上清并感染肾癌细胞株ACHN，5d后采用Western blot检测TD-IκBα蛋白的表达，使用四唑氮化合物（MTS）和Transwell侵袭实验方法检测细胞增殖、迁移和侵袭能力的变化。结果成功构建pLVX—TD—IκBα—IRES—ZsGreenl慢病毒表达载体，包装病毒后成功感染ACHN细胞株，感染组细胞株中的TD—IκBα蛋白显著表达增高。感染组细胞在490nm处吸光度值明显下降（P〈0．05）。细胞迁移和侵袭实验中，相对于阴性对照组穿膜细胞数[（361．400±42．379）个和（328．500±27．031）个]，感染组穿膜细胞数[（252．200±26．142）个和（127．300±35．572）个]显著减少（P〈0．05）。结论成功构建pLVX—TD—IκBα-IRES—ZsGreenl重组慢病毒表达载体并包装病毒，过表达TD—IκBα蛋白可显著降低ACHN细胞株的增殖、迁移和侵袭能力。Objective To construct the pLVX-TD-IκBα-IRES-ZsGreenl lentiviral expression vector and package virus, and to investigate the effect of its infection on proliferation, migration and invasion of clear cell renal cell carcinoma cell line ACHN. Methods The target gene sequence was cloned into pLVX-IRES-ZsGreenl vector. The lentiviral vector and the packaging vector delta8.91, p-VSVG were cotransfected into 293T cells to package virus. After ACHN cell line was infected by the virus supernatant, Western blotting was used to measure the IκBα expression, and 3-（4, 5-Dimethyhhiazol-2-yl）-5-（3-car-boxymethoxyphenyl）-2-（4-sulfophenyl）-2H-tetrazolium （MTS） and transwell were used to assess cell proliferation, migration and invasion. Results After construction of recombinant pLVX-TD-IκBα-IRES-Zs- Greenl lentiviral expression vector, the lentiviral particles were successfully packaged. ACHN ceil line was successfully infected and TD-IκBα protein expression significantly increased in the infection group. MTS assay demonstrated the absorbance in TD-IκBα infected group was significantly decreased at 24, 48, 72 and 96 h after infection （P 〈 0. 05 ） , and Transwell assay showed that the number of migratory and invasive cells in TD-IκBα infection group （252. 200±26. 142 and 127. 300±35. 572） was significantly reduced as compared with negtive control group （361. 400± 42. 379 and 328. 500 ± 27.031 ）. Conclusion Recombinant pLVX-TD-IκBα-IRES-ZsGreenl lentiviral expression vector was successfully constructed and the lentiviral particles were successfully packaged. Infection of TD-IκBα lentivirus could significantly decrease cell proliferation, migration and invasion of ACHN cell line.

关 键 词：IΚBΑ 病毒包装 肾透明细胞癌 增殖 侵袭 

分 类 号：R737.11[医药卫生—肿瘤]