HNF6重组慢病毒载体的构建及对大肠癌SW620细胞侵袭转移能力的影响  

Effect of a recombinant lentiviral vector carrying hepatocyte nuclear factor 6 gene on migration and invasion abilities of SW620 cells

作　　者：耿海涛[1,2] 肖乾[1,2] 徐邓勇[1,2] 胡立锋[1,2] 丁克峰[1,2] 

机构地区：[1]浙江大学肿瘤研究所 [2]浙江大学医学院附属第二医院肿瘤外科,浙江杭州310009

出　　处：《南方医科大学学报》2012年第1期66-70,共5页Journal of Southern Medical University

基　　金：国家自然科学基金(81071801);浙江省自然科学基金杰出青年基金(R2100071)~~

摘　　要：目的构建HNF6的慢病毒载体pLeno-DCE-HA-HNF6,建立稳定表达HNF6的大肠癌细胞系并观察HNF6对大肠癌细胞系SW620的侵袭转移能力的影响。方法构建HNF6慢病毒载体pLeno-DCE-HA-HNF6,感染293T细胞,收集上清测定病毒滴度;病毒颗粒转染SW620细胞,通过荧光显微镜及流式细胞仪观察转染效率;定量PCR(qPCR)及Western blotting检测HNF6的表达;应用Transwell和划痕试验观察SW620细胞侵袭转移能力的变化。结果通过PCR及酶切鉴定成功构建pLeno-DCE-HA-HNF6载体;包装并得到高滴度的病毒颗粒;转染SW620后,qPCR及Western blotting检测到HNF6表达明显升高;HNF6导致SW620明显的细胞形态变化,Transwell及划痕试验证明HNF6使SW620细胞迁移能力降低。结论成功构建并包装得到HNF6的慢病毒颗粒,建立稳定表达HNF6的大肠癌细胞系SW620-HNF6,证明过表达HNF6后可以抑制大肠癌侵袭转移能力。Objective To construct a recombinant lentiviral vector that stably express hepatocyte nuclear factor 6（HNF6） in colorectal cancer cell line and examine its effects on the invasive ability of SW620 cells.Methods The lentiviral vector pLeno-DCE-HA-HNF6 was constructed and transfected into 293T cells.The supernatant containing the lentivirus particles was harvested to determine the virus titer.A stable cell line was established by infecting SW620 cells with the lentivirus particles,and the transfection efficiency was examined by fluorescence microscopy and flow cytometry.The invasion ability of the transfected SW620-HNF6 cells was assessed by wound healing and transwell assays.Results The recombinant lentiviral vector was correctly constructed and verified by sequencing.SW620-HNF6 cell line with stable HNF6 expression was established successfully,and the transfection efficiency reached 82.3%.Western blotting and quantitative PCR demonstrated significantly upregulated HNF6 expression in SW620-HNF6 cells,which showed obviously suppressed invasive ability in wound healing and transwell assays.Conclusion We have successfully established a colorectal cancer cell line SW620-HNF6 stably expressing HNF6,which shows a lowered migration activity in vitro.

关 键 词：肝细胞核因子6 SW620 慢病毒 大肠癌 转移 

分 类 号：R735.34[医药卫生—肿瘤]