应用RNA干扰技术建立PRL-3和CDH22双基因表达抑制的结直肠癌细胞模型  

Establishment of a colorectal cancer cell line with PRL-3 and CDH22 gene knock-down by lentivirus-mediated RNA interference

作　　者：姚娟[1] 丁彦青[1] 周军[1] 柳玉红[1] 李建明[1] 

机构地区：[1]南方医科大学南方医院病理科,南方医科大学基础医学院病理学系,广东省分子肿瘤病理重点实验室,教育部广东省共建人类重大疾病转录组学和蛋白组学重点实验室,广东广州510515

出　　处：《南方医科大学学报》2009年第4期593-597,共5页Journal of Southern Medical University

基　　金：国家自然科学基金资助项目(30500241;30670968和30700286);广东省自然科学基金重点项目(5200512)

摘　　要：目的建立稳定干扰PRL-3及CDH-22基因的结直肠癌细胞株,为探讨PRL-3、CDH22及其相互作用在结直肠发生及转移中的作用提供理想的细胞模型。方法以稳定干扰人PRL-3基因大肠癌SW480细胞的克隆为基础,以靶向人CDH22基因的RNAi慢病毒再次感染该细胞株,将经慢病毒二次感染的细胞悬液通过有限稀释法制备细胞单克隆,各细胞克隆扩大培养,荧光定量PCR检测各细胞克隆PRL-3及CDH22mRNA表达水平。结果应用于二次感染的慢病毒悬液的滴度为8×105U/ml。综合分析荧光定量PCR结果,所获得的细胞克隆中1号克隆PRL-3及CDH22mRNA水平的表达显著降低。结论成功建立PRL-3及CDH-22基因稳定敲低的大肠癌细胞克隆,探索同时敲低2个基因的慢病毒RNAi技术。Objective To establish a colorectal cancer cell line with stable PRL-3 and CDH22 gene knock-down for investigating the role of PRL-3 and CDH22 genes in the carcinogenesis and metastasis of eolorectal cancer. Methods A recombinant lentiviral vector targeting CDH22 gene was obtained using the pENTRTM/U6 construct and pLenti6/BLOCK-iT TM-DEST vector. The recombinant lentivirus was harvested from 293FT cells cotransfected with the optimized ViraPowerTM Packaging Mix and the pLenti6/BLOCK-iT^TM-DEST expression construct. SW480/PRL-3- cells were infected with the recombinant lentivirus targeting CDH22, and SW480 cells with stable PRL-3 and CDH22 knock-down were screened by blasticidin selection. PRL-3 expression in the cells was determined by real-time RT-PCR. Results The titer of the lentivirus for the second infection was 8 ×10^5 U/ml. Seventeen positive clones were selected, among which the Clone 1 exhibited substantially down-regulated CDH22 and PRL-3 mRNA expressions. Conclusion A human coloreetal cancer cell line with stable PRL-3 and CDH22 gene knock-down has been established.

关 键 词：PRL-3 CDH22 RNA干扰 慢病毒 结直肠癌 

分 类 号：R735.3[医药卫生—肿瘤]