稳定过表达、干扰RN181的MHCC97L细胞系建立及RN181功能的初步研究  

Establishment of MHCC97L cell line stably expressing RN181 and study on RN181 function in MHCC97L cells

作　　者：王小波[1] 彭迎霞[1] 张贤[1] 尹江丽[1] 李明[1] 

机构地区：[1]南方医科大学生物技术学院,广东广州510515

出　　处：《热带医学杂志》2013年第4期393-396,F0004,共5页Journal of Tropical Medicine

基　　金：国家自然科学基金(81171959)

摘　　要：目的构建过表达和干扰RN181基因的肝癌MHCC97L重组细胞系,并研究RN181对该细胞体外增殖的影响。方法基于基因克隆技术,构建plncx2-GFP-IRES/RN181重组载体,常规鉴定后,与pVSV-G共转染GP2-293细胞包装逆转录病毒。该病毒与RN181干扰慢病毒分别感染MHCC97L细胞,流式分选表达绿色荧光的细胞,RT-PCR、Western blotting鉴定后,CCK-8实验和软琼脂克隆形成实验检测细胞增殖情况。结果重组载体构建成功,重组细胞荧光良好,RT-PCR、Western blotting分别证实重组细胞中RN181的mRNA和蛋白表达水平在重组细胞系间均出现差异有统计学意义(P<0.05)。增殖实验显示RN181上调后细胞生长减慢,克隆数也显著少于对照组(P<0.05),而RN181干扰后上述现象可得到逆转。结论成功构建了过表达和干扰RN181的MHCC97L重组细胞系,初步证实了RN181在体外对MHCC97L细胞增殖的抑制作用。Objective To establish MHCC97L cell line stably expressing RN181 and study the effect of RN181 on the proliferation of MHCC97L in vitro. Methods Gene cloning techniques were used to constructed plncx2-GFP-IRES/ RN181 recombinant vector, which was subsequently co-tansfected into GP2-293 cells with pVSV-G plasmid to package retrovirus after routine identification.Then,the MHCC97L cells infected respectively by this retrovirus and RN181 knocking-down lentivirus were sorted by FACS.After identification by RT-PCR and Western blotting,CCK-8 and Soft agarose cell clone formation assays were used to evaluate the effect of RN181 on MHCC97L growth. Results We verified correct construction of the recombinant plasmid.RT-PCR and Western blotting confirmed both mRNA and protein levels of RN181 existed significant differences among the recombinant cell lines （P〈0.05）. Proliferation assays showed a remarkable growth suppression in MHCC97L-RNI81 cell line and a notable acceleration in MHCC97L-KD （P〈0.05）. Conclusions We have successfully constructed recombinant MHCC97L cell line with different RN181 expression levels of individual clones. RN181 could inhibit the proliferation of MHCC97L cell in vitro.

关 键 词：RN181 基因克隆 病毒包装 稳定表达 

分 类 号：R735.7[医药卫生—肿瘤]