shRNA靶向干扰COX-2稳定细胞系的建立及鉴定  

Construction and Identification of Cell Lines of COX-2 Inhibited by shRNA

作　　者：汪先桃[1] 董晋豫[1] 郭变琴[1] 马婷婷[1] 熊海玉[1] 涂植光[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016

出　　处：《中国细胞生物学学报》2013年第6期774-779,共6页Chinese Journal of Cell Biology

基　　金：国家自然科学基金面上项目(批准号:81172016)资助的课题~~

摘　　要：脂质体法将COX-2-shRNA载体转染肝癌细胞株SMMC-7721,G418筛选获得稳定细胞系COX-2-shRNA-SMMC-7721,通过RT-PCR和Western blot分别检测细胞系中COX-2基因mRNA转录水平和蛋白质表达水平,通过MTT、流式细胞术和Transwell等观察细胞恶性生物学行为的改变。建立COX-2-shRNA-SMMC-7721(干扰组)、HK-SMMC-7721(阴性对照组)细胞株。干扰组的COX-2基因mRNA的转录水平、蛋白表达水平和细胞增殖能力较阴性对照组和SMMC-7721(空白组)明显下降(P<0.05),而阴性对照组与空白组间无明显差异(P>0.05);干扰组、阴性对照组和空白组处于G0/G1期的细胞分别为(68.85±0.27)%、(53.05±0.35)%和(53.54±0.33)%;细胞凋亡率分别为(9.60±0.20)%、(1.79±0.23)%和(1.75±0.20)%;穿膜细胞数分别为(117.60±5.30)、(338.40±11.50)和(347.40±12.80)个。干扰组与阴性对照组和空白组有显著差异(P<0.05),而阴性对照组与空白组间无明显差异(P>0.05)。利用RNA干扰技术成功构建了靶向干扰COX-2的稳定肝癌细胞系COX-2-shRNA-SMMC-7721,为COX-2分子的作用机制研究提供了细胞模型。The constructed recombinant interference plasmid pshRNA-COX-2 was transfected into SMMC7721 cells by lipofectamine mediation, then monoclonal cell line was selected by G418 pressure. The expressions of COX-2 mRNA and protein were measured by reverse transcription polymerase chain reaction （RT-PCR） and Western blot respectively, and the changes of cell proliferation and apoptosis were assessed by MTT assay and flow cytometry, respectively. The Transwell test was used to detect the invasion ability of cell lines. COX-2-shRNA-SMMC-7721 （interference group）, HK-SMMC-7721 group （negative control group） cell lines were established. The levels of interference group＇s COX-2 mRNA transcription, COX-2 protein expression and cell proliferation significantly decreased. There were significant differences compared with those of the control group and SMMC-7721 （blank group） （P〈0.05）, while those of control group and blank control group were not significantly different （P〉0.05）. Interference group, control group and blank group of cells in G0√G1 phase were （68.85±0.27）%, （53.05±0.35）% and （53.54±0.33）%, respectively; the percentage of apoptotic cells were （9.60±0.20）%, （1.79±0.23）% and （1.75±0.20）% separately; transmembrane cell number were （117.60±5.30）, （338.40±11.50） and （347.40±12.80） cells, respectively. There were significant differences comparing interference group with control group and blank group （P〈O.05）, while the controlgroup and blank control group were not significantly different （P〉0.05）. The cell line COX-2-shRNA-SMMC-7721,in which COX-2 expression was inhibited using RNA interference technology, was successfully established, whichcould provide the cell model for fflrther study of C0X-2 gene function.

关 键 词：COX-2 SHRNA 稳定细胞系 肝癌 

分 类 号：R735.7[医药卫生—肿瘤]