siArid2重组腺病毒载体的构建及其对肝癌细胞增殖的影响  

Construction of si Arid2 recombinant adenovirus and its effect on prolife ration of hepatocarcinoma cells

作　　者：段玉洁[1] 聂丽珠[1] 田玲[1] 李治[1] 陈震[1] 唐霓[1] 

机构地区：[1]重庆医科大学感染性疾病分子生物学教育部重点实验室,重庆400016

出　　处：《中国病理生理杂志》2014年第11期1998-2003,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81371827;No.31171307);"十二五"传染病国家科技重大专项(No.2012ZX10002005-003-002;No.2013ZX10002002-005-003)

摘　　要：目的：通过RNA干扰技术沉默内源性抑癌基因Arid2（si Arid2）的表达,观察其对肝癌细胞生长及细胞周期的影响。方法：设计3对特异性沉默Arid2基因的shRNA序列,分别克隆到腺病毒骨架载体上形成重组腺病毒质粒。将质粒转染到HEK293细胞中,包装和扩增形成完整高滴度的重组腺病毒Adsi Arid2-1-3。用构建好的腺病毒感染人肝癌SMMC-7721细胞,Western blotting方法检测Arid2蛋白的表达,筛选干扰效果最佳的重组腺病毒。MTS技术和流式细胞术观察干扰Arid2基因后肝癌细胞增殖和细胞周期的变化。结果：成功构建了高滴度的重组腺病毒Adsi Arid2-1-3。经Western blotting鉴定,Adsi Arid2-3为抑制效果最佳的重组腺病毒。MTS结果显示,Adsi Arid2组细胞在72 h、96 h的吸光度值与空细胞组和Adsicontrol组相比增高,细胞增殖速率明显加快。流式细胞术检测结果显示,Adsi Arid2组处于G1期、S期的细胞百分比与空细胞组和Adsicontrol组相比,G1期细胞百分比明显减少,S期细胞百分比明显增多。结论：成功构建干扰Arid2的重组质粒及重组腺病毒。沉默Arid2可以促进肝癌细胞的增殖,促进其由G1期向S期的转换。AIM： To observe the effects of Arid2 gene on cell proliferation and cell cycle by interference of endogenous Arid2 expression in hepatoma cells. METHODS： Three pairs of shRNA targeting Arid2 gene were cloned into a shuttle vector to construct recombinant adenovirus plasmids. HEK293 cells were transfected with the recombinant adenovirus plasmids. After several rounds of the package and amplification,the high-titer adenoviruses Adsi Arid2-1 - 3 were obtained. To verify the inhibitory effects of Adsi Arid2 adenoviruses,Western blotting was used to detect the endogenous Arid2 protien expression in SMMC-7721 cells. Cell growth and cell cycle analysis were carried out by MTS assay and flow cytometry. RESULTS： High- titer recombinant adenovirus of si Arid2 were successfully obtained,and named Adsi Arid2-1 - 3,among which the Adsi Arid2-3 had the best inhibitory effects. MTS assay showed that the absorbance values at 490 nm were increased at 72 h and 96 h after transduction compared with the mock and Adsicontrol groups. These data indicated that knockdown of Arid2 promoted the proliferation rate of SMMC-7721 cells（ P〈0. 05）. Moreover,the flow cytometry analysis revealed that the G1-phase distribution at 72 h in Adsi Arid2 group was lower than that in mock group and Adsicontrol group. In contrast,the S-phase distribution in Adsi Arid2 group was much higher than that in mock group and Adsicontrol group. CONCLUSION： The recombinant plasmids and recombinant adenovirus were successfully constructed. shRNAmediated knockdown of Arid2 promotes the proliferation and the transition from G1 phase to S phase of hepatoma cells.

关 键 词：基因 Arid2 肝细胞癌 SMMC-7721细胞 细胞增殖 细胞周期 

分 类 号：R73-3[医药卫生—肿瘤]