CDK2干扰RNA对人肝癌细胞HepG_2细胞周期和增殖的影响  

EFFECTS OF CDK2 siRNA ON CELL CYCLE AND PROLIFERATION OF HUMAN HEPATOMA CELLS HepG_2

作　　者：王文礼[1] 李丽梅[1] 关则红[1] 王震宇[1] 郝兴霞[1] 姜丽丽[1] 

机构地区：[1]内蒙古医学院生物化学与分子生物学教研室,内蒙古呼和浩特010059

出　　处：《内蒙古医学院学报》2008年第3期153-157,共5页Acta Academiae Medicinae Neimongol

基　　金：内蒙古医学院重大科研项目(NY2004ZD002)

摘　　要：目的:探讨CDK2 siRNA对人肝癌细胞株HepG2细胞CDK2 mRNA表达及其对HepG2细胞周期和增殖的影响。方法:利用脂质体转染法将特异性的携带绿色荧光蛋白的CDK2干扰RNA的重组质粒PGenesil-1-CDK2导入HepG2细胞,同时设立阴性对照空载体转染组PGenesil-1-HK。G418筛选稳定表达细胞株扩增培养。并用倒置荧光显微镜观察转染细胞的绿色荧光蛋白。反转录-聚合酶链反应(RT-PCR)和流式细胞术(FCM),分别检测转染后的HepG2细胞CDK2 mRNA和细胞周期的变化,噻唑蓝(MTT)法检测癌细胞生长增殖率。结果:经1mo的G418筛选得到稳定表达细胞株,与阴性对照组及空白对照组相比,转染组CDK2 mRNA水平明显下降,细胞周期明显改变,细胞从G1期到S期发生阻滞。结论:RNA干扰技术可明显抑制CDK2基因的表达,引起HepG2细胞周期阻滞及肿瘤细胞生长抑制作用,为CDK2介导的肿瘤基因沉默疗法提供实验依据。Objective：To investigate the effects of CDK2 siRNA on CDK2 mRNA , cell cycle and proliferation of human hepatoma cells HepG2. Methods：The special recombinant plasmid p^Genesil-1 -CDK2 and P^Genesil- 1-HK were transfected into cultured human hepatoma cells HepG2 via lipofectamine2000. The stably expressed cells were selected by G418 and proliferated through cuhivating. The green fluorescence protein was observed by inverted fluorescence microscope. The mRNA contents of CDK2 was detected by reverse transcription polymerase chain reaction （RT- PCR） and the changes of cell cycle was assayed by flow cytometry （FCM）. The proliferation rate of tumor ceils was examined by MTT. Results：After I month G418 selection,the stably expressed cells were successfully obtained, mRNA copy numbers of CDK2 in transfected group was significantly decreased compared with the p^Gencsil-1-HK and control group, the cell cycle progression was arrested from G1 to S phase. Conelusion：RNA interference （RNAi） induced by recombinant plasmid P^Genesil-1- CDK2 could significantly suppress endogenous CDK2 gene expression , arrest cell cycle and atatiproliferate in HepG2. These results provide experimental basis for CDK2 mediated gene silencing anti - cancer therapeutics .

关 键 词：CDK2 HEPG2细胞 RNA干扰 细胞周期 

分 类 号：R349.3[医药卫生—基础医学] R322.81