真核表达载体pcDNA3.1-Beclin1的构建及其对子宫内膜癌HEC细胞增殖的影响  

The Effect of Building Eukaryotic Expression Vector pcDNA3.1-Beclin1 on the HEC Cell Growth of Endometrial Carcinoma

作　　者：何珏[1] 张文书[1] 王珍玲[1] 

机构地区：[1]赣南医学院,江西赣州341000

出　　处：《中国医学创新》2014年第9期18-20,共3页Medical Innovation of China

基　　金：江西省教育厅青年科学基金项目(GJJ12551)

摘　　要：目的:构建自噬基因Beclin1的真核表达载体pcDNA3.1-Beclin1,检测人子宫内膜癌HEC细胞转染pcDNA3.1-Beclin1后,外源性基因Beclin1在蛋白水平的表达,并观察其对HEC细胞增殖的影响。方法:通过RT-PCR和T/A克隆等方法,构建自噬基因Beclin1的真核表达载体pcDNA3.1-Beclin1,并通过脂质体介导的方法将外源性Beclin1基因导入人类子宫内膜癌细胞株HEC中,通过实时PCR检测转染后Beclin1在HEC中表达的情况,并设立实验组(转染pcDNA3.1-Beclin1真核载体)、空质粒对照组(转染pcDNA3.1空载体)、空白脂质体对照组,用Western blot法检测子宫内膜癌HEC细胞中Beclin1的表达情况,MTT法检测Beclin1对HEC细胞体外生长的影响等,研究Beclin1基因与子宫内膜癌的关系。结果:经限制性内切酶鉴定及测序,证明Beclin1基因真核质粒的序列完全正确,将其转染HEC细胞,转染后的HEC细胞在体外能继续增殖,但增殖能力明显下降。结论:Beclin1基因pcDNA3.1-Beclin1真核表达载体构建成功,并能在HEC细胞中表达,转染HEC细胞体外增殖能力明显降低,Beclin1基因用于子宫内膜癌基因治疗具有重要靶向价值。Objective：To build the eukaryotic expression vector pcDNA3.1-Beclin1 of the autophagy gene Beclind1 and to examin the pcDNA3.1-Beclin1 transfected by the human endometrial carcinoma HEC cells,the expression of exogenous gene Beclin1 in protein,together with its effect to the HEC cell multiplication will be examined.Method：By way of RT-PCR＆amp;T/A cloning,built the eukaryotic expression vector pcDNA3.1-Beclin1,transferred the exogenous genes Beclin1 into the human endometrial carcinoma cell line HEC based on the lipidosome mediation,examined the expression of the transfected Beclin1 in HEC based on RT-PCR,set up the experimental group（transfected eukaryotic vector pcDNA3.1-Beclin1）,empty plasmid control group（transfected empty blank load group pcDNA3.1）and lipidosome control group, Western Blot and MTT were employed to research the interrelationship between Beclin1 and endometrial carcinoma,which Western Blot was employed to examine the expression of Beclin1 in endometrial carcinoma HEC cell and MTT was employed to examine the effect of Beclin1 to HEC cell external growth.Result：Being identified and sequenced by restriction enzyme, DNA sequence in the eukaryotic plasmid of Beclin1 was proved to be exactly correct.The transfected HEC cells could continue to multiply in vitro,but the multiplication capabilities remarkably declined.Conclusion：Beclin1 gene is successfully built in eukaryotic expression vector pcDNA3.1-Beclin1 and can be expressed in HEC cells.The multiplication capabilities of transfected HEC cells remarkably decline in vitro.It is quite significant to apply Beclin1 genes into the endometrial carcinoma gene treatment.

关 键 词：BECLIN1 真核表达载体 细胞 增殖 

分 类 号：R737.33[医药卫生—肿瘤]