survivin启动子调控肿瘤干细胞标记CD133基因siRNA增殖型溶瘤腺病毒的构建及对肝癌细胞生长的抑制作用  

Construction and identification of replication-competent adenovirus expressing siRNA targeting CD133 gene regulated by survivin promoter and its inhibition of liver cancer cell growth

作　　者：牛坚[1] 王月[1] 刘斌[1] 王人颢[1] 朱志军[2] 申海莲[3] 

机构地区：[1]徐州医学院附属医院肝脏病研究中心,徐州221002 [2]北京友谊医院肝胆外科,北京100000 [3]上海交通大学附属同济医院干细胞研究中心,上海200000

出　　处：《安徽医科大学学报》2016年第7期926-930,共5页Acta Universitatis Medicinalis Anhui

基　　金：天晴甘美基金项目资助(编号:CFHPC20132020);江苏省333人才项目(编号:Ⅲ-2290);徐州市重大科研项目(编号:KC14SX011)

摘　　要：目的构建survivin启动子调控的靶向CD133基因的siRNA增殖型溶瘤腺病毒,研究其对肝癌细胞生长的影响。方法 RT-PCR法扩增survivin启动子,测序鉴定,双酶切连接,获得p H-XC2-survivin。酶切p H-XC2-survivin、p ZD55-CD133-siRNA获得survivin启动子表达框的亚克隆和CD133-siRNA基因表达框的亚克隆,连接获得survivin启动子调控的siRNA增殖型溶瘤腺病毒表达载体质粒p T-ZD55-CD133-siRNA。增殖型溶瘤腺病毒survivin-T-ZD55-CD133-siRNA经PCR和测序鉴定。qRT-PCR法检测CD133表达,Western blot法检测E1A,CCK-8法检测细胞生长,流式细胞术检测细胞凋亡。结果成功构建增殖型溶瘤腺病毒survivin-T-ZD55-CD133-siRNA。qRT-PCR法检测CD133 mRNA明显下降,Western blot证实survivin-T-ZD55-CD133-siRNA在肿瘤细胞中表达E1A能抑制肝癌细胞CD133表达及生长。结论构建的增殖型溶瘤腺病毒可有效降低肝癌细胞CD133的表达,用于肝癌基因治疗的进一步研究。Objective To construct a replication-competent adenovirus expressing siRNA targeting CD133 gene regulated by survivin promoter and investigate its inhibitory effect on Hep3 B cells. Methods The fragment of the survivin promoter was amplified by PCR and inserted into pH-XC2 to reconstruct a recombinant plasmid pH-XC2-survivin. Complete digestion pH-XC2-survivin and p ZD55-CD133-siRNA,combinational joining the subclones,then getting replication-competent adenovirus expressing short interference RNA targeting CD133 gene regulated by survivin promoter, replication-competent adenovirus was constructed. The recombined adenoviruses（ T-ZD55-CD133-siRNA） were verified by PCR and sequencing. The effect of T-ZD55-CD133-siRNA on CD133 expression in Hep3 B cells was detected by qRT-PCR. The expression of E1 A was detected by Western blot. The antitumor potential of replication-competent adenovirus in Hep3 B cells were evaluated by CCK-8 assay and cell apoptosis was detected by Flow cytometry. Results Replication-competent adenovirus were constructed successfully. Western blot analyses indicated that T-ZD55-CD133-siRNA might express E1 A in adenovirus-infected Hep3 B cells. TZD55-CD133-siRNA were more effective to inhibit CD133 mRNA expression and Hep3 B cells proliferation. Apoptosis was significantly increased in the interference group compared with the control group. Conclusion SurvivinT-ZD55-CD133-siRNA expressing CD133-siRNA can inhibit CD133 expression and may be used for further investigation of gene therapy for liver cancer.

关 键 词：肝癌 干细胞 CD133 细胞增殖 基因表达 

分 类 号：R349.5[医药卫生—基础医学]