中文摘要：

目的 构建人动粒蛋白-H（CENP-H） shRNA反转录病毒表达载体,获得稳定转染的人乳腺癌细胞株.方法 合成CENP-H基因干扰序列,定向插入反转录病毒表达载体pSUPE-Rretro-puro质粒并测序鉴定;干扰质粒经磷酸钙介导转染293FT细胞制备反转录病毒;对人乳腺癌MCF-7和MDA-MB-435细胞进行反转录病毒感染,嘌呤霉素筛选获得稳定转染的细胞株.Real-time PCR和Western blot分别检测稳定转染细胞株的CENP-H mRNA和蛋白的表达.结果 成功构建和筛选出靶向CENP-H基因的两对shRNA质粒表达载体.稳定转染细胞CENP-H mRNA表达显著下调,CENP-H的蛋白表达水平明显低于对照.结论 稳定沉默CENP-H表达的人乳腺癌细胞株的建立,为以CENP-H基因为靶点的人乳腺癌基因研究提供了实验基础.

英文摘要：

Objective To establish breast cancer cell lines with the centromere protein-H （CENP-H） stably silenced by applying the retroviral expression system.Methods The interference sequences targeting CENP-H gene were synthesized and insetted into the retroviral vector pSUPE-Rretro-puro.The recombinant plasmids were transfected into the packing cells 293FT with calcium phosphate and the retroviral supernatant was collected to infect MCF-7 and MDA-MB-435 breast cancer cells.After selection by puromycin and culture expansion,the stable cell clones were obtained.The levels of CENP-H mRNA and protein in the selected clones were detected by Real-time PCR and Western blot.Results Two CENP-H shRNA retroviral vectors were constructed successfully.After infection,CENP-H-knocked down breast cancer stable cell lines were successfully established.The mRNA and protein expression levels of CENP-H were significantly down regulated in CENP-H-knocked down cells compared to control.Conclusion The establishment of breast cancer stable cell lines in which the expression of endogenous CENP-H is knocked down will provide an original route for further exploring the mechanism of CENP-H in human breast cancer.