CAJ | 학술논문

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.
목적 구건인동립단백-H(CENP-H) shRNA반전록병독표체재체,획득은정전염적인유선암세포주.방법 합성CENP-H기인간우서렬,정향삽입반전록병독표체재체pSUPE-Rretro-puro질립병측서감정;간우질립경린산개개도전염293FT세포제비반전록병독;대인유선암MCF-7화MDA-MB-435세포진행반전록병독감염,표령매소사선획득은정전염적세포주.Real-time PCR화Western blot분별검측은정전염세포주적CENP-H mRNA화단백적표체.결과 성공구건화사선출파향CENP-H기인적량대shRNA질립표체재체.은정전염세포CENP-H mRNA표체현저하조,CENP-H적단백표체수평명현저우대조.결론 은정침묵CENP-H표체적인유선암세포주적건립,위이CENP-H기인위파점적인유선암기인연구제공료실험기출.
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.