逆转录病毒介导的SHP-1基因在乳腺癌MDA-MB-231细胞中的表达  

Retrovirus-mediated SHP-1 gene expression in human breast cancer MDA-MB-231 cells

作　　者：梁继珍[1] 李荣[1] 张军一[1] 郑航[1] 罗荣城[1] 

机构地区：[1]南方医科大学南方医院肿瘤中心,广东广州510515

出　　处：《南方医科大学学报》2009年第5期902-905,共4页Journal of Southern Medical University

基　　金：广东省科技计划项目(2008B030301143);中国临床肿瘤学科学基金(Y-2006-0009)

摘　　要：目的构建逆转录病毒表达载体pLNCX2-SHP-1,获取重组逆转录病毒,并将SHP-1基因转导入乳腺癌MDA-MB-231细胞株。方法采用RT-PCR方法从高表达SHP-1的人乳腺癌细胞株MCF-7中克隆出SHP-1基因全长cDNA,构建重组逆转录病毒表达载体pLNCX2-SHP-1,通过脂质体介导将其转染入包装细胞系PT67,G418筛选建立稳定产病毒的细胞株,将病毒感染人乳腺癌细胞MDA-MB-231。采用Western blotting方法检测SHP-1基因在MDA-MB-231细胞中的表达情况。结果成功构建重组逆转录病毒表达载体pLNCX2-SHP-1,转染包装细胞PT67,筛选出对G418具有稳定抗性的克隆PT67/SHP-1,获取重组逆转录病毒上清液,并感染人乳腺癌细胞MDA-MB-231。从蛋白水平证实,感染后的MDA-MB-231有SHP-1基因的表达。结论将SHP-1基因定向克隆入逆转录病毒载体的方法可成功获取重组逆转录病毒,感染MDA-MB-231细胞得到稳定表达SHP-1基因MDA-MB-231/SHP-1,为进一步研究奠定了基础。Objective To construct a retrovirus-mediated expression system carrying human SHP-1 gene to transfer SHP-1 gene in human breast cancer MDA-MB-231 cells. Methods The full-length SHP-1 gene fragment was amplified by RT-PCR from the total RNA extracted from human breast cancer cell line MCF-7 over-expressing SHP-1 protein. The gene fragment was inserted into the vector pLNCX2 to construct the recombinant retroviral plasmid, which-was transfected into the packaging cell PT67 via Lipofectamine2000. A cell line stably producing the virus was selected with G418. MDA-MB-231 cells was infected with the virus, . and the expression of SHP-1 gene in the positive cell clone was detected with Western blotting. Results A 1.8 kb eDNA fragment ofSHP-1 gene was obtained from MCF-7 cells and successfully inserted into the pLNCX2. A stable cell clone PT67/SHP-1 and virus supernatant were obtained. Expression of SHP-I protein was detected in the cells infected with the virus. Conclusion The recombinant retroviral vector carrying SHP-1 gene has been successfully constructed and MDA- MB-231/SHP-I cell line expressing SHP-1 has been obtained to allow further functional study of SHP-1 in breast cancer.

关 键 词：SHP-1 酪氨酸磷酸酶 乳腺肿瘤 基因重组 逆转录病毒 

分 类 号：R737.9[医药卫生—肿瘤]