人KiSS-1和p27^(Kip1)基因在骨肉瘤MG63细胞株中的共表达  

The Coexpression of KiSS-1 and p27^(kip1) Genes in the Osteosarcoam Cell Line MG63

作　　者：白国昌[1] 林建华[1] 

机构地区：[1]福建医科大学附属第一医院骨科,福州350004

出　　处：《福建医科大学学报》2009年第3期198-201,共4页Journal of Fujian Medical University

基　　金：福建省卫生教育联合攻关计划项目(WKJ2008-2-42)

摘　　要：目的克隆肿瘤转移抑制基因KiSS-1和细胞周期依赖性激酶抑制剂基因p27Kip1的表达序列,应用内部核糖体插入位点(IRES)序列构建共表达载体pIRES-KiSS-1-p27Kip1,测序鉴定,并获得稳定表达目的蛋白的细胞株。方法利用RT-PCR从人正常胎盘组织中获得KiSS-1和p27Kip1基因开放阅读框的cDNA序列,将两目的基因的cDNA序列重组至pIRES载体,构建共表达载体pIRES-KiSS-1-p27Kip1,应用脂质体转染骨肉瘤MG63细胞,并通过RT-PCR和Westernblot检测目的蛋白的表达情况。结果经PCR、酶切鉴定和基因序列测定,证实重组入pIRES载体上的两个片段均为目的基因开放阅读框的核苷酸序列,KiSS-1和p27Kip1蛋白在转染后的细胞中有表达。结论成功构建共表达载体pIRES-KiSS-1-p27Kip1,获得稳定表达KiSS-1和p27Kip1蛋白的细胞株,为进一步观察目的基因对骨肉瘤细胞体内外效应奠定基础。Objective To clone the metastasis suppressor gene KiSS-1 and cell cycle dependent kinase inhibitor gene p27^Kip1 from human placenta tissue, construct a bicistronic expression vector to express KiSS-1 along wiht p27^Kip1 via the internal ribosome entry site （IRES）, and establish stable cell line ex pressing these two genes. Methods Total RNA was extracted from human placenta tissue. The two opening reading frames of KiSS-1 and p27^Kip1 cDNA were amplified by RT-PCR, and were subeloned into shuttle vector pIRES. The plasmid construct was transfected into the osteosarcoma MG63 cells. The expressions of KiSS-1 and p27^Kip1 were detected in the transfected cells by RT-PCR and western blot. Results The recombinant plasmid construct was confirmed by methods of polymerase chain reaction （PCR）, restriction enzyme digestion and sequence analysis. The interested genes were detected in the transfected cells. Conclusion The successful construction of the reeomhinant plasmid plRES-KiSS-1-p27^Kip1 and the establishment of cell line with stable expression of these two genes will benefit the further study about the effect of these genes on osteosarcoma cells in vitro and in vivo.

关 键 词：骨肉瘤 重组 遗传 克隆 分子 基因 肿瘤抑制 细胞周期蛋白质类 肿瘤细胞 培养的 

分 类 号：R738.1[医药卫生—肿瘤] R346[医药卫生—临床医学]