人成骨肉瘤MG-63细胞雌激素相关基因的分离  

Isolation of estrogen-responsive genes of human osteosarcoma MG-63 cells

作　　者：彭依群[1] 廖二元[1] 邓小戈[1] 

机构地区：[1]中南大学湘雅二医院代谢内分泌研究所,长沙410011

出　　处：《湖南医科大学学报》2003年第5期459-462,共4页Bulletin of Hunan Medical University

基　　金：国家"九五"攻关项目 ( 96 90 6 0 5 0 5 );卫生部临床学科重点建设项目 ( 0 8 960 0 2 16);湖南省社会发展科研计划 ( 99SSY10 0 9 10 )

摘　　要：目的 :筛查 17β雌二醇 (17β estradiol,E2 )诱导人成骨肉瘤MG 6 3细胞株差异表达cDNA片段 ,寻找雌激素相关基因。方法 :改良的cDNA代表性差异分析法 (cDNArepresentationaldifferenceanalysis,cDNARDA)分离E2 干预MG 6 3细胞株表达上调cDNA片段 ,经Southern和快速Northern杂交证实表达上调的cDNA片段来源后 ,将其克隆到 pGEM Teasy载体 ,蓝白筛选后挑取阳性克隆进行测序和同源比较分析 ,最后选取其中部分克隆行Northern印迹杂交。结果 :第 4轮消减杂交得到 5个E2 诱导人成骨肉瘤MG 6 3细胞表达上调的差异cDNA条带 ,Southern和快速Northern杂交证明表达上调的cDNA片段来自E2 干预的MG 6 3细胞 ;随机选 2 5个克隆测序得到13个序列 ,7个与已知基因高度同源 ;其中 2个克隆经Northern杂交证实E2 干预后表达上调。结论 :cDNARDA能有效筛查差异表达基因 ,人成骨肉瘤MG 6 3细胞中某些基因受雌激素诱导表达上调 ,可能与绝经后骨质疏松的发病相关。Objective To obtain a series of differentially expressed cDNA fragments from osteosarcoma MG 63 cells induced by 17 beta estradiol and to find some estrogen responsive genes. Methods Optimized cDNA representational difference analysis (RDA) was performed to isolate up regulated expressed sequences between cDNAs from MG 63 cells treated with and without 17 beta estradiol. The sources of up regulated expressed cDNA fragments were proved by Southern blot and 'shotgun' Northern blot. The fragments were cloned into the pGEM T easy vector. After blue white selection, white colonies were picked up and individually grown in LB/Amp + medium. Some white colonies were randomly selected for DNA sequencing, homology analysis,and Northern blot analysis. Results Five up regulated expressed fragments were isolated in the 4th subtraction hybridization by cDNA RDA. These fragments proved to be really up regulated expressed and really came from the cDNAs from 17 beta estradiol treated MG 63 cells by Southern blot and 'shotgun' Northern blot. Twenty five clones were sequenced and 13 sequences were obtained. Seven sequences were highly homologous to known genes and 2 of them proved to be differentially expressed through Northern blot. Conclusion cDNA RDA is one of the most effective methods to isolate differentially expressed genes. Some genes expression of human osteosarcoma MG 63 cells are up regulated and induced by 17 beta estradiol, and it may be related to the pathogenesis of postmenopausal osteoporosis.

关 键 词：cDNA代表性差异分析法 雌激素 MG-63细胞 差异表达基因 

分 类 号：R738.1[医药卫生—肿瘤]