基因捕获筛选TGF-β1诱导间充质干细胞C3H／10T1／2平滑肌分化的差异表达基因  

Identification of differentially expressed genes upon TGF-β1-induced smooth muscle differentiation of mesenchymal stem cell line C3H/10T1/2 by gene trap screening

作　　者：王明科[1,2] 姜帆[3] 孙慧勤[1] 叶枫[3] 程晋[3] 粟永萍[1] 邹仲敏[3] 

机构地区：[1]第三军医大学军事预防医学院,全军复合伤研究所,创伤、烧伤与复合伤国家重点实验室,重庆400038 [2]海军医学研究所,上海200433 [3]第三军医大学军事预防医学院毒理学研究所,重庆400038

出　　处：《第三军医大学学报》2014年第2期92-97,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(31201035);国家重点基础研究发展计划(973计划,2005CB522605);全军“十一五”计划一般课题(06H029)~~

摘　　要：目的利用基因捕获方法,分离转化生长因子-β1(transforming growth factor-β1,TGF-β1)诱导小鼠胚胎间充质干细胞C3H/10T1/2(简称10T1/2细胞)平滑肌分化前后差异表达基因。方法 5 ng/mL TGF-β1诱导10T1/2细胞平滑肌分化,倒置显微镜下观察细胞形态学改变,RT-PCR鉴定平滑肌分化相关基因alpha平滑肌肌动蛋白(alpha smooth muscle actin,αSMA)、平滑肌22 alpha(smooth muscle 22 alpha,SM22α)、平滑肌肌球蛋白重链(smooth muscle myosin heavy chain,SM-MHC)和血清应答因子(serum response factor,SRF),免疫细胞化学实验检测αSMA。TGF-β1诱导建立的10T1/2细胞基因捕获阳性克隆库分化前后进行LacZ染色。cDNA末端快速扩增法(rapid amplification of cDNA ends,RACE)及电子克隆分离差异表达序列,在GenBank网站nBLAST进行生物信息学分析,用GenBank网站在线软件BankIt提交GenBank获取ID号,用pI/Mw软件计算蛋白分子量、等电点。Real-time PCR检测新基因mgt-16在BALB/c小鼠小肠、骨骼肌及心肌的表达。结果细胞形态学、RT-PCR及免疫细胞化学实验鉴定TGF-β1成功诱导10T1/2细胞平滑肌分化。LacZ染色筛选获得2个平滑肌分化后差异表达克隆。RACE、电子克隆和nBLAST分析后发现为线粒体核糖体蛋白S6(mitochondrial ribosomal protein S6,Mrps6)和新基因mgt-16。mgt-16基因位于19号染色体,编码一个93个氨基酸的蛋白(命名为mgt-16),相对分子质量为9 772.02,等电点为6.04,提交后ID号为GU266552。新基因mgt-16在BALB/c小鼠小肠表达较高,而在骨骼肌、心肌表达较低。结论利用基因捕获分离获得了TGF-β1诱导平滑肌分化前后2个差异表达基因:Mrps6和新基因mgt-16。Objective To identify genes involved in transforming growth factor beta-1 （TGF-β1）-in- duced smooth muscle differentiation of murine embryonic mesenchyma] stem cell line C3H/10T1/2 （10T1/2 cells） by gene trap screening. Methods 10T1/2 cells were induced by 5 ng/mL TGF-[31 to differentiate into smooth muscle cells （ SMCs）, which were identified by cell morphology, RT-PCR identification of smooth muscle-related genes including alpha smooth muscle actin （ctSMA）, smooth muscle 22 alpha （SM22ot）, smooth muscle myosin heavy chain （SM-MHC）, serum response factor （SRF）, and immunocytochemical detection of ctSMA. LacZ staining was used before and after TGF-131 induced phenotypic modulation of gene trapped 10T1/2 clones to SMCs. Differential]y expressed gene sequences were obtained by rapid amplification of cDNA ends （RACE） and in silico cloning. Then the sequences were searched in GenBank using BLAST algorithm for bioinformatics analysis and submitted to GenBank by its online software BankIt. The molecular weight and isoelectrie point of differentially expressed genes were calculated by pI/Mw software. The mRNA expression of a novel gene （mgt-16） in the intestine, skeletal muscle and heart in BALB/c mice were detectedby real-time PCR. Results The model of SMCs differentiation was successfully established based on cell morphology, RT-PCR and immunocytochemical staining. Two gene trapped clones with differential expression patterns of trapped genes were obtained by LacZ staining before and after TGF-~I treatment. It was found that the trapped genes were mitochondrial ribosomal protein S6 （ Mrps6 ） and a novel gene （ mgt-16 ） when the RACE-obtained sequences were searched in GenBank using BLAST algorithm, mgt-16 （GenBank accession No. GU266552） was mapped to mouse chromosome 19, encoded a protein whose length was 93 amino acids, molecular weight was 9 772.02 and isoeleetrie point was 6.04. Real-time PCR results showed that mgt-16 was highly expressed in the intestine, while lo

关 键 词：基因捕获 转化生长因子-Β1 间充质干细胞 平滑肌分化 

分 类 号：R322.74[医药卫生—人体解剖和组织胚胎学] R394-33[医药卫生—基础医学]