Titin基因在骨髓间充质干细胞体外分化为心肌细胞过程中的干预研究  

Effect of titin gene on the procession of MSCs differentiating into cardiomyocytes in vitro

作　　者：王金菊[1] 田杰[1] 吕铁伟[1] 张小飞[1] 李扬[1] 朱静[2] 陈沅[1] 

机构地区：[1]重庆医科大学附属儿童医院心血管内科,重庆400014 [2]重庆医科大学附属儿童医院儿科研究所,重庆400014

出　　处：《重庆医科大学学报》2007年第11期1121-1125,1130,共6页Journal of Chongqing Medical University

基　　金：重庆市教委资助课题(2004);国家自然科学基金资助项目(批准号30271381)。

摘　　要：目的:探讨结构蛋白即肌联蛋白titin基因在骨髓间充质干细胞(Mesenchymal stem cells,MSCs)体外分化为心肌细胞过程中的作用及其对收缩蛋白基因β-肌球蛋白重链(β-myson heavy chain,β-MHC)和肌钙蛋白(ICardiac troponinI,cTnI)基因表达的影响。方法:无菌条件下冲洗Wistar大鼠双侧股骨和胫骨的骨髓腔获得细胞,并进行体外纯化、扩增,后加入5-氮杂胞苷(5-azacytidine,5-aza)进行诱导,分别在诱导后1天、4天、7天、14天在倒置显微镜下观察其形态变化,同时将携带有针对大鼠titinN2B区的siRNA重组质粒pSITITIN和针对人β-actin的siRNA重组质粒pSIACTIN转染入诱导后的MSCs,转染后24h,采用RT-PCR技术检测各时间点干扰前后结构蛋白titin基因和收缩蛋白β-MHC、cTnI基因在MSCs体外心肌细胞样分化过程中的动态表达。结果:1.诱导前titin、β-MHC、cTnI基因都有弱的表达,诱导后titin、β-MHC、cTnI基因的表达增强。2.重组质粒转染诱导后的MSCs,titin基因的表达减弱。3.titin基因的表达被干扰后,β-MHC基因的表达无明显变化,而cTnI基因的表达有减弱。结论:1.MSCs是一种具有心肌源性的种子细胞2.titin基因可能是MSCs向心肌细胞分化发育过程中有重要意义的基因,对cTnI基因的表达有调控作用。Objective：To investigate the effect of the titin gene and its influence on the contraction protein gene such as β-MHC and cTnI in the procession of MSCs differentiating into cardiomyocytes in vitro. Methods：MSCs were isolated from bilateral thighbones and tibias of Wistar rats,purified,expanded and then exposed to 5-aza.we observed the morphology of MSCs with phase-contrast microscope at 1^st,4^th 7^th,14^th day after induction. Then we transfected the recombination plasmid vector （including pSITITIN and pSIACTIN） into MSCs after induction, detected the expression of titin, β-MHC and cTnI at interval times by RT-PCR. Results： 1. we obtained the mild expression of titin, β-MHC and cTnI gene before 5-aza treament,but the expression of them increased after induction. 2. After recombination plasmid were transfected into MSCs which have been induced by 5-aza,the expression of titin gene was decreased 3. The expression of β-MHC gene kept invariant,while the expression of cTnI gene was weakened after the interference. Conclusion：1, titin gene probably play an important role in the procession of MSCs differentiating into cardiomyocytes in vitro. 2. titin gene may regulate the expression of cTnI.

关 键 词：骨髓间充质干细胞 RNA干扰 分化 TITIN 基因 

分 类 号：R725.4[医药卫生—儿科]