hhlim促进DMSO诱导的P19细胞向心肌分化  

Overexpression of hhlim Enhances Cardiomyogenesis of Embryonic Stem Cells

作　　者：郑斌[1] 温进坤[1] 韩梅[1] 周爱儒[2] 

机构地区：[1]河北医科大学基础医学研究所 [2]北京大学医学部生物化学与分子生物学系,北京100083

出　　处：《生物化学与生物物理进展》2004年第6期506-511,共6页Progress In Biochemistry and Biophysics

基　　金：国家重点基础研究发展规划项目 (973)(G2 0 0 0 0 5 690 5 );国家自然科学基金资助项目 (3 0 3 0 0 13 2);河北省自然科学基金资助项目(2 0 0 40 0 0 64 4)~~

摘　　要：为了确定hhlim是否参与胚胎期的心肌分化和发育过程 ,用可表达hhlim蛋白和hhlim反义RNA的真核表达质粒转染P19胚胎干细胞 ,经G4 18筛选得到稳定表达hhlim和hhlim反义RNA的P19细胞克隆后 ,观察hhlim对P19细胞向心肌分化和发育的影响 .结果显示 ,Nkx2 5和GATA 4在未被外源性hhlim基因转染的P19细胞中不表达 .DMSO刺激细胞 2天后 ,GATA 4开始表达 ,3天后Nkx2 5的表达活性显著升高 .hhlim的过表达不但有利于P19细胞的存活和生长 ,而且还可以使Nkx2 5和GATA 4的表达比对照细胞提前 1天 .反义hhlim细胞株被DMSO诱导 5天后 ,细胞仍呈集落化生长 .同时 ,Nkx2 5和GATA 4开始表达的时间明显延滞 .结果表明 ,hhlim能促进P19细胞向心肌细胞分化 ,其作用是通过促进转录因子GATA 4和Nkx2In order to determine the importance of hhlim in cardiomyocyte differentiation and to define the hierarchy of transcription factors during cardiac development, the cardiac differentiation profile of the pluripotent P19 cells was characterized at cellular and molecular level. Genes that express early in the cardiogenesis of the embryo include GATA-4 and Nkx2. 5. The time course of expression of these genes during in vitro differentiation of P19 cells into cardiomyocytes was established using RT-PCR. GATA-4 is expressed at day 2 after treatment with DMSO, and Nkx2. 5 at day 3. hhlim is almost undetected until day 4 after DMSO treatment. Western blot had the same results, which showed that hhlim might participate in the cardiac differentiation pathway. P19-hhlim-overexpressing cells did not show morphological or biochemical evidence of cardiomyogenesis without DMSO treatment, but overexpression of hhlim had a benefit effect on cell aggregates that were especially obvious in the absence of DMSO. Moreover, when hhlim-overexpressing cells were treated with DMSO, the expression of Nkx2. 5 and GATA-4 appeared earlier compared with the control. hhlim-deficient P19 cells induced by DMSO retained the ability to differentiate into cardiomyocytes. The time course of expression of Nkx2. 5 and GATA-4 in the hhlim-deficient clones was significantly reduced, whereas these clones treated by RA produced neuroectodermal derivatives. Then it was concluded that lack of hhlim expression specifically inhibits cardiomyocyte formation of P19 cells without affecting neuronal differentiation pathway. These data showed that overexpression of hhlim could enhance cardiogenesis of embryonic stem cells whose mechanism might be involved in upregulation of the expression of Nkx2.5 and GATA-4.

关 键 词：hhlim基因 心肌细胞 转录因子 P19细胞 分化 

分 类 号：Q78[生物学—分子生物学]