摘要
目的探讨转染生肌调节因子(myoblast determination,MyoD)基因和连接蛋白43(Connexin,Cx43)基因的大鼠真皮成纤维细胞(der-mal fibroblast,DFs)生物学功能的变化及MyoD和Cx43基因转染DFs细胞治疗心力衰竭的可行性。方法采用Gateway技术,构建真核质粒表达载体,利用慢病毒(LV)表达系统,将大鼠MyoD cDNA和Cx43cDNA转入大鼠成纤维细胞中,经Blasticidin筛选培养,通过RT-PCR,Western印迹法等方法检测MyoD及Cx43蛋白及mRNA表达情况,并且通过显微镜观察转染后生长情况,膜片钳技术检测离子电流变化。结果RT-PCR,Western印迹法检测出MyoD及Cx43蛋白及相应mRNA表达,膜片钳检测到转染后钙离子电流,显微镜观察到基因转染筛选后培养1w细胞的融合现象,并有多核肌管形成。结论MyoD和Cx43基因转染使DFs分化为成肌细胞,为进一步研究基因治疗心力衰竭奠定基础。
Objective To study the changes of the differentiation growth and biological function of cultured rat fibroblasts （FB） transfected by myoblast determining （MyoD） and connexin 43 （Cx43） genes in order to explore preliminarily the possible mechanism and way by which MyoD and Cx43 genes on treatment of heart failure （HF）. Methods Gene cloning technology was used to construct the eukaryotic expressed plasmid vector pLenti6/V5-DEST-MyoD and cells were pLenti6/V5-DEST- Cx43 in which MyoD cDNA or Cx43 cDNA was inserted. Dermal fibroblasts （DFs） transfected with exogenetic MyoD cDNA or Cx43 cDNA via lipofectamine, and followed by blasticidin selection, according to lentiviral expression system （ViraPower） protocol. Expression and its biological functions of MyoD and Cx43 in the transfectants were testified by RT-PCR, Western blot, molecular and clamp patch methods. The morphological structure changes of cells before and after transfection were observed with microscopy. Results The expressions of MyoD and Cx43 were detected in the MyoD and Cx43 genes transfected DFs with RT-PCR and Western blot. Myotube were found from cultures incubated for a week in differentiation medium, which the transfected cells were of characteristic of filaments in their cytoplasm and presented myoblast morphlogy. Conclusions MyoD cDNA could induce cultured DFs to differentiate into myoblasts and Cx43 cDNA could enhance gap junctional intercellular communication between cell and cell, furtherly providing an experimental foundation for the therapy of HF.
出处
《中国老年学杂志》
CAS
CSCD
北大核心
2008年第5期417-420,共4页
Chinese Journal of Gerontology
基金
国家自然科学基金项目(30471719)
上海市浦东新区优秀学科带头人项目(PWR2004-1)
上海市曙光计划项目(04SG25)
