心肌干细胞c-kit基因表达的甲基化研究

C-kit is a specific surface markers of CSCs . In myocardium after AMI,the density of c-kit+CSCs and c-kit expression are increased significantly,which is a good repair effect to the injury of the myocardium. Our preliminary experiments show SDF-1 can increase the surface expression of c-kit,and the methylation differences between c-kit-CSCs and c-kit+CSCs are significant. However,its precise mechanism is unknown. This project intends to①transfecting plasmid DNMT and DNMT-siRNA plasmid into CSCs, observation on c-kit,DNMT expression and so on. ②CSCs is intervented by SDF-1 ,and ade in LY294002,which is specific inhibitor of PI3K / Akt signaling pathway,observation on c-kit,DNMT, phosphorylated Akt expression,and so on. ③CSCs which is intervented by SDF-1 and LY294002 are transplanted into the c-kit gene knockout models of AMI rat myocardium,observation on myocardial infarct size, heart function and c-kit, DNMT expression. Demonstration of PI3K / AKT signaling pathway activation results in DNMT reduction,c-kit gene hypomethylation, and the expression of c-kit increased, then enhance migration ability,proliferation ability of CSCs,and the regeneration and repair of cardiac injury potential. This study perspective to explore the effect of c-kit to CSCs and the related molecular mechanism, for the treatment of ischemic heart disease with a new target.

c-kit是心肌干细胞（CSCs）特异性表面标志，心梗后c-kit表达增高，对损伤心肌有修复作用。我们的前期试验显示基质细胞衍生因子-1（SDF-1）能上调CSCs表面c-kit表达；c-kit+与c-kit-CSCs存在明显甲基化差异。但机制不明。本课题拟①将DNMT质粒及DNMT-siRNA质粒转染CSCs，观察c-kit、DNA甲基转移酶（DNMT）表达②SDF-1及PI3K/Akt信号通路特异性抑制剂LY294002干预CSCs，观察DNMT、磷酸化Akt表达③将SDF-1及LY294002干预的CSCs移植入心梗模型大鼠心肌内，观察心梗面积、心功能及c-kit、DNMT表达。论证PI3K/AKT信号通路激活致DNMT减少，使c-kit基因低甲基化，c-kit表达增加，增强CSCs迁移、增殖潜能。本研究探索c-kit基因表达对CSCs影响及分子机制，为缺血性心脏病的治疗提供新靶点。

CPCs 能自我更新并且可以自发的增殖分化为心肌细胞、平滑肌细胞和内皮细胞等，能够程序性地重建心脏。探索基质细胞衍生因子-1α（SDF-1α）影响小鼠心肌祖细胞（CPCs）c-kit基因表达的相关机制研究。从小鼠心肌中分离培养出CPCs，并用免疫磁珠法筛选出c-kit+和c-kit- CPCs。使用不同浓度的SDF-1α干预c-kit+ 和c-kit- CPCs 不同时间，Western blot法检测c-kit蛋白的表达，qPCR观察c-kit mRNA的表达。然后用SDF-1α和AMD3100干预c-kit+ 和c-kit- CPCs后，CCK-8试剂盒检测细胞增殖能力， Transwell 小室检测细胞迁移能力。最后用SDF-1α和AMD3100干预c-kit+ 和c-kit- CPCs后，qPCR检测DNA甲基转移酶 ( DNMT) mRNA的表达，DNMT活性检测试剂盒检测DNMT的活性，基质辅助激光解吸附电离飞行时间质谱分析技术检测DNA甲基化水平。结果 消化后的心肌组织块经过10天左右的培养，可见小、圆、亮的细胞出现在组织块周围爬出的成纤维细胞层上。流式细胞分析显示经过磁珠分选后c-kit+ CPCs c-kit阳性率为 82.06%，Sca-1阳性率为82.87%。Western blot及qPCR结果显示SDF-1α干预后c-kit+ CPCs c-kit蛋白及mRNA表达均显著增加，c-kit- CPCs c-kit蛋白及mRNA从阴性转为阳性，并且均呈浓度及时间依赖性，AMD3100可以拮抗SDF-1α对c-kit表达的影响。SDF-1α干预后c-kit+ 和c-kit- CPCs增殖和迁移能力较对照组显著增加。最后，SDF-1α可以抑制DNMT1、DNMT3β mRNA的表达以及DNMT活性，并且导致c-kit基因发生去甲基化。因此，SDF-1α可以通过抑制DNMTs的表达与活性，下调c-kit基因启动子区CpG岛的甲基化水平，从而提高c-kit基因的表达，促进CPCs的增殖和迁移能力。

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