DNA甲基化异常介导高糖刺激TGFβ表达激活机制及在DN肾小球硬化中的作用

The mesangial cells phenotype transformation and glomerulosclerosis is the main pathological alteration of diabetic nephropathy,transforming growth factor β(TGFβ),a Pleiotropic cytokine, plays an important role in this pathogenesis induced by hyperglycemia.Based on our recently research progress,we put forward a hypothesis that DNA hypomethylation is the new molecular mechanism meidating upregulation of TGFβ in mesangial cells induced by hyperglycemia.We designed a project to prove the theory.First, we should examine the alteration of DNA methyltransferases(DNMTs) and TGFβ expression at the level of mRNA and protein,the genomic methylation and promoter DNA methylation of TGFβ,in mesangial cells under the condition of high glucose in vitro and in vivo,the purpose is to clarify which DNMT(dnmt1,dnmt3a and dnmt3b) expression is downregulated by hyperglycemia. Second, we should investigate that whether or not expression of TGFβ could be affected by overexpression and downexpression of DNMT through infection of mouses and mesangial cells with reorganization lentviral.Third,by in vivo research,we want to know that the effect of silencing DNMT of DN mouse model by infected with lentiviral in which siRNA of DNMT has been inserted on the expression of TGFβ and glomerulosclerosis.Meanwhile, by infection of lentiviral enhancing DNMT expression,We also want to demonstrate that whether the expression of TGFβ and DN occurrence and development could be inhibited or not through infecting the DN mouses with lentiviral containing the overexpression of DNMT. Finally, if the results of the abovementioned experiment are positive,that is to say, hyperglycemia could induce the upregulation of TGFβ expression by decreasing or suppressing DNMT expression and DNA methylation,we should further evaluate the concrete its molecular mechanism by applications of subcloning the different lengths of TGFβ promoter region, point mutation, CHIP technology. The results will provide new ideas and contribute to the evaluation of DN mechanism,provide new candidate drug targets for the prevention and treatment of DN.

TGFβ是介导糖尿病肾病（DN）肾小球硬化的主要分子，但其表达激活机制不清。本项目在前期研究基础上，提出"DNA甲基化异常是高糖诱导TGFβ表达激活机制"的假说。拟以DNA甲基转移酶（DNMTs）为切入点，首先分析DN进程中DNMTs、TGFβ的表达规律，鉴定出表达下调的DNMT；再此基础上，利用重组慢病毒感染小鼠和系膜细胞，使DNMTs超表达或沉默，确认DNMTs对TGFβ表达的调控和肾脏的病理作用；并通过构建DNMT沉寂的DN小鼠模型，从整体水平观察DNMT表达缺失对糖尿病肾小球系膜细胞TGFβ表达和肾小球硬化的影响；感染超表达DNMT的慢病毒，观察其对TGFβ表达、抑制DN发生发展的效应；进一步应用亚克隆TGFβ不同长度的启动子区、位点突变、预甲基化修饰和CHIP技术，探讨DNMT对TGFβ表达激活的具体机制。研究结果将为揭示DN机理和为DN的防治提供新的思路、新的候选药物靶标分子。

糖尿病患者长期的高糖状态是其主要的生理改变，这种长期的高糖状态影响了患者体内多种生物学过程，也是其并发症的原因之一。在解释其并发症的机制中，表观遗传学作为一个新兴的领域发挥着重要作用。转化生长因子-β1（TGFβ1）在糖尿病肾病（DN）的发生发展中起着重要作用。本课题主要研究高糖环境下加氧酶TET在启动TGFβ1的表达过程中的作用机制，并从细胞和动物水平进行了验证，试图解释加氧酶TET在DN发生发展中的重要作用。.我们首先利用人系膜细胞为实验对象，发现在高糖环境中TGFβ1表达确有升高，且TGFβ1表达调控区的甲基化水平呈降低趋势，同时也造成了系膜细胞增殖增加及表型转换标志物的表达升高。进一步，我们检测了DNA甲基转移酶（DNMTs）和加氧酶TET（TETs），发现DNMT3B和TET2在高糖环境下表达呈增加趋势，但只有TET2在TGFβ1表达调控区的结合呈增加趋势。我们在高糖环境中敲减人系膜细胞的TET2基因后，发现TGFβ1表达、系膜细胞增殖及表型转化标志物表达均降低。.在db/db小鼠的肾脏组织中，我们得到了与人系膜细胞同样的结果，证实了在高糖环境中TET2对TGFβ1表达、系膜细胞增殖及表型转化标志物表达影响的重要作用，并且我们还采用了PJ-34作为小鼠的口服TET2抑制剂，在体内证实了以上观点。小鼠的肾脏病理改变也提示，随着口服TET2抑制剂服用时间延长，其糖尿病肾病病理改变呈部分缓解趋势。最后我们利用临床血液标本，证实了在人体中，糖尿病肾病患者组TGFβ1表达调控区的甲基化水平与单纯糖尿病无并发症患者组、对照租相比，都呈下降趋势。以上数据表明，在糖尿病肾病的发生发展中，TET2通过在TGFβ1表达调控区的更多的结合，导致了其甲基化水平降低，从而增加了细胞中TGFβ1表达，进而导致了糖尿病肾病的发生发展。这项发现可帮助我们在表观遗传学角度进一步理解糖尿病肾病的发生机制，并为其治疗方法开辟新的途径。