糖尿病视网膜病变中乙酰肝素酶对VEGF基因转录调控的研究

Diabetic retinopathy (DR) is a major cause of blindness. VEGF is the most important factor in the pathogenesis of DR, but the regulatory mechanism of VEGF expression is still unclear. We have found the expression of heparanase (Hpa) was increased accompanying with VEGF expression in the DR patients, human retinalendothelial cells (HRECs) cultured under high glucose and the DR rat model. The VEGF expression was reduced by Hpa inhibitor. Follow-up studies have shown that high Hpa expression promoted the migration and proliferation of HRECs. Based on the series of studies, the applicant has continuously published three SCI articles as a common corresponding author.. Recently, it was showed that a novel class of signal transduction kinases translocate into the nucleus and associate with chromatin to directly modulate transcription of target genes. Our latest study also showed that Hpa could translocate into the nucleus of HRECs, and bind to the promoter region of the VEGF gene. When treated with high glucose, this Hpa-VEGF promoter binding was remarkably increased. Therefore, at the first time we propose that Hpa is directly involved in the VEGF gene transcription regulation in HRECs in the development of DR. In this project, Hpa-DNA binding in HRECs is determined by chromatin immunoprecipitation (ChIP) assay, after transfected with Hpa cDNA or Hpa siRNA. The role of histone acetylation and methylation in this binding is also observed to obtain the direct evidence of how Hpa was involved in VEGF transcriptional regulation. The potential mechanism will be elucidated.

糖尿病性视网膜病变（DR）是一种主要致盲眼病，VEGF是引起DR病理改变的最重要因子，但其调控机制不清。我们先后在DR患者、高糖培养人视网膜内皮细胞（HREC）、DR大鼠模型中证实了乙酰肝素酶（Hpa）和VEGF表达同步升高，Hpa的抑制剂可降低VEGF表达；Hpa表达升高促进了HREC的迁移和增殖。.最近研究表明某些细胞信号传导蛋白激酶能进入细胞核参与目标基因转录的调控。我们的前期研究也发现，Hpa能进入HREC胞核内，与VEGF基因的启动子结合，高糖状态下这一结合增加。因此我们首次提出：在DR中Hpa在HREC胞核内参与了VEGF基因转录调控。本项目分别以Hpa-cDNA和Hpa siRNA转染HRECs，采用ChIP法检测与Hpa结合的VEGF-DNA含量改变，以获得Hpa参与VEGF转录调控的直接证据，在此基础上进一步研究组蛋白乙酰化和甲基化修饰在其中的作用，以探讨其可能的机制。

糖尿病性视网膜病变（DR）是一种主要致盲眼病，血管内皮生长因子（VEGF）是引起DR病理改变的最重要因子，但其调控机制不清。我们前期证实了乙酰肝素酶（Hpa）表达升高促进了人视网膜血管内皮细胞（HREC）的迁移和增殖，也发现Hpa能进入HREC胞核内。本研究在此基础上，采用体外高糖及低氧培养的HREC作为DR模型，应用多种实验方法，获得Hpa直接参与VEGF基因转录调控的证据，在观察组蛋白（H3K9ac）修饰对这一作用影响的基础上，阐明DR发展过程中Hpa调控VEGF表达的机制。.重要实验结果：.1.证明了在高糖及低氧诱导的HRECs中，核内HPA与VEGF基因启动子结合，参与VEGF基因转录调控.2.筛选出H3赖氨酸残基9乙酰化（H3K9ac）与VEGF和HPA表达相关.3.IF证实高糖、低氧条件下HRECs中的HPA和H3K9ac在细胞核内共定位；.4.染色质免疫沉淀（ChIP）联合real-time PCR实验结果表明在高糖和低氧诱导的HRECs细胞核内H3K9ac及 RNA Pol II与VEGF启动子的结合率明显高于正常组.5.Co-IP发现高糖、低氧诱导HRECs中HPA及 H3K9ac相互结合共同作用，而正常组中HPA及H3K9ac则无结合.6.Re-ChIP联合PCR证实在高糖和低氧组的HRECs细胞核内H3K9ac与HPA形成复合物，结合在VEGF基因启动子上；正常组中的VEGF启动子序列上未检测到此结合.综上实验结果我们得出结论：HPA通过与H3K9ac相互结合共同作用于VEGF基因启动子区域，并且招募大量RNA Pol II形成复杂的转录起始复合物，启动VEGF基因转录，调控了VEGF基因表达。.本研究的创新性及意义：首次提出并证实了HPA通过与H3K9ac相互结合共同作用于VEGF基因启动子区域，招募大量RNA Pol II调控VEGF基因表达。阐明了DR发展过程中Hpa调控了VEGF表达的机制。这将对我们寻找DR更有效的治疗手段具有积极意义。.在此基金资助下，现今已发表文章3篇（均为第一标注），其中SCI1篇，中华核心期刊2篇，另有1篇相关SCI文章已进入二次修改环节。培养研究生5名，其中4名已毕业。