转录因子KLF15抑制静脉血栓形成的机理研究

Deep venous thrombosis (deep vein thrombosis,abbreviated to DVT) is a common clinical disease, which currently lack of a sensitive, accurate and effective method for forecasting diagnosis. So the diagnosis and treatment have become the focus of clinical attention in which it is the difficult and key factor to elucidatethe pathophysiological mechanism. Kruppel-like factors transcription regulatory factors of the zinc finger family and they are critical for hypertension, atherosclerosis and other cardiovascular diseases. .In our previous research we found the expression of KLF15 mRNA was upregulated in the vein walls of mouse DVT group. When KLF15siRNA was injected into tail veins with transfect reagent, KLF15 mRNA expression was decreased and the weight of thrombus increased. HUVECs were transfected with KLF15 RNA interferance vector and the expression of TM and MCP-1 in thrombin-treated cells decreased. Then C57 mice DVT model of inferior vena cava stenosis was established and the up-experssion of TM and MCP-1 mRNA in vein walls were found. We speculated that at the early stage of DVT, KLF15 could regulate the expression of and MCP-1、VCAM-1 and TM to promote the formation of DVT..We will study the role of KLF15 in DVT formation in vivo and in vitro, genes and mechanism regulated by KLF15 in the pathophysiological progress. The results may provide new theoretical basis for future development of DVT predictive diagnosis and drugs.

深静脉血栓（deep vein thrombosis，DVT）缺乏灵敏性高、准确有效的预测诊断方法，其诊断和治疗等观测指标一直是临床关注的热点，探明发病机理成为揭示该疾病的重难点。KLF15(Kruppel-like factors 15)是一种锌指家族转录调控因子，在心血管、动脉粥样硬化中起重要作用，但在深静脉血栓中尚无报道。我们发现小鼠DVT模型静脉内皮组织中KLF15、血栓调节蛋白TM的表达出现差异，在HUVEC上干扰KLF15的表达后发现TM的表达也随之下降。KLF15干扰病毒尾静脉注射小鼠，发现抑制血管内皮细胞中KLF15的表达可影响形成血栓的重量。本课题将在体内和体外利用CHIP-seq、RNA-seq、小鼠敲减模型等方法，寻找KLF15 调控TM等基因参与血栓形成的证据，揭示其参与DVT形成的机制,为临床深静脉血栓形成的早期预防、诊断治疗等监测等提供有实际意义的实验理论依据。

深静脉血栓（Deep vein Thrombosis，DVT），是常发生于下肢的深静脉栓塞，与肺栓塞统称为静脉血栓栓塞症（VTE），是继急性心肌梗塞和中风之后的第三大主要血管疾病。由于DVT发病机制复杂，尚未被完全阐释，目前临床上缺乏特异、灵敏的早期诊断标记物，因此DVT的发生机制成为当前研究的热点。KLF15(Kruppel-like factor 15)是一种锌指蛋白家族的转录调控因子，在心血管、动脉粥样硬化中起重要作用，但在深静脉血栓中尚无研究报道。在本研究中我们首次发现并报道了通过使用Klf15干扰片段尾静脉注射C57BL/6小鼠，抑制内皮细胞中Klf15的表达可影响小鼠DVT模型形成的血栓重量。我们在小鼠深静脉血栓模型DVT组和空白对照组中鉴定出2206个差异表达基因（DEGs），包括1330个上调基因和876个下调基因。在Klf15-siRNA组和Klf15阴性对照NC组中鉴定出1041个DEGs（235个上调，806个下调）。同时，还进行了GO和KEGG富集分析。最终我们从RNA-seq结果中总共筛选出了七个重要的DEG，分别是基质金属蛋白酶Mmp12，Mmp13，Mmp19，Arg1、Ccl2、Hmox1和Fmo3，与正常对照组相比，它们在DVT组中的表达水平显著升高。其中，Klf15-siRNA组Mmp13的表达还显著高于Klf15-NC组，静脉内皮细胞实验也证实了这一结果。结合尾静脉siRNA抑制实验和细胞实验，我们推测KLF15调控DVT形成可能是通过抑制Mmp13等基因来实现。本项目通过小鼠DVT模型、RNA-seq、尾静脉注射和细胞模型等手段，得到小鼠DVT形成过程中Klf15调控DVT形成的候选基因，初步证实小鼠DVT模型中Klf15可能通过抑制Mmp13等基因调控血栓形成。同时得到了一批小鼠DVT形成时差异表达的基因以供后续研究DVT的发生机制，初步为今后DVT的预防和治疗提供了理论依据及可能的药物新靶点。