基于VHL病iPS细胞模型的VHL基因突变影响血管内皮细胞成血管功能及其机制的研究

Von Hippel-Lindau disease (VHL) is an inherited disease caused by VHL gene mutations, and is characterized by predisposition to tumors with abnormal angiogenesis, including renal cell carcinoma (RCC). However, its pathogenesis remains unclear. The influence of VHL gene mutations on the vascular endothelial cell (ECs) angiogenesis function and its mechanisms, is an important scientific issue in the etiology for abnormal angiogenesis of VHL disease tumor. However, the lack of VHL-hECs carrying VHL gene mutations of VHL patients limits the study of this scientific issue. In our preliminary experiment, we obtained VHL-hECs for the first time in the world, through in vitro differentiation from the VHL disease specific induced pluripotent stem cell lines (VHL-hiPSCs). And we found, VHL-hECs exhibited increased proliferation and decreased apoptosis under the stimulation of VEGF, as well as increased angiogenic activity in vitro under the stimulation of bFGF. These results suggested VHL-hECs had increased angiogenic activity. In order to acquire the reliable evidences to prove that VHL gene mutations enhance the angiogenic activity of VHL-hECs and interpret its mechanisms, we will carry out experiments including angiogenesis assay, RNA-Seq, Western blot, PCR, etc., and study the gene expression profiling, the expression and degradation of VEGFR and FGFR in VHL-hECs. This program will interpret the influence of VHL gene mutations on the ECs angiogenesis and its mechanisms, and lay the foundation for the new anti-angiogenic therapies studies of VHL disease.

VHL病是VHL基因突变引起的遗传病，容易产生肾癌等血管生成异常的肿瘤，但是其发病机制仍不清楚。VHL基因突变对于血管内皮细胞（ECs）成血管功能的影响及其机制，是VHL病肿瘤血管生成异常病因学的重要科学问题。但是缺乏携带病人VHL基因突变的ECs（VHL-hECs）限制了该问题的研究。我们前期已建立了VHL病人特异的诱导多能干细胞系（VHL-hiPSCs），通过体外诱导分化首次获得了VHL-hECs，并发现其在VEGF刺激下的增殖增强和凋亡减弱，在bFGF刺激下的成管能力增强，提示其成血管功能增强。本项目拟进一步采用血管生成分析实验、RNA-Seq和PCR等方法，旨在获得VHL基因突变增强VHL-hECs成血管功能的可靠证据，并通过研究其基因表达谱、VEGFR和FGFR的表达和降解情况，阐明VHL基因突变增强ECs成血管功能的机制，为研究VHL病肿瘤的抗血管生成新疗法的提供科学依据。

项目的背景：VHL 病是 VHL 基因突变引起的遗传病，容易产生肾癌等血管生成异常的肿瘤，但是其发病机制仍不清楚。VHL 基因突变对于血管内皮细胞（ECs）成血管功能的影响及其机制，是 VHL 病肿瘤血管生成异常病因学的重要科学问题。但是缺乏携带病人 VHL 基因突变的ECs（ VHL-hECs）限制了该问题的研究。主要研究内容、重要结果、关键数据及其科学意义：①我们首先通过重编程技术，获得了携带VHL病人VHL基因突变的VHL-hiPSCs细胞系。然后通过体外诱导分化技术将VHL-hiPSCs 在体外诱导分化为VHL-hECs，进而利用VHL-hECs进行成血管功能分析实验，建立了VHL病血管生成异常的体外人类疾病模型。为研究VHL基因突变影响血管内皮细胞功能、VHL病肿瘤血管生成异常的机制和抗VHL病血管生成特异性药物的筛选提供很好的研究工具，具有较大的科学意义和良好的应用前景。②我们发现与正常对照组相比较，动物体内实验结果显示VHL-hECs可以在体内形成更多的血管；体外实验结果显示，与正常对照组相比较，VHL-hECs在VEGF刺激下表现出增殖增强（细胞增殖标志物ki67表达升高）和凋亡减弱（细胞凋亡标志物caspas3表达下降），在FGF刺激下的成管能力增强；这些实验结果提示VHL-hECs存在对FGF和VEGF的敏感性增强和成血管能力增强，获得了VHL基因突变促使hECs成血管功能增强的证据，从VHL-hECs成血管功能变化方面揭示了VHL病肿瘤血管生成增加的机制。③我们发现VHL基因突变会造成 VHL-hECs 的FGFR和VEGFR的表达增加，揭示了VHL-hECs对于VEGF和bFGF刺激的敏感性增强和成血管功能增强的机制。④通过RNA-Seq等方法，我们找到了一些影响VHL-hECs成血管功能的FGFR和VEGFR下游相关信号通路基因，包括PKC、Ras/Raf/MEK/ERK、JAK/STAT和PI3K等，为进一步深入研究提供了线索和依据。

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