截短型rhtBIGH3-(RGD)2蛋白对大鼠角膜新生血管的抑制和分子机制研究

The elevated expression level of vascular endothelial growth factor (VEGF) is widely believed to be associated with corneal neovascularization (CNV), although the specific mechanism is not fully illustrated. The fourth FAS1 domain of the human BIGH3 (transforming growth factor-β1-inducible gene-h3) protein has been reported to play an important role in mediating tumor angiogenesis. An Arg-Gly-Asp (RGD) motif can enhance its gene function by competitive binding with integrin. We first truncated the C terminal fragment of BIGH3 protein which includes the fourth FAS1 domain, and modified it with an RGDRGD motif, which is named rhtBIGH3-(RGD)2 protein. In our previous study, rhtBIGH3-(RGD)2 inhibited vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cell (HUVEC) proliferation, adhesion and migration far more effectively than did wild-type C-terminal fragment BIGH3. We speculated that rhtBIGH3-(RGD)2 protein mediated corneal neovascularization by inhibiting the biological activities of HUVECs. In order to proof this hypothesis, we investigated the effect and molecular mechanism of rhtBIGH3-(RGD)2 protein on the biological activities of VEGF-HUVECs in vitro and the effect and corneal location of rhtBIGH3-(RGD)2 protein on CNV model in vivo, which might provide a novel target for treating corneal neovascularization.

角膜新生血管（CNV）确切机制尚未阐明，多认为与VEGF表达高有关。人角膜上皮素（BIGH3）的第四个Fastalin（FAS）结构有明确的抑制新生血管作用。RGDRGD串联肽能通过与整合素竞争结合而增强其所在基因功能。本课题组首次截短了BIGH3 C末端基因，含有第四个FAS结构，并改良使其含有RGDRGD结构域，命名为rhtBIGH3-(RGD)2蛋白 。在前期研究中发现，rhtBIGH3-(RGD)2抑制VEGF-HUVECs的增殖、黏附和迁移优于改良前。我们推测CNV发生时，rhtBIGH3-(RGD)2蛋白抑制HUVECs生物学活性，从而抑制新生血管发生。为证实这一假说，体外研究rhtBIGH3-(RGD)2蛋白对VEGF-HUVECs生物学功能影响及分子机制，通过体内大鼠CNV模型观察rhtBIGH3-(RGD)2蛋白抑制CNV情况及在角膜组织定位,为CNV的防治提供新靶点。

VEGF是公认的导致角膜新生血管发生主要病因，抑制VEGF抑制角膜新生血管的关键因素。人角膜上皮素（BIGH3）的第四个Fastalin（FAS）结构有明确的抑制新生血管作用。RGDRGD串联肽能通过与整合素竞争结合而增强其所在基因功能。我们课题组成功改良了人角膜上皮素（BIGH3）的第四个Fastalin（FAS）结构，使其含有双RGD结构域，命为rhtBIGH3-(RGD)2蛋白 。在体外，通过表达、纯化，研究其蛋白在调节HUVECs细胞增殖、黏附和迁移等活性，结果显示改良后的rhtBIGH3-(RGD)2蛋白，抑制HUVECs活性功能明显优于改良前。进一步我们已经基本摸清了rhtBIGH3-(RGD)2蛋白抑制VEGF-HUVECs分子机制的信号传导通路。证明 rhtBIGH3-(RGD)2蛋白在100ug/ml浓度下，发挥功能最强，在由于V EGF激活的在HUVEC细胞中，其是通过能抑制PI3K/AKT和ERK信号通路，而导致细胞凋亡，抑制HUVEC细胞活性，从而抑制细胞管道形成，抑制角膜新生血管的发生。近几年，mir-126在新生血管发生领域中发挥了重要的作用。为了进一步研究，mir-126是否参与rhtBIGH3-(RGD)2抑制HUVECS功能，体外培养VEGF孵化的人脐静脉内皮细胞（VEGF-HUVEC）,加入rhtBIGH3-(RGD)2蛋白终浓度为100ug/ml时，截短型rhtBIGH3-(RGD)2蛋白通过上调miR-126表达从而促进细胞凋亡、抑制HUVEC生物活性，从而抑制角膜新生血管的发生。进一步，从体内实验证实了rhtBIGH3-(RGD)2蛋白能抑制角膜新生血管的发生。rhtBIGH3-(RGD)2蛋白来源于人体，将成为一种新的治疗角膜新生血管的药物。