应用转基因斑马鱼模型研究Tie2-R849W 突变体导致静脉发育畸形的分子机制

Venous malformations (VM) are the most common symptomatic vascular anomalies, and head and neck regions make up about 40%. These lesions are often chronically painful, cause disfigurement and dysfunction, and are difficult to treat. Histologically, VMs are characterized by enlarged venous channels lined by a single flattened layer of endothelial cells (ECs)surrounded by sparse, irregularly distributed vascular smooth muscle cells(VSMCs). It's believed the interaction between ECs and VSMCs plays an important role in the formation of VM. Angiogenesis is a complex process requiring endothelial cell migration, proliferation and tube formation. The formation of blood vessel networks is completed by remodeling through interaction between ECs and surrounding mural or VSMCs. The crosstalk between ECs and VSMCs in vascular wall is thus critical for the vascular maturation. Moreover, VSMCs provide structural and functional support for the endothelium. This entire process is tightly controlled by various factors. PDGF-BB is the most potent know chemoattractant for VSMCs in culture cells. In several cell types including VSMCs, increased FAK tyrosine phosphorylation correlate with the chemotactic response to migration factors, including PDGF-BB. A missense mutation at position 849 in the kinase domain of the receptor tyrosine kinase TIE2(Tie2-R849W) is commonly identified in about 40% inherited and sporadic forms of VM that lead to Tie2 auto-hypo-phosphorylation. The mechanistic action of Tie2-R849W variant expression on angiogenic cascades including smooth muscle cell recruitment, however, remains elusive. To gain insight into the cause of VM, based on the above-mentioned facts, we hypothesized:1.The expression of Tie2-R849W mutant lead to PDGF hypo-phosphorylation.2. PDGF-BB hypo-phosphorylation induce FAK dephosphorylation, and decrease its ability to recruit VSMCs which lead to the formation of VM. To test above hypothesis in vivo and ex vivo, this research is designed. We construct transgenic zebrafish model and cell lines which express Tie2-R849W targeted to ECs, and observe the development of vascular system and migration of VSMCs, as well as analyzed the expression level of PDGF-BB and FAK through fluorescence microscope、Western blotting、immunoprecipitation 、RT-PCR and Whole-mount hybridization in situ. This research is significant to explore the pathogenesis of VM, and can provide molecular target for treatment of VM，especially inherited VM.

静脉畸形（VM）是最常见先天性脉管畸形。研究表明，Tie2突变并过度磷酸化与VM的发生密切相关，但具体机制不明。血管发育生物学研究多集中于其初级阶段，而对平滑肌细胞（VSMCs）参与的血管成熟及稳定阶段的研究不多。本研究提出假说：1、Tie2突变并过度磷酸化，使PDGF信号高度激活。2、高度激活的PDGF信号抑制FAK磷酸化，使其介导VSMCs迁移、增殖障碍，从而导致VM发生。为验证此假说，建立ECs特异性表达Tie2突变（R849W）转基因斑马鱼模型，荧光显微镜观察斑马鱼发育各阶段血管系统的发育及VSMCs迁移及增殖情况。应用免疫组化及Whole-Mount原位杂交、RT-PCR、Westen-blotting等技术，检测Tie2、PDGF-BB及FAK的表达及磷酸化水平，并在人细胞水平验证。探讨PDGF、FAK信号与Tie2-R849W相互作用及其与VM发病关系，为VM的治疗探寻新靶点

家族性皮肤粘膜静脉畸形（VMCM）病变范围广，且广泛存在TIE2-R849W突变，然而该突变在静脉畸形中的致病机制尚不完全明确，相关裸鼠模型也难以满足深入研究的目的。本项目通过合成TIE2-R849W突变基因表达体系，利用显微注射技术，基于fli1a: EGFP转基因斑马鱼构建TIE2-R849W过表达斑马鱼模型，荧光显微镜下记录斑马鱼的早期发育改变并定量比较，随后提取斑马鱼mRNA，针对目标基因行实时定量PCR（qRT-PCR），利用已有基因芯片进一步验证结论。基于对TIE2-R849W过表达斑马鱼模型的观察，TIE2-R849W显著影响了其早期尾静脉丛的发育，窦腔数量和面积均存在近50%的减少。此外，该突变还显著引发了多种颅颌面畸形表型（眼、前脑、下颌骨畸形）。眼部发育缺陷表现为单侧眼缺失、双侧眼缺失、单侧眼发育不足、双侧眼融合，而前脑则隆起明显，下颌骨体宽度明显缩窄且前伸明显。经组织学研究发现，突变组斑马鱼的眼球存在晶状体缺失、视网膜网状层紊乱，下颌骨软骨异常增殖，前脑脊髓区域异位发育，神经胶质细胞分布紊乱。经qRT-PCR显示，TIE2-R894W可引发pik3r2、foxo1b的表达上调，而与尾静脉丛发育相关的基因，除显著高表达的egfl7外，nr2f2、nr2f1a和s1pr1均无显著异常的表达。根据既往研究，wnt通路相关分子的异常表达可引发相似的颅面部畸形，qRT-PCR也进一步证实该猜测，TIE2-R849W不仅引发了经典wnt通路配体（wnt8a及wnt9a）的明显上调，也导致了wnt通路相关受体及胞内下游分子的上调。通过表达TIE2-R849W的人脐静脉内皮细胞（HUVEC）模型，基于既往基因芯片，部分突变组内的tie2、pik3r2、foxo1b、egfl7、wnt9a、lrp5、dkk1均存在一致的高表达。综上，基于该项目的相关研究结果，TIE2-R849W引发斑马鱼尾静脉丛发育异常与Egfl7的高表达关系密切，同时，首次发现了TIE2-R849W对于EGFL7及WNT相关信号的调控作用，拓展了静脉畸形的相关理论，也揭示了TIE2-R849W对眼、脑、颌骨等相关发育过程的深度参与，相关成果对于颌面部发育畸形的研究具有十分重要的科研及临床意义。