MMP9介导静脉壁Sca-1干细胞在静脉血栓内募集的机制研究

No reports were currently available about the role of vein wall derived stem cells playing in the resolution of venous thrombus. Our recent study shows that: After venous thrombosis, MMP9 highly expressed in the vein wall with thrombus, and lots of Sca-1 stem cells proliferated in vein wall with part of stem cells moved across vein wall and accumulated in venous thrombus.Sca-1 stem cells, derived from vein wall, enhanced the resolution of venous thrombus by neovascularization. However, the mechanism is unclear for the mobilization of vein wall derived Sca-1 stem cells and its accumulation into venous thrombus. It is well elucidated that the mobilization of bone marrow stem cell is mediated by active MMP9-dependent C-X-C chemokine receptor type 4 (CXCR4) expression on bone marrow stem cell. The activation of MMP9 is induced by plasmin which activated by mobilization agent of bone marrow stem cell. Venous thrombosis would activate the fibrinolytic system and the atctive MMP9 would degrade matrix of vein wall. We hypothesize that vein wall derived Sca-1 stem cells are mobilized by active MMP9 after venous thrombosis, and the degradation of matrix by active MMP9 facilitate Sca-1 stem cells moving across vein wall and accumulation into the venous thrombus. In this protocol, we will culture the vein wall derived Sca-1 stem cells with thrombus and study the mechanisms of MMP9 in directing accumulation of vein wall derived Sca-1 stem cells into venous thrombus by gene knockout,antibody blockage,receptor antagonist, stem cells transplantation in the adventitia of vein wall, stem cells tracing,Trans-well co-culture and Bioreactor. It would contribute to further illustrate the mechanism of natural resolution of venous thrombus and explore the new treatment of venous thrombus.

目前国内外没有关于静脉壁干细胞溶解静脉血栓的报道。前期研究我们发现静脉血栓形成后静脉壁MMP9表达增加，其静脉壁Sca-1干细胞大量增殖并跨过静脉壁在血栓内募集，通过血管新生溶解血栓，但不清楚静脉壁Sca-1干细胞如何被动员并在血栓内募集。已证实干细胞动员剂通过纤溶酶活化MMP9，后者调控CXCR4的表达而动员骨髓干细胞。因静脉血栓形成后纤溶激活，活化的MMP9降解静脉壁基质。我们推测血栓形成后静脉壁活化的MMP9不仅动员静脉壁Sca-1干细胞，而且通过降解静脉壁基质而利于Sca-1干细胞跨过静脉壁在血栓内募集。本项目以血栓形成后的静脉壁Sca-1干细胞为研究对象，应用基因敲出、抗体封闭、受体拮抗、干细胞移植及示踪、Bioreactor等，研究MMP9介导静脉壁Sca-1干细胞在静脉血栓内募集的机制。对进一步阐明静脉血栓自然溶解机理及探寻血栓新的疗法有重要意义。

静脉血栓是一严重疾病，促进静脉血栓早期溶解可显著减轻静脉瓣膜纤维化，减轻血栓后综合症发生。我们研究发现人静脉壁外膜存在大量干细胞，流式分选后获得CD34和CD117双阳性干细胞，同时在人大隐静脉内血栓中也发现大量CD34和CD117干细胞。小鼠静脉血栓形成后，大量干细胞经血管壁跨过内膜进入血栓内。进入血栓的干细胞主要出现在血栓的周边区域，局部大量新生血管形成。尽管14天的血栓几乎完全机化，但免疫组化发现干细胞仍然主要出现在血栓的周边区域，与7天血栓并没显著区别，这是因为进入血栓中心区域的干细胞已经分化。大隐静脉壁干细胞经原代培养及CD34和CD117干细胞流式细胞技术分选后体外培养。移植于裸鼠血栓静脉壁外膜后，血栓的溶解机化显著增快，血栓中细胞密度、巨噬细胞数量等显著增加。为了明确进入血栓的干细胞是否为血管壁外膜干细胞，体外培养的人大隐静脉外膜干细胞经体外扩增标记后移植于血栓静脉壁外膜，发现标记的干细胞很快进入血栓内的中心区域，并分化为内皮细胞，显示进入血栓的干细胞可分化为新生血管内皮细胞。进入血栓的干细胞通过分化为新生血管内皮细胞促进血栓的溶解。为了明确进入血栓的干细胞分化为新生血管内皮细胞的机制。我进行了质谱分析，发现与干细胞分化为内皮细胞关系明显的ITGA6显著升高。为了研究静脉壁干细胞跨过静脉壁进入血栓内的能力，我们获取人大隐静脉，脱细胞处理大隐静脉后移植于兔腹主动脉，发现大隐静脉壁的内膜和外膜最先细胞化，并逐渐完成血管塑型，脱细胞大隐静脉可以维持长期通畅。提示静脉壁外膜干细胞可以跨过静脉壁进入中膜、内膜。.研究中发现进入血栓的干细胞通过分化为内皮细胞促进血栓的新生血管形成，初期结构显示进入血栓的干细胞可能通过ITGA6途径分化为新生血管内皮细胞，但其具体的信号通路尚需要进一步研究。我们拟进一步研究ITGA6的相关配体laminin在血栓中的表达及其可能通过FAK/PI3K通路调控血栓内干细胞分化为新生血管内皮细胞的过程。