【作者】 李俊；

【导师】 郑启昌；

【作者基本信息】 华中科技大学 ， 普外科， 2012， 博士

【摘要】 目的：移植物动脉硬化是以弥漫性内膜增生为特征的慢性血管增殖性疾病,也是导致远期移植器官功能丧失、限制其远期疗效的主要原因。移植物动脉中膜平滑肌细胞凋亡是器官移植后的早期事件,有研究提示移植物动脉中膜平滑肌细胞凋亡可能通过募集骨髓间充质干细胞参与移植物动脉硬化。本研究拟探讨移植物动脉中膜平滑肌细胞凋亡对移植物动脉硬化的促进作用及其分子机制。方法：原代培养大鼠血管平滑肌细胞,构建并转染携带SM-22a启动子的野生型p53基因慢病毒载体,采用DAPI细胞核染色和流式细胞术检测过表达p53对血管平滑肌细胞凋亡的诱导作用,采用Western blot和ELISA分析检测观察血管平滑肌细胞凋亡对细胞因子SDF-1a,TGF-β和PDGF-BB表达的影响。体外培养大鼠骨髓间充质干细胞,制备p53过表达血管平滑肌细胞条件培养液,采用Transwell细胞迁移分析和BrdU标记法检测血管平滑肌细胞凋亡对骨髓间充质干细胞迁移和增殖的影响；并应用选择性细胞信号阻断剂对骨髓间充质干细胞进行预处理,以探讨血管平滑肌细胞凋亡诱导骨髓间充质干细胞迁移和增殖的分子机制。建立大鼠主动脉移植模型,构建并转染携带SM-22a启动子的BclxL基因慢病毒载体,采用TUNEL分析检测移植动脉中膜平滑肌细胞凋亡情况；采用免疫组化染色检测移植动脉壁中SDF-1a的蛋白水平；采用HE和Masson染色进行组织病理学分析,探讨BclxL基因转染对移植动脉新生内膜形成的影响；并应用免疫双荧光染色探讨BclxL基因转染对骨髓间充质干细胞参与内膜增生的影响。同时,建立异系大鼠主动脉移植模型,对受体大鼠分别给予mTOR抑制剂rapamycin和MAPK/ERK抑制剂PD98059治疗,检测其对移植动脉新生内膜形成的影响,以探讨PI3K/Akt/mTOR和MAPK/ERK信号通路在移植物动脉硬化中的作用。结果：慢病毒介导p53过表达有效诱导血管平滑肌细胞凋亡,凋亡血管平滑肌细胞释放趋化因子SDF-1a,并诱导周围血管平滑肌细胞表达SDF-la,引起p53过表达血管平滑肌细胞培养液中SDF-1a水平明显升高。血管平滑肌细胞凋亡可诱导骨髓间充质干细胞的迁移和增殖,以SDF-la中和抗体可阻断该作用。Western blot分析显示血管平滑肌细胞凋亡活化骨髓间充质干细胞的PI3K/Akt/mTOR和MAPK/ERK信号通路,且该作用是由SDF-1a介导。用PI3K/Akt/mTOR抑制剂Ly294002和rapamycin或MAPK/ERK抑制剂PD98059对骨髓间充质干细胞进行预处理,可有效阻断血管平滑肌细胞凋亡诱导的骨髓间充质干细胞迁移和增殖。与同系移植相比,异系移植动脉中膜平滑肌细胞大量凋亡,SDF-1a蛋白水平明显升高,8周后异系移植动脉新生内膜形成并伴有新生内膜细胞PI3K/Akt/mTOR和MAPK/ERK信号通路的活化。慢病毒介导转染BclxL基因,在SM-22a启动子调控下目的基因选择性在移植动脉中膜平滑肌细胞高表达,使中膜平滑肌细胞凋亡明显减少,移植动脉中膜SDF-1a的表达下调及其在血清中的含量下降；并且,伴随早期移植动脉中膜平滑肌细胞凋亡抑制,8周后异系移植动脉新生内膜中CD90和CD105双阳性细胞的数量明显减少,且血管新生内膜面积和管腔狭窄程度均显著减少。同样,rapamycin和PD98059均能有效减少骨髓间充质干细胞募集至新生内膜以及移植动脉硬化。结论：移植动脉中膜平滑肌细胞凋亡通过产生SDF-1a募集骨髓间充质干细胞,从而在移植物动脉硬化的发生发展中发挥关键作用。PI3K/Akt/mTOR和MAPK/ERK信号传导通路活化是血管平滑肌细胞凋亡诱导骨髓间充质干细胞迁移和增殖的分子机制。以血管平滑肌细胞凋亡为干预靶点将是防治移植物动脉硬化的一个有效策略。更多还原

【Abstract】 Objective-Allograft arteriosclerosis is a vascular proliferative disease characterized by diffuse intima hyperplasia and is also a leading cause of late allograft loss, limiting the long-term success of organ transplantation. Allograft medial vascular smooth muscle cell (VSMC) apoptosis is the early event after organ transplantation and is involved in allograft arteriosclerosis by recruitment of bone marrow-mesenchymal stem cells (BM-MSCs). The present study elucidates the underlying molecular mechanism and the contribution of medial VSMC apoptosis to allograft arteriosclerosis. Methods-Recombinant lentivirus vector of wild-type p53 carrying a SM-22αpromoter was constructed and transferred into primarily cultured VSMCs. Nuclei staining with DAPI and flow cytometry was used to detect the roles of overexpressing p53 in inducing VSMC apoptosis. The effect of VSMC apoptosis on expression of SDF-1α, TGF-βand PDGF-BB was assessed by Western blot and ELISA. BM-MSCs were primarily cultured in vitro and conditioned medium was prepared from VSMCs with overexpressing p53. The influence of VSMC apoptosis on BM-MSCs migration and proliferation was further evaluated by Transwell and BrdU labeling. BM-MSCs were pretreated with selective inhibitor of cell signaling pathway to explore the molecular mechanism that VSMC apoptosis promoted migration and proliferation of BM-MSCs. Rat aortic transplant models were established. Recombinant lentivirus vector of BclxL carrying a SM-22αpromoter was constructed and transferred into rat aortic allografts. TUNEL assay was performed to detect the medial VSMC apoptosis in allograft aorta. Expression of SDF-1αin allograft arteries was detected by immunostaining. Histopathology of allograft arteries was assessed by HE and Masson staining to explore the influence of BclxL gene transfer in allograft arterial neointima formation. On the other hand, the allograft recipient rats were treated with rapamycin and PD98059, the inhibitor of mTOR and MAPK/ERK signaling, to investigate their effect on allograft arterial neointima formation thereby exploring the roles of PI3K/Akt/mTOR and MAPK/ERK signaling in allograft arteriosclerosis. Results-Lentivirus-mediated p53 overexpression effectively induced VSMC apoptosis. Apoptotic VSMCs released chemokine SDF-1αand induced neighbor vital VSMCs express SDF-1αthereby elevating SDF-1αlevel in conditioned medium from VSMCs with overexpressing p53. VSMC apoptosis was able to promote BM-MSCs migration and proliferation however, neutralizing antibody of SDF-1αwas capable of abolishing this effect. Western blot showed that VSMC apoptosis activated PI3K/Akt/mTOR and MAPK/ERK signaling in BM-MSCs by SDF-1α. Pretreatment of BM-MSCs with inhibitor of PI3K/Akt/mTOR signaling or MAPK/ERK signaling, Ly294002 and rapamycin or PD98059 obviously prevented VSMC apoptosis-induced migration and proliferation of BM-MSCs. Compared with isograft arteries, medial VSMC apoptosis and expression of SDF-1αsignificantly revealed in allograft arteries. After 8 weeks transplantation, neointima with activation of PI3K/Akt/mTOR and MAPK/ERK signaling obviously formed in allograft arteries. BclxL was selectively overexpressed in medial VSMCs by lentivirus-mediated BclxL gene transfer under regulation of SM-22αpromoter. Overexpression of BclxL significantly inhibited medial VSMC apoptosis and reduced SDF-1αexpression level both in allograft arterial media and serum. Inhibition of early medial VSMC apoptosis in allograft arteries accompanied notable decrease of CD90+CD105+ double-positive cells in neointima as well as attenuation of neointima areas and luminal stenosis for 8 weeks transplantation. Likewise, both of rapamycin and PD98059 was able to suppress recruitment of bone marrow-mesenchymal stem cells (BM-MSCs) to neointima and prevent allograft arteriosclerosis. Conclusions-Allograft arterial medial VSMC apoptosis plays crucial roles in development of allograft arteriosclerosis through recruitment of BM-MSCs by inducing the production of SDF-1α. Activation of PI3K/Akt/mTOR or MAPK/ERK signaling is the molecular mechanism for VSMC apoptosis-induced migration and proliferation of BM-MSCs. VSMC apoptosis represents a relevant target of therapeutic interventions for allograft arteriosclerosis.更多还原