巨噬细胞通过刺激血管壁细胞MicroRNA表达促进移植血管内膜重塑

发布时间：2018-04-30 03:38

本文选题：移植性血管病 + 内膜　；参考：《山东大学》2017年硕士论文

【摘要】：研究背景血管及脏器移植术后往往会发生移植血管再狭窄即移植性血管病(transplant vasculopathy,TV),是造成移植器官慢性失去功能的主要原因,目前TV发生发展的生物学机制尚未完全清楚。前期研究发现,移植术后早期即存在血管外膜炎症,外膜的巨噬细胞浸润可能在移植性血管病发生中起关键作用。因此,本课题采用动脉血管移植模型,通过体内、外实验证明血管外膜早期的单核巨噬细胞浸润在促进TV病变发生过程中具有因果作用,并以microRNA(miR)-21为例,进一步证实外膜巨噬细胞通过调节血管壁细胞(主要针对平滑肌细胞和成纤维细胞)miR的表达促进移植血管内膜重塑的相关机制。研究目的1.确定抑制早期外膜巨噬细胞浸润聚集能否抑制移植血管的内膜重塑。2.明确巨噬细胞调节血管壁细胞miR表达的机制。3.检测通过外膜途径抑制miR的表达,能否抑制TV的发生发展。研究方法体内实验:建立大鼠胸-腹主动脉移植模型,氯膦酸二钠脂质体(clodronate liposomes)用于体内巨噬细胞耗竭。对移植大鼠进行随机分组:巨噬细胞耗竭组(clodronate liposomes)、miR-21 抑制组(miR-21 inhibitor)、及其相应对照组(control liposomes/control miRNA)。取 0、1、2、3、7、14 天移植血管进行HE染色。测量移植血管的各层增生情况;免疫组织化学染色检测CD3,CD68,proliferating cell nuclear antigen(PCNA),monocyte chemotactic protein-1(MCP-1),vascular cell adhesion molecule-1(VCAM-1),intercellular adhesion molecule-1(ICAM-1),α-smooth muscle actin(α-SM actin)的表达。分离血管外膜和中膜组织,QRT-PCR检测miR-21,MCP-1,VCAM-1,ICAM-1的表达。体外实验:分离大鼠胸主动脉外膜和中膜组织,分别做原代细胞培养。制作RAW264.0巨噬细胞条件培养基,ELISA检测条件培养基内各类细胞因子的含量,如:tumor necrosis factor-α(TNF-α),transforming growth factor-β1(TGF-β)等。用TNF-α细胞因子刺激外膜的成纤维细胞/中膜的平滑肌细胞,QRT-PCR检测miR-21表达;条件培养基培养外膜成纤维细胞/中膜平滑肌细胞,实验组加入TNF-α中和抗体,QRT-PCR检测miR-21表达。TNF-α刺激外膜成纤维细胞/中膜平滑肌细胞,实验组转染miR-21 inhibitor,EDU和Transwell检测细胞的增殖、迁移能力,QRT-PCR 检测 miR-21,MCP-1,VCAM-1,ICAM-1,CCL-5。外膜成纤维细胞/中膜平滑肌细胞转染miR-21 mimic,QRT-PCR检测MCP-1,VCAM-1,ICAM-1。研究结果1.巨噬细胞耗竭能够减轻血管内膜的增生和移植导致的外膜炎症。巨噬细胞耗竭显著降低血管壁miR-21的表达。QRT-PCR结果显示,体内干预耗竭巨噬细胞后MCP-1,VCAM-1,ICAM-1的表达均降低。与其空白对照组相比,miR-21抑制剂干预后血管内膜增生也显著减弱;免疫组化结果显示,与其对照组相比,巨噬细胞耗竭组和miR-21抑制组CD68,PCNA,MCP-1,VCAM-1,ICAM-1的表达均减少。2.巨噬细胞通过旁分泌TNF-α调节miR-21在外膜的成纤维细胞和中膜的平滑肌细胞中的表达。ELISA结果显示,巨噬细胞条件培养基中含有TNF-α等细胞因子;QRT-PCR结果显示,TNF-α可上调miR-21在外膜的成纤维细胞和中膜的平滑肌细胞中的表达;条件培养基培养外膜的成纤维细胞及中膜的平滑肌细胞,QRT-PCR结果显示,添加TNF-α中和抗体组miR-21的表达下调。3.沉默miR-21可抑制TNF-α导致的外膜的成纤维细胞及中膜的平滑肌细胞增殖、迁移及炎症反应。TNF-α刺激外膜成纤维细胞及中膜平滑肌细胞,EDU和Transwell结果显示,转染miR-21 inhibitor组外膜的成纤维细胞及中膜的平滑肌细胞的增殖和迁移能力均减弱;QRT-PCR结果显示,转染miR-21 inhibitor组外膜的成纤维细胞/中膜的平滑肌细胞的miR-21,MCP-1,VCAM-1,ICAM-1表达均降低。4.miR-21可以促进外膜的成纤维细胞/中膜的平滑肌细胞的炎症反应。QRT-PCR结果显示,转染miR-21 mimic组,外膜成纤维细胞MCP-I,VCAM-1表达上调,中膜平滑肌细胞MCP-1表达上调。研究结论1.移植血管外膜巨噬细胞在新生内膜发生发展中具有重要作用。2.巨噬细胞通过旁分泌TNF-α调节血管壁细胞miR-21的表达,进而调节血管壁细胞的功能变化及炎症反应。3.血管壁细胞中表达的miR-21可能通过影响移植血管的炎症反应及其功能变化,进一步促进新生内膜重塑。
[Abstract]:Background blood vessel restenosis, transplant vasculopathy (TV), is the main cause of chronic loss of function of transplanted organs. The biological mechanism of TV development is not completely clear at present. Early study found that angioepicarditis existed early after transplantation. The infiltration of macrophages in the outer membrane may play a key role in the occurrence of transplantation of vascular disease. Therefore, this subject uses an arterial vessel transplantation model. Through the body and in vitro experiments, it is proved that the early mononuclear macrophage infiltration in the epicardial membrane plays a causal role in promoting the occurrence of TV lesions, and is further confirmed by microRNA (miR) -21 as an example. Epicardium macrophages regulate the remodeling of vascular intima by regulating the expression of miR in vascular wall cells (mainly for smooth muscle cells and fibroblasts). Objective 1. to determine whether inhibition of infiltration and aggregation of early membrane macrophages inhibits the remodeling of the intima of the transplanted blood vessels.2. clearly regulates the miR surface of the vascular wall cells by macrophages The mechanism.3. detects the inhibition of the expression of miR through the outer membrane pathway and inhibits the development of TV. In vivo experiments in vivo: a rat thoracic abdominal aorta transplantation model was established, and chlorphosphonic acid two liposomes (clodronate liposomes) was used for the depletion of macrophages in the body. A randomized grouping of transplanted rats: a macrophage depletion group (clodronate L). Iposomes), the miR-21 inhibition group (miR-21 inhibitor), and its corresponding control group (control liposomes/control miRNA). Take the 0,1,2,3,7,14 day transplant vessels for HE staining and measure the proliferation of the various layers of the transplanted vessels. CP-1), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), alpha -smooth muscle (alpha -smooth muscle) expression. RAW264.0 macrophage conditioned medium was produced, and the contents of all kinds of cytokines in the conditioned medium were detected by ELISA, such as tumor necrosis factor- alpha (TNF- alpha), transforming growth factor- beta 1 (TGF- beta), etc., and the smooth muscle cells of the fibroblasts / middle membranes were stimulated with TNF- alpha cytokine, and the QRT-PCR detected the expression; The culture medium cultured outer membrane fibroblast / middle membrane smooth muscle cells, the experimental group added TNF- alpha neutralizing antibody, and QRT-PCR detected miR-21 expression.TNF- alpha stimulated outer membrane fibroblasts / middle membrane smooth muscle cells. The experimental group transfected miR-21 inhibitor, EDU and Transwell detected the cell proliferation, migration ability, QRT-PCR detected miR-21, MCP-1, VCAM-1, etc. -5. adventitial fibroblasts / medium membrane smooth muscle cells transfected with miR-21 mimic, QRT-PCR detection MCP-1, VCAM-1, ICAM-1. study results 1. macrophage depletion can reduce the proliferation of vascular intima and transplant the outer membrane inflammation. Macrophage depletion significantly reduces the expression of miR-21 in the vascular wall and the results show that the body intervention depleted macrophages in vivo The expression of post MCP-1, VCAM-1 and ICAM-1 decreased. Compared with the blank control group, the intima hyperplasia of the vascular endothelial cells decreased significantly after the intervention of miR-21 inhibitors. The immunohistochemical results showed that the expression of the macrophage depletion group and the miR-21 inhibition group CD68, the expression of PCNA, MCP-1, VCAM-1, ICAM-1 reduced the.2. macrophage through the paracrine TNF- alpha. The expression of miR-21 in the fibroblasts of the outer membrane and the smooth muscle cells of the middle membrane showed that the macrophage conditioned medium contained TNF- alpha and other cytokines. QRT-PCR results showed that TNF- alpha could increase the expression of miR-21 in the smooth muscle cells of the fibroblasts and middle membranes of the outer membrane; the conditioned medium cultured the outer membrane of the fibroblasts. The QRT-PCR results showed that the expression of TNF- alpha neutralizing antibody group miR-21 down regulated.3. silencing miR-21 could inhibit the proliferation, migration and inflammatory response of TNF- alpha induced fibroblasts and middle membrane smooth muscle cells, and.TNF- alpha stimulated the outer membrane fibroblasts and middle membrane smooth muscle cells, EDU and Transwell junction. The results showed that the proliferation and migration ability of the fibroblasts and middle membrane cells in the outer membrane of the miR-21 inhibitor group were weakened. The results of QRT-PCR showed that miR-21, MCP-1, VCAM-1, and ICAM-1 expressed in the fibroblasts / middle membranes of the outer membrane of the miR-21 inhibitor group and the.4.miR-21 could promote the fibroblasts of the outer membrane. The inflammatory response of cell / medium membrane smooth muscle cells.QRT-PCR results showed that the transfection of miR-21 mimic group, outer membrane fibroblast MCP-I, VCAM-1 expression up-regulated and the expression of MCP-1 in middle membrane smooth muscle cells up regulated. Conclusion 1. transplantation of vascular outer membrane macrophages plays an important role in the development of neointima by.2. macrophages passing through the paracrine TNF - alpha regulates the expression of miR-21 in vascular wall cells, and then regulates the functional changes of vascular wall cells and the expression of miR-21 in the inflammatory response.3. cell wall cells may further promote the remodeling of neointima by affecting the inflammatory response and its function changes in the transplanted blood vessels.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R543

【相似文献】