远隔预处理对大鼠脑缺血后血管生成及其相关因子表达影响的研究

发布时间：2018-06-04 10:22

本文选题：远隔预处理 + 血管生成　；参考：《复旦大学》2014年硕士论文

【摘要】：背景与目的：远隔预处理(Remote ischemic preconditioning, RPC)是继缺血预处理(IPC)后发现的一种在不同器官之间诱导缺血耐受的方法。它通过预先给予远隔部位的器官或组织亚致死性的缺血刺激,来诱导另一器官或组织对致死性缺血损伤的耐受力。远隔预处理的器官保护作用自发现以来,备受神经学界的关注。目前,动物实验以及临床实验均已证实远隔预处理是一种有效且可行的脑保护诱导方式。但是远隔预处理的分子生物学机制仍不明确。血管生成是从先存血管产生新生血管的过程,它受血管生成因子和血管生成抑制因了的精细调节,是机体一项重要的修复再生程序,也是脑缺血后神经功能修复的主要途径之一。本实验的目的在于研究远隔预处理对局部脑缺血后缺血半暗带区血管生成过程以及相关因子表达模式的影响,进一步揭示远隔预处理介导脑保护的分子机制。方法：雄性SD大鼠(鼠龄11-12周,体重270g-330g),随机分为远隔预处理(RPC)组、缺血对照(ISC)组和假手术(SHO)组。脑缺血模型采用远端大脑中动脉闭塞模型。远隔预处理以同侧股动脉夹闭15分钟、复流15分钟,循环三次的方式诱导。采用免疫组化CD31染色法计数RPC组、ISC组、SHO组缺血后1天、2天、3天、7天、14天缺血半暗带区的微血管密度。免疫荧光CD31/BrdU双标计数各小组缺血后2天、7天缺血半暗带区新生血管数目。免疫组化法半定量计算各实验小组缺血半暗带区膜受体VEGFR-2、Tie-2的表达水平,Western Blotting法半定量计算血管生成因子VEGF-A、Ang-1、Ang-2、HIF-lalpha的表达水平。图片分析采用Image Proplus、Image J软件,统计分析采用SPSS19.0软件,p0.05显示差异有统计学意义。结果：RPC组缺血半暗带区微血管密度在缺血后1天、2天、7天、14天均高于ISC对照组,两组差异有统计学意义,RPC组与ISC组半暗带区微血管密度均于缺血后7天达峰。RPC组半暗带区VEGFR-2蛋白表达水平亦高于ISC组,两组差异在缺血后1天、2天、3天、7天有统计学意义,且RPC与ISC组VEGFR-2表达量也在缺血后7天达峰,其时间演变规律与微血管密度相仿。缺血后半暗带区Tie-2的上调在RPC组更为明显,两组蛋白水平差异在缺血后2天、3天、7天、14天有统计学意义,且RPC组蛋白表达上调较ISC组出现更早且持续时间更长。在缺血后2天、7天时RPC组VEGF-A、Ang-2蛋白表达量均高于ISC组,与之相反,Ang-1的表达量在缺血2天时RPC组低于ISC组,7天时两组无差异。RPC组与ISC组缺血后2天、7天半暗带区HIF-lalpha蛋白的表达量无差异,但在缺血早期(缺血后6小时内),RPC组HIF-lalpha表达的上升较ISC组略滞后但明显增强。结论：远隔预处理显著增强了缺血半暗带区血管生成过程,并改变了半暗带区血管生成因子的表达模式。这很可能是远隔预处理介导脑保护的主要的分子生物学机制
[Abstract]:Background & AIM: remote ischemic preconditioning, RPC) is a method to induce ischemic tolerance between different organs after ischemic preconditioning. It induces the tolerance of another organ or tissue to fatal ischemic injury by giving sublethal ischemic stimulation to the distant organ or tissue in advance. The role of remote preconditioning in organ protection has been concerned by neuroscientists since it was discovered. At present, animal experiments and clinical trials have proved that remote preconditioning is an effective and feasible way to induce brain protection. However, the molecular biological mechanism of remote preconditioning is still unclear. Angiogenesis is a process of angiogenesis from pre-existing blood vessels, which is carefully regulated by angiogenic factors and angiogenesis inhibition, and is an important procedure for the body to repair and regenerate. It is also one of the main ways to repair nerve function after cerebral ischemia. The purpose of this study was to investigate the effects of remote preconditioning on angiogenesis and expression patterns of related factors in ischemic penumbra after local cerebral ischemia, and to further reveal the molecular mechanism of remote preconditioning mediated brain protection. Methods: male Sprague-Dawley rats (11-12 weeks old, weight 270g-330g) were randomly divided into remote preconditioning (RPC) group, ischemic control group (ISCC) and sham operation group. The middle cerebral artery occlusion model was used in cerebral ischemia model. Distal preconditioning was induced by ipsilateral femoral artery occlusion for 15 minutes, reflow for 15 minutes and circulation for three times. The microvessel density of ischemic penumbra in RPC group was counted by immunohistochemical CD31 staining method. The number of neovascularization in ischemic penumbra was counted by double labeling immunofluorescence CD31/BrdU 2 days after ischemia and 7 days after ischemia. The expression level of VEGFR-2Tie-2 in ischemic penumbra was semi-quantitatively calculated by immunohistochemical method and the expression level of angiogenic factor VEGFR-2HIF-lalpha was semi-quantitatively calculated by Western Blotting method. Image Proplusus Image J software was used for image analysis, and SPSS19.0 software for statistical analysis showed significant difference. Results the microvessel density of the ischemic penumbra in the control group was significantly higher than that in the control group on the 1st day, 2nd day, 7th day and 14th day after ischemia. There was significant difference between the two groups. The microvessel density of penumbra in RPC group and ISC group was significantly higher than that in ISC group 7 days after ischemia. The difference between the two groups was statistically significant at 1 day, 2 days, 3 days and 7 days after ischemia, and the expression level of VEGFR-2 protein in the penumbra region of the two groups was significantly higher than that in the ISC group on the 7th day after ischemia, and the difference between the two groups was statistically significant. The expression of VEGFR-2 in RPC and ISC groups reached the peak 7 days after ischemia, and its temporal evolution was similar to that of microvessel density. The upregulation of Tie-2 in the posterior penumbra of ischemia was more obvious in the RPC group. The difference of protein level between the two groups was statistically significant 2 days after ischemia and 7 days and 14 days after ischemia, and the up-regulation of RPC protein expression appeared earlier and lasted longer than that in the ISC group. The expression of VEGF-AfAng-2 protein in RPC group was higher than that in ISC group at 7 days after ischemia. On the contrary, the expression of Ang-1 in the RPC group was lower than that in the ISC group on the 7th day after ischemia. There was no difference in the expression of HIF-lalpha protein in the penumbra between the RPC group and the ISC group on the 7th day after ischemia. However, in the early stage of ischemia (within 6 hours after ischemia), the increase of HIF-lalpha expression in ISC group was slightly slower than that in ISC group. Conclusion: the process of angiogenesis in ischemic penumbra was significantly enhanced by remote preconditioning and the expression pattern of angiogenic factors in penumbra was changed. This may be the main molecular biological mechanism of remote preconditioning mediating brain protection.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3

【共引文献】