Rock信号通路参与TNF-α刺激兔基底动脉平滑肌增殖的研究

发布时间：2018-03-24 20:17

本文选题：Rho激酶　切入点：血管平滑肌细胞　出处：《第三军医大学》2014年硕士论文

【摘要】：研究背景和目的：蛛网膜下腔出血(subarachnoid hemorrhage,SAH)是出血性卒中较为常见的疾病，除了发病前期的高病死率外，SAH后期较多因素引起的脑血管痉挛(cerebralvasospasm,CVS)的发生率也很高[1]。CVS引起的缺血性脑损害，成为SAH后期致死和致残的重要原因。因此预防和治疗CVS是临床上亟待解决的问题。迄今为止，已有比较多的学者对SAH后CVS的发生发展进行了研究，力图阐明其发生机制从而探索出有效的治疗方法，但至今其具体发生机制仍未能够完全阐明。但是随着相关研究的深入，，我们对于SAH后CVS发生发展机制的认识也逐渐加深。以往大多数研究是对于SAH后的致痉挛因子展开，并尝试应用相应致痉因子阻滞剂来防治CVS，但效果不佳。因此SAH后受累相关动脉血管平滑肌细胞(vascular smoothmuscle cells,VSMCs)增殖引发的管腔狭窄及管壁增厚等病理形态改变引起了相关学者专家的注意，并且成为了目前研究SAH后CVS发生的新热点。在SAH后CVS发生的脑血管壁增殖过程中，炎性因子与其关系密切。 Rock信号通路在生物体内发挥着广泛的作用。有研究表明Rock信号可能通过诱导血管平滑肌收缩而与CVS密切相关，临床上也使用一种Rock抑制剂法舒地尔治疗CVS。但目前对于SAH后Rock信号是否参与炎性因子引起的VSMCs异常增殖和CVS的发生鲜有报道。所以进一步阐明Rock信号通路在炎性因子引起的VSMCs增殖中的具体作用，对于明确SAH后CVS发生的具体机制，从而在临床上能够采取相应措施阻断其发生发展具有重要的意义。方法： 1.组织块法培养家兔VSMCs并鉴定； 2.将细胞进行分组：阴性对照组，TNF-α处理组，Y-27632干扰组，检测各组细胞的增殖及细胞周期； 3.应用免疫细胞化学及Western-blot检测各分组VSMCs中PCNA蛋白的表达及分布。结果： 1.成功利用组织块法原代培养出家兔基底动脉VSMCs，同时完成鉴定工作； 2. CCK-8法检测提示TNF-α显著促进VSMCs的增殖(P0.01)，Y-27632呈浓度依赖性的抑制TNF-α的促增殖作用(P0.01)； 3.流式结果显示：TNF-α刺激VSMCs后与对照组相比，处于G0/G1期细胞比例明显降低，S期+G2/M期细胞比例明显增高(P0.05)，Y-27632预处理后此种变化被逆转； 4.免疫细胞化学及Western-blot检测结果显示PCNA蛋白表达于细胞核内，TNF-α显著增加PCNA于细胞核内的表达(P0.01)，Y-27632具有显著的逆转TNF-α促PCNA表达的作用(P0.05)；结论： 1.应用组织贴块法可成功培养出家兔基底动脉VSMCs； 2. TNF-α可以通过加速细胞周期的进程来促进VSMCs的增殖，这一促增殖作用中Rock信号通路发挥了重要的作用； 3. Y-27632通过抑制Rock活性使得VSMCs阻滞于G0/G1期，从而发挥其逆转TNF-α的促进增殖以及促PCNA表达的作用。
[Abstract]:Background and objectives of the study:. Subarachnoid hemorrhagesia (SAH) is a common disease in hemorrhagic stroke. In addition to the high mortality in the early stage of SAH, the incidence of cerebral vasospasm caused by cerebral vasospasm (CVS) in the later stage of SAH is also very high [1] .CVS causes ischemic brain damage. It is an important cause of death and disability in the later stage of SAH, so the prevention and treatment of CVS is an urgent problem to be solved in clinic. Up to now, more and more scholars have studied the occurrence and development of CVS after SAH, trying to clarify its pathogenesis and explore effective treatment methods. However, with the deepening of relevant studies, our understanding of the pathogenesis of CVS after SAH has gradually deepened. Most of the previous studies have focused on spasmodic factors after SAH. We tried to use the corresponding spasmodic factor blocker to prevent and treat CVS, but the effect was not good. Therefore, the pathological changes such as lumen stenosis and wall thickening caused by the proliferation of vascular smooth muscle cells (VSMCs) and vascular smooth muscle cells (VSMCs) associated with SAH caused the correlation. The attention of scholars and experts, It has become a new focus in the study of the occurrence of CVS after SAH. Inflammatory factors are closely related to the proliferation of cerebrovascular wall in CVS after SAH. Rock signaling pathway plays a wide range of roles in organisms. Some studies have shown that Rock signal may be closely related to CVS by inducing contraction of vascular smooth muscle. A Rock inhibitor, fasudil, was also used in the treatment of SAH. However, there are few reports on whether the Rock signal after SAH is involved in the abnormal proliferation of VSMCs induced by inflammatory factors and the occurrence of CVS. Therefore, we further elucidate the role of Rock signal pathway in inflammation. The specific role of factors in VSMCs proliferation, It is of great significance to clarify the mechanism of occurrence of CVS after SAH and to take corresponding measures to block the occurrence and development of CVS in clinic. Methods:. 1. Rabbit VSMCs was cultured and identified by tissue mass method. 2. The cells were divided into two groups: negative control group treated with TNF- 伪 and Y-27632 interference group, cell proliferation and cell cycle were detected. 3. The expression and distribution of PCNA protein in VSMCs were detected by immunocytochemistry and Western-blot. Results:. 1. VSMCs of rabbit basilar artery were cultured successfully by tissue mass method, and the identification work was completed at the same time. 2. The detection of CCK-8 showed that TNF- 伪 significantly promoted the proliferation of VSMCs. P0.01TNF- 伪 inhibited the proliferation of TNF- 伪 in a concentration-dependent manner. 3. The results of flow cytometry showed that the proportion of cells in G0/G1 phase was significantly lower than that in control group after stimulation of VSMCs with TNF- 伪. The percentage of cells in G _ 2 / M phase in S phase was significantly increased after pretreatment with P0.05TNF- 伪, and the change was reversed after pretreatment. 4. The results of immunocytochemistry and Western-blot detection showed that the expression of PCNA protein in the nucleus of TNF- 伪 significantly increased the expression of PCNA in the nucleus. P0.01- Y-27632 significantly reversed the effect of TNF- 伪 on the expression of PCNA. Conclusion:. 1. VSMCs of rabbit basilar artery could be cultured successfully by using tissue patch method. 2. TNF- 伪 can accelerate the proliferation of VSMCs by accelerating the process of cell cycle. The Rock signaling pathway plays an important role in the proliferation; 3. Y-27632 blocked VSMCs at G0/G1 stage by inhibiting the activity of Rock, thus reversing the proliferation of TNF- 伪 and promoting the expression of PCNA.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.35

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