本文選題：Rho激酶　切入點：血管平滑肌細(xì)胞　出處：《第三軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：研究背景和目的： 蛛網(wǎng)膜下腔出血(subarachnoid hemorrhage,SAH)是出血性卒中較為常見的疾病，除了發(fā)病前期的高病死率外，SAH后期較多因素引起的腦血管痙攣(cerebralvasospasm,CVS)的發(fā)生率也很高[1]。CVS引起的缺血性腦損害，成為SAH后期致死和致殘的重要原因。因此預(yù)防和治療CVS是臨床上亟待解決的問題。 迄今為止，已有比較多的學(xué)者對SAH后CVS的發(fā)生發(fā)展進(jìn)行了研究，力圖闡明其發(fā)生機制從而探索出有效的治療方法，但至今其具體發(fā)生機制仍未能夠完全闡明。但是隨著相關(guān)研究的深入，，我們對于SAH后CVS發(fā)生發(fā)展機制的認(rèn)識也逐漸加深。以往大多數(shù)研究是對于SAH后的致痙攣因子展開，并嘗試應(yīng)用相應(yīng)致痙因子阻滯劑來防治CVS，但效果不佳。因此SAH后受累相關(guān)動脈血管平滑肌細(xì)胞(vascular smoothmuscle cells,VSMCs)增殖引發(fā)的管腔狹窄及管壁增厚等病理形態(tài)改變引起了相關(guān)學(xué)者專家的注意，并且成為了目前研究SAH后CVS發(fā)生的新熱點。在SAH后CVS發(fā)生的腦血管壁增殖過程中，炎性因子與其關(guān)系密切。 Rock信號通路在生物體內(nèi)發(fā)揮著廣泛的作用。有研究表明Rock信號可能通過誘導(dǎo)血管平滑肌收縮而與CVS密切相關(guān)，臨床上也使用一種Rock抑制劑法舒地爾治療CVS。但目前對于SAH后Rock信號是否參與炎性因子引起的VSMCs異常增殖和CVS的發(fā)生鮮有報道。所以進(jìn)一步闡明Rock信號通路在炎性因子引起的VSMCs增殖中的具體作用，對于明確SAH后CVS發(fā)生的具體機制，從而在臨床上能夠采取相應(yīng)措施阻斷其發(fā)生發(fā)展具有重要的意義。 方法： 1.組織塊法培養(yǎng)家兔VSMCs并鑒定； 2.將細(xì)胞進(jìn)行分組：陰性對照組，TNF-α處理組，Y-27632干擾組，檢測各組細(xì)胞的增殖及細(xì)胞周期； 3.應(yīng)用免疫細(xì)胞化學(xué)及Western-blot檢測各分組VSMCs中PCNA蛋白的表達(dá)及分布。 結(jié)果： 1.成功利用組織塊法原代培養(yǎng)出家兔基底動脈VSMCs，同時完成鑒定工作； 2. CCK-8法檢測提示TNF-α顯著促進(jìn)VSMCs的增殖(P0.01)，Y-27632呈濃度依賴性的抑制TNF-α的促增殖作用(P0.01)； 3.流式結(jié)果顯示：TNF-α刺激VSMCs后與對照組相比，處于G0/G1期細(xì)胞比例明顯降低，S期+G2/M期細(xì)胞比例明顯增高(P0.05)，Y-27632預(yù)處理后此種變化被逆轉(zhuǎn)； 4.免疫細(xì)胞化學(xué)及Western-blot檢測結(jié)果顯示PCNA蛋白表達(dá)于細(xì)胞核內(nèi)，TNF-α顯著增加PCNA于細(xì)胞核內(nèi)的表達(dá)(P0.01)，Y-27632具有顯著的逆轉(zhuǎn)TNF-α促PCNA表達(dá)的作用(P0.05)； 結(jié)論： 1.應(yīng)用組織貼塊法可成功培養(yǎng)出家兔基底動脈VSMCs； 2. TNF-α可以通過加速細(xì)胞周期的進(jìn)程來促進(jìn)VSMCs的增殖，這一促增殖作用中Rock信號通路發(fā)揮了重要的作用； 3. Y-27632通過抑制Rock活性使得VSMCs阻滯于G0/G1期，從而發(fā)揮其逆轉(zhuǎn)TNF-α的促進(jìn)增殖以及促PCNA表達(dá)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- 偽 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- 偽 significantly promoted the proliferation of VSMCs. P0.01TNF- 偽 inhibited the proliferation of TNF- 偽 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- 偽. The percentage of cells in G _ 2 / M phase in S phase was significantly increased after pretreatment with P0.05TNF- 偽, 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- 偽 significantly increased the expression of PCNA in the nucleus. P0.01- Y-27632 significantly reversed the effect of TNF- 偽 on the expression of PCNA. Conclusion:. 1. VSMCs of rabbit basilar artery could be cultured successfully by using tissue patch method. 2. TNF- 偽 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- 偽 and promoting the expression of PCNA.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743.35

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相關(guān)期刊論文 前1條

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