miR-145介导阿司匹林抗血管平滑肌细胞增殖及抗炎作用的研究

发布时间：2019-05-31 20:01

【摘要】：研究背景：血管平滑肌细胞(vascular smooth muscle cells, VSMCs)的表型变化引起的VSMCs异常增殖是动脉粥样硬化(atherosclerosis,AS)、脑梗死等心脑血管疾病的重要病理基础,但其表型调节的机制仍未被完全阐释清楚。miR-145是一种与心血管疾病密切相关的microRNA,主要在VSMCs中表达,对VSMCs具有表型调节作用。CD40、CD40L在VSMCs中均有表达,且CD40-CD40L系统激活后具有促炎及促VSMCs增殖作用,在AS的发生发展过程中起重要作用。生物信息学软件分析CD40可能是miR-145重要的靶基因,miR-145可能通过对CD40的调节作用介导VSMCs的增殖及炎症因子生成。 阿司匹林是临床上用于治疗脑梗死的常用药物。文献报道阿司匹林具有抗血栓及抗炎作用,还可以抑制VSMCs增殖。但是阿司匹林抑制VSMCs增殖的机制仍不清楚。阿司匹林抗血栓及抗炎作用与调节机体CD40-CD40L系统的活性有关,但尚无阿司匹林是否能影响VSMCs中CD40的水平及其与miR-145关系的报道。 研究目的：1.探讨miR-145是否通过对CD40的调节介导VSMCs的增殖及炎症因子生成,为miR-145在心血管疾病中的研究提供参考；2.探讨miR-145是否通过对CD40的调节参与阿司匹林的抗VSMCs增殖及抗炎作用,为阐述阿司匹林的新作用机制提供理论依据。 实验方法：1.miR-145通过调节CD40介导VSMCs的增殖及炎症因子生成：培养人血管平滑肌细胞(human vascular smooth muscle cells,HAVSMCs),应用TNF-α建立细胞增殖模型,运用miR-145的模拟物转染细胞,分组为：(1)空白对照组(Control)、(2)造模组(TNF-α)、(3)转染miR-145模拟物对照造模组(miR-145mimic control+TNF-α)、(4)转染miR-145模拟物造模组(miR-145mimic+TNF-α)。EdU检测细胞增殖情况,Real-time PCR法检测miR-145、CD40、VSMC分化标志基因Calponin的mRNA表达,Western-blot法检测CD40的蛋白表达,ELISA检测细胞上清炎性因子IL-6的浓度；2.miR-145通过对CD40的调节参与阿司匹林的抗VSMCs增殖及抗炎作用：培养HAVSMCs,应用TNF-α建立细胞增殖模型,进行药物研究。分组为：(1)溶媒对照组(DMSO)、(2)造模组(TNF-α)、(3)转染miR-145抑制剂对照造模组(miR-145inhibitor control+TNF-α)、(4)阿司匹林预处理造模组(ASA+TNF-α)、(5)转染miR-145抑制剂及给药阿司匹林预处理造模组(miR-145inhibitor+ASA+TNF-α)。EdU检测细胞增殖情况,Real-time PCR法检测miR-145、CD40、VSMC分化标志基因Calponin的mRNA表达,Western-blot法检测CD40的蛋白表达,ELISA检测细胞上清炎性因子IL-6的浓度。 实验结果：1.miR-145通过调节CD40介导VSMCs的增殖及炎症因子生成：过表达miR-145能抑制TNF-α诱导的HAVSMCs增殖,逆转TNF-α引起的平滑肌细胞分化标志基因Calponin的下调及逆转TNF-α引起的CD40的升高,可以降低VSMCs激活后释放的IL-6水平。2.miR-145通过对CD40的调节参与阿司匹林的抗VSMCs增殖及抗炎作用：TNF-α引起VSMCs增殖,平滑肌细胞分化标志基因Calponin下调,miR-145水平下调及CD40水平的上调,并伴有IL-6水平显著升高；预先使用20μg/mL的阿司匹林处理VSMCs后可明显抑制TNF-a诱导的VSMCs增殖并升高Calponin的表达,同时阿司匹林还能够升高miR-145的水平,下调CD40及IL-6的水平 结论：1.miR-145通过抑制CD40的表达起抗VSMCs增殖及抗VSMCs炎症因子生成的作用；2.miR-145通过抑制HAVSMCs中CD40的表达参与阿司匹林的抗VSMCs增殖及抗炎作用。
[Abstract]:Background: The abnormal proliferation of VSMCs caused by the phenotypic change of vascular smooth muscle cells (VSMCs) is an important pathological basis for cardiovascular and cerebrovascular diseases such as atherosclerosis (AS) and cerebral infarction, but the mechanism of phenotypic regulation is not fully explained. MiR-145 is a microRNA which is closely related to cardiovascular diseases, is mainly expressed in VSMCs, and has a phenotypic regulation effect on VSMCs. CD40 and CD40L are expressed in VSMCs, and it plays an important role in the development of AS after activation of the CD40-CD40L system. The analysis of CD40 by bioinformatics software may be an important target gene for miR-145, and miR-145 may mediate the proliferation of VSMCs and the production of inflammatory factors through the regulation of CD40. Aspirin is a common drug used in the treatment of cerebral infarction It is reported that aspirin has antithrombotic and anti-inflammatory effects and can inhibit the proliferation of VSMCs. However, the mechanism of aspirin to inhibit the proliferation of VSMCs is still unclear. The anti-thrombotic and anti-inflammatory effects of aspirin are related to the regulation of the activity of CD40-CD40L system in the body, but no aspirin can affect the level of CD40 in the VSMCs and its relationship with the miR-145. A. The study 1. To investigate whether miR-145 is produced by regulating the proliferation and inflammatory factors of the VSMCs mediated by the regulation of CD40, and to provide reference for the study of miR-145 in the cardiovascular disease. 2. To investigate whether miR-145 has been involved in the anti-VSMCs proliferation and anti-inflammatory effects of aspirin on the regulation of CD40, and to provide a basis for elucidating the new mechanism of aspirin. On the basis of the experimental method,1. The expression of miR-145 by regulating the proliferation of the CD40-mediated VSMCs and the production of the inflammatory factors: the human vascular smooth muscle cells (HAVSMCs) were cultured, the cell proliferation model was established by using the TNF-145, the cells were transfected with the mimetic of the miR-145, and the group was: (1) the blank control group (Con (l), (2) Construction module (TNF-1), (3) Transfection of miR-145 mimetic control (miR-145 mic control + TNF-1), (4) Transfection of miR-145 mimetic (miR-145 mic + T) The expression of the expression of the expression of the expression of the expression of the expression of calcin in the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the expression of the VSMCs, the application of TNF-1 to establish a cell proliferation model, The group was: (1) Vehicle control group (DMSO), (2) Construction module (TNF-1), (3) Transfection of miR-145 inhibitor control building module (miR-145 in control + TNF-1), (4) Aspirin pre-treatment module (ASA + T NF-1, (5) Transfection of miR-145 inhibitor and administration of aspirin pre-treatment module (miR-145 in hibitor + ASA + T The expression of the expression of the expression of calcin in the expression of miR-145, CD40, and VSMC was detected by the real-time PCR, and the expression of the protein of CD40 was detected by Western-blot. -6. Experimental results:1. miR-145 is generated by regulating the proliferation and the inflammatory factors of the CD40-mediated VSMCs: the overexpression of the miR-145 can inhibit the proliferation of the TNF-1-induced HAVSMCs, reverse the down-regulation of the differentiation marker gene Calcin of the smooth muscle cells induced by the TNF-1 and the reversal of the TNF-1 induced by the TNF-145. The increase of CD40 can reduce the level of IL-6 released after activation of VSMCs.2. miR-145 is involved in the anti-VSMCs proliferation and anti-inflammatory effects of aspirin on the regulation of CD40: TNF-1 causes the proliferation of VSMCs, the down-regulation of the differentiation marker gene of smooth muscle cells, the down-regulation of miR-145 and the up-regulation of the level of CD40, and is accompanied by an IL- 6. The level of VSMCs induced by TNF-a was significantly inhibited after treatment with 20. m u.g/ mL of aspirin, and the expression of Calcin was increased, while aspirin was able to increase the level of miR-145 and down-regulate CD40. and IL -6. Horizontal conclusion:1. miR-145 acts to inhibit the proliferation of the anti-VSMCs and the anti-VSMCs inflammatory factors by inhibiting the expression of CD40;2. miR-145 inhibits the anti-VSM of aspirin by inhibiting the expression of CD40 in the HAVSMCs
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R969

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