【摘要】：研究背景:慢性心力衰竭是多種心血管疾病的終末階段。國內(nèi)外研究者普遍認(rèn)為心室重構(gòu)是慢性心力衰竭的主要發(fā)病機(jī)制。血管緊張素II(AngII)可通過激活神經(jīng)內(nèi)分泌系統(tǒng)而導(dǎo)致心室重塑的發(fā)生。但越來越多的證據(jù)表明,AngII還可通過激活白介素-6(IL-6)、轉(zhuǎn)化生長因子-β(TNF-β)等炎癥細(xì)胞因子導(dǎo)致心室重塑的發(fā)生。NLRP3炎性小體及其下游產(chǎn)物參與了各種病因所導(dǎo)致的心肌炎癥,進(jìn)而可導(dǎo)致心室重塑。但是目前關(guān)于AngII能否直接通過NLRP3炎性小體誘導(dǎo)心肌細(xì)胞發(fā)生炎癥的報道較少,本研究擬對此作一些探討。目的:探討血管緊張素II對H9C2細(xì)胞(大鼠心肌細(xì)胞)中NLRP3的作用。方法:培養(yǎng)H9C2細(xì)胞株,選用對數(shù)生長期的H9C2細(xì)胞進(jìn)行試驗,用10-6mol/l的AngII予以刺激,于不同的時間點(0小時(0h)、1小時(1h)、3小時(3h)、6小時(6h)、12小時(12h))收集細(xì)胞,通過實時熒光定量PCR(RT-qPCR)及蛋白質(zhì)印跡法(Western blot)分別檢測細(xì)胞中NLRP3、ASC、Caspase-1、IL-1β的基因和蛋白水平;通過酶聯(lián)免疫吸附法(ELISA)檢測細(xì)胞培養(yǎng)液上清液中IL-1β的含量。結(jié)果:1.AngII分別刺激H9C2細(xì)胞0h、1h、3h、6h及12h后,NLRP3、ASC、Caspase-1及IL-1β的基因相對表達(dá)量在各處理組與0h組相比,除1h組的Caspase-1差異無統(tǒng)計學(xué)意義外(P0.05),余各處理組差異均有統(tǒng)計學(xué)意義(P0.05)。1h組、3h組、6h組、12h組的基因相對表達(dá)量進(jìn)行兩兩比較,除1h組與3h組之間NLRP3的基因相對表達(dá)量差異無統(tǒng)計學(xué)意義(P0.05)外,余各組間差異均有統(tǒng)計學(xué)意義(P0.05)。2.AngII分別刺激H9C2細(xì)胞0h、1h、3h、6h及12h后,NLRP3、ASC、Caspase-1及IL-1β的蛋白相對表達(dá)量在各處理組與0h組相比,差異有統(tǒng)計學(xué)意義(P0.05),且1h組、3h組、6h組及12h組間差異有統(tǒng)計學(xué)意義(P0.05)。3.AngII分別刺激H9C2細(xì)胞0h、1h、3h、6h及12h后,細(xì)胞培養(yǎng)液上清液中IL-1β的含量在各處理組與0h組相比,差異有統(tǒng)計學(xué)意義(P0.05),且1h組、3h組、6h組及12h組間差異有統(tǒng)計學(xué)意義(P0.05)。結(jié)論:1.經(jīng)濃度為10-6mol/l的血管緊張素II刺激后,H9C2細(xì)胞生長狀態(tài)良好,細(xì)胞形態(tài)未見明顯異常。2.濃度為10-6mol/l的血管緊張素II可促使H9C2細(xì)胞中NLRP3、ASC、Caspase-1和IL-1β的基因及蛋白表達(dá)水平增加,同時也可導(dǎo)致細(xì)胞培養(yǎng)液上清液中IL-1β的含量增加。3.AngII可通過直接激活NLRP3炎性小體而導(dǎo)致心肌細(xì)胞炎癥的發(fā)生,這一發(fā)病機(jī)制可能與AngII激活心力衰竭相關(guān),為臨床上藥物治療心肌炎癥所致心功能不全及心肌重構(gòu)提供理論依據(jù)。
[Abstract]:Background: chronic heart failure is the terminal stage of many cardiovascular diseases. Researchers at home and abroad generally believe that ventricular remodeling is the main pathogenesis of chronic heart failure. Angiotensin II (AngII) can induce ventricular remodeling by activating the neuroendocrine system. But there is growing evidence that AngII can also cause ventricular remodeling by activating inflammatory cytokines such as interleukin-6 (IL-6) and transforming growth factor- 尾 (TNF- 尾). NLRP3 inflammatory bodies and their downstream products are involved in various etiological causes of myocarditis. In turn, it can lead to ventricular remodeling. However, there are few reports on whether AngII can induce cardiomyocyte inflammation directly through inflammatory corpuscles of NLRP3. Aim: to investigate the effect of angiotensin II on NLRP3 in H9C2 cells (rat cardiomyocytes). Methods: H9C2 cell lines were cultured. H9C2 cells in logarithmic growth phase were tested and stimulated with AngII of 10-6mol/l. The cells were collected at different time points (0 h (0 h), 1 hour (1 h), 3 h (3 h), 6 h (6 h), 12 h (12 h). The gene and protein levels of NLRP3,ASC,Caspase-1,IL-1 尾 were detected by real-time fluorescence quantitative PCR (RT-qPCR) and Western blot (Western blot), and the content of IL-1 尾 in the supernatant of cell culture medium was detected by enzyme-linked immunosorbent assay (ELISA). Results: the relative expression of NLRP3,ASC,Caspase-1 and IL-1 尾 genes in H9C2 cells stimulated by 1.AngII for 6 h and 12 h were compared with those in 0 h group. There was no significant difference in Caspase-1 between 1h group and 6h group (P0.05), but there were significant differences among the other treatment groups (P0.05). The relative gene expression levels of 1h group, 3h group, 6h group and 12h group were compared. There was no significant difference in the relative expression of NLRP3 gene between 1h group and 3h group (P0.05), but there was significant difference between the other groups (P0.05). The relative expression of NLRP3,ASC,Caspase-1 and IL-1 尾 protein in the treatment group was higher than that in the 0h group after 2.AngII stimulation for 6 h and 12 h, respectively. The difference was statistically significant (P0.05), and there was significant difference between 1h group, 3h group, 6h group and 12h group (P0.05). After 3.AngII stimulated H9C2 cells for 6 h and 12 h, the content of IL-1 尾 in supernatant of cell culture medium was higher than that of 0 h group. The difference was statistically significant (P0.05), and there was significant difference between 1h group, 3h group, 6h group and 12h group (P0.05). Conclusion: 1. After stimulation of angiotensin II with concentration of 10-6mol/l, H9C2 cells grew well and the morphology of H9C2 cells was not abnormal. 2. Angiotensin II at concentration of 10-6mol/l increased the gene and protein expression of NLRP3,ASC,Caspase-1 and IL-1 尾 in H9C2 cells. It can also increase the content of IL-1 尾 in the supernatant of cell culture medium. 3.AngII can directly activate the inflammatory body of NLRP3 and induce the inflammation of cardiomyocytes, which may be related to the activation of heart failure by AngII. To provide a theoretical basis for the clinical treatment of cardiac insufficiency and myocardial remodeling caused by myocardial inflammation.
【學(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R541.6

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