【摘要】：目的:研究巨噬細(xì)胞冠蛋白-1(Coronin-1)不同表達水平對i NOS、TLRs表達和凋亡的影響及其可能機制。方法:1.據(jù)Coronin-1表達水平的差異,將實驗組細(xì)胞分為RAW264.7-Cor.Plus(過表達)、RAW264.7(正常表達)和RAW264.7-Cor.Minus(抑制表達)三組。用Realtime PCR法檢測Coronin-1不同表達水平巨噬細(xì)胞誘導(dǎo)性一氧化氮合酶(inducible nitric oxide synthase,i NOS)m RNA水平;Western-blot法檢測細(xì)胞Toll樣受體(Toll-like receptors,TLRs),其中主要檢測TLR2、TLR4、TLR9的蛋白表達水平;流式細(xì)胞術(shù)檢測各組細(xì)胞凋亡百分率。2.各組細(xì)胞經(jīng)4μmol/L的鈣調(diào)磷酸酶抑制劑環(huán)孢素A(cyclosporin A,Cs A)處理24h,用Western-blot法檢測Cs A處理前后Coronin-1不同表達水平巨噬細(xì)胞鈣調(diào)磷酸酶的蛋白質(zhì)表達。RAW264.7(正常表達)組細(xì)胞經(jīng)2μmol/L肌動蛋白聚合抑制劑細(xì)胞松弛素D(cytochalasin D,cyt D)處理48h后,用四甲基異硫氰酸熒光素標(biāo)記的鬼筆環(huán)肽(TRITC-phalloidin)對細(xì)胞染色,計算Coronin-1不同表達水平巨噬細(xì)胞以及cyt D處理前后RAW264.7細(xì)胞纖維型肌動蛋白(F-actin)重排率。3.各組細(xì)胞經(jīng)4μmol/L環(huán)孢素A(Cs A)和2μmol/L細(xì)胞松弛素D(cyt D)分別處理24h和48h。用Realtime PCR法檢測i NOS m RNA水平;Western-blot法檢測細(xì)胞TLR2、TLR4、TLR9的蛋白質(zhì)水平;流式細(xì)胞術(shù)檢測細(xì)胞凋亡百分率。結(jié)果:1.RAW264.7-Cor.Minus(抑制表達)組細(xì)胞,其i NOS、TLR2、TLR4、TLR9的表達以及細(xì)胞凋亡率與RAW264.7(正常表達)組細(xì)胞相比沒有顯著差異(P0.05)。RAW264.7-Cor.Plus(過表達)組細(xì)胞與RAW264.7組細(xì)胞相比,i NOS、TLR2、TLR4、TLR9表達以及細(xì)胞凋亡率顯著降低(P0.05)。各項指標(biāo)趨勢為:RAW264.7RAW264.7-Cor.Plus(P0.05)。2.巨噬細(xì)胞鈣調(diào)磷酸酶表達水平與Coronin-1不同表達水平呈正相關(guān)。RAW264.7-Cor.Plus(過表達)、RAW264.7(正常表達)和RAW264.7-Cor.Minus(抑制表達)三組細(xì)胞的鈣調(diào)磷酸酶表達水平依次降低。經(jīng)Cs A處理后,RAW264.7和RAW264.7-Cor.Plus組細(xì)胞鈣調(diào)磷酸酶表達水平顯著低于未處理組(P0.05)。鈣調(diào)磷酸酶表達水平呈現(xiàn)趨勢:RAW264.7-Cor.PlusRAW264.7RAW264.7-Cor.Minus;RAW264.7RAW264.7+Cs A;RAW264.7-Cor.PlusRAW264.7-Cor.Plus+Cs A(P0.05)。經(jīng)TRITC-phalloidin染色后,各組細(xì)胞F-actin重排率從高至低依次為RAW264.7-Cor.Plus、RAW264.7、RAW264.7-Cor.Minus(P0.05)。cyt D處理組細(xì)胞,其F-actin重排率顯著低于未處理組(P0.05)。3.RAW264.7-Cor.Plus組細(xì)胞經(jīng)Cs A處理后,其i NOS、TLR2、TLR4、TLR9表達以及細(xì)胞凋亡率顯著高于未處理組(P0.05)。各項指標(biāo)趨勢為:RAW264.7-Cor.Plus+Cs ARAW264.7-Cor.Plus(P0.05)。cyt D處理前后,RAW264.7-Cor.Plus組細(xì)胞i NOS m RNA表達和細(xì)胞凋亡率并無顯著差異,但RAW264.7和RAW264.7-Cor.Plus組細(xì)胞TLR2、TLR4、TLR9的表達都被顯著抑制,這種效應(yīng)與Coronin-1的表達水平無關(guān)。結(jié)論:1.Coronin-1過表達抑制巨噬細(xì)胞i NOS、TLR2、TLR4、TLR9表達和細(xì)胞凋亡。2.Coronin-1過表達可促進巨噬細(xì)胞Ca N蛋白表達和F-actin重排。Cs A可抑制巨噬細(xì)胞Ca N的表達;cyt D對細(xì)胞F-actin重排具有顯著抑制效應(yīng),但與Coronin-1表達水平無關(guān)。3.Coronin-1可能通過激活鈣調(diào)磷酸酶信號通路,抑制巨噬細(xì)胞i NOS、TLRs表達和細(xì)胞凋亡,這一過程并不依賴于F-actin調(diào)節(jié)。
[Abstract]:Objective: To study the effect of different expression levels of macrophage-crown-1 (Coronin-1) on the expression and apoptosis of i-NOS and TLRs and its possible mechanism. Method:1. The cells of the experimental group were divided into three groups: RAW264.7-Cor. Plus (overexpressing), RAW264.7 (normal expression) and RAW264.7-Cor. Minus (suppressed expression) according to the difference of the expression level of Coronin-1. The expression levels of Toll-like receptors (TLRs) were detected by Western-blot, and the expression of TLR2, TLR4 and TLR9 was detected by Western-blot. The percentage of apoptosis in each group was detected by flow cytometry. Cyclosporin A (Cyclosporin A, Cs A) was treated with 4. m u.mol/ L of calcineurin inhibitor, Cyclosporin A (Cs A) for 24 h, and the protein expression of calcium-regulated phosphatase was detected by Western-blot. RAW264.7 (normal expression) group cells were treated with a 2. m u.mol/ L actin polymerization inhibitor cell relaxin D (cytchalkasin D, cyt D) for 48 h, and the cells were stained with a tetramethyl isothiocyanate-labeled ghost cyclopeptide (TRITC-phalloidin), The cell-type actin (F-actin) rearrangement rate of RAW264.7 cells before and after the treatment of Coronin-1 different expression levels of macrophages and cyt D was calculated. The cells of each group were treated with 4. m u.mol/ L of cyclosporin A (Cs A) and 2. m u.mol/ L of cell relaxin D (cyt D) for 24 h and 48 h, respectively. The protein levels of TLR2, TLR4 and TLR9 were detected by the real time PCR, and the percentage of cell apoptosis was detected by flow cytometry. Results:1. The expression of i-NOS, TLR2, TLR4, TLR9 and the apoptosis rate of RAW264.7-Cor. Minus (suppressed expression) group were not significantly different from those in the RAW264.7 (normal expression) group (P0.05). The expression of TLR9 and the cell apoptosis rate were significantly lower (P0.05). The trend of the indicators is: RAW264.7 RAW264.7-Cor. Plus (P0.05). The level of the expression of calcineurin in macrophages was positively correlated with that of Coronin-1. RAW264.7-Cor. Plus (over-expression), RAW264.7 (normal expression) and RAW264.7-Cor. Minus (inhibition of expression) of the three groups of cells decreased in turn. The expression of calcineurin in RAW264.7 and RAW264.7-Cor. Plus group was significantly lower than that of untreated group (P0.05). The expression level of calcineurin: RAW264.7-Cor. PlusRAW264.7 RAW264.7-Cor. Minus; RAW264.7 RAW264.7 + Cs A; RAW264.7-Cor. PlusRAW264.7-Cor. Plus + Cs A (P0.05). After TITC-phaloidin staining, the rate of F-actin rearrangement in each group was RAW264.7-Cor. Plus, RAW264.7, RAW264.7-Cor. Minus (P0.05). The trend of various indexes was: RAW264.7-Cor. Plus + Cs ARAW264.7-Cor. Plus (P0.05). Before and after the treatment of cyt D, the expression of NOS mRNA and the cell apoptosis rate of RAW264.7-Cor. Plus group were not significantly different, but the expression of the cells TLR2, TLR4 and TLR9 in RAW264.7 and RAW264.7-Cor. Plus was significantly inhibited, which was not related to the level of expression of Coronin-1. Conclusion:1. The overexpression of Coronin-1 inhibits the expression of iNOS, TLR2, TLR4, TLR9 and apoptosis of macrophages. Cs A can inhibit the expression of calcium N in macrophages, and cyt D has a significant inhibitory effect on cell F-actin rearrangement, but not related to the level of Coronin-1.3. Coronin-1 may inhibit the expression of iNOS, TLRs and apoptosis of macrophages i by activating the signaling pathway of calcineurin, which is not dependent on the regulation of F-actin.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R392

【參考文獻】

相關(guān)期刊論文 前1條

1 周芳妮;陳全;周靜瑤;劉革力;張路渝;;冠蛋白1參與分枝菌酸誘導(dǎo)巨噬細(xì)胞的泡沫化[J];細(xì)胞與分子免疫學(xué)雜志;2016年04期

，

本文編號：2483044