發(fā)布時(shí)間：2019-05-22 15:46

【摘要】：目的:研究巨噬細(xì)胞冠蛋白-1(Coronin-1)不同表達(dá)水平對(duì)i NOS、TLRs表達(dá)和凋亡的影響及其可能機(jī)制。方法:1.據(jù)Coronin-1表達(dá)水平的差異,將實(shí)驗(yàn)組細(xì)胞分為RAW264.7-Cor.Plus(過(guò)表達(dá))、RAW264.7(正常表達(dá))和RAW264.7-Cor.Minus(抑制表達(dá))三組。用Realtime PCR法檢測(cè)Coronin-1不同表達(dá)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(cè)細(xì)胞Toll樣受體(Toll-like receptors,TLRs),其中主要檢測(cè)TLR2、TLR4、TLR9的蛋白表達(dá)水平;流式細(xì)胞術(shù)檢測(cè)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(cè)Cs A處理前后Coronin-1不同表達(dá)水平巨噬細(xì)胞鈣調(diào)磷酸酶的蛋白質(zhì)表達(dá)。RAW264.7(正常表達(dá))組細(xì)胞經(jīng)2μmol/L肌動(dòng)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(duì)細(xì)胞染色,計(jì)算Coronin-1不同表達(dá)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(dòng)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(cè)i NOS m RNA水平;Western-blot法檢測(cè)細(xì)胞TLR2、TLR4、TLR9的蛋白質(zhì)水平;流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡百分率。結(jié)果:1.RAW264.7-Cor.Minus(抑制表達(dá))組細(xì)胞,其i NOS、TLR2、TLR4、TLR9的表達(dá)以及細(xì)胞凋亡率與RAW264.7(正常表達(dá))組細(xì)胞相比沒(méi)有顯著差異(P0.05)。RAW264.7-Cor.Plus(過(guò)表達(dá))組細(xì)胞與RAW264.7組細(xì)胞相比,i NOS、TLR2、TLR4、TLR9表達(dá)以及細(xì)胞凋亡率顯著降低(P0.05)。各項(xiàng)指標(biāo)趨勢(shì)為:RAW264.7RAW264.7-Cor.Plus(P0.05)。2.巨噬細(xì)胞鈣調(diào)磷酸酶表達(dá)水平與Coronin-1不同表達(dá)水平呈正相關(guān)。RAW264.7-Cor.Plus(過(guò)表達(dá))、RAW264.7(正常表達(dá))和RAW264.7-Cor.Minus(抑制表達(dá))三組細(xì)胞的鈣調(diào)磷酸酶表達(dá)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(dá)水平顯著低于未處理組(P0.05)。鈣調(diào)磷酸酶表達(dá)水平呈現(xiàn)趨勢(shì):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(dá)以及細(xì)胞凋亡率顯著高于未處理組(P0.05)。各項(xiàng)指標(biāo)趨勢(shì)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(dá)和細(xì)胞凋亡率并無(wú)顯著差異,但RAW264.7和RAW264.7-Cor.Plus組細(xì)胞TLR2、TLR4、TLR9的表達(dá)都被顯著抑制,這種效應(yīng)與Coronin-1的表達(dá)水平無(wú)關(guān)。結(jié)論:1.Coronin-1過(guò)表達(dá)抑制巨噬細(xì)胞i NOS、TLR2、TLR4、TLR9表達(dá)和細(xì)胞凋亡。2.Coronin-1過(guò)表達(dá)可促進(jìn)巨噬細(xì)胞Ca N蛋白表達(dá)和F-actin重排。Cs A可抑制巨噬細(xì)胞Ca N的表達(dá);cyt D對(duì)細(xì)胞F-actin重排具有顯著抑制效應(yīng),但與Coronin-1表達(dá)水平無(wú)關(guān)。3.Coronin-1可能通過(guò)激活鈣調(diào)磷酸酶信號(hào)通路,抑制巨噬細(xì)胞i NOS、TLRs表達(dá)和細(xì)胞凋亡,這一過(guò)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(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R392

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本文編號(hào)：2483044