巨噬细胞Coronin-1不同表达水平对iNOS、TLRs表达以及凋亡的影响
发布时间：2019-05-22 15:46
【摘要】：目的:研究巨噬细胞冠蛋白-1(Coronin-1)不同表达水平对i NOS、TLRs表达和凋亡的影响及其可能机制。方法:1.据Coronin-1表达水平的差异,将实验组细胞分为RAW264.7-Cor.Plus(过表达)、RAW264.7(正常表达)和RAW264.7-Cor.Minus(抑制表达)三组。用Realtime PCR法检测Coronin-1不同表达水平巨噬细胞诱导性一氧化氮合酶(inducible nitric oxide synthase,i NOS)m RNA水平;Western-blot法检测细胞Toll样受体(Toll-like receptors,TLRs),其中主要检测TLR2、TLR4、TLR9的蛋白表达水平;流式细胞术检测各组细胞凋亡百分率。2.各组细胞经4μmol/L的钙调磷酸酶抑制剂环孢素A(cyclosporin A,Cs A)处理24h,用Western-blot法检测Cs A处理前后Coronin-1不同表达水平巨噬细胞钙调磷酸酶的蛋白质表达。RAW264.7(正常表达)组细胞经2μmol/L肌动蛋白聚合抑制剂细胞松弛素D(cytochalasin D,cyt D)处理48h后,用四甲基异硫氰酸荧光素标记的鬼笔环肽(TRITC-phalloidin)对细胞染色,计算Coronin-1不同表达水平巨噬细胞以及cyt D处理前后RAW264.7细胞纤维型肌动蛋白(F-actin)重排率。3.各组细胞经4μmol/L环孢素A(Cs A)和2μmol/L细胞松弛素D(cyt D)分别处理24h和48h。用Realtime PCR法检测i NOS m RNA水平;Western-blot法检测细胞TLR2、TLR4、TLR9的蛋白质水平;流式细胞术检测细胞凋亡百分率。结果:1.RAW264.7-Cor.Minus(抑制表达)组细胞,其i NOS、TLR2、TLR4、TLR9的表达以及细胞凋亡率与RAW264.7(正常表达)组细胞相比没有显著差异(P0.05)。RAW264.7-Cor.Plus(过表达)组细胞与RAW264.7组细胞相比,i NOS、TLR2、TLR4、TLR9表达以及细胞凋亡率显著降低(P0.05)。各项指标趋势为:RAW264.7RAW264.7-Cor.Plus(P0.05)。2.巨噬细胞钙调磷酸酶表达水平与Coronin-1不同表达水平呈正相关。RAW264.7-Cor.Plus(过表达)、RAW264.7(正常表达)和RAW264.7-Cor.Minus(抑制表达)三组细胞的钙调磷酸酶表达水平依次降低。经Cs A处理后,RAW264.7和RAW264.7-Cor.Plus组细胞钙调磷酸酶表达水平显著低于未处理组(P0.05)。钙调磷酸酶表达水平呈现趋势:RAW264.7-Cor.PlusRAW264.7RAW264.7-Cor.Minus;RAW264.7RAW264.7+Cs A;RAW264.7-Cor.PlusRAW264.7-Cor.Plus+Cs A(P0.05)。经TRITC-phalloidin染色后,各组细胞F-actin重排率从高至低依次为RAW264.7-Cor.Plus、RAW264.7、RAW264.7-Cor.Minus(P0.05)。cyt D处理组细胞,其F-actin重排率显著低于未处理组(P0.05)。3.RAW264.7-Cor.Plus组细胞经Cs A处理后,其i NOS、TLR2、TLR4、TLR9表达以及细胞凋亡率显著高于未处理组(P0.05)。各项指标趋势为:RAW264.7-Cor.Plus+Cs ARAW264.7-Cor.Plus(P0.05)。cyt D处理前后,RAW264.7-Cor.Plus组细胞i NOS m RNA表达和细胞凋亡率并无显著差异,但RAW264.7和RAW264.7-Cor.Plus组细胞TLR2、TLR4、TLR9的表达都被显著抑制,这种效应与Coronin-1的表达水平无关。结论:1.Coronin-1过表达抑制巨噬细胞i NOS、TLR2、TLR4、TLR9表达和细胞凋亡。2.Coronin-1过表达可促进巨噬细胞Ca N蛋白表达和F-actin重排。Cs A可抑制巨噬细胞Ca N的表达;cyt D对细胞F-actin重排具有显著抑制效应,但与Coronin-1表达水平无关。3.Coronin-1可能通过激活钙调磷酸酶信号通路,抑制巨噬细胞i NOS、TLRs表达和细胞凋亡,这一过程并不依赖于F-actin调节。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392
本文编号：2483044
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392
【参考文献】
相关期刊论文 前1条
1 周芳妮;陈全;周静瑶;刘革力;张路渝;;冠蛋白1参与分枝菌酸诱导巨噬细胞的泡沫化[J];细胞与分子免疫学杂志;2016年04期
，本文编号：2483044
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