【摘要】：目的:本研究進(jìn)一步探討NADPH氧化酶NOX4介導(dǎo)的氧化應(yīng)激對高糖刺激足細(xì)胞損傷及產(chǎn)生微囊泡過程中的作用。方法:體外培養(yǎng)MPC-5細(xì)胞,高糖組處理因素為30mM葡萄糖。1.采用RT-qPCR、Western Blot技術(shù)分別檢測高糖刺激MPC-5細(xì)胞2小時、4小時、6小時、12小時、24小時后NOX4 mRNA、蛋白的水平,確定高峰時間點(diǎn)。2.對MPC-5細(xì)胞進(jìn)行FAM-si RNA轉(zhuǎn)染,濃度范圍為0-150nM,熒光顯微鏡觀察熒光,確定最佳轉(zhuǎn)染濃度。3.以最佳濃度對MPC-5細(xì)胞進(jìn)行siRNA轉(zhuǎn)染,采用RT-qPCR技術(shù)篩選轉(zhuǎn)染率最高的siRNA序列。4.將MPC-5細(xì)胞分為6組,分別為正常組、NOX4 siRNA組、陰性siRNA組、高糖組、高糖+NOX4 siRNA組、高糖+陰性siRNA組,高糖干預(yù)時間為方法1中NOX4蛋白水平高峰時間點(diǎn),采用Western Blot技術(shù)檢測各組NOX4蛋白的水平,采用ELISA技術(shù)檢測各組MPC-5細(xì)胞產(chǎn)生MVs的含量。5.統(tǒng)計學(xué)方法:采用SPSS 18.0統(tǒng)計軟件分析數(shù)據(jù)。結(jié)果用均數(shù)±標(biāo)準(zhǔn)差(sx?)表示。兩組以上比較采用單因素方差分析(ANOVA)。P0.05為差異有統(tǒng)計學(xué)意義。結(jié)果:1.NOX4 mRNA檢測結(jié)果:隨高糖刺激時間的延長,NOX4 m RNA表達(dá)水平呈先升高后降低的趨勢,與正常對照(NC)組相比,2小時、4小時、6小時、12小時、24小時分別是NC組的2.61、1.94、1.83、1.52、1.11倍,可見2小時達(dá)高峰,隨后逐漸下降,差異有統(tǒng)計學(xué)(P0.01)。2.NOX4蛋白檢測結(jié)果:隨高糖刺激時間延長,NOX4蛋白表達(dá)量呈先升高后降低的趨勢,與正常對照(NC)組相比,2小時、4小時、6小時、12小時、24小時分別是NC組的1.14、2.08、1.63、1.51、0.96倍,可見4小時達(dá)高峰,隨后逐漸下降,差異有統(tǒng)計學(xué)(P0.01)。3.FAM-siRNA轉(zhuǎn)染MPC-5細(xì)胞結(jié)果:熒光顯微鏡下觀察足細(xì)胞內(nèi)可見綠色熒光,當(dāng)siRNA濃度在50-125nM之間時,隨siRNA濃度增加,含有綠色熒光的細(xì)胞數(shù)目逐漸增多,當(dāng)si RNA濃度達(dá)150nM時,可見多數(shù)細(xì)胞被殺傷,說明siRNA濃度為125nM時轉(zhuǎn)染效率最佳。4.不同序列siRNA轉(zhuǎn)染足細(xì)胞NOX4 mRNA檢測結(jié)果:與正常對照組、lipo2000組、陰性siRNA組、siRNA-A組、siRNA-C組相比,siRNA-B組NOX4mRNA表達(dá)水平明顯降低(P0.05),說明siRNA-B序列干擾NOX4基因的效率最佳。5.NOX4 siRNA轉(zhuǎn)染后高糖刺激足細(xì)胞NOX4蛋白檢測結(jié)果:NOX4 siRNA組(2組),與正常組(1組)相比,NOX4蛋白表達(dá)降低了54.1%(P0.01);高糖組(3組),較1、2組NOX4蛋白表達(dá)升高,分別為1、2組的2.20、4.79倍(P0.01);高糖+NOX4 siRNA組(4組),與3組相比,NOX4蛋白表達(dá)降低了59.7%,為2組的1.93倍(P0.01);陰性siRNA組、高糖+陰性siRNA組,分別與1、3組相比,差異無統(tǒng)計學(xué)意義(P0.05)。6.NOX4 siRNA轉(zhuǎn)染后高糖刺激足細(xì)胞產(chǎn)生MVs檢測結(jié)果:NOX4 siRNA組,較正常組(1組)MVs濃度降低17.6%(P0.05),高糖組(3組),較1、2組MVs濃度升高,分別為1、2組的2.1、2.55倍(P0.01);高糖+NOX4 siRNA組(4組),與3組相比,MVs濃度降低了48.0%(P0.01),較2組升高24.5%(P0.01),與1組差異無統(tǒng)計學(xué)意義(P0.05);陰性siRNA組、高糖+陰性siRNA組,分別與1、3組相比,差異無統(tǒng)計學(xué)意義(P0.05)。結(jié)論:1.高糖可以增加足細(xì)胞NOX4 mRNA及蛋白的表達(dá),呈時間依賴性。2.高糖可以刺激足細(xì)胞MVs產(chǎn)生增加,NOX4 siRNA干擾后足細(xì)胞MVs產(chǎn)生減少。3.高糖可能主要通過增加NADPH氧化酶NOX4來源的氧化應(yīng)激而促進(jìn)足細(xì)胞產(chǎn)生MVs。
[Abstract]:Objective: To study the role of NADPH oxidase NOX4-mediated oxidative stress on the injury of high-sugar-stimulated foot cells and the production of microvesicles. Methods: MPC-5 cells were cultured in vitro, and the processing factors of high sugar group were 30mM glucose. The peak time point was determined by RT-qPCR and Western Blot technique respectively for the 2-hour, 4-hour, 6-hour, 12-hour, 24-hour NX4 mRNA and protein level of the high-sugar-stimulated MPC-5 cells. The MPC-5 cells were transfected with FAM-si RNA with a concentration range of 0-150 nM, and fluorescence was observed with a fluorescence microscope to determine the optimal transfection concentration. The siRNA sequence with the highest transfection efficiency was selected by RT-qPCR. The MPC-5 cells were divided into 6 groups, the normal group, the NOX4 siRNA group, the negative siRNA group, the high sugar group, the high sugar + NOX4 siRNA group, the high sugar + negative siRNA group, the high sugar intervention time is the NOX4 protein level peak time point in the method 1, and the level of the NOX4 protein in each group is detected by the Western Blot technique, The content of MVs in each group of MPC-5 cells was detected by ELISA. Statistical method: SPSS 10.0 was used to analyze the data. Results average standard deviation (sx?) a representation. One-factor analysis of variance (ANOVA) was used for the comparison between the two groups. Results: 1. The results of NX4 mRNA detection showed that the expression level of NOX4 mRNA in the NOX4 mRNA was decreased with the time of high sugar stimulation. Compared with the normal control (NC) group, the expression level of NOX4 mRNA was 2 hours, 4 hours, 6 hours, 12 hours and 24 hours, respectively, 2.61, 1.94, 1.83, 1.52, 1.11 times of the NC group, and the peak at 2 hours and then gradually decreased. The difference was statistically significant (P0.01). The results of NOX4 protein detection showed that, with the time of high sugar stimulation, the expression of NOX4 protein was decreased first and then decreased, compared with the normal control (NC) group, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours were 1.14, 2.08, 1.63, 1.51, 0.96 times of the NC group, and the peak was reached at 4 hours. Subsequently, the difference was statistically significant (P0.01). 3. FAM-siRNA was transfected into the MPC-5 cell result: the visible green fluorescence in the foot cells was observed under the fluorescence microscope, and the number of cells containing the green fluorescence increased as the siRNA concentration was between 50 and 125nM, and when the si RNA concentration was 150 nM, Most of the cells were seen to be killed, indicating that the transfection efficiency was the best when the siRNA concentration was 125nM. The expression of NOX4mRNA in the siRNA-B group was significantly lower than that of the normal control group, the lipo2000 group, the negative siRNA group, the siRNA-A group and the siRNA-C group (P0.05). The results showed that the efficiency of the siRNA-B sequence to the NOX4 gene was the best. The expression of NOX4 in the NOX4 siRNA group (group 2) decreased by 54. 1% (P0.01) than in the normal group (group 1), and the expression of NOX4 in the high-sugar group (group 3) was higher than that of the normal group (group 1). The expression of NOX4 in the high-sugar group (group 3) was 2.20, 4.79-fold (P0.01), and the expression of NOX4 in the high-sugar + NOX4 siRNA group (group 4) decreased by 59.7%. The results showed that the MVs concentration in the normal group (1 group) decreased by 17. 6% (P0.05) and the high sugar group (3 groups). The concentration of MVs in group 1 and 2 was higher than that of group 1 and group 2 (P 0.01). The concentration of MVs decreased by 48. 0% (P 0.01) in the high sugar + NOX4 siRNA group (P 0.01). Compared with group 3, the concentration of MVs decreased by 25.5% (P0.01), and there was no significant difference with group 1 (P0.05). The negative siRNA group, high sugar + negative siRNA group, respectively, were compared with group 1 and 3. The difference was not significant (P0.05). Conclusion: 1. High sugar can increase the expression of NOX4 mRNA and protein in the foot cell, and it is time-dependent. high sugar can stimulate that increase of MVs of the foot cell, and the MVs of the foot cell after the NOX4 siRNA is interfered by the siRNA can be reduced. High sugar may promote the generation of MVs by increasing the oxidative stress of the NADPH oxidase NOX4 source.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R587.2

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