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siRNA干扰NOX4基因对高糖刺激小鼠肾小球足细胞产生微囊泡的影响

发布时间:2018-12-19 10:34
【摘要】:目的:本研究进一步探讨NADPH氧化酶NOX4介导的氧化应激对高糖刺激足细胞损伤及产生微囊泡过程中的作用。方法:体外培养MPC-5细胞,高糖组处理因素为30mM葡萄糖。1.采用RT-qPCR、Western Blot技术分别检测高糖刺激MPC-5细胞2小时、4小时、6小时、12小时、24小时后NOX4 mRNA、蛋白的水平,确定高峰时间点。2.对MPC-5细胞进行FAM-si RNA转染,浓度范围为0-150nM,荧光显微镜观察荧光,确定最佳转染浓度。3.以最佳浓度对MPC-5细胞进行siRNA转染,采用RT-qPCR技术筛选转染率最高的siRNA序列。4.将MPC-5细胞分为6组,分别为正常组、NOX4 siRNA组、阴性siRNA组、高糖组、高糖+NOX4 siRNA组、高糖+阴性siRNA组,高糖干预时间为方法1中NOX4蛋白水平高峰时间点,采用Western Blot技术检测各组NOX4蛋白的水平,采用ELISA技术检测各组MPC-5细胞产生MVs的含量。5.统计学方法:采用SPSS 18.0统计软件分析数据。结果用均数±标准差(sx?)表示。两组以上比较采用单因素方差分析(ANOVA)。P0.05为差异有统计学意义。结果:1.NOX4 mRNA检测结果:随高糖刺激时间的延长,NOX4 m RNA表达水平呈先升高后降低的趋势,与正常对照(NC)组相比,2小时、4小时、6小时、12小时、24小时分别是NC组的2.61、1.94、1.83、1.52、1.11倍,可见2小时达高峰,随后逐渐下降,差异有统计学(P0.01)。2.NOX4蛋白检测结果:随高糖刺激时间延长,NOX4蛋白表达量呈先升高后降低的趋势,与正常对照(NC)组相比,2小时、4小时、6小时、12小时、24小时分别是NC组的1.14、2.08、1.63、1.51、0.96倍,可见4小时达高峰,随后逐渐下降,差异有统计学(P0.01)。3.FAM-siRNA转染MPC-5细胞结果:荧光显微镜下观察足细胞内可见绿色荧光,当siRNA浓度在50-125nM之间时,随siRNA浓度增加,含有绿色荧光的细胞数目逐渐增多,当si RNA浓度达150nM时,可见多数细胞被杀伤,说明siRNA浓度为125nM时转染效率最佳。4.不同序列siRNA转染足细胞NOX4 mRNA检测结果:与正常对照组、lipo2000组、阴性siRNA组、siRNA-A组、siRNA-C组相比,siRNA-B组NOX4mRNA表达水平明显降低(P0.05),说明siRNA-B序列干扰NOX4基因的效率最佳。5.NOX4 siRNA转染后高糖刺激足细胞NOX4蛋白检测结果:NOX4 siRNA组(2组),与正常组(1组)相比,NOX4蛋白表达降低了54.1%(P0.01);高糖组(3组),较1、2组NOX4蛋白表达升高,分别为1、2组的2.20、4.79倍(P0.01);高糖+NOX4 siRNA组(4组),与3组相比,NOX4蛋白表达降低了59.7%,为2组的1.93倍(P0.01);阴性siRNA组、高糖+阴性siRNA组,分别与1、3组相比,差异无统计学意义(P0.05)。6.NOX4 siRNA转染后高糖刺激足细胞产生MVs检测结果:NOX4 siRNA组,较正常组(1组)MVs浓度降低17.6%(P0.05),高糖组(3组),较1、2组MVs浓度升高,分别为1、2组的2.1、2.55倍(P0.01);高糖+NOX4 siRNA组(4组),与3组相比,MVs浓度降低了48.0%(P0.01),较2组升高24.5%(P0.01),与1组差异无统计学意义(P0.05);阴性siRNA组、高糖+阴性siRNA组,分别与1、3组相比,差异无统计学意义(P0.05)。结论:1.高糖可以增加足细胞NOX4 mRNA及蛋白的表达,呈时间依赖性。2.高糖可以刺激足细胞MVs产生增加,NOX4 siRNA干扰后足细胞MVs产生减少。3.高糖可能主要通过增加NADPH氧化酶NOX4来源的氧化应激而促进足细胞产生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.
【学位授予单位】:天津医科大学
【学位级别】:硕士
【学位授予年份】:2015
【分类号】:R587.2

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