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
[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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