FoxO1转录因子在巨噬细胞中的表达及其与炎症的关系

发布时间：2018-05-05 16:10

本文选题：FoxO1 + 炎症反应　；参考：《山西医科大学》2017年硕士论文

【摘要】：研究目的:1.观察Fox O1转录因子在巨噬细胞活化前后的表达情况。2.观察Fox O1转录因子敲低后对巨噬细胞炎症过程的影响。3.检测Fox O1是否通过TGF-β来影响炎症反应。研究方法:1.应用脂多糖(Lipopolysaccharide,LPS)激活巨噬细胞,酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)测LPS活化巨噬细胞的最适浓度和时间。收集LPS各个浓度、培养时间的细胞上清液。ELISA试剂盒检测各个浓度和时间段收集上清液中炎症因子IL-6和TNF-α的含量,推出LPS活化巨噬细胞的最适时间及浓度。琼脂糖凝胶电泳检测Foxo1、Foxo3a、Foxo4、Foxo6四个基因在活化前后巨噬细胞的表达变化。实时荧光定量PCR检测Foxo1基因的mRNA水平,Western blot检测FOXO1蛋白表达。2.敲低活化组巨噬细胞Foxo1基因,应用流式细胞术、实时荧光定量PCR和Western blot检验小干扰RNA转染效率。同时使用ELISA试剂盒检验两组细胞上清液IL-6、TNF-α的含量,比较活化组巨噬细胞在转染前后IL-6、TNF-α含量变化情况。3.巨噬细胞敲低Foxo1表达,分两组。一组不处理,另一组LPS激活细胞。应用Western blot实验方法比较四组巨噬细胞中TGF-β蛋白的表达情况。研究结果:1.脂多糖激活巨噬细胞后,上清液中IL-6和TNF-α的含量逐渐升高。在LPS浓度50 ng/ml,培养36 h时测得炎症因子含量均高于其他浓度和时间的LPS刺激下细胞上清液中炎症因子IL-6和TNF-α含量,表明此时是巨噬细胞活化的最适条件。采用实时荧光定量PCR和Western blot实验得出活化组FoxO1转录因子在m RNA水平和蛋白水平表达均低于未活化组,且差异具有统计学意义(p0.05)。2.活化组敲低Foxo1,应用流式细胞术、实时荧光定量PCR、Western blot方法检验转染效率。结果表明FoxO1转录因子在mRNA水平和蛋白水平的表达量是转染前的0.47倍,转染成功。ELISA实验结果显示:活化组巨噬细胞IL-6的含量为151.2 ng/ml±49.1 ng/ml,TNF-α含量为746.2 ng/ml±68.2 ng/ml,转染组IL-6的含量上升到247.2 ng/ml±56 ng/ml、TNF-α的含量上升到1282 ng/ml±26.7 ng/ml。转染组炎症因子IL-6和TNF-α含量明显升高,且差异有统计学意义(p0.01)。3.将正常巨噬细胞分为两组:一组不处理,另一组敲低Foxo1。然后分别将两组巨噬细胞再分为两组:一组不做处理,另一组用LPS激活。采用Western blot分析四组细胞中TGF-β蛋白相对表达量。实验结果显示:在活化组、活化组+siRNA组中TGF-β蛋白均有表达。活化组TGF-β蛋白的相对表达量为0.37,活化组+siRNA组的相对表达量是0.54。说明巨噬细胞敲低Foxo1后活化会引起TGF-β因子的增加,说明Fox O1转录因子有可能通过TGF-β因子影响炎症反应。研究结论:1.Fox O1有可能参与炎症过程。2.转录因子Fox O1可能具有负性调控炎症反应的作用。3.Fox O1转录因子可以通过增加TGF-β表达影响炎症反应。
[Abstract]:Objective: 1. To observe the expression of Fox O 1 transcription factor before and after macrophage activation. To observe the effect of Fox O 1 transcription factor knockdown on macrophage inflammation. Determine whether Fox O 1 affects inflammation through TGF- 尾. Research method: 1. Lipopolysaccharide (LPS) was used to activate macrophages and enzyme linked immunosorbent Assay Elisa was used to determine the optimal concentration and time of LPS activated macrophages. The concentration of inflammatory cytokines IL-6 and TNF- 伪 in supernatant of culture time was determined by Elisa kit. The optimal time and concentration of LPS to activate macrophage were deduced. Agarose gel electrophoresis (agarose) was used to detect the expression of four genes of Foxo1, Foxo3, Foxo4 and Foxo6 in macrophages before and after activation. Real time fluorescence quantitative PCR was used to detect the mRNA level of Foxo1 gene. Western blot was used to detect the expression of FOXO1 protein. The transfection efficiency of small interfering RNA was detected by flow cytometry, real-time fluorescence quantitative PCR and Western blot. At the same time, ELISA kit was used to detect the content of IL-6 TNF- 伪 in the supernatant of the two groups, and to compare the changes of IL-6 TNF- 伪 content in macrophages of activated group before and after transfection. Macrophages knocked down Foxo1 expression and were divided into two groups. One group was not treated, the other group LPS activated cells. The expression of TGF- 尾 protein in macrophages was compared by Western blot assay. The result of the study was: 1. After lipopolysaccharide activated macrophages, the content of IL-6 and TNF- 伪 in supernatant increased gradually. At the concentration of 50 ng / ml of LPS, the levels of inflammatory factors IL-6 and TNF- 伪 in the supernatant stimulated by other concentrations and time of LPS were higher than those in the supernatants stimulated by LPS for 36 h, which indicated that this was the best condition for the activation of macrophages. The results of real-time quantitative PCR and Western blot showed that the expression of FoxO1 transcription factors in activated group was lower than that in non-activated group at m RNA level and protein level, and the difference was statistically significant. The activation group knocked down Foxo1 and the transfection efficiency was detected by flow cytometry and real-time fluorescence quantitative PCR blot method. The results showed that the expression of FoxO1 transcription factors at mRNA and protein levels was 0.47 times higher than that before transfection. The results of successful transfection and Elisa showed that the IL-6 content of activated macrophages was 151.2 ng/ml 卤49.1 ng / ml, the content of TNF- 伪 was 746.2 ng/ml 卤68.2 ng / ml, and the content of IL-6 in transfection group increased to 247.2 ng/ml 卤56 ng / ml TNF- 伪 to 1282 ng/ml 卤26.7 ng / ml. The levels of inflammatory factor IL-6 and TNF- 伪 in transfection group were significantly higher than those in control group (P < 0.01). Normal macrophages were divided into two groups: one without treatment and the other with low Foxo 1. Then two groups of macrophages were divided into two groups: one group was not treated, the other group was activated by LPS. The relative expression of TGF- 尾 protein in four groups of cells was analyzed by Western blot. The results showed that TGF- 尾 protein was expressed in activated group and siRNA group. The relative expression of TGF- 尾 protein was 0.37 in activated group and 0.54 in siRNA group. It was suggested that activation of macrophages after knocking down Foxo1 could induce the increase of TGF- 尾 factor, which suggested that Fox O1 transcription factor might affect the inflammatory response through TGF- 尾 factor. Conclusion: 1. Fox O1 may be involved in the inflammatory process. 2. The transcription factor Fox O1 may play a negative role in the regulation of inflammatory response. 3. Fox O1 transcription factor may influence the inflammatory response by increasing the expression of TGF- 尾.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R363

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