miR-146a调控Th17细胞分化和功能的体外研究

发布时间：2018-06-02 12:06

本文选题：miR-146a + 初始T细胞　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的研究miR-146a对Th17细胞分化过程和功能的影响并探索可能的调控机制,为调控Th17细胞参与的免疫应答寻找新方法。方法应用磁珠法分选小鼠脾脏CD4+CD25-初始T细胞,流式细胞术检测目的细胞的纯度;应用抗小鼠CD3/CD28单克隆抗体刺激CD4+CD25-初始T细胞活化增殖后,用miR-146a agomir和antagomir试剂转染调控CD4+CD25-初始T细胞中miR-146a表达水平,实验设置为上调组、下调组以及空白对照组,根据课题组前期实验结果,每组分别设置不同转染浓度亚组,并选取最佳转染浓度;根据文献选取3个不同浓度的IL-6、TGF-β、抗小鼠IL-4抗体以及抗小鼠IFN-γ抗体组合诱导CD4+CD25-初始T细胞向Th17细胞方向分化,验证各方案的诱导效果并选取最佳诱导方案;流式检测各组Th17细胞数目并用ELISA方法检测各组细胞培养体系上清中IL-17水平,QRT-PCR和Western-blot方法检测筛选出的miR-146a可能发挥作用的靶基因的转录及蛋白表达水平。结果1.磁珠分选前小鼠脾脏CD4+CD25-初始T细胞的比例为24.9%;磁珠分选后CD4+CD25-初始T细胞纯度为92.3%,细胞活性可达到98%。2.成功转染调控CD4+CD25-初始T细胞中mi R-146a的表达水平,并且成功诱导CD4+CD25-初始T细胞向Th17细胞方向分化。3.用最佳浓度miR-146a调控试剂转染以及最佳浓度诱导后,上调组、下调组与空白对照组相比Th17细胞数目和上清中IL-17水平不同。流式细胞学检测结果提示,上调组较下调组及空白对照组Th17数目显著增多(P0.01),下调组较空白组Th17细胞数目明显减少(P0.01);ELISA检测结果显示,上调组较下调组及空白对照组上清中IL-17水平显著升高(P0.01),下调组较空白对照组上清中IL-17水平明显降低(P0.01)。4.QRT-PCR和Western blot结果提示,Th17细胞关键转录因子ROR-γt和IL-17基因的mRNA和蛋白表达水平在上调组与下调组和空白对照组相比,明显升高(P0.01),在下调组显著降低(P0.01);STAT1的表达水平在各组无明显差异(P0.05);IRAK1表达水平在上调组较下调组和空白组明显增高(P0.01);在下调组中其表达水平明显降低(P0.01)。结论1.CD4+CD25-初始T细胞中mi R-146a的表达水平与Th17细胞的分化及其分泌的IL-17水平呈平行关系,miR-146a表达上调可以促使Th17细胞数目及IL-17分泌量增多;而miR-146a的表达下调后,Th17细胞数目以及IL-17分泌量也显著降低。2.在Th17参与的炎性环境中,调控miR-146a表达水平后,其靶基因之一STAT1的表达水平无显著变化;miR-146a与另一个靶基因IRAK1不再是负性调控关系,miR-146a表达上调后,IRAK1的表达水平也随之升高,这可能与促进Th17细胞的分化及其分泌IL-17能力的有关。
[Abstract]:Objective to investigate the effect of miR-146a on the differentiation process and function of Th17 cells and to explore the possible regulatory mechanism, and to find a new method for regulating the immune response of Th17 cells. Methods CD4 CD25- initial T cells were isolated from mouse spleen by magnetic bead method, the purity of target cells was detected by flow cytometry, and the activation and proliferation of CD4 CD25- initial T cells were stimulated by monoclonal antibody against mouse CD3/CD28. MiR-146a agomir and antagomir reagents were used to transfect and regulate the expression of miR-146a in CD4 CD25- initial T cells. The cells were divided into three groups: up-regulation group, down-regulation group and blank control group. According to the results of previous experiment, each group was divided into different transfection concentration subgroups. Three different concentrations of IL-6 TGF- 尾, anti-mouse IL-4 antibody and anti-mouse IFN- 纬 antibody combination were selected to induce the differentiation of CD4 CD25- initial T cells into Th17 cells. Verify the induction effect of each scheme and select the best induction scheme; Flow cytometry was used to determine the number of Th17 cells in each group and ELISA method was used to detect the level of IL-17 in the supernatant of cell culture system. QRT-PCR and Western-blot methods were used to detect the transcriptional and protein expression of the target genes that miR-146a might play a role in. Result 1. The percentage of CD4 CD25- initial T cells in mouse spleen was 24.9before magnetic bead sorting, and the purity of CD4 CD25- initial T cell was 92.3g after magnetic bead sorting. The expression of mi R-146a in CD4 CD25- initial T cells was successfully regulated by transfection, and the differentiation of CD4 CD25- initial T cells into Th17 cells was induced successfully. The number of Th17 cells and the level of IL-17 in supernatant of up-regulated group and down-regulated group were different from those of blank control group after transfection and induction with the best concentration of miR-146a regulatory reagent. The results of flow cytometry showed that the number of Th17 in the up-regulated group was significantly higher than that in the down-regulated group and the blank control group, and the number of Th17 cells in the down-regulated group was significantly decreased than that in the blank group. The level of IL-17 in supernatant of down-regulated group and blank control group was significantly higher than that of control group, and the level of IL-17 in supernatant of down-regulated group was significantly lower than that of blank control group. 4. The results of RT-PCR and Western blot indicated that mRNA and protein of ROR-纬 t and IL-17 gene in Th17 cells were significantly decreased by RT-PCR and Western blot. Compared with the down-regulated group and the blank control group, the expression level was higher in the upregulation group. In the down-regulated group, there was no significant difference between the two groups in the expression of P0.05 and IRAK1. The expression level of IRAK1 in the up-regulated group was significantly higher than that in the down-regulated group and the blank group, and the expression level in the down-regulated group was significantly lower than that in the down-regulated group. The expression level of IRAK1 in the down-regulated group was significantly lower than that in the down-regulated group. Conclusion the expression level of miR-146a in 1.CD4 CD25- initial T cells is parallel to the differentiation of Th17 cells and the level of IL-17 secreted by Th17 cells. Upregulation of miR-146a expression can increase the number of Th17 cells and the secretion of IL-17. After down-regulation of miR-146a expression, the number of Th17 cells and the secretion of IL-17 decreased significantly. In the inflammatory environment involved in Th17, the expression level of STAT1, one of its target genes, did not change significantly after regulating the level of miR-146a expression. The expression of simiR-146a and another target gene IRAK1 was no longer a negative regulatory relationship. The expression level of IRAK1 increased after the up-regulation of the expression of miR-146a. This may be related to promoting the differentiation of Th17 cells and their ability to secrete IL-17.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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