本文選題：miR-146a + 初始T細(xì)胞�。� 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的研究miR-146a對(duì)Th17細(xì)胞分化過程和功能的影響并探索可能的調(diào)控機(jī)制,為調(diào)控Th17細(xì)胞參與的免疫應(yīng)答尋找新方法。方法應(yīng)用磁珠法分選小鼠脾臟CD4+CD25-初始T細(xì)胞,流式細(xì)胞術(shù)檢測(cè)目的細(xì)胞的純度;應(yīng)用抗小鼠CD3/CD28單克隆抗體刺激CD4+CD25-初始T細(xì)胞活化增殖后,用miR-146a agomir和antagomir試劑轉(zhuǎn)染調(diào)控CD4+CD25-初始T細(xì)胞中miR-146a表達(dá)水平,實(shí)驗(yàn)設(shè)置為上調(diào)組、下調(diào)組以及空白對(duì)照組,根據(jù)課題組前期實(shí)驗(yàn)結(jié)果,每組分別設(shè)置不同轉(zhuǎn)染濃度亞組,并選取最佳轉(zhuǎn)染濃度;根據(jù)文獻(xiàn)選取3個(gè)不同濃度的IL-6、TGF-β、抗小鼠IL-4抗體以及抗小鼠IFN-γ抗體組合誘導(dǎo)CD4+CD25-初始T細(xì)胞向Th17細(xì)胞方向分化,驗(yàn)證各方案的誘導(dǎo)效果并選取最佳誘導(dǎo)方案;流式檢測(cè)各組Th17細(xì)胞數(shù)目并用ELISA方法檢測(cè)各組細(xì)胞培養(yǎng)體系上清中IL-17水平,QRT-PCR和Western-blot方法檢測(cè)篩選出的miR-146a可能發(fā)揮作用的靶基因的轉(zhuǎn)錄及蛋白表達(dá)水平。結(jié)果1.磁珠分選前小鼠脾臟CD4+CD25-初始T細(xì)胞的比例為24.9%;磁珠分選后CD4+CD25-初始T細(xì)胞純度為92.3%,細(xì)胞活性可達(dá)到98%。2.成功轉(zhuǎn)染調(diào)控CD4+CD25-初始T細(xì)胞中mi R-146a的表達(dá)水平,并且成功誘導(dǎo)CD4+CD25-初始T細(xì)胞向Th17細(xì)胞方向分化。3.用最佳濃度miR-146a調(diào)控試劑轉(zhuǎn)染以及最佳濃度誘導(dǎo)后,上調(diào)組、下調(diào)組與空白對(duì)照組相比Th17細(xì)胞數(shù)目和上清中IL-17水平不同。流式細(xì)胞學(xué)檢測(cè)結(jié)果提示,上調(diào)組較下調(diào)組及空白對(duì)照組Th17數(shù)目顯著增多(P0.01),下調(diào)組較空白組Th17細(xì)胞數(shù)目明顯減少(P0.01);ELISA檢測(cè)結(jié)果顯示,上調(diào)組較下調(diào)組及空白對(duì)照組上清中IL-17水平顯著升高(P0.01),下調(diào)組較空白對(duì)照組上清中IL-17水平明顯降低(P0.01)。4.QRT-PCR和Western blot結(jié)果提示,Th17細(xì)胞關(guān)鍵轉(zhuǎn)錄因子ROR-γt和IL-17基因的mRNA和蛋白表達(dá)水平在上調(diào)組與下調(diào)組和空白對(duì)照組相比,明顯升高(P0.01),在下調(diào)組顯著降低(P0.01);STAT1的表達(dá)水平在各組無明顯差異(P0.05);IRAK1表達(dá)水平在上調(diào)組較下調(diào)組和空白組明顯增高(P0.01);在下調(diào)組中其表達(dá)水平明顯降低(P0.01)。結(jié)論1.CD4+CD25-初始T細(xì)胞中mi R-146a的表達(dá)水平與Th17細(xì)胞的分化及其分泌的IL-17水平呈平行關(guān)系,miR-146a表達(dá)上調(diào)可以促使Th17細(xì)胞數(shù)目及IL-17分泌量增多;而miR-146a的表達(dá)下調(diào)后,Th17細(xì)胞數(shù)目以及IL-17分泌量也顯著降低。2.在Th17參與的炎性環(huán)境中,調(diào)控miR-146a表達(dá)水平后,其靶基因之一STAT1的表達(dá)水平無顯著變化;miR-146a與另一個(gè)靶基因IRAK1不再是負(fù)性調(diào)控關(guān)系,miR-146a表達(dá)上調(diào)后,IRAK1的表達(dá)水平也隨之升高,這可能與促進(jìn)Th17細(xì)胞的分化及其分泌IL-17能力的有關(guān)。
[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- 緯 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-緯 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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R392

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