MBL对调节性T细胞诱导分化及功能的影响

发布时间：2018-11-26 16:06

【摘要】：背景甘露聚糖结合凝集素(mannan-binding lectin,MBL)是一种能够激活补体并促进调理吞噬作用的蛋白质。调节性T细胞(regulatory T cells,Tregs)是维持外周耐受的CD4+T细胞亚群。与MBL同为C型凝集素家族的肺表面活性蛋白A(surfactant protein A,SP-A)能够促进诱导性Tregs(iTregs)的表达。那么MBL是否同样促进调节iTregs的表达?是否对iTregs功能产生影响?至今仍不清楚。因此,深入研究MBL对Tregs诱导分化及功能影响,对阐明MBL在获得性免疫方面的新角色,具有重要意义。目的探讨MBL对调节性T细胞数目和功能的干预,为自身免疫性疾病和炎性疾病的治疗提供新思路方法1.Ficoll密度梯度离心法:采用Ficoll密度梯度离心法分离人脐血单个核细胞(CBMCs)和人外周血单个核细胞(PBMCs)。2.免疫磁珠分选法:采用免疫磁珠分选法从CBMCs中分选出CD4+CD25-T细胞;采用相同的方法分别从MBL诱导的CBMCs组和MBL诱导的CD4+CD25-T细胞组分选出CD4+CD25+T细胞(即iTregs)。3.流式细胞术(FCM)检测:分别收集新鲜提取的CMBCs、不同浓度MBL诱导的CBMCs、不同浓度MBL诱导的CD4+CD25-T细胞,检测诱导前后Foxp3的表达情况。4.RT-PCR法:分别收集不同浓度MBL诱导的CBMCs、不同浓度MBL诱导的CD4+CD25-T细胞,检测诱导前后Foxp3 mRNA的表达情况。5.CCK-8法:CCK-8法检测iTregs对PBMCs和CD4+CD25-T细胞增殖的影响。结果1.新鲜CBMCs中低表达Foxp3。2.MBL可以显著提高CBMCs中CD4+CD25+Foxp3+T细胞的比例。3.MBL促进CD4+CD25-T细胞向iTregs诱导分化。4.MBL不仅促进CBMCs中高表达Foxp3 m RNA,而且诱导CD4+CD25-T细胞高表达Foxp3 m RNA。5.iTregs不仅以浓度依赖方式抑制PBMCs的增殖,而且以浓度依赖的方式抑制CD4+CD25-T细胞的增殖。6.加入MBL后不仅增强iTregs对PBMCs增殖的抑制作用,而且增强iTregs对CD4+CD25-T细胞增殖的抑制作用。结论MBL不仅能够协同促进调节性T细胞的诱导分化,而且对调节性T细胞抑制效应T细胞的增殖具有一定的调控作用。
[Abstract]:Background mannan-binding lectin (mannan-binding lectin,MBL) is a protein that activates complement and promotes phagocytosis. Regulatory T cell (regulatory T cells,Tregs is a CD4 T cell subgroup that maintains peripheral tolerance. Pulmonary surfactant protein (A (surfactant protein Agna SP-A), which is a C-type lectin family with MBL, can promote the expression of inducible Tregs (iTregs). Does MBL also promote the regulation of iTregs expression? Do you have an impact on iTregs functionality? It is still unclear. Therefore, it is of great significance to study the effects of MBL on the differentiation and function of Tregs in order to clarify the new role of MBL in acquired immunity. Objective to investigate the effect of MBL on the number and function of regulatory T cells. 1.Ficoll density gradient centrifugation was used to isolate human umbilical cord blood mononuclear cells (CBMCs) and human peripheral blood mononuclear cells (PBMCs). 2). Immunomagnetic bead sorting method: CD4 CD25-T cells were isolated from CBMCs by immunomagnetic bead sorting, CD4 CD25 T cells (iTregs). 3) were isolated from CBMCs induced by MBL and CD4 CD25-T cells induced by MBL by the same method. Flow cytometry (FCM) (FCM) analysis: CD4 CD25-T cells induced by different concentrations of CBMCs, and MBL were collected from freshly extracted CMBCs, with different concentrations of MBL. The expression of Foxp3 was detected before and after induction. 4.RT-PCR method: the CD4 CD25-T cells induced by CBMCs, and MBL with different concentrations of MBL were collected, respectively. 5.CCK-8 method: CCK-8 method was used to detect the effect of iTregs on the proliferation of PBMCs and CD4 CD25-T cells. Result 1. The low expression of Foxp3.2.MBL in fresh CBMCs could significantly increase the proportion of CD4 CD25 Foxp3 T cells in CBMCs. 3.MBL promoted the differentiation of CD4 CD25-T cells into iTregs. 4.MBL not only promoted the high expression of Foxp3 m RNA, in CBMCs, but also promoted the differentiation of CD4 CD25-T cells. Moreover, overexpression of Foxp3 m RNA.5.iTregs in CD4 CD25-T cells not only inhibited the proliferation of PBMCs in a dose-dependent manner, but also inhibited the proliferation of CD4 CD25-T cells in a concentration-dependent manner. The addition of MBL not only enhanced the inhibitory effect of iTregs on PBMCs proliferation, but also enhanced the inhibitory effect of iTregs on the proliferation of CD4 CD25-T cells. Conclusion MBL can not only co-promote the induction and differentiation of regulatory T cells, but also regulate the proliferation of regulatory T cells.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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