MBL對調(diào)節(jié)性T細胞誘導分化及功能的影響
發(fā)布時間：2018-11-26 16:06
【摘要】：背景甘露聚糖結(jié)合凝集素(mannan-binding lectin,MBL)是一種能夠激活補體并促進調(diào)理吞噬作用的蛋白質(zhì)。調(diào)節(jié)性T細胞(regulatory T cells,Tregs)是維持外周耐受的CD4+T細胞亞群。與MBL同為C型凝集素家族的肺表面活性蛋白A(surfactant protein A,SP-A)能夠促進誘導性Tregs(iTregs)的表達。那么MBL是否同樣促進調(diào)節(jié)iTregs的表達?是否對iTregs功能產(chǎn)生影響?至今仍不清楚。因此,深入研究MBL對Tregs誘導分化及功能影響,對闡明MBL在獲得性免疫方面的新角色,具有重要意義。目的探討MBL對調(diào)節(jié)性T細胞數(shù)目和功能的干預(yù),為自身免疫性疾病和炎性疾病的治療提供新思路方法1.Ficoll密度梯度離心法:采用Ficoll密度梯度離心法分離人臍血單個核細胞(CBMCs)和人外周血單個核細胞(PBMCs)。2.免疫磁珠分選法:采用免疫磁珠分選法從CBMCs中分選出CD4+CD25-T細胞;采用相同的方法分別從MBL誘導的CBMCs組和MBL誘導的CD4+CD25-T細胞組分選出CD4+CD25+T細胞(即iTregs)。3.流式細胞術(shù)(FCM)檢測:分別收集新鮮提取的CMBCs、不同濃度MBL誘導的CBMCs、不同濃度MBL誘導的CD4+CD25-T細胞,檢測誘導前后Foxp3的表達情況。4.RT-PCR法:分別收集不同濃度MBL誘導的CBMCs、不同濃度MBL誘導的CD4+CD25-T細胞,檢測誘導前后Foxp3 mRNA的表達情況。5.CCK-8法:CCK-8法檢測iTregs對PBMCs和CD4+CD25-T細胞增殖的影響。結(jié)果1.新鮮CBMCs中低表達Foxp3。2.MBL可以顯著提高CBMCs中CD4+CD25+Foxp3+T細胞的比例。3.MBL促進CD4+CD25-T細胞向iTregs誘導分化。4.MBL不僅促進CBMCs中高表達Foxp3 m RNA,而且誘導CD4+CD25-T細胞高表達Foxp3 m RNA。5.iTregs不僅以濃度依賴方式抑制PBMCs的增殖,而且以濃度依賴的方式抑制CD4+CD25-T細胞的增殖。6.加入MBL后不僅增強iTregs對PBMCs增殖的抑制作用,而且增強iTregs對CD4+CD25-T細胞增殖的抑制作用。結(jié)論MBL不僅能夠協(xié)同促進調(diào)節(jié)性T細胞的誘導分化,而且對調(diào)節(jié)性T細胞抑制效應(yīng)T細胞的增殖具有一定的調(diào)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.
【學位授予單位】：新鄉(xiāng)醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R392
本文編號：2359013
[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.
【學位授予單位】：新鄉(xiāng)醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R392
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