【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(bǔ)體并促進(jìn)調(diào)理吞噬作用的蛋白質(zhì)。調(diào)節(jié)性T細(xì)胞(regulatory T cells,Tregs)是維持外周耐受的CD4+T細(xì)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(jìn)誘導(dǎo)性Tregs(iTregs)的表達(dá)。那么MBL是否同樣促進(jìn)調(diào)節(jié)iTregs的表達(dá)?是否對(duì)iTregs功能產(chǎn)生影響?至今仍不清楚。因此,深入研究MBL對(duì)Tregs誘導(dǎo)分化及功能影響,對(duì)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(duì)調(diào)節(jié)性T細(xì)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(gè)核細(xì)胞(CBMCs)和人外周血單個(gè)核細(xì)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(xì)胞;采用相同的方法分別從MBL誘導(dǎo)的CBMCs組和MBL誘導(dǎo)的CD4+CD25-T細(xì)胞組分選出CD4+CD25+T細(xì)胞(即iTregs)。3.流式細(xì)胞術(shù)(FCM)檢測(cè):分別收集新鮮提取的CMBCs、不同濃度MBL誘導(dǎo)的CBMCs、不同濃度MBL誘導(dǎo)的CD4+CD25-T細(xì)胞,檢測(cè)誘導(dǎo)前后Foxp3的表達(dá)情況。4.RT-PCR法:分別收集不同濃度MBL誘導(dǎo)的CBMCs、不同濃度MBL誘導(dǎo)的CD4+CD25-T細(xì)胞,檢測(cè)誘導(dǎo)前后Foxp3 mRNA的表達(dá)情況。5.CCK-8法:CCK-8法檢測(cè)iTregs對(duì)PBMCs和CD4+CD25-T細(xì)胞增殖的影響。結(jié)果1.新鮮CBMCs中低表達(dá)Foxp3。2.MBL可以顯著提高CBMCs中CD4+CD25+Foxp3+T細(xì)胞的比例。3.MBL促進(jìn)CD4+CD25-T細(xì)胞向iTregs誘導(dǎo)分化。4.MBL不僅促進(jìn)CBMCs中高表達(dá)Foxp3 m RNA,而且誘導(dǎo)CD4+CD25-T細(xì)胞高表達(dá)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(xì)胞的增殖。6.加入MBL后不僅增強(qiáng)iTregs對(duì)PBMCs增殖的抑制作用,而且增強(qiáng)iTregs對(duì)CD4+CD25-T細(xì)胞增殖的抑制作用。結(jié)論MBL不僅能夠協(xié)同促進(jìn)調(diào)節(jié)性T細(xì)胞的誘導(dǎo)分化,而且對(duì)調(diào)節(jié)性T細(xì)胞抑制效應(yīng)T細(xì)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(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 王凡平;王明永;郭曉芳;石如玲;徐素玲;馬淑君;李海斌;郭繼強(qiáng);楊秀麗;;MBL抑制LPS誘導(dǎo)DC成熟機(jī)制的研究[J];中國(guó)實(shí)驗(yàn)血液學(xué)雜志;2013年03期

2 王凡平;王明永;楊建斌;趙東方;王紅坡;邵峰;孫瑞利;郭慶合;凌明智;趙永新;宋士軍;郭繼強(qiáng);;甘露聚糖結(jié)合凝集素誘導(dǎo)DC成熟機(jī)制分析[J];中國(guó)免疫學(xué)雜志;2012年12期

3 王凡平;王明永;楊建斌;趙東方;連榮;徐素玲;邵峰;孫瑞利;郭慶合;李海斌;郭繼強(qiáng);宋志善;;甘露聚糖結(jié)合凝集素抑制肽聚糖刺激THP-1/CD14細(xì)胞產(chǎn)生的炎性反應(yīng)及機(jī)制[J];中華微生物學(xué)和免疫學(xué)雜志;2012年11期

4 王明永;王凡平;郭曉芳;孫瑞利;郭慶合;張婧婧;楊景瑞;張新富;段巨洪;;MBL抑制白假絲酵母菌刺激THP1/CD14細(xì)胞產(chǎn)生TNF-α和IL-8[J];中華微生物學(xué)和免疫學(xué)雜志;2011年01期

5 王明永;張雅妮;雷鳴;張麗蕓;盧曉;陳政良;;MBL抑制Jurkat細(xì)胞增殖和分泌IL-2[J];現(xiàn)代免疫學(xué);2010年06期

6 王明永;張雅妮;張麗蕓;盧曉;雷鳴;李瑞芳;陳政良;;MBL與Raji細(xì)胞結(jié)合特性的研究[J];生物化學(xué)與生物物理進(jìn)展;2008年03期

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