B10细胞在NOD小鼠1型糖尿病发生发展中作用机制的探究

发布时间：2018-03-24 11:28

本文选题：B10细胞　切入点：1型糖尿病　出处：《南京医科大学》2015年硕士论文

【摘要】：第一部分NOD小鼠糖尿病发生发展过程中B10细胞数量水平的研究目的:观察NOD小鼠T1D自然病程中B10细胞及Th1、Th17、Treg细胞的数量变化,探讨B10细胞数量与NOD小鼠T1D发生发展的关系。方法:分离4周、8周及糖尿病NOD小鼠胰腺组织固定并作病理切片HE染色评估胰岛炎;流式细胞术检测4周(A组)、8周(B组)、糖尿病(C组)NOD小鼠及同龄WT C57BL/6小鼠(对照组)脾脏、肠系膜淋巴结、外周淋巴结、胰腺淋巴结中B10、Th1、Th17及Treg细胞的比例。结果:(1)不同年龄段NOD小鼠胰岛炎比较:4周NOD小鼠无胰岛炎;8周NOD小鼠发生胰岛炎;糖尿病NOD小鼠发生明显胰岛炎且无完整胰岛;(2)不同病程阶段NOD小鼠B10细胞比较:B及C组B10细胞较A组增高,而C组B10细胞则较B组显著下降,差异有统计学意义(P0.01);A组B10细胞各组织间无明显差异(P0.05),B组B10细胞以胰腺淋巴结最高,C组B10细胞以外周淋巴结最高且胰腺淋巴结B10细胞较B组显著下降,差异有统计学意义(P0.01);(3)不同病程阶段NOD小鼠Th1及Th17细胞比较:C组Th1及Th17细胞较A、B组显著增加,差异有统计学意义(P0.01);(4)不同病程阶段NOD小鼠Treg细胞比较:C组Treg细胞较A、B组无明显变化,且NOD小鼠Treg细胞较同龄WT C57BL/6小鼠亦无明显变化,差异无统计学意义(P0.05)。结论:随NOD小鼠年龄增长及自身免疫性胰岛炎进展,B10细胞存在数量变化及组织分布的差异;糖尿病NOD小鼠B10细胞与致病性Th1、Th17细胞之间存在明显的免疫失衡,B10细胞数量减少可能参与了NOD小鼠T1D的发生发展。第二部分NOD小鼠糖尿病发生发展过程中B10细胞功能的研究目的:观察NOD小鼠T1D自然病程中B10细胞免疫调节功能的变化,探究B10细胞参与NOD小鼠T1D发生发展的可能机制。方法:流式细胞术分选4周、8周、糖尿病NOD小鼠、8-12周野生型(阳性对照组)和IL-10-/-小鼠(阴性对照组)脾脏B10及CD19+CD5-CD1dlow B细胞;磁活性标记细胞分选术分选4周、8周、糖尿病NOD小鼠、8-12周WT C57BL/6小鼠(阳性对照组)及IL-10-/-小鼠(阴性对照组)脾脏和淋巴结CD4+CD25-T细胞,利用体外淋巴细胞反应观察B10细胞对CFSE标记的CD4+CD25-T细胞增殖的抑制情况,对CD4+CD25-T细胞分泌IFN-γ、IL-4、IL-17等细胞因子及Foxp3表达的影响。结果:(1)与CD19+CD5-CD1dlow B细胞相比,野生型小鼠B10细胞能明显抑制CD4+CD25-T细胞3-4代子细胞增殖(P0.01),而4周、8周、糖尿病NOD小鼠及IL-10-/-小鼠B10细胞均不能抑制CD4+CD25-T细胞增殖(P0.05);与对照组相比,野生型小鼠、IL-10-/-小鼠及4周、8周、糖尿病NOD小鼠B10和CD19+CD5-CD1dlow B细胞均明显促进CD4+CD25-T细胞增殖(P0.01);(2)野生型小鼠B10细胞明显抑制IFN-γ分泌、促进IL-4分泌、增加Foxp3表达(P0.05),IL-10-/-小鼠B10细胞仍可促进IL-4分泌(P0.05),但对IFN-γ分泌及Foxp3表达水平无明显影响(P0.05),而4周、8周、糖尿病NOD小鼠B10细胞对IFN-γ、IL-4分泌及Foxp3的表达水平均无明显影响(P0.05);野生型、IL-10-/-小鼠及4周、8周、糖尿病NOD小鼠B10细胞均不能抑制IL-17分泌(P0.05);(3)Transwell分隔B-T细胞接触后,野生型小鼠B10细胞能完全抑制CD4+CD25-T细胞增殖(P0.01),而4周、8周、糖尿病NOD小鼠B10细胞则不能抑制CD4+CD25-T细胞增殖(P0.05);(4)体外混合淋巴细胞共培养后,NOD小鼠B10细胞分泌IL-10较野生型小鼠明显减少(P0.01);(5)体外混合淋巴细胞共培养后,野生型小鼠及NOD小鼠B10和CD19+CD5-CD1dlow B细胞均可活化表达高水平协同刺激分子CD80、CD86,且NOD小鼠B10细胞CD80、CD86表达水平较野生型小鼠无明显差异(P0.05)。结论:(1)NOD小鼠B10细胞存在免疫抑制功能缺陷,B10细胞抑制功能缺陷可能参与了NOD小鼠自身免疫性胰岛炎及T1D的发生与进展;(2)NOD小鼠B10细胞对IFN-γ、IL-4分泌及Foxp3表达水平的调节功能存在缺陷;(3)NOD小鼠B10细胞免疫抑制功能缺陷主要由IL-10分泌减少所致;(4)野生型及NOD小鼠B10细胞均可活化表达协同刺激分子CD80、CD86,但NOD小鼠B10细胞功能缺陷与CD80、CD86表达水平无相关性。
[Abstract]:Objective to study the first part NOD diabetes in mice B10 cells during the development of the number of levels: Observation of B10 cells and Th1, NOD mice in the natural history of T1D Th17, Treg cells, and explore the relationship between the number of NOD and T1D in mice B10 cell development. Methods: 4 weeks, 8 weeks and pancreatic tissue of diabetic NOD mice fixed and pathological HE staining to evaluate insulitis; flow cytometry 4 weeks (A group), 8 weeks (group B), diabetes mellitus (C group) NOD mice and age-matched WT C57BL/6 mice (control group) of spleen, mesenteric lymph node, peripheral lymph nodes, pancreatic lymph nodes in B10, Th1 Th17, and the proportion of Treg cells. Results: (1) comparison of different age of insulitis in NOD mice: NOD mice 4 weeks without insulitis; occurrence of insulitis 8 weeks NOD mice; diabetic NOD mice have obvious insulitis and no intact islets; (2) comparison of different stages of mouse B10 cells: B and NOD C group B10 The cells were higher than the A group, C group and B10 cells were significantly decreased compared with B group, the difference was statistically significant (P0.01); no significant difference between B10 cell A group (P0.05), B10 cells in B group pancreatic lymph node is highest, B10 cell group C peripheral lymph node and pancreatic lymph node B10 the cell was lower than that in B group, the difference was statistically significant (P0.01); (3) comparison of different stages of NOD mouse Th1 and Th17 cells: Th1 and Th17 cells in C group than in A group, B increased significantly, the difference was statistically significant (P0.01); (4) comparison of different stages of NOD mouse Treg cells: Treg C cells compared with group A, group B had no obvious change, and the Treg cells of NOD mice compared with age-matched WT C57BL/6 mice also had no obvious change, the difference was not statistically significant (P0.05). Conclusion: with the development of age NOD mice and autoimmune insulitis, there is quantity variation and tissue distribution of B10 cells; B10 cells in diabetic rats NOD mice Th1 and pathogenicity, obvious immune imbalance between Th17 cells and B10 cells may be involved in the occurrence and development of the decrease in the number of NOD mice T1D. The research purpose of the second part of diabetes in NOD mice B10 cell function in the process of development: To observe the changes of B10 cells in NOD mice were immunized with T1D in the natural history of the regulatory function of B10 cells may be involved in the inquiry. The mechanism of the occurrence and development of T1D in NOD mice. Methods: flow cytometry sorting for 4 weeks, 8 weeks, diabetic NOD mice, 8-12 weeks of wild type (positive control group) and IL-10-/- mice (negative control group) and CD19+ CD5-CD1dlow B B10 spleen cells; magnetic activated cell sorting sorting marker for 4 weeks, 8 weeks, diabetes NOD mice, 8-12 weeks of WT C57BL/6 mice (positive control group) and IL-10-/- mice (negative control group) the spleen and lymph nodes of CD4+CD25-T cells, B10 cells were observed on CFSE labeled CD4+CD25-T cells by in vitro lymphocyte reaction The inhibition of proliferation, IFN- gamma, on the secretion of CD4+CD25-T IL-4 cells, expression of cytokines such as IL-17 and Foxp3. Results: (1) compared with CD19+CD5-CD1dlow B cells, wild-type mice B10 cells could inhibit the proliferation of CD4+CD25-T cells of 3-4 generation cells (P0.01), and 4 weeks, 8 weeks, mice and IL-10-/- mouse B10 cells of diabetic NOD could not inhibit the proliferation of CD4+CD25-T cells (P0.05); compared with the control group, IL-10-/- mice and wild type mice, 4 weeks, 8 weeks, diabetic NOD mice and CD19+CD5-CD1dlow B10 B cells were significantly promoted CD4+CD25-T cell proliferation (P0.01); (2) wild type mice B10 cells inhibit IFN- gamma secretion, promote the secretion of IL-4, increase the expression of Foxp3 (P0.05), IL-10-/- cells can promote IL-4 secretion of mouse B10 (P0.05), but had no significant effect on IFN- secretion and Foxp3 expression level (P0.05), and 4 weeks, 8 weeks, the IFN- gamma B10 cells in diabetic NOD mice, IL- There were no significant effects of the expression level of 4 secretion and Foxp3 (P0.05); wild type IL-10-/- mice and 4 week, 8 weeks, B10 cells in diabetic NOD mice can inhibit the secretion of IL-17 (P0.05); (3) Transwell separated B-T cells after contact with wild type mice B10 cells can inhibit the proliferation of CD4+CD25-T cells (P0.01), 4 weeks, 8 weeks, B10 cells in diabetic NOD mice can inhibit the proliferation of CD4+CD25-T cells (P0.05); (4) mixed lymphocyte co culture in vitro, B10 cells secrete IL-10 NOD mice than wild type mice significantly reduced (P0.01); (5) mixed lymphocyte co culture in vitro after activation of wild-type mice B10 and CD19+CD5-CD1dlow and NOD mice B cells can express high levels of costimulatory molecules CD80, CD86, CD80 and B10 cells in NOD mice, CD86 expression level was higher than that of wild type mice showed no significant difference (P0.05). Conclusion: (1) NOD mice B10 cells suppress immunity function defect, B10 Inhibition of dysfunctional cells may be involved in the occurrence and development of autoimmune insulitis in NOD mice and T1D; (2) the IFN- gamma NOD mouse B10 cells, regulating function level defects of secretion and expression of IL-4 Foxp3; (3) NOD mice B10 cells function defects mainly secreted by IL-10 decreased; (4 the activation of B10 cells) are wild type NOD mice and expression of costimulatory molecules CD80, CD86, but NOD B10 cells of mice with CD80 deficiency, there is no correlation between the expression level of CD86.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1

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