天然免疫在小鼠肾移植模型及预致敏心脏移植模型中的作用及机制的初步探讨

发布时间：2018-04-17 18:18

本文选题：天然免疫 + MyD88　；参考：《华中科技大学》2012年博士论文

【摘要】：第一部分TLR信号通路中重要转接蛋白MyD88对小鼠肾移植存活时间及排斥反应的影响目的：探讨天然免疫反应TLR通路中转接蛋白分子MyD88对小鼠肾移植存活时间及移植肾排斥反应的影响方法：建立小鼠肾移植模型,移植小鼠分为同基因移植组：(1)供受体均为B6WT小鼠(n=3),(2)供受体均为BALB/c WT小鼠(n=3)；异基因移植组：(1)供体为B6WT小鼠,受体为BALB/cWT小鼠(n=10),(2)供体为B6WT小鼠,受体为BALB/c MyD88-/-小鼠(n=10)。观察移植小鼠存活时间；移植后第7天、14天检测外周血中血肌酐水平。移植后第7天、14天组织病理学检测移植肾淋巴细胞浸润及肾小球形态。结果：B6WT→BALB/c MyD88-/-组中90%的移植小鼠存活时间超过100天,B6WT→BALB/c WT组的平均生存时间为36.8天,两组间比较差异有统计学意义(P0.05)。移植后第7天,B6WT→BALB/c MyD88-/-组和B6WT→BALB/c WT组血肌酐水平分别是(0.302±0.07)mg/dL,(1.780±0.40)mg/dL,两组间比较差异有统计学意义(P0.05)；移植后第14天两组的血肌酐水平分别是(0.860±0.2)mg/dL,(2.778±0.4)mg/dL,两组间比较差异有统计学意义(P0.05)。在移植后第7天及14天,B6WT→BALB/c MyD88组移植物中炎症细胞浸润的程度明显低于B6WT→BALB/c WT组。B6WT→BALB/c MyD88-/-组移植肾间质中CD4+T细胞数量明显少于B6WT→BALB/c WT组,而血管周围CD4+T细胞数量,两组间无明显差异。在术后第3周,B6WT→BALB/c MyD88士组中肾小球结构完整,而B6WT→BALB/cWT组肾小球多数呈萎缩,硬化状态。结论：敲除受体MyD88基因能明显延长小鼠移植肾脏的存活时间,改善移植肾功能,可能与移植肾中淋巴细胞浸润减少和肾小球结构保持相对完整相关第二部分MyD88分子在小鼠肾移植中作用机制的初步研究目的：从T细胞功能、B细胞功能及免疫调节等方面探讨MyD88分子在小鼠肾移植中的作用。方法：建立小鼠肾移植模型,移植小鼠分为对照组：供体为B6WT小鼠,受体为BALB/cWT小鼠(n=5)。实验组：供体为B6WT小鼠,受体为BALB/c MyD88-1小鼠(n=5)。小鼠肾移植后第7天,第14天时,分离小鼠脾脏淋巴细胞进行混合淋巴细胞培养,同时用Luminex系统检测培养液上清中细胞因子含量；留取受体小鼠血清,流式细胞术检测供者特异性抗体水平；分离脾脏及移植肾脏淋巴细胞,流式细胞术检测Treg细胞比例。结果：移植后第14天,当用与供体相同种系的小鼠APC刺激时,MyD88-/-组小鼠脾脏淋巴细胞增殖能力较WT组小鼠降低(P0.05)。当用无关第三方小鼠APC刺激时,MyD88-/-组小鼠与WT小鼠组脾脏淋巴细胞增殖能力无显著性差异(P0.05)。在Naive状态下,MyD88-/-小鼠和WT小鼠脾脏淋巴细胞的增殖能力没有明显差异(P0.05)；移植后第7天及第14天MLR上清液中, MyD88-/-小鼠组IL-17,IL-6,TNF-α水平较WT组小鼠下降(P0.05),而IL-4水平升高(P0.05)。血清中DSA水平亦是MyD88-/-小鼠较WT小鼠降低,尤其以IgG2为著(P0.05),IgG1及IgG3水平亦降低,但是差异没有统计学意义(P0.05)；移植小鼠脾脏中CD4+FoxD3+T细胞及CD8+Foxp3+T细胞,MyD88-/-组与WT组之间差异无统计学意义(P0.05)。而移植肾脏中,MyD88-/-组中CD8+FOxp3+T细胞比例较WT组明显升高,差异有统计学意义(P0.05)。结论：肾移植后,与WT小鼠比较,MyD88-/-小鼠脾脏淋巴细胞的增殖能力及炎症因子的分泌能力下降；B细胞抗体产生能力下降。移植物中CD8+FOXp3+T细胞比例升高。第三部分：TLR信号通路在预致敏小鼠心脏移植模型中的作用目的探讨MyD88及Trif在小鼠预致敏模型中对血清中供体特异性抗体(Donor specific-antibodies DSA)及脾脏记忆性T细胞的影响。方法实验动物分为Naive组(不做移植手术),对照组(供体为C3H小鼠,受体为C57BL/6小鼠)和实验组(供体为C3H小鼠,受体为MyD88及Trif基因敲除小鼠)。采用皮肤移植对受体小鼠进行预致敏,2周后检测受体小鼠血清中DSA的水平,然后采用与皮肤移植相同的供体对受体小鼠行心脏移植,观察移植心脏存活时间,在观察终点时再次检测受体小鼠血清中的DSA水平,同时检测受体小鼠脾脏中记忆性T细胞的比例。结果皮肤移植2周后,实验组DSA IgG2水平低于对照组,差异有统计学意义(P＜0.05), DSA IgG1和IgG3的水平亦降低,但无显著性差异(P0.05)。心脏移植3天后,与对照组相比,实验组DSA IgG2水平明显降低(P0.01)。脾脏中记忆性T细胞的比例,实验组亦比对照组明显降低(CD4阳性记忆性T细胞P0.001,CD8阳性记忆性T细胞P0.05)。然而两组的移植心脏存活时间无明显差异。结论敲除受体MyD88及Trif虽然不能延长二次心脏移植的存活时间。但是能显著降低受体血清中DSA的水平及脾脏中记忆性T细胞的比例。
[Abstract]:The effect of important transfer protein MyD88 in the TLR signal pathway on the survival time and rejection of renal transplantation in mice
Objective: To investigate the effect of MyD88 on the survival time and graft rejection of kidney transplantation in the TLR pathway of natural immune response
Methods: to establish the mouse model of kidney transplantation, transplantation of syngeneic mice were divided into transplantation group (1) as donor and recipient B6WT mice (n=3), (2) as donor and recipient WT mice (BALB/c n=3); allogeneic transplantation group: (1) from B6WT mice, BALB/cWT mice (n=10) receptor, (2) donor B6WT mice, MyD88-/- mice of BALB/c receptor (n=10). To observe the survival time of transplanted mice; seventh days after transplantation, blood creatinine levels in peripheral blood were detected 14 days. Seventh days after transplantation, 14 days of histopathological diagnosis of renal graft infiltrating lymphocytes and glomerular morphology.
Results: B6WT and BALB/c in the MyD88-/- group of 90% mice survived more than 100 days, the average survival time of B6WT and BALB/c in WT group was 36.8 days, there were significant differences between the two groups (P0.05). Seventh days after transplantation, B6WT BALB/c, MyD88-/- B6WT, BALB/c group and WT group respectively, serum creatinine level (0.302 + 0.07) mg/dL, (1.780 + 0.40) mg/dL, there are significant differences between the two groups (P0.05); fourteenth days after transplantation in two groups of serum creatinine levels were (0.860 + 0.2) mg/dL, (2.778 + 0.4) mg/dL, there are significant differences between the two groups (P0.05). In seventh days and 14 days after transplantation, B6WT, BALB/c, MyD88 group of graft infiltrating inflammatory cells was significantly lower than the B6WT, BALB/c.B6WT, BALB/c WT group MyD88-/- group of renal transplantation between CD4+T cells in B6WT BALB/c, the number was significantly less than WT group, and the blood vessels around the number of CD4+T cells between the two groups of ignorance The glomerular structure in B6WT to BALB/c MyD88 group was complete at third weeks after the operation, and most of the glomeruli in group B6WT to BALB/cWT were atrophied and sclerosis.
Conclusion: knockout receptor MyD88 gene can significantly prolong the survival time of transplanted kidney and improve the function of transplanted kidney, which may be related to the decrease of lymphocyte infiltration and the structure of glomerulus in transplanted kidney.
Preliminary study on the mechanism of the second part of MyD88 molecule in mouse kidney transplantation
Objective: To explore the role of MyD88 in renal transplantation in mice from the function of T cell, function of B cell and immunoregulation.
Methods: to establish the mouse model of kidney transplantation, transplantation of mice were divided into control group: donor B6WT mice, BALB/cWT mice as receptor (n=5). The experimental group: donor B6WT mice, BALB/c receptor (n=5) in MyD88-1 mice. Mice after renal transplantation in seventh days, fourteenth days, isolated from mouse spleen lymphocytes were mixed lymphocyte at the same time training, Luminex system for the detection of cell culture factor in supernatant; specimens from recipient mice serum specific antibody levels were detected by flow cytometry for separation of spleen and kidney; lymphocyte, flow cytometry detection ratio of Treg cells.
Results: fourteenth days after transplantation, mice with the same APC as donor strains of stimulation, MyD88-/- group decreased mouse lymphocyte proliferation in mice than in WT group (P0.05). When using the unrelated third party mouse APC stimulation, MyD88-/- group mice and WT mice spleen lymphocyte proliferation had no significant difference (P0.05). In Naive, there was no significant difference between MyD88-/- and WT mice spleen lymphocyte proliferation (P0.05); seventh and 14 days after transplantation of MLR in the supernatant of MyD88-/- mice in groups IL-17, IL-6, TNF- decreased alpha level compared with WT mice (P0.05), and increased the level of IL-4 (P0.05) DSA level. The serum is reduced in MyD88-/- mice compared with WT mice, especially for IgG2 (P0.05), IgG1 and IgG3 levels were decreased, but the difference was not statistically significant (P0.05); CD4+FoxD3+T cells and CD8+Foxp3+T cells of spleen transplantation in mice, MyD88-/- group and WT There was no significant difference between the groups (P0.05). In the transplanted kidney, the proportion of CD8+FOxp3+T cells in MyD88-/- group was significantly higher than that in WT group, the difference was statistically significant (P0.05).
Conclusion: compared with WT mice, the proliferation and secretion of inflammatory cytokines in spleen lymphocytes of MyD88-/- mice were decreased after kidney transplantation. The antibody production ability of B cells decreased. The proportion of CD8+FOXp3+T cells in grafts increased.
The third part: the role of TLR signaling pathway in presensitized mouse heart transplantation model
Objective to investigate the effects of MyD88 and Trif on donor specific antibody (Donor specific-antibodies DSA) and spleen memory T cells in a mouse pre sensitized model.
Methods the experimental animal were divided into Naive group (no transplantation), control group (donor C3H mice, C57BL/6 receptor mice) and experimental group (donor C3H mice, MyD88 receptor and Trif gene knockout mice). The skin transplantation in recipient mice were pre sensitized to detect the level of receptor in serum of mice DSA after 2 weeks, and then using the same and skin transplantation of donor recipient mice underwent heart transplantation, observe the survival time of the transplanted heart, again was used to detect the expression of serum DSA level in the observation end point, simultaneous detection of memory T cells in recipient mice spleen ratio.
Results 2 weeks after skin transplantation, the experimental group DSA IgG2 level lower than the control group, the difference was statistically significant (P < 0.05), DSA IgG1 and IgG3 levels were decreased, but no significant difference (P0.05). 3 days after heart transplantation, compared with the control group, the experimental group DSA IgG2 were significantly lower (P0.01). Memory T cells in the spleen ratio of experimental group than the control group also decreased significantly (CD4 + memory T cells P0.001, CD8 positive memory T cell P0.05). However, no significant difference between the two groups of the cardiac allograft survival time.
Conclusion the knockout receptor MyD88 and Trif can not prolong the survival time of the two heart transplantation, but it can significantly reduce the level of DSA in the serum and the proportion of memory T cells in the spleen.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R-332

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