發(fā)布時(shí)間：2018-04-17 18:18

  本文選題：天然免疫 + MyD88�。� 參考：《華中科技大學(xué)》2012年博士論文

【摘要】：第一部分TLR信號(hào)通路中重要轉(zhuǎn)接蛋白MyD88對(duì)小鼠腎移植存活時(shí)間及排斥反應(yīng)的影響 目的：探討天然免疫反應(yīng)TLR通路中轉(zhuǎn)接蛋白分子MyD88對(duì)小鼠腎移植存活時(shí)間及移植腎排斥反應(yīng)的影響 方法：建立小鼠腎移植模型,移植小鼠分為同基因移植組：(1)供受體均為B6WT小鼠(n=3),(2)供受體均為BALB/c WT小鼠(n=3)；異基因移植組：(1)供體為B6WT小鼠,受體為BALB/cWT小鼠(n=10),(2)供體為B6WT小鼠,受體為BALB/c MyD88-/-小鼠(n=10)。觀察移植小鼠存活時(shí)間；移植后第7天、14天檢測(cè)外周血中血肌酐水平。移植后第7天、14天組織病理學(xué)檢測(cè)移植腎淋巴細(xì)胞浸潤(rùn)及腎小球形態(tài)。 結(jié)果：B6WT→BALB/c MyD88-/-組中90%的移植小鼠存活時(shí)間超過(guò)100天,B6WT→BALB/c WT組的平均生存時(shí)間為36.8天,兩組間比較差異有統(tǒng)計(jì)學(xué)意義(P0.05)。移植后第7天,B6WT→BALB/c MyD88-/-組和B6WT→BALB/c WT組血肌酐水平分別是(0.302±0.07)mg/dL,(1.780±0.40)mg/dL,兩組間比較差異有統(tǒng)計(jì)學(xué)意義(P0.05)；移植后第14天兩組的血肌酐水平分別是(0.860±0.2)mg/dL,(2.778±0.4)mg/dL,兩組間比較差異有統(tǒng)計(jì)學(xué)意義(P0.05)。在移植后第7天及14天,B6WT→BALB/c MyD88組移植物中炎癥細(xì)胞浸潤(rùn)的程度明顯低于B6WT→BALB/c WT組。B6WT→BALB/c MyD88-/-組移植腎間質(zhì)中CD4+T細(xì)胞數(shù)量明顯少于B6WT→BALB/c WT組,而血管周圍CD4+T細(xì)胞數(shù)量,兩組間無(wú)明顯差異。在術(shù)后第3周,B6WT→BALB/c MyD88士組中腎小球結(jié)構(gòu)完整,而B6WT→BALB/cWT組腎小球多數(shù)呈萎縮,硬化狀態(tài)。 結(jié)論：敲除受體MyD88基因能明顯延長(zhǎng)小鼠移植腎臟的存活時(shí)間,改善移植腎功能,可能與移植腎中淋巴細(xì)胞浸潤(rùn)減少和腎小球結(jié)構(gòu)保持相對(duì)完整相關(guān) 第二部分MyD88分子在小鼠腎移植中作用機(jī)制的初步研究 目的：從T細(xì)胞功能、B細(xì)胞功能及免疫調(diào)節(jié)等方面探討MyD88分子在小鼠腎移植中的作用。 方法：建立小鼠腎移植模型,移植小鼠分為對(duì)照組：供體為B6WT小鼠,受體為BALB/cWT小鼠(n=5)。實(shí)驗(yàn)組：供體為B6WT小鼠,受體為BALB/c MyD88-1小鼠(n=5)。小鼠腎移植后第7天,第14天時(shí),分離小鼠脾臟淋巴細(xì)胞進(jìn)行混合淋巴細(xì)胞培養(yǎng),同時(shí)用Luminex系統(tǒng)檢測(cè)培養(yǎng)液上清中細(xì)胞因子含量；留取受體小鼠血清,流式細(xì)胞術(shù)檢測(cè)供者特異性抗體水平；分離脾臟及移植腎臟淋巴細(xì)胞,流式細(xì)胞術(shù)檢測(cè)Treg細(xì)胞比例。 結(jié)果：移植后第14天,當(dāng)用與供體相同種系的小鼠APC刺激時(shí),MyD88-/-組小鼠脾臟淋巴細(xì)胞增殖能力較WT組小鼠降低(P0.05)。當(dāng)用無(wú)關(guān)第三方小鼠APC刺激時(shí),MyD88-/-組小鼠與WT小鼠組脾臟淋巴細(xì)胞增殖能力無(wú)顯著性差異(P0.05)。在Naive狀態(tài)下,MyD88-/-小鼠和WT小鼠脾臟淋巴細(xì)胞的增殖能力沒(méi)有明顯差異(P0.05)；移植后第7天及第14天MLR上清液中, MyD88-/-小鼠組IL-17,IL-6,TNF-α水平較WT組小鼠下降(P0.05),而IL-4水平升高(P0.05)。血清中DSA水平亦是MyD88-/-小鼠較WT小鼠降低,尤其以IgG2為著(P0.05),IgG1及IgG3水平亦降低,但是差異沒(méi)有統(tǒng)計(jì)學(xué)意義(P0.05)；移植小鼠脾臟中CD4+FoxD3+T細(xì)胞及CD8+Foxp3+T細(xì)胞,MyD88-/-組與WT組之間差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。而移植腎臟中,MyD88-/-組中CD8+FOxp3+T細(xì)胞比例較WT組明顯升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論：腎移植后,與WT小鼠比較,MyD88-/-小鼠脾臟淋巴細(xì)胞的增殖能力及炎癥因子的分泌能力下降；B細(xì)胞抗體產(chǎn)生能力下降。移植物中CD8+FOXp3+T細(xì)胞比例升高。 第三部分：TLR信號(hào)通路在預(yù)致敏小鼠心臟移植模型中的作用 目的探討MyD88及Trif在小鼠預(yù)致敏模型中對(duì)血清中供體特異性抗體(Donor specific-antibodies DSA)及脾臟記憶性T細(xì)胞的影響。 方法實(shí)驗(yàn)動(dòng)物分為Naive組(不做移植手術(shù)),對(duì)照組(供體為C3H小鼠,受體為C57BL/6小鼠)和實(shí)驗(yàn)組(供體為C3H小鼠,受體為MyD88及Trif基因敲除小鼠)。采用皮膚移植對(duì)受體小鼠進(jìn)行預(yù)致敏,2周后檢測(cè)受體小鼠血清中DSA的水平,然后采用與皮膚移植相同的供體對(duì)受體小鼠行心臟移植,觀察移植心臟存活時(shí)間,在觀察終點(diǎn)時(shí)再次檢測(cè)受體小鼠血清中的DSA水平,同時(shí)檢測(cè)受體小鼠脾臟中記憶性T細(xì)胞的比例。 結(jié)果皮膚移植2周后,實(shí)驗(yàn)組DSA IgG2水平低于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P＜0.05), DSA IgG1和IgG3的水平亦降低,但無(wú)顯著性差異(P0.05)。心臟移植3天后,與對(duì)照組相比,實(shí)驗(yàn)組DSA IgG2水平明顯降低(P0.01)。脾臟中記憶性T細(xì)胞的比例,實(shí)驗(yàn)組亦比對(duì)照組明顯降低(CD4陽(yáng)性記憶性T細(xì)胞P0.001,CD8陽(yáng)性記憶性T細(xì)胞P0.05)。然而兩組的移植心臟存活時(shí)間無(wú)明顯差異。 結(jié)論敲除受體MyD88及Trif雖然不能延長(zhǎng)二次心臟移植的存活時(shí)間。但是能顯著降低受體血清中DSA的水平及脾臟中記憶性T細(xì)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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R-332

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Regulation of Toll-like receptor signaling in innate immunity[J];Science China(Life Sciences);2010年01期

，

本文編號(hào)：1764725