本文選題：IgA腎病 + Peyer小結(jié)　； 參考：《華中科技大學(xué)》2012年博士論文

【摘要】：第一部分IgAN動(dòng)物模型的制備、鑒定及免疫組化 目的用黏膜免疫誘導(dǎo)法構(gòu)建IgAN動(dòng)物模型，以此模型為基礎(chǔ)，探討IgAN的發(fā)病機(jī)制及Peyer小結(jié)在IgAN中的作用。方法雄性BALB/C小鼠，給予牛血清白蛋白、注射葡萄球菌腸毒素B誘導(dǎo)IgAN模型。第0周、6周、12周分別用代謝籠收集24小時(shí)尿液測(cè)定24小時(shí)尿蛋白，并尿沉渣紅細(xì)胞計(jì)數(shù)。12周去眼球收集血清，測(cè)定血清總蛋白（TP）、白蛋白（Alb）、尿素氮（BUN）、肌酐（Cr）、半胱氨酸蛋白酶抑制劑C（CysC）；腎組織IgA免疫熒光；腎組織HE、PAS及DAB染色；Peyer小結(jié)IgA、Bcl-6免疫組化。結(jié)果(1)IgAN組和Control組第0w、6w、12w24h尿蛋白定量分別為（0.61±0.26）mg vs （0.67±0.22）mg、（3.61±0.75）mg vs （0.77±0.27）mg、（4.15±0.82）mg vs （1.09±0.51）mg，IgAN組第0w24h尿蛋白定量與對(duì)照相比無(wú)統(tǒng)計(jì)學(xué)差異(P＞0.05)，第6w、12w24h尿蛋白定量分別高于Control組(P＜0.01)。IgAN組和Control組第0w、6w、12w尿沉渣紅細(xì)胞計(jì)數(shù)分別為（1.90±1.20）個(gè)/HP vs （1.30±0.95）個(gè)/HP、（1.80±0.92）個(gè)/HP vs （1.50±1.08）個(gè)/HP、（2.50±1.18）個(gè)/HP vs （2.00±0.82）個(gè)/HP，IgAN組第0w、6w、12w尿沉渣紅細(xì)胞計(jì)數(shù)與Control組相比均無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05)。（2）IgA免疫熒光顯示IgAN組40只小鼠有33只系膜區(qū)有IgA沉積，Control組40只小鼠僅有2只腎小球有少量IgA沉積，IgA沉積于腎在介導(dǎo)B細(xì)胞分化成為分泌IgA的漿細(xì)胞中起調(diào)節(jié)作用；Peyer小結(jié)B細(xì)胞發(fā)育并向分泌IgA漿母細(xì)胞生成明顯占優(yōu)勢(shì)，是IgA漿母細(xì)胞生成的主要部位，為B細(xì)胞向分泌IgA的漿母細(xì)胞轉(zhuǎn)化和IgAN發(fā)病提供了微環(huán)境。 第三部分Peyer小結(jié)中Tfh細(xì)胞功能測(cè)定 目的測(cè)定Peyer小結(jié)中抗牛血清白蛋白（抗-BSA）抗體水平，間接反映Peyer小結(jié)Tfh細(xì)胞功能，探討IgA腎病Peyer小結(jié)中Tfh細(xì)胞功能的改變。方法分離Peyer小結(jié)，磁珠分選Peyer小結(jié)中的CXCR5+CD4+細(xì)胞，與自體脾細(xì)胞共培養(yǎng)，第8天Western blot測(cè)定上清液抗-BSA含量反映Tfh細(xì)胞的功能。結(jié)果IgAN模型組和Control組細(xì)胞培養(yǎng)上清液抗-BSA表達(dá)水平分別為0.58±0.10vs0.31±0.08。IgAN組Peyer小結(jié)抗－BSA濃度較Control組升高，差異有統(tǒng)計(jì)學(xué)意義(t=2.97，P=0.01)。結(jié)論IgAN中，Peyer小結(jié)Tfh細(xì)胞不僅百分率增加，而且功能明顯增強(qiáng)，進(jìn)一步提示Tfh細(xì)胞在介導(dǎo)B細(xì)胞分化成為分泌IgA的漿細(xì)胞中起重要的調(diào)節(jié)作用。 第四部分Peyer小結(jié)中Tfh細(xì)胞相關(guān)轉(zhuǎn)錄因子和細(xì)胞因子的表達(dá) 目的測(cè)定Peyer小結(jié)Tfh細(xì)胞分泌因子白細(xì)胞介素-21(IL-21)和轉(zhuǎn)化生長(zhǎng)因子-β1(TGF-β1) mRNA的表達(dá)，測(cè)定IL-21、Bcl-6、Blimp-1蛋白質(zhì)的表達(dá)。探討Tfh細(xì)胞相關(guān)細(xì)胞因子的功能及它們?cè)贗gAN中的作用。方法分離Peyer小結(jié)，提取總RNA、合成cDNA及進(jìn)行熒光定量PCR反應(yīng)，測(cè)定IL-21和TGF-β1mRNA的表達(dá)。分離Peyer小結(jié)，提取組織蛋白，Western blot測(cè)定IL-21、Bcl-6、Blimp-1蛋白質(zhì)的表達(dá)。結(jié)果IgAN組和Control組IL-21和TGF-β1mRNA相對(duì)表達(dá)值分別為1.67±0.13vs1.49±0.13、1.21±0.09vs1.10±0.10。IgAN組IL-21和TGF-β1mRNA相對(duì)表達(dá)值較Control組升高，差異分別有統(tǒng)計(jì)學(xué)意義(t=2.730，P=0.016；t=2.416，P=0.03)。IgA組IL-21，Bcl-6和Blimp-1相對(duì)蛋白質(zhì)的表達(dá)值分別為0.67±0.21，0.60±0.19和1.03±0.07；Control組IL-21, Bcl-6和Blimp-1相對(duì)蛋白質(zhì)的表達(dá)值分別為0.45±0.10，0.34±0.21和0.67±0.07。IgAN組IL-21、Bcl-6和Blimp-1蛋白質(zhì)相對(duì)表達(dá)值較Control組升高，差異分別有統(tǒng)計(jì)學(xué)意義(t=2.628，P=0.025；t=2.665，P=0.019；t=10.128，P=0.000)。結(jié)論IgA腎病Peyer小結(jié)Tfh細(xì)胞相關(guān)轉(zhuǎn)化因子和細(xì)胞因子表達(dá)增加，促進(jìn)B細(xì)胞向分泌IgA的漿細(xì)胞分化，Peyer小結(jié)中Tfh細(xì)胞及其相關(guān)細(xì)胞因子可能參與IgA腎病的發(fā)病。 第五部分Tfh細(xì)胞相關(guān)細(xì)胞因子促進(jìn)初始B細(xì)胞分化成熟并分泌半乳糖缺乏的IgA1 目的觀察IgA腎病患兒初始B細(xì)胞在Tfh細(xì)胞相關(guān)因子作用下的誘導(dǎo)成熟過(guò)程，探討Tfh細(xì)胞在IgAN發(fā)病機(jī)制中的作用。方法磁珠分選IgA腎病患兒外周血初始B細(xì)胞（CD27-IgD+），加IL-21和TGF-β1等細(xì)胞因子共培養(yǎng)，測(cè)定細(xì)胞培養(yǎng)上清液J鏈的表達(dá)、IgA分泌、半乳糖缺乏的IgA1（Gd-IgA1）水平。結(jié)果IgA腎病組和Control組J鏈的表達(dá)相對(duì)值、IgA分泌、異常糖基化的IgA1分別為（0.85±0.16）vs（0.63±0.28）、（6.64±0.85） pg/ml vs （6.43±0.51） pg/ml、（85.93±7.91）U/ml vs （73.1±8.24）U/ml。IgAN組J鏈的表達(dá)和IgA分泌水平與對(duì)照組相比較無(wú)統(tǒng)計(jì)學(xué)意義(t=1.914，P=0.076；t=0.592，P=0.563)，而IgAN組Gd-IgA1水平較Control組顯著升高，差異有顯著的統(tǒng)計(jì)學(xué)意義(t=3.182，P=0.007)。結(jié)論IgA腎病患兒外周血初始B細(xì)胞可分化成熟，并產(chǎn)生異常糖基化的IgA1。Tfh細(xì)胞及相關(guān)細(xì)胞因子在誘生B細(xì)胞成為分泌IgA的漿母細(xì)胞及半乳糖缺乏的IgA1過(guò)程中起關(guān)健的調(diào)節(jié)作用，，參與IgAN發(fā)病。
[Abstract]:Part one: preparation, identification and immunohistochemistry of IgAN animal models.
Objective to construct IgAN animal model with mucosal immune induction, based on this model, the pathogenesis of IgAN and the role of Peyer nodules in IgAN were discussed. Methods male BALB/C mice were given bovine serum albumin and staphylococcal enterotoxin B induced IgAN model. Zeroth weeks, 6 weeks and 12 weeks respectively, the urine samples were collected by metabolic cage for 24 hours, respectively. Urine protein, and urine sediment red cell count for.12 weeks to collect blood serum, serum total protein (TP), albumin (Alb), urea nitrogen (BUN), creatinine (Cr), cysteine protease inhibitor C (CysC), renal tissue IgA immunofluorescence, renal tissue HE, PAS and DAB staining; Peyer nodules, immunohistochemistry. Results (1) subordinate group and subordinate group Societies The quantitative urine protein of 12w24h was (0.61 + 0.26) mg vs (0.67 + 0.22) Mg, (3.61 + 0.75) mg vs (0.77 + 0.27) Mg, (4.15 + 0.82) mg vs (1.09 + 0.51) Mg, IgAN group of 0w24h proteinuria was not significantly different from that of photography. The red cell count of the sediment was (1.90 + 1.20) /HP vs (1.30 + 0.95) /HP, (1.80 + 0.92) /HP vs (1.50 + 1.08) /HP, (2.50 + 1.18) /HP vs (2 + 0.82) /HP, IgAN group 0W. There were IgA deposits in the mesangial region, in group Control, only a small amount of IgA was deposited in 2 glomeruli in 40 mice, and IgA was deposited in the kidneys that mediate the differentiation of B cells into IgA secreting plasma cells; Peyer nodules, B cells developed, were dominant in the production of IgA plasma mother cells, which were the main parts of IgA plasma mother cells, and the B cells secreted I. GA provides a microenvironment for plasma cell transformation and IgAN pathogenesis.
Determination of Tfh cell function in the third part of Peyer nodules
Objective to determine the level of anti bovine serum albumin (anti -BSA) antibody in Peyer nodules, to indirectly reflect the function of Peyer nodules in Tfh cells and to explore the changes of Tfh cell function in Peyer nodules of IgA nephropathy. Methods to separate Peyer nodules, magnetic beads were used to separate CXCR5+CD4+ cells from Peyer nodules, co culture with autologous splenocytes, and the eighth day Western blot assay supernatant was resistant to inhibition. The content of SA reflected the function of Tfh cells. Results the anti -BSA expression level of the supernatant in the IgAN model group and the Control group was 0.58 + 0.10vs0.31 + 0.08.IgAN group, and the anti BSA concentration was higher than that of the Control group, and the difference was statistically significant (t=2.97, P=0.01). These results suggest that Tfh cells play an important role in regulating the differentiation of B cells into plasma cells secreting IgA.
The fourth part is the expression of Tfh cell related transcription factors and cytokines in Peyer summary.
Objective to determine the expression of interleukin -21 (IL-21) and transforming growth factor - beta 1 (TGF- beta 1) mRNA, the secretory factor of Tfh cells, to determine the expression of IL-21, Bcl-6, and Blimp-1 protein. The function of Tfh cell related cytokines and their role in IgAN were investigated. The expression of IL-21 and TGF- beta 1mRNA was measured by PCR reaction. The expression of IL-21, Bcl-6, Blimp-1 protein was determined by separating Peyer nodules, extracting tissue protein and Western blot. Compared with the Control group, the difference was statistically significant (t=2.730, P=0.016, t=2.416, P=0.03).IgA group IL-21, Bcl-6 and Blimp-1 relative protein expression values were 0.67 + 0.21,0.60 + 0.19 and 1.03 + 0.07, respectively, Control group IL-21, 0.45 + 0.21 and 0.67 + 1, the relative expression values of Bcl-6 and Blimp-1 were higher than those in the Control group, and the differences were statistically significant (t=2.628, P=0.025; t=2.665, P=0.019; t=10.128, P=0.000). Conclusion IgA nephropathy Peyer nodule Tfh cell related conversion factor and cytokine expression are increased. Its related cytokines may be involved in the pathogenesis of IgA nephropathy.
The fifth part of Tfh cell related cytokines promotes the differentiation and maturation of initial B cells and secretes IgA1 of galactose deficiency.
Objective To observe the induction of maturation of initial B cells under the action of Tfh cell related factors in children with IgA nephropathy, and to explore the role of Tfh cells in the pathogenesis of IgAN. Methods magnetic beads were used to separate initial B cells (CD27-IgD+) of peripheral blood of children with IgA nephropathy and co culture of IL-21 and TGF- beta 1, and the expression of J chain in cell culture supernatant was determined, and IgA fractions were determined. IgA1 (Gd-IgA1) level of lacked galactose. Results the relative value of expression of J chain in IgA nephropathy group and Control group, IgA secretion, the Abnormal Glycosylated IgA1 was (0.85 + 0.16) vs (0.63 + 0.28), (6.64 + 0.85) pg/ml vs (6.43 + 0.51) pg/ml, (85.93 + 7.91) (85.93 + 7.91) (73.1 + 8.24)) expression and secretion level compared with the control group There was no statistical significance (t=1.914, P=0.076, t=0.592, P=0.563), while the Gd-IgA1 level in IgAN group was significantly higher than that in the Control group (t=3.182, P=0.007). Conclusion the initial B cells in peripheral blood of children with IgA nephropathy can differentiate and mature, and produce abnormal glycosylated IgA1.Tfh cells and related cytokines in the induced cells. Guan Jian plays a regulatory role in the process of IgA1 secreting IgA's plasma cells and galactose deficiency, and is involved in the pathogenesis of IgAN.
【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;R726.9

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