Peyer小结中滤泡辅助性T细胞诱导B细胞分化成分泌IgA的浆细胞参与IgA肾病机制

发布时间：2018-06-09 20:55

  本文选题：IgA肾病 + Peyer小结　； 参考：《华中科技大学》2012年博士论文

【摘要】：第一部分IgAN动物模型的制备、鉴定及免疫组化 目的用黏膜免疫诱导法构建IgAN动物模型，以此模型为基础，探讨IgAN的发病机制及Peyer小结在IgAN中的作用。方法雄性BALB/C小鼠，给予牛血清白蛋白、注射葡萄球菌肠毒素B诱导IgAN模型。第0周、6周、12周分别用代谢笼收集24小时尿液测定24小时尿蛋白，并尿沉渣红细胞计数。12周去眼球收集血清，测定血清总蛋白（TP）、白蛋白（Alb）、尿素氮（BUN）、肌酐（Cr）、半胱氨酸蛋白酶抑制剂C（CysC）；肾组织IgA免疫荧光；肾组织HE、PAS及DAB染色；Peyer小结IgA、Bcl-6免疫组化。结果(1)IgAN组和Control组第0w、6w、12w24h尿蛋白定量分别为（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组第0w24h尿蛋白定量与对照相比无统计学差异(P＞0.05)，第6w、12w24h尿蛋白定量分别高于Control组(P＜0.01)。IgAN组和Control组第0w、6w、12w尿沉渣红细胞计数分别为（1.90±1.20）个/HP vs （1.30±0.95）个/HP、（1.80±0.92）个/HP vs （1.50±1.08）个/HP、（2.50±1.18）个/HP vs （2.00±0.82）个/HP，IgAN组第0w、6w、12w尿沉渣红细胞计数与Control组相比均无统计学意义(P＞0.05)。（2）IgA免疫荧光显示IgAN组40只小鼠有33只系膜区有IgA沉积，Control组40只小鼠仅有2只肾小球有少量IgA沉积，IgA沉积于肾在介导B细胞分化成为分泌IgA的浆细胞中起调节作用；Peyer小结B细胞发育并向分泌IgA浆母细胞生成明显占优势，是IgA浆母细胞生成的主要部位，为B细胞向分泌IgA的浆母细胞转化和IgAN发病提供了微环境。 第三部分Peyer小结中Tfh细胞功能测定 目的测定Peyer小结中抗牛血清白蛋白（抗-BSA）抗体水平，间接反映Peyer小结Tfh细胞功能，探讨IgA肾病Peyer小结中Tfh细胞功能的改变。方法分离Peyer小结，磁珠分选Peyer小结中的CXCR5+CD4+细胞，与自体脾细胞共培养，第8天Western blot测定上清液抗-BSA含量反映Tfh细胞的功能。结果IgAN模型组和Control组细胞培养上清液抗-BSA表达水平分别为0.58±0.10vs0.31±0.08。IgAN组Peyer小结抗－BSA浓度较Control组升高，差异有统计学意义(t=2.97，P=0.01)。结论IgAN中，Peyer小结Tfh细胞不仅百分率增加，而且功能明显增强，进一步提示Tfh细胞在介导B细胞分化成为分泌IgA的浆细胞中起重要的调节作用。 第四部分Peyer小结中Tfh细胞相关转录因子和细胞因子的表达 目的测定Peyer小结Tfh细胞分泌因子白细胞介素-21(IL-21)和转化生长因子-β1(TGF-β1) mRNA的表达，测定IL-21、Bcl-6、Blimp-1蛋白质的表达。探讨Tfh细胞相关细胞因子的功能及它们在IgAN中的作用。方法分离Peyer小结，提取总RNA、合成cDNA及进行荧光定量PCR反应，测定IL-21和TGF-β1mRNA的表达。分离Peyer小结，提取组织蛋白，Western blot测定IL-21、Bcl-6、Blimp-1蛋白质的表达。结果IgAN组和Control组IL-21和TGF-β1mRNA相对表达值分别为1.67±0.13vs1.49±0.13、1.21±0.09vs1.10±0.10。IgAN组IL-21和TGF-β1mRNA相对表达值较Control组升高，差异分别有统计学意义(t=2.730，P=0.016；t=2.416，P=0.03)。IgA组IL-21，Bcl-6和Blimp-1相对蛋白质的表达值分别为0.67±0.21，0.60±0.19和1.03±0.07；Control组IL-21, Bcl-6和Blimp-1相对蛋白质的表达值分别为0.45±0.10，0.34±0.21和0.67±0.07。IgAN组IL-21、Bcl-6和Blimp-1蛋白质相对表达值较Control组升高，差异分别有统计学意义(t=2.628，P=0.025；t=2.665，P=0.019；t=10.128，P=0.000)。结论IgA肾病Peyer小结Tfh细胞相关转化因子和细胞因子表达增加，促进B细胞向分泌IgA的浆细胞分化，Peyer小结中Tfh细胞及其相关细胞因子可能参与IgA肾病的发病。 第五部分Tfh细胞相关细胞因子促进初始B细胞分化成熟并分泌半乳糖缺乏的IgA1 目的观察IgA肾病患儿初始B细胞在Tfh细胞相关因子作用下的诱导成熟过程，探讨Tfh细胞在IgAN发病机制中的作用。方法磁珠分选IgA肾病患儿外周血初始B细胞（CD27-IgD+），加IL-21和TGF-β1等细胞因子共培养，测定细胞培养上清液J链的表达、IgA分泌、半乳糖缺乏的IgA1（Gd-IgA1）水平。结果IgA肾病组和Control组J链的表达相对值、IgA分泌、异常糖基化的IgA1分别为（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组J链的表达和IgA分泌水平与对照组相比较无统计学意义(t=1.914，P=0.076；t=0.592，P=0.563)，而IgAN组Gd-IgA1水平较Control组显著升高，差异有显著的统计学意义(t=3.182，P=0.007)。结论IgA肾病患儿外周血初始B细胞可分化成熟，并产生异常糖基化的IgA1。Tfh细胞及相关细胞因子在诱生B细胞成为分泌IgA的浆母细胞及半乳糖缺乏的IgA1过程中起关健的调节作用，，参与IgAN发病。
[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.
【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;R726.9

【参考文献】

中国期刊全文数据库 前2条

1 周金金;薛继强;;IgA肾病动物模型研究的新进展[J];中国中西医结合肾病杂志;2011年10期

2 陆慧瑜;蒋小云;陈丽植;莫樱;张巧玲;孙良忠;陈述枚;;黄芪对IgA肾病大鼠蛋白尿及肾组织表达nephrin、podocin的影响[J];实用儿科临床杂志;2011年17期

本文编号：2000800