视黄酸受体基因在小儿淋巴结的表达与B细胞发育的研究

发布时间：2018-07-04 23:21

本文选题：视黄酸 + 视黄酸受体　；参考：《复旦大学》2005年博士论文

【摘要】：目的与意义:维生素A缺乏导致免疫功能降低仍然是影响贫困地区年幼儿童健康的重要问题。视黄酸是维生素A的活性代谢产物,在体外实验中它可增加B细胞抗体的产量,但其作用的途径及机制还有待研究阐明。淋巴结作为人体最重要的周围免疫器官是B细胞进一步分化成熟,发生免疫应答和产生抗体的重要场所。本课题研究视黄酸对儿童淋巴结B细胞成熟分化的影响,及其与视黄酸受体基因表达水平变化的关系,深入阐明视黄酸促进抗体产生的途径与机制。为合理补充维生素A以增强儿童抗感染免疫力提供理论依据。方法:外科消化道畸形矫治开腹手术患儿共32例,取手术切除组织外周淋巴结,作冰冻切片,采用地高辛素标记6种视黄酸受体基因(RARα、β、γRXRα、β、γ)反义RNA探针作原位杂交,观察视黄酸受体基因在淋巴结的表达与分布,分析其与B细胞分化成熟的关系;采用RT-荧光定量PCR对6种视黄酸受体基因在不同年龄儿童淋巴结的表达水平进行定量分析,分析其与淋巴结B细胞个体发育的关系。淋巴结分离细胞进行体外培养,分为RA组,RA+视黄酸受体α(RARα)拮抗剂(Ro41-5253)组(RA+Ro),脂多糖组(LPS),(LPS+RA)组和对照组。培养24、48h收集细胞,采用流式细胞术检测分析细胞表面标志的变化,观察B细胞的成熟分化;同时细胞提取RNA,用RT-PCR和荧光定量PCR对视黄酸受体基因的表达水平进行定量分析。结果:1.原位杂交结果显示6种视黄酸受体基因在淋巴结中的淋巴细胞和网状细胞中均有表达,分布广泛。2.逆转录—荧光定量PCR显示在不同年龄小儿淋巴结6种视黄酸受体基因表达水平有所差异,1岁以下婴儿受体基因表达水平普遍较低,此后随免疫发育而升高。3.体外培养的儿童淋巴结B细胞在无刺激剂存在时,CD19~+IgM~+的成熟B细胞百分比随培养时间逐渐增加,而相对不成熟的CD19~+IgM~-B细胞百分比逐渐减少,以48小时明显(P0.05);体外培养加入视黄酸可使CD19~+IgM~+B细胞百分比进一步升高,在培养24和48小时均显著高于对照组(P均0.05);同时加入视黄酸受体拮抗剂则能完全拮抗该促进作用。4.体外培养中加入视黄酸后,成熟B细胞(IgM~+IgD~+CD25~-)数量明显高于对照(P0.05),而不成熟的B细胞(IgM~+IgD~-CD25~-)明显低于对照(P0.05),以48小时明显(P0.05)。5.在LPS作用同时加入视黄酸后,活化的B细胞(IgM~+CD25~+)明显高于单加LPS组(P0.05),而静息态的B细胞(IgM~+CD25~-)显著降低(P0.05)。这些作用都是在3岁以下的婴幼儿更为明显。此外,视黄酸在促进淋巴结中不成熟B细胞成熟分化和静息B细胞活化的同时,也使RARα基因表达显著
[Abstract]:Objective and significance: vitamin A deficiency leads to decreased immune function is still an important problem affecting the health of young children in poor areas. Retinoic acid is the active metabolite of vitamin A. it can increase the production of B cell antibody in vitro, but the mechanism of its action remains to be elucidated. As the most important peripheral immune organ, lymph node is an important place for B cell differentiation and maturation, immune response and antibody production. The aim of this study was to investigate the effect of retinoic acid on the maturation and differentiation of B cells in children's lymph nodes and its relationship with the expression level of retinoic acid receptor gene, and to elucidate the pathway and mechanism of retinoic acid promoting antibody production. To provide a theoretical basis for rational supplementation of vitamin A to enhance children's anti-infection immunity. Methods: Thirty-two children with surgical alimentary tract malformation were treated with laparotomy. Peripheral lymph nodes were removed and used as frozen sections. Six antisense RNA probes of retinoic acid receptor genes (RAR 伪, 尾, 纬 RXR 伪, 尾, 纬) were used for in situ hybridization. To observe the expression and distribution of retinoic acid receptor gene in lymph nodes, to analyze the relationship between retinoic acid receptor gene expression and B cell differentiation and maturation, and to quantitatively analyze the expression level of six retinoic acid receptor genes in lymph nodes of children of different ages by RT-PCR. To analyze the relationship between B cell ontogeny and lymph node. Lymph node isolated cells were cultured in vitro and divided into RA group with RA receptor 伪 (RAR 伪) antagonist (Ro41-5253), lipopolysaccharide (LPS-LPS-RA) group and control group. The cells were collected for 24 hours for 48 hours. The changes of cell surface markers were detected by flow cytometry, and the maturation and differentiation of B cells were observed. At the same time, RNA was extracted from the cells, and the expression level of retinoic acid receptor gene was quantitatively analyzed by RT-PCR and fluorescence quantitative PCR. The result is 1: 1. The results of in situ hybridization showed that six retinoic acid receptor genes were expressed in lymphocytic and reticular cells of lymph nodes and were widely distributed. Reverse transcription-fluorescence quantitative PCR (RT-PCR) showed that there were differences in the expression levels of six retinoic acid receptor genes in the lymph nodes of children of different ages. The expression level of receptor genes in infants under 1 year old was generally lower, and then increased with the immune development. The percentage of mature B cells of CD19 ~ IgM~ was increased with the culture time, but the percentage of relatively immature CD19 ~ IgM- B cells decreased gradually in the absence of stimulant. The percentage of CD19 ~ IgMB cells was further increased by adding retinoic acid at 48 hours (P0.05), and the percentage of CD19 ~ IgMB cells was significantly higher at 24 and 48 hours of culture than that of the control group (P 0.05), and retinoic acid receptor antagonist could completely antagonize the effect of retinoic acid receptor antagonist on the proliferation of CD19 ~ IgMB cells (P < 0.05). After the addition of retinoic acid in vitro, the number of mature B cells (IgM- IgD- CD25-) was significantly higher than that of the control (P0.05), while that of immature B cells (IgM- IgD- CD25-) was significantly lower than that of the control (P0.05), especially at 48 hours (P0.05). After the addition of retinoic acid to LPS, the activated B cells (IgM- CD25-) were significantly higher than those of LPS alone (P0.05), while the resting B cells (IgM- CD25-) were significantly decreased (P0.05). These effects were more pronounced in infants under 3 years of age. In addition, retinoic acid not only promoted immature B cell differentiation and resting B cell activation, but also significantly expressed RAR 伪 gene in lymph nodes.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392

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