發(fā)布時間：2018-07-04 23:21

  本文選題：視黃酸 + 視黃酸受體��； 參考：《復(fù)旦大學(xué)》2005年博士論文

【摘要】：目的與意義:維生素A缺乏導(dǎo)致免疫功能降低仍然是影響貧困地區(qū)年幼兒童健康的重要問題。視黃酸是維生素A的活性代謝產(chǎn)物,在體外實驗中它可增加B細(xì)胞抗體的產(chǎn)量,但其作用的途徑及機(jī)制還有待研究闡明。淋巴結(jié)作為人體最重要的周圍免疫器官是B細(xì)胞進(jìn)一步分化成熟,發(fā)生免疫應(yīng)答和產(chǎn)生抗體的重要場所。本課題研究視黃酸對兒童淋巴結(jié)B細(xì)胞成熟分化的影響,及其與視黃酸受體基因表達(dá)水平變化的關(guān)系,深入闡明視黃酸促進(jìn)抗體產(chǎn)生的途徑與機(jī)制。為合理補(bǔ)充維生素A以增強(qiáng)兒童抗感染免疫力提供理論依據(jù)。 方法:外科消化道畸形矯治開腹手術(shù)患兒共32例,取手術(shù)切除組織外周淋巴結(jié),作冰凍切片,采用地高辛素標(biāo)記6種視黃酸受體基因(RARα、β、γRXRα、β、γ)反義RNA探針作原位雜交,觀察視黃酸受體基因在淋巴結(jié)的表達(dá)與分布,分析其與B細(xì)胞分化成熟的關(guān)系;采用RT-熒光定量PCR對6種視黃酸受體基因在不同年齡兒童淋巴結(jié)的表達(dá)水平進(jìn)行定量分析,分析其與淋巴結(jié)B細(xì)胞個體發(fā)育的關(guān)系。淋巴結(jié)分離細(xì)胞進(jìn)行體外培養(yǎng),分為RA組,RA+視黃酸受體α(RARα)拮抗劑(Ro41-5253)組(RA+Ro),脂多糖組(LPS),(LPS+RA)組和對照組。培養(yǎng)24、48h收集細(xì)胞,采用流式細(xì)胞術(shù)檢測分析細(xì)胞表面標(biāo)志的變化,觀察B細(xì)胞的成熟分化;同時細(xì)胞提取RNA,用RT-PCR和熒光定量PCR對視黃酸受體基因的表達(dá)水平進(jìn)行定量分析。 結(jié)果:1.原位雜交結(jié)果顯示6種視黃酸受體基因在淋巴結(jié)中的淋巴細(xì)胞和網(wǎng)狀細(xì)胞中均有表達(dá),分布廣泛。2.逆轉(zhuǎn)錄—熒光定量PCR顯示在不同年齡小兒淋巴結(jié)6種視黃酸受體基因表達(dá)水平有所差異,1歲以下嬰兒受體基因表達(dá)水平普遍較低,此后隨免疫發(fā)育而升高。3.體外培養(yǎng)的兒童淋巴結(jié)B細(xì)胞在無刺激劑存在時,CD19~+IgM~+的成熟B細(xì)胞百分比隨培養(yǎng)時間逐漸增加,而相對不成熟的CD19~+IgM~-B細(xì)胞百分比逐漸減少,以48小時明顯(P0.05);體外培養(yǎng)加入視黃酸可使CD19~+IgM~+B細(xì)胞百分比進(jìn)一步升高,在培養(yǎng)24和48小時均顯著高于對照組(P均0.05);同時加入視黃酸受體拮抗劑則能完全拮抗該促進(jìn)作用。4.體外培養(yǎng)中加入視黃酸后,成熟B細(xì)胞(IgM~+IgD~+CD25~-)數(shù)量明顯高于對照(P0.05),而不成熟的B細(xì)胞(IgM~+IgD~-CD25~-)明顯低于對照(P0.05),以48小時明顯(P0.05)。5.在LPS作用同時加入視黃酸后,活化的B細(xì)胞(IgM~+CD25~+)明顯高于單加LPS組(P0.05),而靜息態(tài)的B細(xì)胞(IgM~+CD25~-)顯著降低(P0.05)。這些作用都是在3歲以下的嬰幼兒更為明顯。此外,視黃酸在促進(jìn)淋巴結(jié)中不成熟B細(xì)胞成熟分化和靜息B細(xì)胞活化的同時,也使RARα基因表達(dá)顯著
[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 偽, 尾, 緯 RXR 偽, 尾, 緯) 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 偽 (RAR 偽) 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 偽 gene in lymph nodes.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R392

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