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Identification of cells producing antibodies specific for Helicobacter pylori in chronic atrophic gastritis : Application of the enzyme-labeled antigen method.

慢性萎缩性胃炎中产生幽门螺杆菌特异性抗体的细胞的鉴定：酶标记抗原方法的应用。

基本信息

批准号：
04670189
负责人：
TSUTSUMI Yutaka
金额：
$ 1.34万
依托单位：
Tokai University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

It has been suggested that infection of Helicobacter pylori is pathogenic for chronic atrophic gastritis. However, no infection is demonstrated on the surface of intestinal metaplasia. The present study was aimed at identifying plasma cells producing antibodies specific for H.pylori in the mucosa with chronic atrophic gastritis by using "the enzyme-labeled antigen method". [Materials and Methods] Fresh gastric mucosa was sampled. We tried to prepare the labeled antigen in the following three approaches : A) Purification and biotinylation of urease, B) synthesis of biotinylated polypeptides from the known urease amino acid sequence. c) Production of recombinant protein of maltose-binding protein (MBP) and bacterial protein. [Results] Methods for histochemical detection of H.pylori was established. However, it was very difficult to prepare the labeled antigen. The methods A) and B) failed to be achieved. A preliminary study of "the enzyme-labeled antigen method" by using a biotinylated crude bacterial extract revealed no specific findings. Thus, with the method C), a recombinant fusion protein (M.W.60kD) was obtained by inserting a bacterial DNA fragment in the MBP-vector. The MBP-labeled protein showed a reactivity with the patient serum, but, regretably enough, it was of no use for detecting the specific antibody-producing cells in frozen sections. [Comments] The present study was unique in that the site of antibody production be visualized in the gastric mucosa by the novel technology. The preparation of the labeled antigen should be the starting point of study. We are now further trying to establish this approach for clarifying the pathogenesis of the bacteria in a chronic inflammatory process in the gastric mucosa.

有人认为幽门螺杆菌感染是慢性萎缩性胃炎的致病因素。但肠化生表面未见感染。本研究旨在利用“酶标抗原法”鉴定慢性萎缩性胃炎粘膜中产生幽门螺杆菌特异性抗体的浆细胞。 [材料与方法] 取新鲜胃粘膜样本。我们尝试通过以下三种方法制备标记抗原：A）脲酶的纯化和生物素化，B）从已知的脲酶氨基酸序列合成生物素化的多肽。 c)麦芽糖结合蛋白(MBP)和细菌蛋白的重组蛋白的生产。 [结果]建立了H.pylori的组织化学检测方法。然而，标记抗原的制备非常困难。方法A)和B)未能实现。使用生物素化粗细菌提取物进行的“酶标记抗原法”的初步研究没有发现任何具体结果。因此，利用方法C)，通过将细菌DNA片段插入MBP-载体中获得了重组融合蛋白(M.W.60kD)。 MBP 标记的蛋白质显示出与患者血清的反应性，但遗憾的是，它无法用于检测冷冻切片中产生特异性抗体的细胞。 [评论] 本研究的独特之处在于通过新技术使胃粘膜中抗体产生的位点可视化。标记抗原的制备应该是研究的起点。我们现在正在进一步尝试建立这种方法，以阐明细菌在胃粘膜慢性炎症过程中的发病机制。