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Expression of Gut Hormone Gene in Gastric Mucosa Obtained by Endoscopic Biopsy

内镜活检胃粘膜中肠道激素基因的表达

基本信息

批准号：
01570386
负责人：
KANEKO Eizo
金额：
$ 1.22万
依托单位：
Hamamatsu 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：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01570386/
关键词：
Peptic ulcer Gastrin mRNA Northern blot hybridization Polymerase chain reaction Polymerase Chain Reaction法ガストリン mRNA 合成DNAプロ-ブ

项目摘要

We developed a plain polymerase chain reaction (PCR) technique sensitive enough to detect gastrin mRNA in gastric antral mucosa obtained from the patients of peptic ulcer by endoscopic biopsy. From only 1 mug of total cellular RNA we can detect gastrin mRNA.Gastric antral mucosas were obtained by endoscopic biopsy in the patients with peptic ulcers. We can obtain 20 mug of total cellular RNA form 40 mg of gastric antral mucosa. We chose beta-actin as our internal control because, as a major component of the cytoskeletal system, it is transcribed in most eukaryotic cells. Northern blot hybridizations for gastrin mRNA and beta-actin were carried out using synthetic oligodeoxyribonucleotides. Beta-actin mRNAs (2.0 kb) were detected in all specimens examined, but gastrin mRNAs (0.5 kb) was detected in half of them by Northern hybridization. We used 1 mug of total RNA in our gastrin PCR assay. Total cellular RNA was treated with DNase to remove any contaminating DNA. We also chose beta-actin as internal control of amplification. Gastrin and beta-actin transcripts can be simultaneously amplified from gastric antral mucosa. The ratio of PCR-amplified gastrin to beta-actin signal was well correlated with the ratio of gastrin to beta-actin signal in Northern blot hybridization.

我们建立了一种简便、灵敏的聚合酶链式反应(PCR)技术，用于检测消化性溃疡患者胃窦黏膜组织中胃泌素的表达。仅从1杯细胞总RNA中即可检测到胃泌素基因的表达。消化性溃疡患者胃窦粘膜经胃镜活检获得胃窦粘膜。从40 mg胃窦粘膜中可获得20µg的细胞总RNA。我们选择β-肌动蛋白作为我们的内部对照，因为作为细胞骨架系统的主要组成部分，它在大多数真核细胞中转录。用人工合成的寡核苷酸进行胃泌素mRNA和β-肌动蛋白的Northern杂交。Northern杂交结果显示，所有受检标本均检测到β-肌动蛋白mRNAs(2.0kb)，但有一半的标本检测到胃泌素mRNAs(0.5kb)。在我们的胃泌素聚合酶链式反应中，我们使用了1杯总RNA。用DNA酶处理细胞总RNA以去除任何污染的DNA。我们还选择了β-肌动蛋白作为扩增的内对照。胃窦粘膜可同时扩增出胃泌素和β-肌动蛋白的转录本。在Northern杂交中，PCR扩增的胃泌素与β-肌动蛋白信号的比值与胃泌素与β-肌动蛋白信号的比值具有很好的相关性。