Toll-like receptor polymorphism and H.pylori-associated gastric mucosal lesions

1.体外细胞动力学评价系统的建立在进行最初发明的5维数字时间推移荧光显微术之前，应用用于在培养细胞的单层片上产生无细胞区域的材料（日本国内专利：2001 - 328556）。应用图像分析软件可以实时、连续地观察和定量地评价创伤后细胞迁移的过程.在自发性TLR4（Toll样受体-4）缺陷的C3H/HeJ小鼠中幽门螺杆菌相关的胃粘膜损伤形成将幽门螺杆菌（悉尼株; SS 1）的液体培养物悬浮液接种到具有TLR4基因点突变的C3H/HeJ小鼠和它们的正常同窝出生的C3H/HeN小鼠中。胃粘膜髓过氧化物酶（MPO）活性（组织相关多形核细胞蓄积的标志物）和胃粘膜硫代巴比妥酸反应物质（TBARS）含量 ...更多信息 然后测量脂质过氧化反应的标志物。感染H.pylori的C3H/HeJ小鼠胃粘膜MPO活性和TBARS含量均高于C3H/HeN小鼠，提示TLR4介导的信号通路可能在胃粘膜保护中发挥作用。两株菌接种幽门螺杆菌源性脂多糖（LPS）的效果无差异. TLR4多态性与幽门螺杆菌相关胃粘膜疾病本临床研究方案于2002年4月2日获得庆应义塾大学伦理委员会批准（No.13-93）。幽门螺杆菌阳性的慢性胃炎患者被招募参加这项研究，在获得他们的书面知情同意书后开始。从患者中获得10 ml血液样品用于DNA提取。基于最近报道的日本SNP（单核苷酸多态性）数据库，JSNP，探讨TLR4基因的遗传多态性。筛选出12个候选的TLR4多态性突变区域。根据异源双链水平，在启动子区检测到一个多态性，即A → G突变（GenBank基因组序列号2209，AF177765），在内含子区检测到一个多态性，即G → T突变（GenBank基因组序列号11771，AF177765）。通过热变性高效液相色谱法检查每种多态性。基因分型结果显示，它们之间有很强的连锁关系。2209A和2209G等位基因频率分别为71%和29%。2209 A的同源双链为47%，2209 A/G的异源双链为47%，2209 G的同源双链为5%，用Hardy-Weinberg平衡测量。对14例胃体优势型胃炎和24例胃窦优势型胃炎患者进行了TLR4基因多态性检测。幽门螺杆菌相关性胃炎的这种地形差异与TLR4基因多态性无显著相关性（p = 0.52）。少

1. Establishment of an in-vitro cell dynamics evaluation system.The materials for producing a cell-free area on a monolayer sheet of cultured cells (applied for Japanese domestic patent : 2001-328556) were applied before performing the originally invented, 5-dimensional digital time-lapse fluorescence microscopy. The real-time continuous process of cell migrartion after the wounding could be visualized and assessed quantitatively by the application of an image-analysis software.2. H.pylori-associated gastric mucosal lesion formation in spontaneously TLR4 (Toll-like receptor-4) -deficient C3H/HeJ miceA liquid-culture suspension of H.pylori (Sydney strain ; SS1) was inoculated into both C3H/HeJ mice, which have a point-mutation of the TLR4 gene, and C3H/HeN mice, their normal littermates. The gastric mucosal myeloperoxidase (MPO) activity, a marker for tissue-associated polymorphonuclear cell accumulation, and the gastric mucosal content of thiobarbituric acid-reactive substances (TBARS) … More , a marker of lipid peroxidation, were then measured. Both the MPO activity and the TBARS content were detected to be higher in H.pylori-colonized C3H/HeJ mice than in C3H/HeN mice, suggesting that signals transduced through TLR4 could play a role in gastric mucosal protection. There were no differences in the effects of inoculation of H.pylori-derived lipopolysaccharide (LPS) between the two strains.3. TLR4 polymorphism and H.pylori-associated gastric mucosal diseasesThe protocol of the present clinical study was approved by the Keio University Ethics Committee on April 2, 2002 (No.13-93). Patients with H.pylori-positve chronic gastritis were recruited for the study, which was begun after obtaining their written informed consent. Ten-ml blood samples were obtained from the patients for DNA extraction. Genetic polymorphism of the TLR4 gene was explored based on the recently reported database for Japanese SNP (single nucleotide polymorphism), JSNP. Twelve candidate regions for TLR4 polymorphic mutation were selected. Finally, according to the heteroduplex levels, one polymorphism in the promoter region, an A-to-G mutation (GenBank genome sequence No.2209, AF177765), and one polymorphism in the intron, a G-to-T mutation (GenBank genome sequence No.11771, AF177765) were detected. Each polymorphism was examined by heat-denatured high performance liquid chromatography. The results of genetic typing revealed that they bore a strong linkage with each other. The frequency of the 2209A allele and 2209G allele was 71 % and 29%, respectively. The homoduplex for 2209A was 47%, the heteroduplex for 2209A/G was 47%, and the homodupelx for 2209G was 5%, as measured with the Hardy-Weinberg balance. Fourteen patients with corpus-predominant gastritis and 24 patients with antral-predominant gastritis were examined for TLR4 genetic polymorphism. Such topographic differences in H.pylori-associated gastritis were not related significantly to TLR4 genetic polymorphism (p=0.52). Less