|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2001: ¥3,000,000 (Direct Cost: ¥3,000,000)
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 mice
A 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)
, 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 diseases
The 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