| Objective:1. To characterize the variants of the 3'variable region of cytotoxin-associated gene A (cagA) in Helicobacter pylori strains isolated from tissues of human gastric mucosa and to express recombinant CagA by prokaryotic expression system in E. coli BL21 (DE3) pLysS, in order to investigate the different effects between the two kinds of recombinant protein in the apoptosis of human gastric epithelial cells.2. To analyze the changes of the mononuclear cells isolated from the peripheral blood of C57BL/6 mice after H.pylori (Sydney strains, SS1) infection and analysis the leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) molecules expressed on the surface of them.Methods:1. Gastric mucosal specimens obtained from different patients with gastroduodenal diseases, were grinded and inoculated onto Kamali plates at 37? for 72 hours under microaerophilic condition. Cultures were identified as H. pylori by colonial morphology, Gram staining, rapid urease test (RUT), oxidase test and Catalase test.2. Genomic DNA of these strains was extracted and primers were designed by Primer Premier 5.0 according to the sequence of the 3'variable regions of cagA genes from the international standard strains. The 3'regions of cagA genes were amplified by PCR and the products were sequenced after gel extraction, then the amino acid sequences were analysis by the program MegAlign of DNAStar 5 software.3. Primers were designed by Primer Premier5.0 and mismatches were introduced into primers, mutagenesis was performed in a three-step PCR. In addition, we also cloned the wild-type cagA gene. The amplified fragments were sequenced and analyzed, sub-cloned into the pET-30a(+) vector and transformed into E. coli BL21(DE3) pLysS. The recombinant transformants were induced by IPTG and detected by SDS-PAGE. The purified CagA proteins (W-CagA and M-CagA) were co-cultured with GES-1 cells in different concentrations and the apoptosis rates of GES-1 cells were detected by flow cytometry at 3h?12h and 24h after co-culture.4. A mouse model of H. pylori infection was constructed by intragastric inoculation of H. pylori SS1 to the C57BL/6 mice, and then peripheral blood mononuclear cells were isolated through the inner canthus sampling. The PBMC and LAIR-1 expression levels on the surface of them were analyzed by flow cytometry in the 6th week after gavage. Isolate and culture of H. pylori in the gastric mucosa and then cultures were identified as H. pylori through several methods, such as the colony characteristics, Gram staining, PCR, rapid urease test, oxidase test and peroxidase test identification.Results:1. Monoclonal cells were grown on the Karmali plates after 72 hours under microaerophilic condition, and which were identified as Gram-negative bacteria by microscope observation. A total of 66 strains of H. pylori were determined by the urease test, oxidase tests and peroxidase test.2. All the 66 H. pylori strains were cagA-positive (cagA+), of which 93.9% (62/66) belonged to East Asian type with tyrosine phosphorylation motifs (TPMs) EPIYA-ABD. The remainder belonged to Western type, in which TPMs were EPIYA-ABC.3.2%(2/62) of the East Asian type were mutated into ESIYA-B, whereas all four Western type (100%) strains were mutated into EPIYT-B. Of the patients with gastric cancer in our study,88.9%(16/18) carried H. pylori with the East Asian cagA and the remaining 11.1%(2/18) had the genotypes of Western type. There were 95.8%(46/48) East Asian and 4.2%(2/48) Western genotype strains isolated from non-gastric cancer patients. The K (lysine) had changed to E (glutamate) in Western-type cagA of gastric cancer patients.3. According to the DNA sequencing, the nucleotide sequence of cagA of H. pylori ATCC26695 was successfully changed from A to G at the target site. And the prokaryotic expression vector pET-30a(+)-CagA was efficiently transformed into E. coli BL21(DE3) pLysS. The recombinant protein has a distinct band at the relative molecular weight of 30 kDa when detected by SDS-PAGE, which is consistent with the fusion protein of 6His-CagA.4. The recombinant protein co-cultured with cells found that M-CagA protein can increase apoptosis rate of the GES-1 cells with time extending. High concentrations of the recombinant protein can induced apoptosis rate, compared with the low concentration group, whether M-CagA or W-CagA. While M-CagA can inhibit cell apoptosis compared with W-CagA at the same time and the same concentration.5. The mice have been infected by H. pylori after 6 weeks, which were determined by Gram staining, urease test, oxidase and peroxidase test were all positive. A total of 21 strains (91.3%,21/23) were identified as H. pylori strains.6. The expression rate of monocytes in mouse model infected with H. pylori was decreased to 22.85±3.56%, compared with PBS group (49.85±0.35%) (P< 0.05); however, the expression rate of lymphocytes in mouse model infected with H. pylori was increased (40.93±10.05%), as compared with PBS group (30.15±0.35%) (P< 0.05). The expression rate of monocyte LAIR-1 in the group infected with H. pylori was decreased to 70.16±7.76%, compared with PBS group (83.4±0.1%) (P< 0.05); the LAIR-1 expression rate of lymphocytes infected with H. pylori was also decreased (56.81±11.37%), as compared with PBS group (72.3±0.6%) (P< 0.05).Conclusion:1. The overwhelming majority cagA type in these H. pylori strains isolated from Weihai patients was East Asian. There exist mutations in both kinds of the East Asian and Western type strains,2 strains with Western-type cagA obtained from gastric cancer patients contained a single nucleotide polymorphism (SNP).2. High concentrations of the recombinant protein can induced apoptosis rate, M-CagA protein can increase apoptosis rate of the GES-1 cells with time extending. While M-CagA can increase cell apoptosis compared with W-CagA at the same time and the same concentration.3. The expression rate of the monocytes in the PBMC of mice were decreased after the H. pylori colonization, but H. pylori infection can increase the expression of the lymophocytes. LAIR-1 expression on the monocytes and the leukomonocytes of PBMC were both decreased after the H. pylori colonization. |
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