Study On The Effects Of Different Types Of Helicobacter Pylori On Expressions Of Cx32, Cx43 And GJIC Function In GES-1 Cells

Part 1 Study on Relationship between the Polymorphism of 3'Region of CagA of Helicobacter Pylori Strains and Gastric Duodenal DiseasesObjective To explore the relationship between the polymorphism of 3'region of CagA of Helicobacter pylori (H.pylori) strains and gastric duodenal Diseases.Methods There are 235 patients with evident upper gastrointestinal diseases in the location in study, including 57 patients with chronic gastritis (CG),62 patients with gastric ulcer (GU),70 patients with duodenal ulcer(DU),46 patients with gastric cancer. Gastric biopsy specimens were obtained by biopsy forceps.89 H.pylori strains were isolated and cultivated. The CagA gene was expanded and checked by PCR. Accroding to the variation in the carboxy terminus of EPIYA sequences of the CagA gene, the CagA positive H.pylori strains were divided into two groups:the East-Asia type and Western type. The sequence was aligned and analyzed by Bio-software.Resuls The positive identification rate of H.pylori was 37.9% (89/235), among which the positive rate of CagA was 91.7%(77/84),The positive identification rate of CagA in the GU, DU and GC group patients were statistically higher than that in CG group (P<0.05). The 3'region of 77 isolated CagA strains possessed three EPIYA motifs, and there are three mutants in the No.2 EPIYA motifs, accounting for the 18.2% of total strains(14/77). The majority of the 3'region of cagA is Eastern-asian type (88.3%). The CagA strains of East-Asia type in GU, DU and GC groups were significantly higher than that in CG group(P<0.05). All expressions of East-Asia types strains are similar to that of the type A reported by Yamaoka.Conclusion The Eastern-asian type of H.pylori cagA in Hunan region may be related to the occurrence of peptic ulcer and gastric cancer. The majority of the 3'region of cagA in Hunan region is to Eastern-asian type. All isolated strains possessed three EPIYA motifs. There are three mutants in the No.2 EPIYA motifs Part 2 Study on the Effects of Different Types of Helicobacter Pylori on Expressions of Cx32 and Cx43 and the GJIC Function in GES-1 CellsObjective To study the effects of different types of H.pylori on expressions of Connexin (Cx) 32 and Cx43 in human gastric epithelial cell line 1 (GES-1) cells and the gap junction intercellular communication (GJIC) function. To investigate the types of H.pylori those are related to the expressions of Cx32 and Cx43, and the dysfunction of GJIC.Methods Different types of H.pylori clinical strains were isolated and cultured, including the Eastern-Asian type of CagA+H.pylori, the Western type of CagA+H.pylori, and the CagA-H.pylori. We co-cultured all these H.pylori strains above with GES-1 cells for 24 hours and 48 hours, respectively. The control groups were cultured for 24 hours and 48 hours without any type of H.pylori. The indirect immunofluorescence assay (IIF) and the computer image analysis technique were used to detect the change of expressions of Cx32 and Cx43 in GES-1 cells. The change of the GJIC function in GES-1 cells was detected by the scrape-loading dye transfer technique (SLDT). The cell proliferation of each group was examined by the Methyl Tetrazolium (MTT) method.Results 1. Change of expressions of Cx32 and Cx43 in GES-1 cells. For the control group, the positive expression rates of both Cx32 and Cx43 are 100% for 24 hours and 48 hours, and the expression intensity for 48 hours was higher than that for 24 hours (P<0.05). For the CagA-H.pylori group, the positive expression rates of Cx32 in CagA-H.pylori group are 100% and 98.5% for 24 hours and 48 hours, respectively. Compared with the control group, there is no statistical difference (P>0.05). However, the expression intensity for 48 hours was lower than that in the control group (P<0.05). The positive expression rates of Cx43 and its expression intensity for 24 hours and 48 hours are lower than that in the control group (P<0.05). For the group of Western type of CagA+H.pylori, the positive expression rates of Cx32 are 100% and 93.5% for 24 hours and 48 hours, respectively. The positive expression rates of Cx43 are 100% and 88.0% for 24 hours and 48 hours, respectively. The positive expression rate for 48 hours and the expression intensity for 24 hours and 48 hours are lower than that of the control group and the CagA-H.pylori group. For the group of Eastern-Asian type of CagA+H.pylori, the positive expression rates of Cx32 are 100% and 85.50% for 24 hours and 48 hours, respectively, while the positive expression rates of Cx43 are 100% and 74.0%. The positive expression rates and expression intensity for 24 hours and 48 hours are lower than that of the control group, the CagA-H.pylori group, and the Western type of H.pylori CagA+group (P<0.05).2. Change of GJIC function. The control group showed higher GJIC function, and the fluorescent dye migrated 4-5 rows to the adjacent cells. The GJIC function of CagA-H.pylori decreased compared with the control group (P<0.05), and the fluorescent dye migrated 3 rows to the adjacent cells. The GJIC function of the Western type of CagA+H.pylori group decreased compared with the control group and CagA-H.pylori, (P<0.05), and the fluorescent dye migrated 1 row to the adjacent cells. The Eastern-Asian type of CagA+H.pylori group has lower GJIC function than that of CagA-H.pylori and the western type of CagA+H.pylori group, (P<0.05), and the fluorescent dye was confined to the area of scratched single row cells.3. Results of cell proliferation:The difference of cell proliferation between CagA-H.pylori group and the control group has no statistical significance (P>0.05). The cell proliferation of the Western type of CagA+H.pylori group for 24 hours and 48 hours are higher than that in the control group with the strain/cell ratios of 100:1 and 200:1 (P<0.05). The rising level for the group with 200:1 strain/cell ratio was more significant than that for the group with 100:1 strain/cell ratio (P<0.05). The cell proliferation of Eastern-Asian type of CagA+H.pylori group for 24 hours and 48 hours are higher than that of the control group with the strain/cell ratios of 100:1 and 200:1 (P<0.05). For the group with the ratio of 200:1, the cell proliferation is much higher than that of the group with the ratio of 100:1 (P<0.05). However, the cell proliferation of the group with the ratio of 400:1 was significantly lower than that in the control group for 48 hours (P<0.05).Conclusions 1. The H.pylori strain can down-regulate expressions of Cx32 and Cx43 in GES-1 cells and weaken the GJIC function. The effects were associated with CagA+H.pylori, especially with the Eastern-Asian type of CagA+H.pylori.2. The attenuation effect of H.pylori on the expressions of Cx32 and Cx43 in GES-1 cells enhanced with longer the culturing time.3. The promotion effect of H.pylori on the proliferation of GES-1 cells was associated with CagA virulence factor. Low density of Eastern-Asian type of H.pylori CagA+ accelerated the cell proliferation, while high density suppressed the cell proliferation.