Experimental And Clinical Study About Effects Of CagA～+ Helicobacter Pylori Strain On Cell Gap Junction Of Gastric Epithelial Cells

Part 1 Experimental study about effects of CagA~+ Helicobacter pylori strain on cell gap junction of gastric epithelial cellsObjective To observe the effects of CagA~+ Helicobacter pylori (H.pylori) strain and anti-H.pylori drugs on expression of Cx32, Cx43, cell proliferation and cell gap junction ultrastructure of gastric epithelial cells in vitro. To investigate the relation between the changes of expression of Cx, cell gap junction ultrastructure of gastric epithelial cells and CagA~+ H.pylori.Methods BGC-823 gastric carcinoma cells were co-cultured with different H.pylori strains(CagA~+ or CagA~-) at bacteria/cells ratio of 20:1, 100:1 and 500:1 for 24 and 48 hours respectively. Intervention with anti-H.pylori drugs was given in the group co-cultured at bacteria/cells ratio of 100:1 after 16 hours. In the control groups, BGC-823 cells were cultured for 24 and 48 hours respectively, but without using H.pylori or anti-H.pylori drugs. Immunocytochemical SABC method and technique of the image analysis of the computer were applied to detect the changes of Cx32, Cx43 expression in BGC-823 cells. The cell proliferation was examined by methyl tetrazolium (MTT) method. The cell junction ultrastructure was investigated under transmission electron microscope and scanning electron microscope with sample preparation of fixation and embedding in situ.Results (1) There was no Cx32 expression in all BGC-823 cells.The expression of Cx43 in the control group for 48 hours was higher than that for 24 hours (p＜0.05). The expression of Cx43 in the groups co-cultured with NCTC J99 for 48 hours was lower than that co-cultured only for 24 hours. There were significant differences in expression of Cx43 between the control group and the groups co-cultured with NCTC J99 for both 24 and 48 hours (p＜0.05). The bacteria/cells ratio of 500:1 was more influential than the ratio of 20:1 and 100:1 for both 24 and 48 hours (p＜0.05). The bacteria/cells ratio of 100:1 was more influential than the ratio of 20:1 for 48 hours (p＜0.05).However, there was no significant difference in expression of Cx43 between 24 and 48 hours in the groups co-cultured with NCTC 12908 (p＞0.05) .The expression of Cx43 in the groups co-cultured with NCTC 12908 at the ratio of 100:1 and 500:1 was lower than that in the control group, and that of the ratio of 500:1 was lower than that of the ratio of 20:1 for 24 and 48 hours. The expression of Cx43 increased after intervention with triple therapy of anti-H.pylori drugs for 48 hours. (2) In the groups co-cultured with NCTC J99, the optical density value of MTT indicated that the cell proliferation at the bacteria/cells ratio of 100:1 was higher than that in the control group, but no significant difference was found in other two groups co-cultured for 24 hours. After co-cultured 48 hours, cell proliferation in the bacteria/cells ratio of 20:1 and 100:1 was significantly accelerated, while the cell proliferation in 500:1 was. inhibited. But in the groups co-cultured with NCTC 12908,there was no change of cell proliferation. Intervention with anti-H.pylori drugs could suppress the cell proliferation. (3) There were many gap junctions and junction complexes of BGC-823 cells in control group without H.pylori. After co-cultured with H.pylori, the number of cell junction became less or no cell junction was discovered, the length of cell junction also became short, and there was no junction complex to form in BGC-823 cells. The number of cell junction and the number of junction/unit perimeter in the groups co-cultured with NCTC J99, GC 01 and NCTC 11637(CagA~+) were less than that of in the groups co-cultured with NCTC 12908(CagA~-) (p＜0.001 or p＜0.05), and the length of junction/unit perimeter in the groups co-cultured with NCTC J99 and GC 01 was shorter than that of in the groups co-cultured with NCTC 12908 (p＜0.001). These changes indicated that CagA~+ H.pylori was more influential on the cell junction than CagA~- Helicobacter. The anti-H.pylori drugs killed H.pylori, made cells to contact each other, and promoted the formation of cell junction, especially on condition that rabeprazole, amoxycillin and clarithromycin were used together.Conclusions (1) CagA~+ H.pylori can down-regulate expression of Cx43 in BGC-823 cell in relation to the density of H.pylori and reaction time. The higher the density of H.pylori and the longer the reaction time, the less is the expression of Cx43.CagA~-H.pylori was less influential on the expression of Cx43 than CagA~+ Helicobacter. (2) Low density of CagA~+ H.pylori suspensions can accelerate the proliferation of BGC-823 cells, while high density can suppress the cell proliferation. The CagA~- H.pylori didn't have the effect on cell proliferation. This indicated that the effect of H.pylori on cell proliferation was associated with CagA. (3) The CagA~+ H.pylori was more influential on the number of cell junction and length of cell junction than CagA~- Helicobacter. This indicated that the changes of cell gap junction of gastric epithelial cells are associated with the CagA~+ H.pylori. (4)Triple therapy of anti-H.pylori drugs can up-regulate the expression of Cx43,suppress the cell proliferation of BGC-823,decrease the effects of H.pylori on cell gap junction, and promote the formation of cell junction.Part 2 Clinical study about effects of CagA~+ Helicobacter pylori strain on cell gap junction of gastric epithelial cellsObjective To observe the changes of expression of connexin 32(Cx32), Cx43 and cell gap junction ultrastructure of gastric epithelial cells in patients with gastric cancer(GC) and precancerous lesion(PL), and the effects of H.pylori eradication on them in patients with PL. To investigate the relation between the changes of expression of Cx32, Cx43 and cell gap junction ultrastructure of gastric epithelial cells and H.pylori infection especially CagA~+ H.pylori infection.Methods 70 patients with GC, 88 with PL, and 33 with chronic superfial gastritis (CSG) were studied. SABC immunohistochemical methods were used to detect the expression of Cx32 and Cx43 in gastic mucosa biopsy specimens. The cell junction ultrastructure was investigated under transmission electron microscope. The CagA gene of H.pylori in the gastric mucosa was determined by PCR. The changes of expression of Cx32, Cx43 and cell junction ultrastructur were observed in patients with H.pylori infection in PL group after eradication therapy of H.pylori. Results (1) The positive expression rates of Cx32 and Cx43 are 15.7% and 32.9% respectively in patients with GC, 51.1% and 54.5% in patients with PL, 100% and 93.9% in patients with CSG. The positive expression rates in patients with GC or PL were lower than that in patients with CSG (p＜0.05). The positive expression rates of Cx32 and Cx43 in relation to the level of cell differentiation in patients with GC were lower than that in relation to the type of PL in patients with PL. In patients with PL, the positive expression rates and expression intensity of Cx32 and Cx43 in H.pylori positive group are lower than that in H.pylori negative group. In patients with GC, the positive expression rates of Cx32 in H.pylori positive group and H.pylori negative group were 16.7% and 13.6% respectively, Cx43 were 25% and 50% respectively. The positive expression rates of Cx43 had significantly difference between the two groups (p＜0.05), but Cx32 had no significantly difference (p＞0.05). In patients with H.pylori infection in CSG group, the positive expression rates of Cx32 and Cx43 are all 100% in the CagA~+ H.pylori group and CagA~- H.pylori group, but the expression intensity in the CagA~+ H.pylori group was lower than that in the CagA~- H.pylori group. In patients with H.pylori infection in GC group, the positive expression rate of Cx43 in the CagA~+ H.pylori group was lower than that in the CagA~- H.pylori group (17.9% and 55.6% respectively, p＜0.05), but Cx32 had no significantly differenc between the two groups (12.8% and 33.3% respectively, p＞0.05). In patients with H.pylori in PL group, the positive expression rates of Cx32 and Cx43 (97.9% and 91.7% respectively) after H.pylori eradication were higher than that before eradication therapy (41.4% and 44.8% respectively, p＜0.05). But the positive expression rates of Cx32 and Cx43 in no eradication patients were still 40% and 50%. There wasn't statistic significance comparing with that before eradication therapy (p＞0.05). (2) Length of junction/unit perimeter gastric epithelial cells in patients with PL was shorter than that in patients with CSG, and the smallest width of the intercellular space was longer than that in patients with CSG. The number of cell junction, the number of junction/unit perimeter and length of junction/unit perimeter in patients with GC were all less than that in patients with CSG or PL, and it's smallest width of the intercellular space was longer than that in patients with CSG. In patients with GC, the number of cell junction, the number of junction/unit perimeter and length of junction/unit perimeter in CagA~+ H.pylori group were less than that in CagA~- H.pylori group, and it's smallest width of the intercellular space was longer than that in CagA~- H.pylori group. In patients with PL, the intercellular space became short, and the length of cell junction of gastric epithelial cells became longer after H.pylori eradication. The length of junction/unit perimeter in patients of H.pylori eradication was longer than that in patients of no eradication, and the smallest width of the intercellular space was smaller than that in patients of no eradication.Conclusions (1) The patients with GC or PL had a lower positive expression of Cx32 and Cx43 in relation to the level of cell differentiation in patients with GC or the type of PL in patients with PL. (2) In patients with GC or PL, the intercellular space of gastric epithelial cells became large, the number of cell junction became less and the length of cell junction also became short. (3) The changes of expression of Cx32 and Cx43 and cell gap junction of gastric epithelial cells were associated with H.pylori infection. (4) In patients with GC, the changes of cell gap junction of gastric epithelial cells in the CagA~+ H.pylori infection group were more significant than in the CagA~- H.pylori infection group. This indicated that the changes of cell gap junction of gastric epithelial cells are associated with CagA~+ H.pylori infection. (5) H.pylori eradication in patients with PL can up-regulate the expression of Cx32 and Cx43, reduce the intercellular space between the gastric epithelial cells, and increase the length of cell junction. This indicated that H.pylori eradication can promote the formation of cell junction, increase GJIC function, delay or stop PL to progress, and prevent GC from developimg.