| Objective:To investigate the effect of Helicobacter pylori (Hp) on human gastric epithelial cells, the changes of cell morphology, apoptosis and proliferation, as well as the expression of p53 and PUMA, the relationship between them, To elucidate the effect and pathogenic mechanisms of Hp on human gastric epithelial cells in vivo and vitro, and establish new target and direction for further researches on treatment of Hp correlation diseases.Methods:In vivo:gastric mucosal lesions came from 153 patients without eradication therapy of Hp treated by endoscopic biopsy from our hospital(2007.1-2009.9).153 patients include Chronic Non-Atrophic Gastritis (CNAG) 37 cases, chronic atrophy gastritis and intestinal metaplasia 39 cases, dysplasia (DYS) 39 cases, gastric cancer (GC) 38 cases. The Hp infection was detected with Giemsa staining and the Expressions of mutant p53, PUMA protein were detected with immunohistochemical technique in different gastric mucosal lesions.In vitro:GES-1 were co-cultured with the supernatants of Hp, and divided into the following groups:(1) Experimental group of NCTC11637(CagA+, VacA+): groups(group A, B, C, D) were treated with different concentrations of Hp supernatants(11mg/ml,5.55mg/ml,2.775mg/ml,1.3875 mg/ml). (2)Experimental group of NCTC11639(CagA+, VacA-):groups(group A, B, C, D) were treated with different concentrations of Hp supernatants (7.5mg/ml,3.75mg/ml,1.875mg/ml, 0.9375 mg/ml). (3) the negative control treated with Hp medium. GES-1 were co-cultured with the supernatants of Hp at different time points (24h,48h and 72h)in Each group. The cell morphology was observed with inverted microscope, The inhibition rates of cell growth were determined by MTT assay, The level of p53 and PUMA protein was determined by Western blot. Results:1. Part of Clinical Reasearch(1) the expression of mutant p53 in different gastric mucosal lesions:In Hp-positive group, compared the level of mutant p53 expression in CNAG, CAG with IM, DYS and GC subgroups, we found that there were not significant differences among these subgroups(P>0.05); In Hp-negative group, compared the level of mutant p53 protein expression in CNAG, CAG with IM, DYS and GC subgroups, we found there were significant differences between subgroups CNAG and DYS, or CNAG and GC(P<0.05), and also between subgroups CAG with IM and DYS, or GC(P<0.05). In the same type of gastric mucosal lesions, the expression of mutant p53 is generally higher in Hp-positive group than that of Hp-negative group, there were significant differences in CAG with IM group(P<0.05).(2) the expression of PUMA in different gastric mucosal lesions:In Hp-positive group, compared the level of PUMA expression in CNAG, CAG with IM, DYS and GC subgroups, we found that there were significant differences between CNAG and CAG with IM, or GC, (P<0.001-0.05), also between DYS and CAG with IM, or GC(P< 0.01-0.05). In Hp-negative group, compared the level of PUMA in CNAG, CAG with IM, DYS and GC subgroups, we found that there were significant differences between CNAG with DYS, or GC(P<0.01-0.05), also between CAG with IM, DYS with GC subgroup (P<0.001); In the same type of gastric mucosal lesions, the expression of PUMA is generally lower in Hp-positive group than that of Hp-negative group, there were significant differences in CAG with IM group(P<0.01).(3) In Hp-positive group, we conduct the correlation analysis of mutant p53 and PUMA in different gastric mucosal lesions:CNAG, CAG with IM, DYS, the correlation coefficent was 0.032,-0.222,-0.012 respectively, P>0.05; In Hp-negative group, we conduct the correlation analysis of mutant p53 and PUMA in different gastric mucosal lesions:CNAG, CAG with IM, DYS, the correlation coefficient was-0.142,0.221,0.041 respectively, P>0.05.2. Part of reasearch in vitro(1) morphological change the GES-1 cultured with the supernatants of Hp (groupNCTC11637, group NCTC11639) for 48 hours were observed to present the typical morphological change of apoptosis by using inverted microscopy.(2)The effect of different genotypes of Hp supernatants on GES-1 cells'activityâ‘ Based on the different concentration of NCTC11637 Hp culture supernatants, at 24h,72h, The inhibition rates of cell growth of groups(A, B, C, D) treated with NCTC11637 Hp supernatants were significantly higher than those of control group (P<0.001), at 48h, the inhibition rates of experimental group A, B, C were significantly higher than that of control group (P<0.001), but there were not significant difference between D group and control group(P>0.05); at 24h,48h,72h, the inhibition rates of cell growth of group A significantly higher than those of B, C, D groups(P<0.001), at 48h,72h, the inhibition rates of cell growth of group B was higher than those of C, D groups (P<0.05), group C was significantly higher than that of group D (P<0.001), at 24h, there were not significant difference among group B,C,D (P>0.05)â‘¡GES-1 inhibition rates were significantly affected with the treatment time of NCTC11637 Hp supernatants,In the concentrations range ofllmg/ml,5.55mg/ml, 2.775mg/ml,1.3875 mg/ml, the GES-1 inhibition rates were significantly affected with the treatment time of Hp culture supernatants (P<0.001). In experimental groupsA, the GES-1 inhibition rates at 72h was significantly higher than those at 24h, 48h (P<0.001), but there was not significant differences between 24h and 48h (P>0.05); In experimental groups B, C, the GES-1 inhibition rates at 48h and 72h were significantly higher than those at 24h (P<0.001-0.05),but we found no differences between 48h and 72h (P>0.05); In experimental groupD, no differences were found among 24h,48h and 72h (P>0.05)â‘¢The inhibition rates of cell growth of groups treated with NCTC11639 Hp culture supernatants were higher than those of control group, and these inhibition rates were concentration-dependent. At 24h, the inhibition rates of cell growth of groups(A, B) were higher than those of control group (P<0.001), but there were no significant differences among group C, D and control group (P>0.05), at 48h,72h, the inhibition rates of cell growth of groups A, B, C were significantly higher than control group (P<0.001-0.05), but there were not significant difference between group D and control group (P>0.05). at 24h,48h,72h, the inhibition rates of cell growth of groups A was significantly higher than those of groups B, C, D, and at 24h, 48h, group B was higher than those of groups C, D (P<0.05),. there were not significant difference between group C and D (P>0.05), at 72h, group B was higher than that of group D (P<0.001), group C was significantly higher than that of group D (P<0.001), there were not significant differences between group B and C (P>0.05).GES-1 inhibition rates were significantly affected with the treatment time of NCTC11639 Hp supernatants, In the concentrations range of 7.5mg/ml,3.75mg/ml, 1.875mg/ml,0.9375 mg/ml, the GES-1 inhibition rates were affected with the treatment time of Hp culture supernatants. In experimental group A, the GES-1 inhibition rates at 48h and 72h were significantly higher than those at 24h (P<0.001), no differences were found between 48h and 72h (P>0.05); In experimental group B, the GES-1 inhibition rates at 72h were significantly higher than those at 24h(P<0.05), but there were not significant differences between 48h and 24h or 72h; In experimental group C, the GES-1 inhibition rates at 72h were significantly higher than those at 24h and 48h (P<0.001-0.05), no significant differences were found between 24h and 48h (P>0.05); In experimental group D, there were not differences among 24h,48h and 72h (P>0.05)(3)the effects of different genetype Hp culture supernatant on the expression of p53, PUMAâ‘ The expression of p53 protein in GES-1 was significantly higher than that of control group when treated with NCTC 11637(2.775 mg/ml) Hp culture supernatants for 48h (P<0.001-0.01), but we found there were not significant differences with NCTC11639(3.75mg/ml) Hp culture supernatants for 48h.â‘¡The expression of PUMA protein in GES-1 was significantly lower than that of control group when treated with NCTC11639(3.75mg/ml). Hp culture supernatants for 48h (P<0.01-0.05), but we found there were not significant differences with NCTC11637(2.775mg/ml) Hp culture supernatants for 48h (P>0.05) Conclusion:1. Abnormal expression of the mutant p53 and PUMA protein involved in the development of gastric musoal lesions together.,and may relate with early pathopoiesis of Hp.2. The growth of human normal gastric epithelial cell line GES-1 were inhibited by both NCTC11637 and NCTC11639 Hp culture supernatants in time and concentration-dependent patterns, which may be one of the mechanisms of Hp causing gastric mucosa impairment.3. NCTC11637 but not NCTC11639 Hp culture supernatant can upregulate the expression of p53, there are some differences which realate with virulence of the Hp strain.4. NCTC11639 but not NCTC11637 Hp culture supernatant can upregulate the expression of PUMA, there are some differences which realate with virulence of the Hp strain. |
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