Objective: To investigate the effects of tumor necrosis factor-alpha-inducing protein-alpha (Tipalpha) released from Helicobacter pylori (Hp) on human gastric epithelial cells.
Methods: Human gastric epithelial cells of the line GES-1 were cultured. Tipalpha gene located in Hp 0596 was extracted from the genome sequence of the Hp strain 26695 and its open reading frame were cloned into the eukaryotic expressing vector pcDNA3.1. The recombinant plasmid pcDNA3.1-Tipalpha thus obtained and the blank plasmid pcDNA3. 1 were transfected into the GES-1 cells, and the G418-resistent clones were screened. The GES-1 cells transfected with blank vector pcDNA3.1 and normal GES-1 cells were used as controls. RT-PCR and Western blotting were used to detect the expression of Tipalpha. The influences of Tipalpha protein on the cells proliferation, apoptosis and cell cycles, concentration of related cytokines such as TNF-alpha, IL-1beta, and IL-8, and the expression of Bcl-2 and P53 genes were respectively observed by MTT assay, flow cytometry, ELISA, and Western blotting.
Results: 1. A eukaryotic expression vector of Hp Tipalpha was successfully constructed. Steadily transfected strains were screened by G418. 2. MTT method showed that the growth curve of the GES-1 cells transfected with GpcDNA3.1-Tipalpha was higher, showing a faster growth. 3. Flow cytometry showed an increase in the proportion of the S-phase and a decrease in the G1-phase in the GpcDNA3.1-Tipalpha-transfected cells [(45.33 +/- 1.03)% vs (38.24 +/- 1.5)%, (33.94 +/- 1.67)%, (41.39 +/- 0.08)% vs (49.74 +/- 0.12)%, (49.27 +/- 0.15)%], and the apoptotic rate of the GpcDNA3.1-Tipalpha-transfected cells was 0.76 +/- 0.04, significantly lower than those of the GpcDNA3.1-transfected cells and non-transfected cells (16.84 +/- 2.16 and 8.36 +/- 1.07 respectively, both P < 0.05). 4. ELISA showed that there was no significant difference in intracellular TNF-alpha concentration among the 3 GES-1 cell groups (all P > 0.05), however, the extra-cellular TNF-alpha level of the GpcDNA3.1-Tipalpha-transfected cells was significantly higher than those of the 2 control groups (both P < 0.05), and that the intra- and extra-cellular IL-1beta and IL-8 concentrations of the GpcDNA3.1-Tipalpha-transfected cells were both significantly higher than those of the 2 control groups (all P < 0.05). 5. The expression level of Bcl-2 gene of the GpcDNA3.1-Tipalphac-transfected cells was obviously higher than those of the 2 control groups, and the expression of P53 gene of the GpcDNA3.1-Tipalphac-transfected cells was lower than those of the 2 control cells.
Conclusion: The GpcDNA3.1-Tipalpha-transfected cells steadily and highly express Tipalpha protein, that induces the high expression of TNF-alpha, IL-1beta, and IL-8, proinflammatory cytokines, enhances cell proliferation, and upregulates Bcl-2 gene and down-regulates P53 gene. Tipalpha may play an important carcinogenic role in gastric cancer progression.