The Relativity Between Helicobacter Species And Liver Cancer And Effect Of Helicobacter Pylorion Cell Vacuole Denaturalization And Apoptosis Of HepG2 Cells In Vitro

Objectives : The aim of this study was to determine whether Helicobacter infection was associated with primary hepatic carcinomas (PHC) in humans and to establish a cell model of H. pylori infection, to investigate the effects of alive bacterium on the HepG2 cell apoptosis and expression of apoptosis-associated genes . Methods: 1. Resected liver tissues from 30 patients with PHC were placed in brucella broth before microaerobic culture and the remainder of samples were subjected to polymerase chain reaction (PCR) analysis using two sets of Helicobacter-specific 16S ribosomal RNA primers. Control 1 group contain 10 liver tissues with liver cirrhosis and 15 normal liver tissues containing 4 cavernous hemangioma and 10 samples of liver cyst and 1 sample of liver traumatism. 2. (1)H. pylori (NCTC11637 and NCTC11639) diluted 5 times continiously from 1.0 108 CFU/ml to 3. 2 104CFU/ml were coincUbated with HepG2 cells, then vacuole denaturalization of HepG2 cells was observed in discrepancy microscope and electron microscope, and TCID50(50 percent tissure culture infection dose) was calculated. (2) H. pylori (NCTC11637 and NCTC11639) diluted 10 times continiously from 4. 0 108 CFU/ml to 4.0 105 CFU/ml were coincubated with HepG2 cells, and then the relative cell inhibitory effects were measured by MTT and cell count. The different phase of cell apoptosis after incubation with H. pylori observed by AO/EB stain and in electron microscope. The effects of NCTC11637 atvarious concentrations on apotosis rate after 24 hours coincubation with HepG2 cells with TUNEL stain and flow cytometry . (3)The expression of cell apoptosis-associated genes was investigated by immunohistochemical method.Results: J. The Helicobacter spp. were not successfully cultured. The expected 400-bp fragments of Helicobacter 16S rRNA were amplified from 11/30 (33.3% ) liver cancer samples and 2/10 (20%) liver cirrhosis controll speciment. The normal liver samples were negative. The difference is significant(P<0. 05). In 3 patient samples from which 400-bp nucleotides were sequenced, the closest match was H. pylori. 2. There is no difference of the relative cell inhibitory effects measured by cells count after 36 hours coincubation with HepG2 cells until the concentration of H. pylori increased to 4. 0 106 CFU/ml (P<0. 05) .And the the relative cell inhibitory effects increased according to the growing concentration (P<0. 05) .The data are similar to that measured by MTT. The different phase of cell apoptosis after incubation with H.pylori were observed by AO/EB stain and in electron microscope. H. pylori can promote cell apoptosis of HepG2 and the effect was enhanced according to the increasing concentration. NCTC11637 and NCTC11639 have the same effect. The effects of NCTC11637 on apotosis rate after 24 hours coincubation with HepG2 cells with TUNEL stain and flow cytometry were significant according to the concentration. 3. The expression of Bax protein increased and that of Bcl-2 decreased after 24 hours coincubation of HepG2 cells with increasing number of the concentration of NCTC11637 to 4. O 106 CFU/ml.Conclusions : 1. These data suggest an association of Helicobacter spp. with primary 1iver carcinoma . Further studiesare needed to ascertain whether Helicobactcr spp. infection plays a role in the development of liver cancer. 2. The coincubation of HepG2 cells with H. pylori led to increase of cell apoptosis with a H. pylori concentration-dependent way. 3. The effect of H. pylori on HepG2 cell apoptosis could be associated with alterated expression of Bax and Bcl-2 protein.