The Damage Effect Of The Different Genotype Helicobacter Pylori Distributed In High And Low-risk Area Of Gastric Cancer To Human Gastric Epithelial Cell Line GES-1

PrefaceThere is evidence that the genetic difference may play an important role in the clinical outcome of the Hp infection, particularly Hp virulence associated genes such as cagA,vacA and ice A genes. Zhuanghe in Liaoning province is a high - risk area of gastric cancer of North China. In the early study we had reported that more than 60.70% individuals in the high - risk area of gastric cancer in Zhuanghe infected with H. pylori; and the infection have significant association with gastric disease. There is significant difference in the distribution of Hp genotypes between Zhuanghe high - risk area and Shenyang low - risk area of gastric cancer. The difference genotypes combination in Zhuanghe is cagA~+ , vacAsl Vmlb ~+ . The study has shown there may be some relationship between H. pylori infection and gastric cancer incidence in Zhuanghe area. But whether or not the high incidence of gastric cancer in the high - risk area has relationship with the different genotype of H. pylori genotype is still puzzled. This study was designed to invested the damage effect of the different genotype Helicobacter pylori distributed in high and low - risk area of gastric cancer to human gastric epithelial cell line GES - 1 induced by difference genotype helicobacter pylori in human gastric epithelial cells GES - 1, to investigated the mechanism of DNA damage and malignant transformation, the 8 - OHdG were examined by S - P immunohistochemistry method.Materials and MethodsHp strains were grown at 37℃ on brain heart infusion plates containing 7%sheep blood in a 10% CO2 incubator with 95% relative humidity. Standard phenol - chloroform - isoamyl method was used to extract DNA from Hp strain. PCR was used to exam cagA, vac As 1, s2, ml a, mlb, and m2 genotypes of Hp DNA. According to the result of PCR,we classified the Hp into three groups, added strain A(cagA+ ,vacAsl +/mlb+)to experiment group A,added strain B (cagA+ , vacAsl ~/mlb~ ) to experiment group B,added strain C (cagA~ ,va-cAsl +/mlb+ ) to experiment group C, group D ,only added PBS as blank control. Then using the method of cell and Hp coculture,we observed the damage effect of Hp by morphology, and detected the expressions of 8 - OHdG by S - P immunohistochemistry method.?Results1. Identify of Hp and its genotype: According to the bacterial culture and staining of HE, it was verified that the bacterial was helicobacter pylori, and the genotype was accord with the early record of the laboratry, genotype of A group: cagA + , vacAsl Vmlb+, genotype of B group -. cagA + , vacAsl Vmlbl ~ , genotype of C group: cagA ~ , vacAsl+ /ml b + o2. Morphology observation of cocultured GES — 1 cells 2.1 The damage effect of Hp to GES - 1 cellsUnder invert microscope , the normal GES -1 cells are spindle, polygon , adhering to wall, floating cell is almost never seen.1) With the prolongation of coculture time, the morphology of GES - 1 cells were changed from spindle to round, the ability of GES - 1 cells adhering to wall were decreased.2) With the prolongation of coculture time, the nuclei of GES - 1 cells showed chromatin pyknosis, and clustered on the inner border of karyoteca, condense cytoplasm with many vacuoles.3) With the prolongation of coculture time, the dead and damaged cells were increasing.4) With the prolongation of coculture time, the liquid of cell culture changed to yellow, and feculent.