胃癌高低发区差异基因型幽门螺杆菌菌株对人胃上皮细胞系GES-1的损伤作用

目的:研究胃癌高、低发区差异基因型幽门螺杆(H pylori) 菌株对永生化的非肿瘤胃上皮细胞GES-1的损伤作用;并阐明H pylori致细胞损伤的分子机制．方法:通过常规HE染色、Gimsa染色、W-S银染色鉴定H pylori菌株,采用蛋白酶K及酚-氯仿抽提法提取DNA,PCR法扩增cagA、vacAsl、s2、mla、mlb、m2基因,对H pylori 因亚型进行检测．利用细胞和幽门螺杆菌共培养技术,观察胃癌高低发区差异基因亚型H pylori以及其他两种非差异基因型H pylori对胃上皮细胞系GES-1的损伤作用,并通过免疫细胞化学方法检测GES-1细胞8-OHdG的表达．结果:幽门螺杆菌对GES-1细胞具有损伤作用,随着作用时间的延长,GES-1细胞形态由贴壁的梭形变为圆形．细胞核染色质浓缩,呈新月形聚集于核缘,细胞质浓缩,可见空泡变性,死亡细胞逐渐增多．差异基因型H pylori对GES-1细胞的损伤程度比非差异基因型的损伤作用重．正常对照组 8-OHdG表达基本阴性(0．5%),而H pylori处理组GES-1细胞 8-OHdG表达阳性达98．5％,二者差异显著(P<0．01),有统计学意义．结论:胃癌高发区差异基因型H pylori即cagA ,vacAs1 ／m1b 对胃上皮细胞GES-1具有更强损伤作用,其损伤具有时间依赖关系;H pylori可能通过增加胃上皮细胞8-OhdG表达而导致正常胃黏膜上皮细胞恶性转化．

Damage effect of different genotype of Helicobacter pylori on human gastric epithelial cell line GES-1 in high- and low-risk areas of gastric cancer

AIM: To investigate the damage effect of the different genotypes of Helicobacter pylori on human gastric epithelial cell line GES-1 in high- and low-risk areas of gastric cancer, and to explore its related mechanism. METHODS: H pylori were identified by hematoxylin-eosin (HE) staining, Gimsa staining, and Warthin-Starry silver staining. The DNA was obtained by proteinase K and phenol-chloroform extraction method. The cagA, vacAs1/s2, m1a, m1b, and m2 gene were amplified by polymerase chain reaction (PCR). The damage effects of H pylori with differential sub-genotypes and 2 other non-differential genotypes on GES-1 cells were observed by cell and H pylori co-culture. The expression of 8-OHdG in GES-1 cells was detected by S-P immun-ohistochemistry. RESULTS: GES-1 cells were seriously damaged by H pylori. With the prolongation of the co-culture time, the morphology of GES-1 cells were changed from spindle to round, and the nuclei showed chromatin pyknosis and clustered on the inner border of karyon. The cytoplasm condensed and blebbing appeared. The numbers of the dead and damaged cells were increasing. The damage effect of H pylori with differential genotypes on GES-1 cells was more serious than that of the non-differential genotypes. The expression of 8-OHdG were almost all negative (0.5% positive) in GES-1 cells of the normal controls, while the positive rate was 98.5% in the H pylori treatment cells (P <0.01). CONCLUSION: H pylori with cagA , vacAs1 /m1b genotypes in the high-risk area of gastric cancer have more serious damage effects on gastric cancer cell line GES-1, and they can promote the transformation of normal gastric epithelial cells to malignant cells by up-regulation of 8-OHdG expression.