| Background and objective:Helicobacter pylori is a persistent colonization of gram-negative bacteria in the humangastric mucosa. Its colonization can cause gastric inflammation, is closely related with peptic ulcer, gastric cancer and gastric mucosa-associated lymphoid tissue(MALT) lymphoma. More than half of people worldwide infected with H.pylori, in which 60 to 70% with asymptomatic, 15 to 25% with symptoms of indigestion, 15% with peptic ulcer, <1% occurred with gastric malignancy. These different outcomes mainly affected by bacterial factors, host factors and environmental factors, which highly polymorphic genetic factors play an important role in bacterial diversity in the outcome of H.pylori infection, H.pylori virulence factor gene polymorphism is also an important reason. Helicobacter outer membrane B(Hom B), outer inflammatory protein A(Oip A), blood-group antigenbinding adhesin(Bab A), sialic acid-binding adhesin(Sab A) closely related with H.pylori colonization, gastric mucosal injury, secretion of inflammatory mediators and neutrophil infiltration, plays an important role in the pathogenesis of H.pylori. This paper selects the 188 H.pylori strains isolated from clinical samples in Jiangxi province, to observe gene sequence characteristics of the four genes, analysis of sequence features of different regions. In order to analyze the relationship between these fourgenes and their gene polymorphism and digestive diseases. Methods:1. Source strains : Isolates from January 2012- December 2013 in the Jiangxi province during the First Affiliated Hospital of Nanchang University Endoscopy Center patients underwent endoscopy gastric digestion H.pylori 188 isolated and cultured isolates. 51 from chronic gastritis, 79 from duodenal ulcer, 20 from gastric ulcer, 38 from gastric cancer. Wherein gastritis, gastric ulcer and gastric cancer patients were taken were pathological examination, a total of 105 H.pylori patients have histopathologic findings, which 42 came from chronic superficial gastritis, 25 from intestinal metaplasia and dysplasia, 38 from gastric cancer.2. resuscitate and culture H.pylori strains: The pre-separation of 188 clinical isolates of H.pylori were resuscitated, cultured and identificated.3. Genomic DNA, and H.pylori OMP oip A, bab A, sab A and hom B gene was amplified by PCR and feed positive product to huada biological sequenced.4. Bioinformatics Analysis(1) Compare and proofreading DNA sequences of four genes.(2) MEGA 5.0 for single genes using cluster analysis in four kinds of disease classification and three kinds of pathological type.(3) Splicing consensus sequences for four gene in four kinds of disease and three kinds of pathological type.(4) Compare the difference between the consensus sequence of different diseases and different pathological type.(5) Analysis of the average mutation rates among different diseases and pathological type.(6) Identify the high risk of mutation points of sequence among different diseases and pathological type. Results:1. PCR sequencing: The pre-separation of 188 H.pylori has been successfully extracted DNA, after PCR amplification, sequencing and successfully acquired 179 oip A gene sequences, 166 bab A gene sequence, 169 sab A gene sequence, 175 hom B gene sequences.2. Analysis clustering contact for four genes with clinical and pathological.There is no significant correlation was found between the four genes and four kinds of clinical disease and four genes with three kinds of pathological type.3. Comparative analysis of consensus sequencesFor three different pathology(cancer, pathological superficial gastritis, intestinal metaplasia and dysplasia), found in oip A gene, the relatively highest mutation rate of gastric cancer, there are 14.46%; in sab A gene in gastric cancer, intestinal metaplasia and atypical the proliferation of relatively higher mutation rates were 17.28% and 9.87%. For hom B and bab A gene, their mutation rate did not differ much.For four different diseases(gastritis, gastric cancer, duodenal ulcer, gastric ulcer), in oip A gene, the mutation rate relative gastric highest with 13.11%, three other genes are not very different.On the whole, if only from the consensus sequence comparison, only oip A gene can effectively distinguish gastric diseases and other non-reference.4. Analysis of the average mutation rateFor the three kinds of different pathological(cancer, pathological superficial gastritis, intestinal metaplasia and dysplasia). On pathological type, the average mutation rate of gastric cancer in four genes are three kinds of pathological type in the highest. Just four genes is, hom B average gene mutation rate in three kinds of pathological type highest, oip A gene in three kinds of pathological type the lowest average mutation rate.For four different diseases(gastritis, gastric cancer, duodenal ulcer, gastric ulcer). Stomach cancer in oip A, sab A, hom B these three genes in the average mutation rate is the highest in four kinds of disease classification; in bab A gene, the average mutation rate of duodenal ulcer are four kinds of diseases classified in the highest.5. Sequence analysis of each disease in populations at high risk mutation point(1) For three pathological type, ①In oip A genes, mutation rate before 10 largest sites from superficial gastritis are concentrated in the 42-49 sites; previous maximum rate of 10 mutation sites from intestinal metaplasia and dysplasia are more dispersed; mutation rate before 10 largest sites from gastric mostly concentrated in the 1436-1477 site. ② In bab A genes, mutation rate before 10 largest sites from superficial gastritis, intestinal metaplasia and dysplasia are more dispersed; previous maximum rate of 10 mutation sites from stomach cancer are more concentrated in the 1459-1638 site. ③ In sab A genes, mutation rate before 10 largest sites from superficial gastritis, intestinal metaplasia and dysplasia are more dispersed; previous maximum rate of 10 mutation sites from gastric mostly concentrated in the 2715-2806 site. ④ In hom B genes, mutation rate before 10 largest sites from superficial gastritis mostly concentrated in the 3196-3216 site; previous maximum rate of 10 mutation sites from intestinal metaplasia and dysplasia are more dispersed; Previous maximum rate of 10 mutation sites from gastric mostly concentrated in the 3102-3215 site.(2) For four disease, ①In oip A genes, mutation rate before 10 largest sites from gastritis and peptic ulcer are dispersed; previous maximum rate of 10 mutation sites from duodenal ulcers, some are mostly concentrated in the 41-51 sites; previous maximum rate of 10 mutation sites from stomach cancer are mostly concentrated in the 50-55 and 1462-1505 site. ②In bab A genes, mutation rate before 10 largest sites from gastritis are more concentrated in the 1315-1346 site; previous maximum rate of 10 mutation sites from stomach ulcers mostly concentrated in the 1315-1345 site; previous maximum rate of 10 mutation sites from duodenal and gastric cancer are more dispersed. ③In sab A genes, mutation rate before 10 largest sites from gastritis, mostly concentrated in the 2396-2431 and 2959-2986 site; previous maximum rate of 10 mutation sites from stomach ulcers mostly concentrated in the 2401-2441 site; previous maximum rate of 10 mutation sites are more dispersed from the ulcer; previous maximum rate of 10 mutation sites from stomach cancer are mostly highly concentrated in the 2896-2985 site. ④In hom B genes, mutation rate before 10 largest sites from gastritis, mostly concentrated in the 2121-2138 site; previous maximum rate of 10 mutation sites from stomach ulcers are more dispersed; Previous maximum rate of 10 mutation sites from duodenal ulcer mostly concentrated in the 255-510 site; previous maximum rate of 10 mutation sites from stomach cancer is very concentrated in the 247-510 site. Conclusion:Although cluster analysis showed that H.pylori OMP oip A, bab A, sab A and polymorphism hom B there is no obvious relationship between the clustering of four different clinical conditions and three different histopathological changes in, but each gene the average mutation rate and high-risk mutations are different in different diseases and different pathological changes in. Diversity of gene polymorphism prompt oip A, bab A, sab A and hom B possible clinical outcomes in H.pylori infection plays a role. |
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