Construction Of Gastric Cancer-associated Genes Library Of Helicobacter Pylori And Screening Of Gene Difference

IntroductionHelicobacter pylori(H pylori)colonizes the human stomach and establishes long-term infection of the gastric and duodenal mucosa.Although H pylori infects approximately half of the world's population,only a small proportion of infected subjects develop symptoms or clinically significant diseases.The reasons for this fact are unknown but may be related to the host's immunological defenses,environmental factors and/or the virulence of different strains of the bacteria.The genomes of H.pylori from different individuals are quite different and each one contains about 1600 genes, among which almost 320 genes are dispensable and 100 genes are strain unique.Genes that are present in one strain and absent or substantially different in the others can be of great biological interest.Several genes have been identified that may play a role in the pathogenicity of H pylori genotypes such as cagA,vacA,and iceA.H.pylori strains with the cag pathogenicity island(PAI)can induce more severe inflammation,proliferation and apoptosis in the gerbil mucosa than do the strains partial or complete lack of the cag PAI.In contrast to the CagA~- H.pylori infection,CagA~+ H.pylori infection is associated with a higher prevalence of p53 mutation in gastric adenocarcinoma.In addition to the cag island,other polymorphic loci that appear clinically relevant have to be identified. In the west,special vacAslml was the marker of bacterial pathogenicity and was associated with severe epithelial damage In Costa Rica age- and sex-adjusted vacAslb and vacAml were associated with GC while only vacAml was significantly associated with GA.In south Africa,iceA1 was associated with GC.Except for vacA,cagPAI,may other polymorphism sites also are associated with gastric cancer.For example,cloning,expression,and purification of JHP940 revealed a novel,approximately 36-kD a protein in a biologically active form which elicited strong and significant levels of tumor necrosis factor alpha and interleukin-8 in human macrophages.Also,JHP940 was able to induce enhanced translocation of the transcription factor NF-kappaB complex in cultured macrophages.The induction of the proinflammatory cytokines by JHP940,therefore,points to its putative role in chronic gastric inflammation and,possibly,the various other outcomes of H.pylori infection, including gastric cancer.It is evidence that there are important significances to identify these special genes for disclosing the pathogenic mechanism of H.pylori,exploring the molecule marker to diagnosed the disease origin of bacterial isolates,and providing clues and evidences for the target of H.pylori treatment.It may establish for the association of inheritance and the research of differentiation and molecule identification system and molecular epidemiology.ObjectiveTo approach the relationship between H.pylori virulence genes and gastric diseases,construction and screening of a gastric cancer-associated genes library of H.pylori and find the sequences associated with gastric cancer.This may furthermore disclose the pathogenic mechanism of H.pylori,explore the molecule marker to diagnosed the disease origin of bacterial isolates,and provide clues and evidences for the target of H.pylori treatment.Materials and MethodsA total of 491 cases were involved in this study including 136 cases from Shenyang(69 men and 57 women,25-78 years,mean age:48.61 years),355 cases from Zhuanghe(174 men and 181 women,21-79 years,mean age:49.33 years).One antrum biopsy was taken for culture and stored at -70 in 0.5 mL of brucella broth(Difco)with 15%glycerol until incubation.Three biopsies were taken for pathology diagnoses,from gastric antrum,corpus,and angularis,respectively.The study protocols were approved by the Human Ethics Review Committee of China Medical University.Written informed consent was obtained from participants in accordance with the Declaration of Helsinki and its later revision.Hematoxylin and Eosin(HE)Staining were taken part in histopathologic examination.H.pylori was cultured on petri plates containing brain heart infusion(BHI) agar.Stain DNA was isolated by standard phenolchloroform extraction.H.pylori genotypes,such as cagA,vacAs,vacAm,iceA1 and iceA2 were determined by PCR.The high- and low-copied gene libraries were constructed from strain L301 and B975 by SSH.And then these genes wene identified by dot hybridization,gene sequencing,and blastn homology analysis.The difference gene screened was determined by polymerase chain reaction(PCR).The x~2 or Fisher's Exact test were used to examine the differences in the different groups.Results1.H.pylori cagA,vacA,iceA genotypes were detected in different groups.There was statistical significant compared H pylori m2 strain infection in GA group(43.14%) with that in GU(18.18%)and GC(17.86%)groups(P=0.015 and P=0.020).There was statistical significant compared H pylori slm2 strain infection in GA group(44.9%) with that in GU(21.42%)and GC(21.42%)groups(P=0.039)but no statistical significant compared with that in GS group(P=0.067).There were no statistical differences of cagA,iceA genotypes in groups.2.The difference genes libraries were constructed by SSH from strain L301,which was cultured from a gastric cancer patient,and B975,which was cultured from a superficial gastritis patient.The homology analysis of high copy genes including:information storage and processing,cellular processes and signaling,replication,recombination and repair,function unknown et al.The homology analysis of low copy genes including:nucleotide transport and metabolism,cellular processes and signaling,metabolism,information storage and processing,function unknown et al.3.Peptidyl-prolyl cis-trans isomerase gene was amplified with primers designed by our own.It was found there were 10 positive cases(52.6%)in total of 19 GC strains and all the strains form GS were negative(p=0.000).Conclusions1.There is positive relationship between H pylori slm2 strain infection and GA in Liaoning area There are no relationship between H pylori cagA,iceA genotype and gastric diseases.2.The difference genes libraries were successfully constructed by SSH from strain L301,which are including:storage and processing,cellular processes and signaling,replication,recombination and repai,nucleotide transport and metabolism et al.Among of the peptidyl-prolyl cis-trans isomerase gene is high copy and may a marker for strain for gastric cancer.