Mice Stomach Were Phenotype Analysis Of CagA Turn

The discovery of Helicobacter pylori（H.pylori）was among the great events ofthe20th century in the field of gastroenterology. It has been confirmed that H.pylori isthe main cause of chronic gastritis, gastric and duodenal ulcer, gastricadenocarcinoma and gastric mucosa-associated lymphoid tissue lymphomas. In the1994, H.pylori was classified as a class I carcinogen by WHO. The pathogenic factorsof H.pylori include urease, flagella, adhesin, lipopolysaccharide, VacA (Vacuolatingcytotoxin A), CagA(cytotoxin-associated protein A). H.pylori strains are groupedinto two basic types based on whether or not they express the VacA and CagAvirulence protein. Type I strains possess cag PAI (cag pathogenicity island), which ischaracterized by cagA. Numerous researches have proved that whether with cag PAIor not is a major characteristic of strong or weak toxicities and cag PAI plays animportant role in the pathogenic process of H.pylori.CagA protein is of polymorphism, and the EPIYA motifs located in thecarboxyl-terminal of CagA differ greatly between western and East-Asia strains.When CagA protein undergoes tyrosine phosphorylated. Cell experimentsdemonstrated the differences of CagA pathogenic ability between western andEast-Asian strains. However, as H.pylori have many kinds of pathogenic factors,animal models chronically infected by H.pylori cannot reveal the pathogenic effect ofa single CagA protein. Moreover, though cagA transfected AGS cell lines can showthe function of CagA protein and avoid the influence of other factors, they cannotimitate the pathogenic process of H.pylori in vivo very well.Based on the above, two strains of CagA transgenic mices of East-Asian(CagA-9810, with a EPIYA-D motif in the carboxyl-terminal) and Western(CagA-Ca52, with a EPIYA-C in the carboxyl-terminal) were successfully constructedin our lab. On the basis of this, I did research in the following three aspects: 1. Detection of cagA copy number and integrity in the genome.To make sure cagA is effectively expressed in cagA transgenic mice, we firstdetected the copy number of cagA inserted into the genome after obtaining theoffspring of cagA-9810and cagA-Ca52strains, each of which has two independentlines. The results indicated that AC155line, GC996line (cagA-Ca52strain) andYB081line,YB053line (cagA-9810strain) have5,8,6,6copies of cagA gene insertedinto one genome respectively. Then, we tested the integrity of the cagA gene intransgenic mice by using the primers of both5-end and3-end cagA gene to amplifythe corresponding fragment. Positive results were found in all4lines and no mutationwas detected by DNA sequencing.2. Determination of cagA expression level in vivo.We next detected the transcription of cagA in RNA level. Total RNA from gastrictissue was isolated and RT-PCR was performed. It showed that cagA gene insertedinto the cagA positive transgenic mice was exactly transcribed and was detected byDNA sequencing. We also performed Western Blot assay to test the expression of HAtag connected to the carboxyl terminal of CagA protein. The result showed that CagAprotein is successfully expressed in the cagA positive transgenic mice.3. Pathological analysis of gastric tissues from cagA transgenic mice.57cagA-ca52,59cagA-9810transgenic mice and32cagA negative transgenicmice for littermate control older than6month were used for pathological dissectionand observation,. littermate control.. Ki-67assay revealed that part of transgenic micehad nucleus anachromasis and strong proliferation in gastric mucosa gland cervicalpart, gastric pit and fundus gland. Alcian blue staining showed that hypertrophicmucous cells appeared in fundus gland of some transgenic mice. The resultdemonstrated that the stomach of cagA transgenic mice has overt inflammation,polypoid proliferation, adenomatoid hyperplasia and intestinal metaplasia, and thelatter three can be regarded as precancerous lesion. By statistics, we found 30%East-Asian and19%Western cagA transgenic mice developed precancerouslesion. Besides, there’s no obvious cancer lesion in the stomach and duodenum of thetwo strains of transgenic mice during the observation time limit, but cagA transgenicmice are more likely to get ill than wild type and appeared precancerous lesion.In conclusion, after obtaining East-Asian cagA-9801(EPIYA-ABD) and WesterncagA-ca52(EPIYA-ABC)transgenic mice,we performed detection in DNA, RNA andproteins levels as well as through pathobiology, pathohistology,immunohistochemistry and special straining for the transgenic mice. We concludedthat both East-Asian cagA-9801(EPIYA-ABD) and Western cagA-ca52(EPIYA-ABC)transgenic mice can induce apparent pathological changes in stomach, moreover, thepathogenic effect of East-Asian CagA is more severe than that of Western CagA.