| Helicobacter pylori (Hp) infection is responsible for various upper gastrointestinal tract diseases. Urease, the key factor for resistance to gastric acid, is essential for the successful survival and infection of Hp in the stomach. Nickel iron (Ni2+) is an important metal activator of urease. Wuwei region in gansu province is high Ni environment and has high incidence of gastric cancer. Long-term high Ni environmental impact on Hp urease genetic characteristics is necessary to further discuss. Research for the role of Notchl, DLL4 and HES1 in the development of gastric cancer, provide new theoretical basis to the treatment of invasion and metastasis of gastric cancer.Objective1. Analyze the genetic diversity and evolutionary features of urease gene (ureABEFGHI) of Hp isolated from Wuwei; detect Ni2+ in human serum, explore the relationship between Ni2+ and Hp infection; analyze the relationship between Hp infection and clinical diseases, living habits; explore the relationship between Hp infection and upper gastrointestinal tract diseases, deep study the specific information about Hp biological property, pathogenic mechanism.2. Detect expression of Notch signaling components Notchl, DLL4 and HES1 in gastric tumor, analyze the relationship between clinicopathological characteristics and survival, explore the development of gastric cancer.Methods1. Design a questionnaire for epidemiological investigation, determine the Hp infection status by 14C breath test. Gastroscope observation gastric mucosa lesions, take a biopsy pathologic examination. Analyze the relationship between Hp infection and patients age, sex, disease, living habits, especially dietary habit.2. The gastric mucosa samples collected from the patients in Wuwei area were cultured under micro aerobic condition, and the Hp strains were identified by bacterial colony features, morphology,and rapid urease test.3. Urease genes (ureABEFGHI) whole sequence of 23 Hp strains isolated from Wuwei area were amplified by PCR and sequenced. Hp urease genes sequences were analyzed by using NCBI database and MEGA software to build the genetic evolutionary trees, to compare similarities and evolutionary distance of ureABEFGHI between the isolated strains and the reported standard strains all around the world, and to discuss the relationship between the ureABEFGHI genetic features and clinical disease types.4. Atomic absorption spectrometric method was used to detect human serum Ni2+ concentration. The relationship between Hp infection and concentration of Ni2+ were Analyzed.5. Collect daily drinking water from patients’ house of Hp strong positive infection. Condensed water samples and PCR was used to amplify the ureA, ureB gene in order to explore the route of Hp transmission.6. Select paraffin specimens postoperatively in patients with gastric cancer(192 cancer tissue, 56 tissue adjacent to carcinoma), Notchl, DLL4 and HES1 expression were detected by immunohistochemistry and the relationship between Notchl, DLL4 and HES1 expression and patient’s clinicopathological features and patients survival time were analyzed.To further explore the role of Notch signaling pathway in gastric cancer development.Results1. The Hp positive rate of general people in Wwei is 51.93%, SPSS analysis showed that the rate of upper gastrointestinal tract diseases of Hp infection positive was significantly different from negative (P=0.014). And Hp infection significantly related to the frequence of eating pickled or mildew food (P=0.009;P=0.047).2.23 Hp strains were separated from Wuwei city, and then amplified urease genes by PCR, successfully got 22 Hp ureA gene samples,18 ureB gene samples,18 Hp ureE gene samples,21 Hp ureF gene samples,23 Hp ureG gene samples,15 Hp ureH gene samples,23 urel gene samples, and all these samples were successfully sequenced the whole sequences.3. MEGA phylogenetic analysis showed that the evolutionary of Hp ureA in Wuwei city was closer to Asian strains like F57, F32 strains than the strains from other continents. The average evolutionary distance was 0.1772982. Hp ureA gene existed a gathered phenomenon on the evolutionary tree in Wuwei city. ureB was closer to Asian strains such as 51, F57 and American strains like shil69, the average evolutionary distance was 0.25467252, it also has gathered phenomenon. ureE in Wuwei city was closer to Asian strains such as 51, F57, XZ274 and American strains like shi169, Puno135, the average evolutionary distance was 0.03673317, it has gathered phenomenon. ureF was closer to Asian strains such as F30, F57, XZ274, the average evolutionary distance was 0.03671494, it has gathered phenomenon. ureG was closer to Asian strains such as F30, Ok310 and European strains like HPAG1, ELS37, the average evolutionary distance was 0.03384030, it has gathered phenomenon too. ureH was closer to Asian strains such as OK113, XZ274 and European strains like P12, the average evolutionary distance was 0.05754463, it also has gathered phenomenon. Urel was closer to Asian strains like 51, F30, OK310, the average evolutionary distance was 0.02708512, it has gathered phenomenon.4. The evolutionary distance of Hp ureB gene collected from Wuwei city between standard strains (0.25467252) was greater than ureA gene genetic distance (0.17729824), the evolutionary distance of structure gene ureAB was greater than accessory gene ureEFGHI. The relationship between the evolution trend of urease gene (ureABEFGH) and the clinical and pathological features of diseases was no significant correlation. The distribution of ureI evolution trend and diseases present a degree of aggregation, there may had a correlation.5. SPSS analysis show that average concentration of nickel iron in serum is 5.814±9.085 μg/L, there are no significant differences between Hp infection and the concentration of Ni2+ in serum (P=0.224). Hp was not founded in drinking water.6. Notchl positive rates in gastric carcinoma, paracancerous tissue, and control group(48.30%、25.00%、16.67%) were significant higher in gastric carcinoma group than that in the other groups(P=0.002). The positive rate in gastric carcinoma with lymph node metastasis group(41.3%) was significant lower than that without lymph node metastasis group(61.82%, P=0.012). DLL4 positive rate in gastric carcinoma, paracancerous tissue, and control group was no significantly difference between different group(55.94%、77.14%、56.67%). HES1 positive rate in gastric carcinoma, paracancerous tissue, and control group also was no significant difference between different group(36.64%、56.25、33.33%). HES1 positive rate in poor differentiation gastric carcinoma was significantly higher than that in high/middle differentiation group (P=0.029).Conclusions1. Upper gastrointestinal tract diseases is associated with Hp infection, the gastrointestinal tract diseases risk of Hp positive was significantly higher than negative. Hp infection significantly related to the frequence of eating pickled or mildew food, there was a degree of correlation between Hp infection and living style. Ni2+ concentration of human serum in Wuwei failed to affect Hp infection rates.2. The evolutionary of Hp urease gene (ureABEFGH1) in Wuwei city were closer to Asian strains; some strains of Hp urease gene were closer to South American strains and European strains. It can prove Hp has hybridization and mutation in human activation. The average evolutionary distance of Hp urease structure gene ureAB popular in Wuwei city was higher than accessory gene ureEFGHI; ureAB has greater variability than ureEFGHI. The mutation of structural gene make the protein structure changed, may lead to the change of urease activity.3. Notchl can be used as a target for gastric cancer diagnosis and treatment. HES1 can be acted as an indicator of the degree of differentiation. |
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