| Background and Objective: Human H.pylori is a Gram-negative bacterium mainly found in the slime layer of gastric mucosa. It has been acknowledged a critical etiological factor causing chronic active gastritis, atrophic gastritis and duodenal ulcer and a high risk causing gastric cancer. It is also has a close relation with mucosa-associated lymphoma. More and more study results have shown that H.pylori infection is statistically related to many non-gastric diseases, e.g., cardiovascular disease and skin disease. Therefore, H.pyiori infection and related diseases has become a crucial subject of public health.The primary drug resistance rates of H.pylori to antibiotics, especially Clarithromycin and Metronidazole, are increasing, having affecting the curative effect of common eradicative therapies. Clarifying drug resistance mechanism, overcoming primary drug resistance, avoiding secondary drug resistance and rescue therapy after treatment failure are hot topics in H.pylori treatment.Versalovic firstly reported that Clarithromycin resistance is associated with point mutations in H.pylori 23S rRNA, causing conformational changes of ribosome and the changes of the binding sites of macrolides antibiotics, and further lowering the compatibility of H.pylori and macrolides. As a result, the drug will not prevent protein synthesis of bacteria and eventually develop drug resistance.DNA chip is actually settling a large amount of target genes or oligonucleotides orderly and in a high density on glass slides, silicon slides or films. These fixed molecular probes form high-density DNA micro-arrays on the base. After sample nucleus acid molecules being marked, they are hybridized with the points in the DNA arrays fixed on the base. The number of sample molecules and array information are acquired by testing the hybridizing information so as to study DNA arrays and functions intensively. DNA chips are incomparably effective, fast and come with multiple parameters. It is a significant innovation and breakthrough in the conventional biotechnology. DNA chips are used wildly in medicine, military affairs, judicatory affairs and agriculture, including gene expression profile, disease diagnosis, drug screening, looking for new genes, re-sequencing, genotyping, gene mapping and gene mutation. It is also used in the studies on gene functions and pathogenesis, disease typing and drug screening.The research chose gastric mucosa biopsy specimen under gastroscope. H.pylori was separated and cultured out of human body and tested on Clarithromycin sensitive test so that investigated Clarithromycine resistance of H.pylori in populations. The research was to test the point mutations at position 2142, 2143 and 2182 in H.pylori 23S rRNA with oligonucleotide micro-array technology and sequencing, analyze the relation between 23S rRNA gene polymorphism and Clarithromycine resistance of H.pylori and probe the molecular mechanism of Clarithromycine resistance of H.pylori so as to instruct medication.Parti Survey on Clarithromycine Resistance of H.pylori in PopulationThe experiment collected gastroscopy cases from hospitals in Zhoushan and Jiaxing, Zhejiang. Took gastric mucosa biopsy specimens to culture H.pylori, separated and identified them and then tested on Clarithromycin sensitive test. Investigated Clarithromycine resistance of H.pylori preliminarily and monitored drug-resistant strains of H.pylori.1. Materials:Gastric mucosa biopsy specimens were obtained from gastroscoped patients of the digestion departments of the hospitals in Zhoushan and Jiaxing. One mucosa organism was taken from gastric antrum and gastric corpus respectively. All patients had signed ICFs before performing gastric mucosa biopsy.2. Methods:Grinded the gastric mucosa biopsy specimens and put them into enrichment tubes. Cultivated them for 48 hours at 37"C microaerophilic atmosphere. Then obtain enrichment medium with an inoculating loop and separated them on isolation medium. Purely cultivated them for 72 hours in microaerophilic atmosphere. Examined them under microscope and then identified them as H.pylori strains by oxidase reaction test, catalase reaction test, urease test. Finally, performed agar disc diffusion test on antibiotic sensitivity to test Clarithromycin resistance of H.pylori.3. Results:(1) H.pylori colonial morphology: round and isolated small colony, grayish-white, translucent, and tear-shaped with a diameter of 0.5-1.5 mm. There was mildhemolysis on the blood agar. The bacteria were found slim and curving in spiral-shape, S-shape or eagle-shape. Gram staining test showed negative. Biochemical-test identified them H.pylori strains.(2) hi 1439 cases, positive rate of bacteria culture was 32.73%.(3) Cultured 471 positive H.pylori strains, in which, drug sensitive test showed 165 strains were Clarithromycin resistant. The drug resistant rate was 35.03 %. There was no age difference and sex difference in Clarithromycine resistance.Part HA study on Single Nucleotide Polymorphism of a 23S rRNA Gene from Helicobacter pylori associated with Clarithromycin Resistance1. Background and aims:Resistance to antibiotics in Helicobacter pylori is increasing and becoming a serious problem in eradication treatment of H. pylori. Resistance of Helicobacter pylori to clarithromycin has been found to be associated with A2142G, A2143G and C2182T point mutations in the 23S rRNA gene. Thus, the purpose of the present study was to develope a new method to analyze single nucleotide polymorphism (SNPs) of 23 S rRNA gene using oligonucleotide microarray and to determine the prevalence of each mutation in 54 H. pylori positive patients.2. Methods:Gastric tissue biopsy specimens were obstained from patients followed by DNA extraction and a PCR assay that amplified a portion of 23 S rRNA from H. pylori isolates. An oligonucleotide microarray system which included oligonucleotide synthesis, preparation of the oligonucleotide microarray, hybridization and signal detection for analysis of the known SNPs of 23S rRNA gene was fabricated. The relevant mutation was confirmed by transformation and culturing of colonies followed by DNA sequencing analysis.3. Results:Fifty-four gastric biopsy specimens yielded H. pylori-positive results and were studied to detect mutations in the 23 S rRNA gene. Our findings show no A-to-G transition at position 2142. Both the A2143G and the C2182T mutations were present respectively in 11.11% and 12.96% of H. pylori strains examined. The relevant mutation in transformants was confirmed by DNA sequencing to be the sames as that described at position 2142,2143 and 2182 using oligonucleotide microarray.Conclusions1. Clarithromycine resistant rate of H.pylori was 35.03%.2. A2142 positions of H.pylori 23S rRNA were all in wild type (54/54). A2143G mutation rate was 11.11% (6/54). No A2143C and A2143T mutations were found. C2182T mutation rate was 12.96% (7/54). No C2182A and C2182G mutations were found. The rest are in wild type.3. The relevant mutation in transformants were confirmed by DNA sequencing to bethe same as that described at position 2142, 2143 and 2182 using oligonucleotide microarray.4. Oligonucleotide chip technology can be used in treating H.pylori infection.
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