| Helicobacter pylori (H pylori) is an important human pathogenic bacteria that is a Gram-negative, spiral and microphilic bacteria that selectively colonizes gastric mucosa, causes gastritis and gastric ulcers. This human pathogen is also strongly associated with gastric adenocarcinomas and mucosa-associated with lymphoid tissue (MALT)lymphomas. In 1994, international cancer institute had determined it being type I carcinogen. H pylori infects about one-half of the world's population and usually persists lifelong unless eradicated by antibi-otic treatment. Its transmission and persisting mechanism is still not illuminated up to now, also the therapeutic methods.During the process of colonizing the host, H pylori induces a strong inflammatory response from infiltrating. This defence is mediated by neutrophils and macrophages, culminating in generation of large amounts of ROS/RNS presented to the persistent pathogen. Nevertheless, H pylori survives these conditions and persistently colonizes the gastric mucosa. Recent studies have shown the oxidative stress response of H pylori is much more vast, and diverse antioxidant systems of H pylori have been discovered. However, the known H pylori regulating genes to combat the ROS were few, and the antioxitive regulating mechanism is unknown. Studies show that the host defense H pylori infection by cell immune, particularly macrophage phagocytosis, however, H pylori can combat of immune cells effectively, especially Th1 immune response, it is well proved that vacA and urease genes in the immune escape take important role. However, regulation mechanism of H.pylori immune escape is raely studied.spoT gene is a transcribe regulating gene in large of system, the role of which was known firstly in controlling stringent response in Escherichia coli. The so-called stringent response is the strongly changes in gene expression patterns of the bacterial responsing to the lack of amino acid, also known as "nutritional stress response". Now it is collectively referred to as"stringent response"that the survival of bacteria in the various adverse circumstances occurring changes in gene expression patterns collectively referred to as "stringent response". It can conclude the spoT gene from H Pylori may be take the same role. But, the regulating mechanism is not very clear. So this thesis is to investigate the regulation role of in H pylori combating the oxidative stress in vivo.METHODS:1. An spoT gene mutant strain of H pylori type strain 26695 was constructed by insertional mutagenesis, and are feeded to Mongolia gerbils respectively. To put to death of the Mongolia after they were feeded 2,4,6,8 weeks respectively harvest bacteria from the stomach mucous, then to cultivate the bacterium and measure ureC gene copies number to detect H pylori by Real Time Quantitative PCR.2. To stimulate the mutant strain and the wild strain respectively by immunocytes from human peripheral blood for 30 minutes and 60 minutes. Then the total RNA was extracted from the bacteriums with Trizol for revorse transcription and poliferation of cDNA according to the instruction. And to detective the mRNA expression level of antioxidant and virulence genes by Real Time PCR.3. To get together the peripheral blood of every Mongolia gerbil through eyeballs removed when the Mongolia gerbil was died.To meaure the 8-hydroxyl deoxyguanosine (8-OHdG) with enzyme linked immunosorbent assay,8-OHdG is the marker of DNA in peripheral blood when it is oxidative damage.TO make a comparison of the datas. The results can show the level of reactive oxygen species, and can indicate the the level of immunocytes induced by bacteriums.4. Flow cytometry detection:H pylori spoT mutant and wild respectively effect on the immunocytes collected from human peripheral blood (the immunocytes have been loaded by DHE), in order to test the level of reactive oxygen species.RESULTS:1. Animal experiment:The bacterium were cultured well. H pylori spoT mutant and wild both colonize gastric mucosa successfully. The amount of spoT gene mutant strain is fewer than the wild strain in the stomach mucous of Mongolia gerbil.2. Real Time PCR confirmed that the genes associated with defense the oxidative stress (sodB,katA,tasA,rocF) and with virulence (cagA,vacA,napA) are disorderly, and in mutant strain and the critical regulation genes clpX and clpP, which degradate the oxidatived proteins, were are down-transcription.3. The level of 8-OHdG:the diffenrence between W group (infected by wild H pylori) and M group (infected by spoT mutant) has not statistically significant at the end of 2,4,6 weeks; while the level of 8-OHdG of M group is fewer significantly than W group.4. In all the time dots the content of reactive oxygen species of W group (infected by wild Hpylori) is higher than M group (infected by spoT mutant)CONCLUSIONS:The potentia of colonizing in the spoT mutant strain is lower than the wild strain, meanwhile the genes associated with combating oxidative stress and with virulence are disordered and the two critical genes about degrading the damaged protains (clpX and clpP) are down-transcription, which can define the phenomenon. As a result we can infer the explanation of the lower potentia of colonizing of spoT mutant strain:it is mainly the phagocytosis bacteria from immune cells, not the antioxidant stress capacity of the spoT mutant changes.
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