外排泵ABC转运白基因msbA和spab在幽门螺杆菌多重耐药中的作用

发布时间：2018-04-15 09:30

本文选题：外排 + 转运　；参考：《郑州大学》2011年硕士论文

【摘要】：目的幽门螺杆菌(Helicobacter pylori, H.pylori)在人群中感染率在40-90%,它是慢性胃炎、消化性溃疡和胃粘膜相关淋巴组织淋巴瘤的主要病因,并与胃癌的发生密切相关.临床中常联合应用抗生素对幽门螺杆菌进行根除治疗.随着抗生素的广泛应用,幽门螺杆菌的耐药率逐年上升,并且已经出现同时对阿莫西林、克拉霉素和甲硝唑耐药的多重耐药(multi-drug resistance)株.目前针对细菌多重耐药的机制研究已成为细菌耐药性研究中的热点问题.其中,细菌外排泵(efflux pump)在多重耐药中起重要作用。外排泵是细菌体内能将有害底物泵出体外的一类转运蛋白,可分为5个家族。国内外和我们前期对RND类外排泵在幽门螺杆菌多重耐药中作用的机制研究已有报道,但对于ABC转运蛋白基因在幽门螺杆菌多重耐药株中的表达及其作用尚需进一步研究.本研究在我们前期研究的基础上,从GenBank筛选出ABC转运蛋白家族基因中的两个典型的多重耐药蛋白基因msbA和spab,测定其在幽门螺杆菌多重耐药株中的表达,并构建对应的基因敲除株,测定敲除前后对应菌株对各种抗生素的敏感性,研究两种基因在幽门螺杆菌多重耐药中的作用.进一步阐明在幽门螺杆菌多重耐药起作用的外排泵的种类和意义,为临床上幽门螺杆菌多重耐药的研究打下理论基础。方法 1.从临床胃炎和消化性溃疡病人胃粘膜标本分离培养出幽门螺杆菌野生菌株,测定菌株对各种抗生素的MIC。 2.应用氯霉素在敏感野生株中诱导出对应的幽门螺杆菌多重耐药株,并采用琼脂二倍稀释法测定敏感株和多重耐药菌株对各种抗生素的MIC。 3.PCR检测幽门螺杆菌野生株中msbA和spab基因的表达。 4.应用RT-PCR分别测定msbA和spab基因在野生敏感株和诱导多重耐药株中的表达。 5.分别构建msbA和spab的基因敲除株,并测定基因敲除前后对9种抗生素的MIC。结果 1.成功从临床上分离培养出幽门螺杆菌野生株50株。将临床分离的4株敏感株和标准株HpNCTC 11637经氯霉素诱导,成功诱导出对应的多重耐药株5株,经琼脂二倍稀释法测定5株多重耐药株对氨苄西林、头孢菌素、四坏素、克拉霉素、氧氟沙星和呋喃哗酮的MIC相对于敏感株均有不同程度的提高。 2.随机选择20株临床分离株,均检测出msbA和spab基因,大小分别为303bp和424bp,未发现两种基因的缺失株。 3.多重耐药株中msbA和spab基因的相对表达量明显高于野生敏感株(msbA:多重耐药株1.8200±0.5310 vs敏感株0.8420±0.0789,P=0.018；spab多重耐药株1.8340±0.2726 vs敏感株1.2180±0.0743,P=0.015)。 4.成功构建了msbA和spab的基因敲除株(HpMZ1006△msbA和HpMZ1006△spab)。HpMZ1006△msbA和HpMZ1006△spab对于四种抗生素(克拉霉素、氯霉素、氧氟沙星和利福平)的MIC相对敲除前野生株有明显下降。结论 1.在氯霉素的长期次抑菌浓度诱导下,敏感的幽门螺杆菌菌株转变为多重耐药菌株,说明抗生素的长期滥用和剂量不足会导致耐药菌株的产生。 2.ABC转运蛋白msbA和spab基因在幽门螺杆菌中普遍存在。 3.ABC转运蛋白msbA和spab基因在幽门螺杆菌多重耐药机制中起重要作用。 4.首次阐明了在幽门螺杆菌多重耐药机制中,除了RND家族外排泵起作用之外,还有ABC转运蛋白外排泵家族参与其中。 5.由于ABC转运蛋白外排泵家族在能量来源与其他外排泵家族不同,为以后新型外排泵抑制剂的研发提供了理论基础。
[Abstract]:objective
Helicobacter pylori (Helicobacter pylori, H.pylori) in the 40-90% infection rate in the population, it is the main cause of chronic gastritis, peptic ulcer and gastric mucosa associated lymphoid tissue lymphoma, and is closely related with the occurrence of gastric cancer. The clinical often combined application of antibiotics on Helicobacter pylori eradication therapy. With the widespread use of antibiotics. The resistance rate of Helicobacter pylori increased year by year, and has appeared at the same time of amoxicillin, clarithromycin and metronidazole resistance of multidrug resistant strains (multi-drug resistance). The mechanism of multi drug resistant bacteria has become a hot issue in the study of bacterial resistance. The bacterial efflux pump (efflux pump) plays an important role in multi drug resistance efflux pump. Bacteria can be harmful to the substrate is a kind of pump transport protein in vitro, can be divided into 5 families.
At home and abroad and our previous studies on RND efflux function in multidrug resistance of Helicobacter pylori mechanism have been reported, but for the ABC transporter gene expression and its role in multidrug resistance of Helicobacter pylori strains need further study. This study is based on our previous studies, selected ABC transporter family gene in two typical multidrug resistance protein gene msbA and SPAB from GenBank, its expression in multidrug resistance of Helicobacter pylori strains in the determination, and construct the corresponding gene knockout strains, determination of knockout strains to various antibiotics and the corresponding sensitivity effect of two genes in multidrug resistance of Helicobacter pylori the variety and significance. To further clarify the role in multidrug resistance of Helicobacter pylori pumps, lay the theoretical foundation for clinical research on multidrug resistance of Helicobacter pylori.
Method
1. from clinical gastritis and peptic ulcer patients with gastric mucosa specimens isolated from wild strains of Helicobacter pylori, determination of MIC. strains to various antibiotics
Multidrug resistance of Helicobacter pylori strains in the 2. application of chloramphenicol sensitive strains induced in the corresponding, and using two - fold agar dilution method strains to various antibiotics MIC.
Detection of 3.PCR expression of Helicobacter pylori msbA and SPAB genes in wild strains.
4. application of RT-PCR were measured by the msbA and SPAB genes in wild sensitive strains and multi drug resistant strains induced expression.
5. construction of msbA and SPAB gene knockout strains and determination of gene knockout of MIC. to 9 kinds of antibiotics before and after
Result
1. successfully isolated Helicobacter pylori strains of 50 wild strains from clinical. The clinical isolates of 4 strains of sensitive strains and standard strain HpNCTC 11637 was induced by chloramphenicol, successfully induced the corresponding multi drug resistant strains of 5, by two fold agar dilution method for determination of 5 strains of multi drug resistant strains to ampicillin, cephalosporins, four bad elements compared with ofloxacin and clarithromycin, furan ketones wow MIC sensitive strains had different degrees of improvement.
2. randomly selected from 20 clinical isolates were detected in msbA and SPAB genes, the size were 303bp and 424bp, were found two gene deletion strains.
The relative expression of 3. multidrug-resistant msbA and SPAB strain gene was significantly higher than that of wild strains (msbA: 1.8200 + 0.5310 multiple drug resistant strains of vs sensitive strains of 0.8420 + 0.0789, P=0.018; 1.8340 SPAB multiple drug resistant strains of vs sensitive strains of 1.2180 + 0.2726 + 0.0743, P=0.015).
4. msbA was successfully constructed and SPAB gene knockout strains (HpMZ1006 msbA and HpMZ1006 SPAB) and HpMZ1006.HpMZ1006 Delta msbA Delta SPAB to four kinds of antibiotics (clarithromycin and ofloxacin, chloramphenicol, rifampicin) relative MIC knockout before the wild strain decreased significantly.
conclusion
1. in the long term of chloramphenicol subinhibitory concentration induced by Helicobacter pylori strains sensitive to multiple antibiotic resistant strains, indicating that the long-term antibiotic abuse and insufficient dose will lead to the emergence of resistant strains.
2.ABC transporter msbA and SPAB gene exists in Helicobacter pylori.
3.ABC transporter msbA and SPAB gene play an important role in the mechanism of multidrug resistance of Helicobacter pylori.
4. for the first time to clarify the mechanism of multidrug resistance in Helicobacter pylori, in addition to the RND family of efflux pump function, and ABC transporter efflux pump family involved.
5. because of the ABC transporter family of efflux pump in energy sources and other efflux pump family, provides a theoretical basis for the development of new type of efflux pump inhibitors.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

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