本文關鍵詞： 篩選 鑒定 雙重 耐藥 幽門 桿菌 HBD-2 融合 白的 體外 抗菌 作用　出處：《浙江大學》2007年博士論文　論文類型：學位論文

【摘要】： 第一部分幽門螺桿菌三聯(lián)根除療法影響因素的logistic回歸分析 幽門螺桿菌(Helicobacterpylori，Hp)是淺表性胃炎、消化性潰瘍的致病因子，與胃癌、胃黏膜相關性淋巴組織淋巴瘤密切相關，世界上約有一半以上的人感染Hp。幽門螺桿菌根除是治療消化性潰瘍和防止復發(fā)的重要手段。國內(nèi)外很多研究提示，PPI聯(lián)合克拉霉素、阿莫西林/甲硝唑是Hp根除的有效治療方案。但由于幽門螺桿菌耐藥性等問題，嚴重的影響了該三聯(lián)療法的療效。另外，研究表明，Hp耐藥問題及患者醫(yī)囑順從性不能完全解釋Hp根治失敗的原因。目前缺乏Hp根除率影響因素綜合評價的研究。本研究的目的是評估在浙江地區(qū)，以質(zhì)子泵抑制劑(PPI)為基礎聯(lián)用克拉霉素、阿莫西林/甲硝唑的三聯(lián)療法根治幽門螺桿菌的療效，探討幽門螺桿菌的耐藥性、患者的性別、年齡、煙酒史、疾病類型和組織學改變對幽門螺桿菌根除率的影響。通過這一研究，也為進一步研究多藥耐藥幽門螺桿菌提供了物質(zhì)基礎。 材料和方法：患者行胃鏡檢查，符合研究入選標準的患者共取4塊組織標本，，一塊胃竇組織做快速尿素酶試驗，一塊胃竇組織行幽門螺桿菌培養(yǎng)和藥敏試驗，2塊胃竇組織行組織學檢查。患者幽門螺桿菌培養(yǎng)陽性或快速尿素酶、組織學檢查陽性被認為是幽門螺桿菌感染。所有患者接受7天幽門螺桿菌根除治療：達克普隆(30mg)、克拉霉素(500mg)、阿莫西林(1g)或甲硝唑(0.5g)，每天2次，口服。治療結(jié)束后4-6周，采用C~(14)-尿素呼氣試驗評估幽門螺桿菌根除情況。同時收集患者的一般情況。所有組織標本由一高年資的病理科醫(yī)生評估。幽門螺桿菌藥敏試驗采用紙片法和二倍平皿稀釋法。運用SAS軟件，分析研究對象的各類數(shù)據(jù)，采用χ~2檢驗和多因素非條件logistic回歸分析的方法，P＜0.05認為有統(tǒng)計學差異。 結(jié)果與結(jié)論：78個幽門螺桿菌感染的患者中，42個男性，36個女性，平均年齡46歲，27個患者有活動性十二指腸潰瘍(占34.6％)，51個患者是非潰瘍性消化不良(NUD)。34個患者有抽煙史，6個患者有較嚴重的喝酒史。幽門螺桿菌根除率為61/78(78.2％)。5個患者出現(xiàn)輕度治療副反應(輕度腹瀉=2，上腹不適=3)。在78株幽門螺桿菌菌株中，對克拉霉素原發(fā)性耐藥率為14.1％，甲硝唑耐藥率為69.2％，阿莫西林耐藥率為1.3％，其中對克拉霉素耐藥的菌株同時存在甲硝唑耐藥，共11株，未發(fā)現(xiàn)同時對3種抗生素耐藥的菌株。患者性別、克拉霉素耐藥、十二指腸潰瘍病變，這三變量是Hp根除失敗的獨立相關因素(P＜0.05)。在本研究中，患者的年齡，煙酒史，胃竇部組織學改變與療效無明顯相關性(P＞0.05)。 第二部分多藥耐藥幽門螺桿菌篩選及鑒定 如何治療幽門螺桿菌反復根除失敗的患者是臨床上一棘手的問題。目前，關于多藥耐藥幽門螺桿菌的耐藥機制、根除等研究罕見報道。有必要從臨床篩選、鑒定多藥耐藥菌株，以便進一步研究其耐藥機制，尋求新的根除手段。通過我們前面的研究，我們分析了幽門螺桿菌根除治療的影響因素，更重要的是，我們初步篩選了一些多藥耐藥的幽門螺桿菌菌株。通過藥敏試驗，雖然沒能發(fā)現(xiàn)存在克拉霉素、甲硝唑、阿莫西林三個藥物同時耐藥的菌株，但我們篩選到了一些克拉霉素、甲硝唑同時耐藥的菌株。從中，我們篩選了5株高MIC的幽門螺桿菌菌株，通過病例的隨訪，這些菌株的感染者三聯(lián)治療失敗后換用四聯(lián)療法，最后依然沒有根除幽門螺桿菌。通過臨床的治療效果和體外藥敏實驗，我們從臨床初步判斷其為多藥耐藥幽門螺桿菌。本實驗目的，就是通過菌株培養(yǎng)，分析甲硝唑、克拉霉素耐藥的相關基因rdxA、23SrRNA，從實驗室角度進一步篩選、鑒定其是否是多藥耐藥幽門螺桿菌。 材料和方法：按傳統(tǒng)的酚—氯仿抽體法提取了幽門螺桿菌基因組DNA，根據(jù)文獻和Hp26695株基因序列，分別設計和合成rdxA、23SrRNA序列的特異性引物，由上海博亞生物技術有限公司(BioAsia)合成，1.5％的瓊脂糖凝膠電泳檢查擴增產(chǎn)物。將10ul 23SrRNA基因PCR產(chǎn)物行RFLP分析，分別用BsaI、BbsI兩種限制性內(nèi)切酶酶切。前者于50℃孵育24hr，后者于37℃孵育24hr。同時將rdxA基因PCR產(chǎn)物行T-A克隆，委托上海申能搏彩生物科技有限公司，采用雙脫氧鏈末端終止法在測序儀上測定含正確大小插入片段的核苷酸序列。 結(jié)果：5株臨床菌株我們命名為Hp20061、Hp20062、Hp20063、Hp20064、Hp20065、rdxA基因測序結(jié)果，Hp20063菌株于193位點額外插入了了一堿基A，Hp20065菌株于549位點額外插入了一堿基A，導致菌株的rdxA基因核苷酸序列出現(xiàn)移碼突變，從而導致翻譯的蛋白質(zhì)結(jié)構(gòu)和功能的改變。而另3個多藥耐藥菌株由于出現(xiàn)多位點堿基突變，也導致相應的氨基酸改變。23SrRNA基因RFLP分析，5株多藥耐藥菌株的PCR擴增物可被BsaI酶切成兩個片段。5株擴增物均未能被BbsI酶切。提示5株多藥耐藥菌株在23S rRNA基因功能區(qū)Ⅴ第2144位點有A→G突變。所有菌株均不存在2143位點A→G突變。 結(jié)論：通過對5株幽門螺桿菌菌株的rdxA基因、23SrRNA基因的分析，我們認為菌株Hp20063、Hp20065是多藥耐藥幽門螺桿菌，而Hp20061、Hp20062、Hp20064菌株臨床為多藥耐藥菌株，但其分子生物學特性有待更進一步的實驗室鑒定。 第三部分HBD-2融合蛋白對多藥耐藥幽門螺桿菌的體外抗菌作用 機體對于幽門螺桿菌感染的自身反應主要是通過中性粒細胞、淋巴細胞引起的一系列免疫反應。研究表明，幽門螺桿菌感染會引起機體一些抗菌肽表達水平升高。抗菌肽是廣泛存在于動、植物體內(nèi)，具有一定抗菌譜的小分子肽。在脯乳動物中發(fā)現(xiàn)的抗菌肽主要是防御素(defensin)，防御素可分為α和β兩類，β-防御素家族在許多上皮組織包括消化道上皮中有更廣泛的表達。在人類中，已發(fā)現(xiàn)4種β-防御素(HBD1～4)。防御素具有廣譜的抗菌活性，HBD-1對革蘭陽性菌、革蘭陰性菌、真菌、螺旋體、分支桿菌及有包膜病毒均具有殺滅作用，HBD-2對革蘭陰性菌、革蘭陽性菌、酵母菌具有較強的殺菌作用，其對革蘭陰性菌殺菌作用更為顯著。已有研究表明，在體外試驗中，HBD-2對于幽門螺桿菌具有較強的殺傷作用，但是對于多藥耐藥幽門螺桿菌的作用如何，尚未有文獻報道。另外，大量研究表明，抗菌藥物的抗菌作用體內(nèi)試驗和體外試驗往往會存在很大差別，因此，有必要開展HBD-2抗幽門螺桿菌的體內(nèi)試驗。但是，由于存在一些客觀原因1)目前HBD-2蛋白質(zhì)來源不多，購買比較昂貴2)胃酸對蛋白質(zhì)的降解作用，很少有關于HBD-2相關的胃腸道體內(nèi)試驗的研究。因此，我們覺得有必要基因重組表達HBD-2蛋白，并研究重組蛋白對多藥耐藥幽門螺桿菌的抗菌活性。 材料和方法：LB固體培養(yǎng)基上分離劃線，在含氨芐青霉素的新鮮LB液體培養(yǎng)基中擴增，經(jīng)IPTG誘導，超聲破碎儀破碎，獲得重組HBD-2融合蛋白。再將樣品過NTA層析柱，收集目的蛋白反復透析，用SDS-PAGE分析確定目的蛋白的分布情況，純化復性后的蛋白質(zhì)濃縮保存。用二倍平皿稀釋法，分別選擇5株多藥耐藥幽門螺桿菌和5株MTZ、C1A敏感菌株，檢測其MIC。用t-test檢驗2組間差別。 結(jié)果： 1、獲得純化的重組融合蛋白HBD-2。 2、HBD-2對于多藥耐藥幽門螺桿菌具有一定的抗菌作用。 3、所有MIC均取對數(shù)后行t檢驗結(jié)果P＞0.05，兩組間無明顯差異。 結(jié)論：大腸桿菌基因重組獲得HBD-2蛋白是一種可行的辦法，重組蛋白對多藥耐藥的幽門螺桿菌具有一定的抗菌作用。我們可以在此基礎上，進一步開展多藥耐藥幽門螺桿菌根除的體內(nèi)研究。
[Abstract]:Logistic regression analysis of factors influencing Helicobacter pylori triad eradication therapy
Helicobacter pylori (Helicobacterpylori, Hp) is superficial gastritis, pathogenic factor, peptic ulcer and gastric cancer and gastric mucosa associated lymphoid tissue lymphoma is closely related to the world about Hp. of Helicobacter pylori infection eradication of more than half of the people is an important means for the treatment of peptic ulcer and prevent recurrence. Many domestic and foreign research suggests PPI, combined with clarithromycin, amoxicillin / metronidazole is effective in treatment of Hp eradication. But due to the problem of Helicobacter pylori resistance, serious impact on the efficacy of triple therapy. In addition, the research shows that the Hp drug resistance problem and patient compliance can not fully explain the reason of Hp radical failure. The lack of influence of the eradication rate of Hp study on the comprehensive evaluation factors. The purpose of this study was to evaluate in the Zhejiang area, with a proton pump inhibitor (PPI) based combined with clarithromycin, amoxicillin / metronidazole triple therapy Effect of root treatment for Helicobacter pylori infection, to investigate the antibiotic resistance of Helicobacter pylori, the patient's sex, age, smoking history, disease type and histological changes of H.pylori eradication rate. Through this research, it provides the material basis for the further study of multidrug resistance of Helicobacter pylori.
Materials and methods: patients who underwent gastroscopy, met study criteria in patients with a total of 4 pieces of tissue from a gastric antrum do rapid urease test, a gastric antrum for Helicobacter pylori culture and drug sensitive test, 2 gastric antrum tissue for histological examination. Patients with Helicobacter pylori positive or rapid urease test, histological examination was considered positive Helicobacter pylori infection. All patients received 7 days of Helicobacter pylori eradication therapy: Takepron (30mg), clarithromycin (500mg), amoxicillin (1g) or metronidazole (0.5g), 2 times per day orally. 4-6 weeks after the treatment, using C~ (14 -) urea breath test evaluation of Helicobacter pylori eradication. At the same time to collect the general condition of the patient. All of the samples by a senior pathologist. Evaluation of Helicobacter pylori susceptibility test by Kirby Bauer method and two fold agar dilution method. Using SAS software, divided The data of the subjects were analyzed by the methods of chi square ~2 test and multiple factor Logistic regression analysis. P < 0.05 thought there was a statistical difference.
Results and conclusion: 78 patients with Helicobacter pylori infection, 42 male, 36 female, mean age 46 years, 27 patients with active duodenal ulcer (34.6%), 51 patients with non ulcer dyspepsia (NUD).34 patients with a history of smoking, 6 of patients have more serious drinking history. The Helicobacter pylori eradication rate was 61/78 (78.2%).5 patients with mild toxicity (=2 mild diarrhea, epigastric discomfort. =3) in 78 strains of Helicobacter pylori strains, primary resistance to clarithromycin was 14.1%, metronidazole resistance rate was 69.2%. The resistance rate for active forest 1.3%, the resistance to clarithromycin and metronidazole resistant strains, 11 strains, and resistant to 3 antibiotics were found. Patients with gender, clarithromycin resistance, duodenal ulcer disease, these three variables are the independent factors associated with Hp eradication failure (P < 0.05) in this. In the study, there was no significant correlation between the age of the patients, the history of tobacco and alcohol, the histological changes of the gastric antrum and the curative effect (P > 0.05).
Screening and identification of multidrug resistant Helicobacter pylori in second parts
How to treat Helicobacter pylori eradication failure in patients with repeated clinical problem. At present, the resistance mechanism of multidrug resistance of Helicobacter pylori eradication, and so on are rare. It is necessary from the clinical screening, identification of multi drug resistant strains, in order to further study the mechanism of drug resistance, to seek new means. Through eradication in our previous studies, we analyzed the influential factors of Helicobacter pylori eradication, and more importantly, we screened the Helicobacter pylori strains are multidrug resistance. The drug sensitivity test, although not found clarithromycin, metronidazole, amoxicillin and three drug resistant strains, we screened some strains resistant to clarithromycin and metronidazole. From this, we screened 5 strains of high MIC of Helicobacter pylori strains, the patients infected with these strains lost triple therapy After the defeat in quadruple therapy, finally still is not the eradication of Helicobacter pylori. Through clinical treatment and drug sensitivity test in vitro, we determine the initial clinical multidrug resistance of Helicobacter pylori. The purpose of this study is through the analysis, strains, metronidazole, clarithromycin resistance related genes rdxA, 23SrRNA, from the perspective of laboratory further screening, identification of whether the multidrug resistance of Helicobacter pylori.
Materials and methods: according to the traditional phenol chloroform extraction method to extract the body of Helicobacter pylori genomic DNA, according to the literature and Hp26695 gene sequence was designed and synthesized rdxA, specific primers of 23SrRNA sequences, by Shanghai Boya Biotechnology Co. Ltd. (BioAsia) synthesis, agarose gel electrophoresis of the PCR products. The 1.5% check the 10ul 23SrRNA PCR gene product RFLP analysis, respectively BsaI, BbsI two kinds of restriction enzymes. The former at the temperature of 50 24hr incubation, the latter at the temperature of 37 24hr. incubation and rdxA PCR gene product T-A clone, commissioned by the Shanghai lottery to Shen biotechnology limited by the dideoxynucleotide chain determination with the correct size of the nucleotide sequence of the cDNA insert in the sequencer termination method.
Results: 5 clinical strains, named Hp20061, Hp20062, Hp20063, Hp20064, Hp20065, rdxA gene sequencing results, Hp20063 strains in 193 additional sites into a base A, Hp20065 strains in 549 additional sites for inserting a base A, leading to rdxA gene nucleotide sequence of strains have frameshift mutations, resulting in protein structure and function of translation changes. While the other 3 multi drug resistant strains due to the emergence of a number of point mutation and.23SrRNA gene RFLP analysis also leads to change of the corresponding amino acid of 5 strains of multi drug resistant strains were amplified by PCR BsaI can be digested into two fragments of.5 strain were amplified by BbsI to digestion. That 5 strains of multi drug resistant strains in 23S rRNA gene locus A 2144th V to G mutation. All strains were there 2143 loci A to G mutation.
Conclusion: the rdxA gene of 5 strains of Helicobacter pylori strains, 23SrRNA gene analysis, we believe that the strains Hp20063, Hp20065 is the multidrug resistance of Helicobacter pylori, Hp20061, Hp20062, Hp20064 strains of clinical multi drug resistant strains, but its molecular biological characteristics to laboratory identification further.
In vitro antibacterial effect of the third part of HBD-2 fusion protein on multidrug resistant Helicobacter pylori
The body for Helicobacter pylori infection and autoimmune reaction mainly by neutrophils, lymphocytes caused by a series of immune reaction. The results showed that some antibacterial peptides could cause the body to increase in the expression level of Helicobacter pylori infection. Antimicrobial peptides are widely existing in animals, plants, small peptides with certain antibacterial spectrum of antibacterial peptide was found. The milk is mainly preserved in animal defensin (defensin), defensins can be divided into two types of alpha and beta, beta defensin family in many epithelial tissues including more extensive expression with the epithelium of the digestive tract. In humans, has found 4 kinds of beta defensins (HBD1 ~ 4) defense. Element has a wide spectrum of antibacterial activity of HBD-1 against gram positive bacteria, gram negative bacteria, fungi, spirochetes, mycobacteria and enveloped viruses have killing effect of HBD-2 on Gram negative bacteria, gram positive bacteria, yeast has a strong bactericidal effect, the leather The bactericidal effect of Gram-negative bacteria is more significant. Studies have shown that in vitro, HBD-2 has strong killing effect for Helicobacter pylori, but the multidrug resistance of Helicobacter pylori to effect, had not been reported in the literature. In addition, a large number of studies show that the antibacterial test and in vitro test in vivo antibacterial drugs often there is a big difference, therefore, it is necessary to carry out HBD-2 test in vivo anti Helicobacter pylori. However, due to the presence of some objective reasons 1) there are few HBD-2 buy more expensive sources of protein, 2) degradation of acid protein, there are few studies on the gastrointestinal tract in vivo on HBD-2 related. Therefore, we feel the need of recombinant expression of HBD-2 protein, and the antibacterial activity of the recombinant protein in multidrug resistance of Helicobacter pylori.
Materials and methods: Based on the separation of crossed LB solid culture in fresh liquid LB medium containing ampicillin was induced by IPTG, ultrasonic cracker, recombinant HBD-2 fusion protein. Then the samples had NTA column, the purpose of collecting protein repeated dialysis, determine the distribution of target protein by using SDS-PAGE analysis. Purification of refolded protein concentrate preservation. Two times of agar dilution method, we selected 5 strains of multidrug resistant Helicobacter pylori and 5 strains of MTZ, C1A sensitive strains, detection of the MIC. test, the difference between the 2 groups by t-test.
Result錛

本文編號：1462454