筛选鉴定双重耐药幽门螺杆菌和HBD-2融合蛋白的体外抗菌作用

发布时间：2018-01-25 08:23

  本文关键词： 筛选 鉴定 双重 耐药 幽门 杆菌 HBD-2 融合 白的 体外 抗菌 作用　出处：《浙江大学》2007年博士论文　论文类型：学位论文

【摘要】： 第一部分幽门螺杆菌三联根除疗法影响因素的logistic回归分析 幽门螺杆菌(Helicobacterpylori，Hp)是浅表性胃炎、消化性溃疡的致病因子，与胃癌、胃黏膜相关性淋巴组织淋巴瘤密切相关，世界上约有一半以上的人感染Hp。幽门螺杆菌根除是治疗消化性溃疡和防止复发的重要手段。国内外很多研究提示，PPI联合克拉霉素、阿莫西林/甲硝唑是Hp根除的有效治疗方案。但由于幽门螺杆菌耐药性等问题，严重的影响了该三联疗法的疗效。另外，研究表明，Hp耐药问题及患者医嘱顺从性不能完全解释Hp根治失败的原因。目前缺乏Hp根除率影响因素综合评价的研究。本研究的目的是评估在浙江地区，以质子泵抑制剂(PPI)为基础联用克拉霉素、阿莫西林/甲硝唑的三联疗法根治幽门螺杆菌的疗效，探讨幽门螺杆菌的耐药性、患者的性别、年龄、烟酒史、疾病类型和组织学改变对幽门螺杆菌根除率的影响。通过这一研究，也为进一步研究多药耐药幽门螺杆菌提供了物质基础。 材料和方法：患者行胃镜检查，符合研究入选标准的患者共取4块组织标本，，一块胃窦组织做快速尿素酶试验，一块胃窦组织行幽门螺杆菌培养和药敏试验，2块胃窦组织行组织学检查。患者幽门螺杆菌培养阳性或快速尿素酶、组织学检查阳性被认为是幽门螺杆菌感染。所有患者接受7天幽门螺杆菌根除治疗：达克普隆(30mg)、克拉霉素(500mg)、阿莫西林(1g)或甲硝唑(0.5g)，每天2次，口服。治疗结束后4-6周，采用C~(14)-尿素呼气试验评估幽门螺杆菌根除情况。同时收集患者的一般情况。所有组织标本由一高年资的病理科医生评估。幽门螺杆菌药敏试验采用纸片法和二倍平皿稀释法。运用SAS软件，分析研究对象的各类数据，采用χ~2检验和多因素非条件logistic回归分析的方法，P＜0.05认为有统计学差异。 结果与结论：78个幽门螺杆菌感染的患者中，42个男性，36个女性，平均年龄46岁，27个患者有活动性十二指肠溃疡(占34.6％)，51个患者是非溃疡性消化不良(NUD)。34个患者有抽烟史，6个患者有较严重的喝酒史。幽门螺杆菌根除率为61/78(78.2％)。5个患者出现轻度治疗副反应(轻度腹泻=2，上腹不适=3)。在78株幽门螺杆菌菌株中，对克拉霉素原发性耐药率为14.1％，甲硝唑耐药率为69.2％，阿莫西林耐药率为1.3％，其中对克拉霉素耐药的菌株同时存在甲硝唑耐药，共11株，未发现同时对3种抗生素耐药的菌株。患者性别、克拉霉素耐药、十二指肠溃疡病变，这三变量是Hp根除失败的独立相关因素(P＜0.05)。在本研究中，患者的年龄，烟酒史，胃窦部组织学改变与疗效无明显相关性(P＞0.05)。 第二部分多药耐药幽门螺杆菌筛选及鉴定 如何治疗幽门螺杆菌反复根除失败的患者是临床上一棘手的问题。目前，关于多药耐药幽门螺杆菌的耐药机制、根除等研究罕见报道。有必要从临床筛选、鉴定多药耐药菌株，以便进一步研究其耐药机制，寻求新的根除手段。通过我们前面的研究，我们分析了幽门螺杆菌根除治疗的影响因素，更重要的是，我们初步筛选了一些多药耐药的幽门螺杆菌菌株。通过药敏试验，虽然没能发现存在克拉霉素、甲硝唑、阿莫西林三个药物同时耐药的菌株，但我们筛选到了一些克拉霉素、甲硝唑同时耐药的菌株。从中，我们筛选了5株高MIC的幽门螺杆菌菌株，通过病例的随访，这些菌株的感染者三联治疗失败后换用四联疗法，最后依然没有根除幽门螺杆菌。通过临床的治疗效果和体外药敏实验，我们从临床初步判断其为多药耐药幽门螺杆菌。本实验目的，就是通过菌株培养，分析甲硝唑、克拉霉素耐药的相关基因rdxA、23SrRNA，从实验室角度进一步筛选、鉴定其是否是多药耐药幽门螺杆菌。 材料和方法：按传统的酚—氯仿抽体法提取了幽门螺杆菌基因组DNA，根据文献和Hp26695株基因序列，分别设计和合成rdxA、23SrRNA序列的特异性引物，由上海博亚生物技术有限公司(BioAsia)合成，1.5％的琼脂糖凝胶电泳检查扩增产物。将10ul 23SrRNA基因PCR产物行RFLP分析，分别用BsaI、BbsI两种限制性内切酶酶切。前者于50℃孵育24hr，后者于37℃孵育24hr。同时将rdxA基因PCR产物行T-A克隆，委托上海申能搏彩生物科技有限公司，采用双脱氧链末端终止法在测序仪上测定含正确大小插入片段的核苷酸序列。 结果：5株临床菌株我们命名为Hp20061、Hp20062、Hp20063、Hp20064、Hp20065、rdxA基因测序结果，Hp20063菌株于193位点额外插入了了一碱基A，Hp20065菌株于549位点额外插入了一碱基A，导致菌株的rdxA基因核苷酸序列出现移码突变，从而导致翻译的蛋白质结构和功能的改变。而另3个多药耐药菌株由于出现多位点碱基突变，也导致相应的氨基酸改变。23SrRNA基因RFLP分析，5株多药耐药菌株的PCR扩增物可被BsaI酶切成两个片段。5株扩增物均未能被BbsI酶切。提示5株多药耐药菌株在23S rRNA基因功能区Ⅴ第2144位点有A→G突变。所有菌株均不存在2143位点A→G突变。 结论：通过对5株幽门螺杆菌菌株的rdxA基因、23SrRNA基因的分析，我们认为菌株Hp20063、Hp20065是多药耐药幽门螺杆菌，而Hp20061、Hp20062、Hp20064菌株临床为多药耐药菌株，但其分子生物学特性有待更进一步的实验室鉴定。 第三部分HBD-2融合蛋白对多药耐药幽门螺杆菌的体外抗菌作用 机体对于幽门螺杆菌感染的自身反应主要是通过中性粒细胞、淋巴细胞引起的一系列免疫反应。研究表明，幽门螺杆菌感染会引起机体一些抗菌肽表达水平升高。抗菌肽是广泛存在于动、植物体内，具有一定抗菌谱的小分子肽。在脯乳动物中发现的抗菌肽主要是防御素(defensin)，防御素可分为α和β两类，β-防御素家族在许多上皮组织包括消化道上皮中有更广泛的表达。在人类中，已发现4种β-防御素(HBD1～4)。防御素具有广谱的抗菌活性，HBD-1对革兰阳性菌、革兰阴性菌、真菌、螺旋体、分支杆菌及有包膜病毒均具有杀灭作用，HBD-2对革兰阴性菌、革兰阳性菌、酵母菌具有较强的杀菌作用，其对革兰阴性菌杀菌作用更为显著。已有研究表明，在体外试验中，HBD-2对于幽门螺杆菌具有较强的杀伤作用，但是对于多药耐药幽门螺杆菌的作用如何，尚未有文献报道。另外，大量研究表明，抗菌药物的抗菌作用体内试验和体外试验往往会存在很大差别，因此，有必要开展HBD-2抗幽门螺杆菌的体内试验。但是，由于存在一些客观原因1)目前HBD-2蛋白质来源不多，购买比较昂贵2)胃酸对蛋白质的降解作用，很少有关于HBD-2相关的胃肠道体内试验的研究。因此，我们觉得有必要基因重组表达HBD-2蛋白，并研究重组蛋白对多药耐药幽门螺杆菌的抗菌活性。 材料和方法：LB固体培养基上分离划线，在含氨苄青霉素的新鲜LB液体培养基中扩增，经IPTG诱导，超声破碎仪破碎，获得重组HBD-2融合蛋白。再将样品过NTA层析柱，收集目的蛋白反复透析，用SDS-PAGE分析确定目的蛋白的分布情况，纯化复性后的蛋白质浓缩保存。用二倍平皿稀释法，分别选择5株多药耐药幽门螺杆菌和5株MTZ、C1A敏感菌株，检测其MIC。用t-test检验2组间差别。 结果： 1、获得纯化的重组融合蛋白HBD-2。 2、HBD-2对于多药耐药幽门螺杆菌具有一定的抗菌作用。 3、所有MIC均取对数后行t检验结果P＞0.05，两组间无明显差异。 结论：大肠杆菌基因重组获得HBD-2蛋白是一种可行的办法，重组蛋白对多药耐药的幽门螺杆菌具有一定的抗菌作用。我们可以在此基础上，进一步开展多药耐药幽门螺杆菌根除的体内研究。
[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.
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