【作者】 周刚；

【导师】 戴宁；

【作者基本信息】 浙江大学 ， 内科学， 2003， 硕士

【摘要】 幽门螺杆菌(Hp)是消化性溃疡的主要病因，是胃癌及原发性胃淋巴瘤的危险因素。目前，Hp的耐药问题正日益突出，已成为根治失败的主要原因之一。甲硝唑Hp感染首次成功治疗的重要成分，也是新三联，四联治疗的主要成分。但甲硝唑的联合治疗也可能由于耐药的产生而受到限制。 临床上幽门螺杆菌的分离，培养是较困难而复杂的工作，从而导致Hp的药敏试验很难在临床广泛开展。有必要深入了解Hp的耐药机制，在此基础上运用分子生物学的方法进行药敏试验，指导临床用药。同时也能为耐药性逆转提供基础。 目前Hp对克拉霉素等药物的耐药机制已基本明确，关于Hp对甲硝唑耐药机制的观点很多。较多学者认为与rdxA基因的突变有关。RdxA基因约630bp长，编码氧不敏感NADPH硝基还原酶(rdxA)，其作用是把甲硝唑的NO基团还原成羟胺衍生物，还原物造成细胞DNA的损伤、断裂、解旋，进而引起细胞的死亡。rdxA基因发生突变会导致蛋白翻译过程提前终止，从而使硝基还原酶失活。但在实验中，也一直没有发现特定的核苷酸序列与Hp耐药表型有关，而在甲硝唑敏感菌株中也存在rdxA基因突变。在国内，尚无对该基因研究的报导。 而甲硝唑是1-(2-羧基)-2-甲基-5-硝基类药物，为一疏水性药物，需通过膜蛋白转运进入细胞内。戴宁等发现维拉帕米能通过抑制外排而在一定程度上逆转Hp甲硝唑耐药，提示膜耐药存在。但至今尚为发现、分离相关的膜耐药蛋白。 本文分离国内Hp临床菌株，进行耐药，敏感Hp rdxA基因的克隆，测序，并同Hp26695，J99及国外报导的菌株进行序列比较。并在此基础上选用无rdxA基因移码突变的敏感、耐药菌株，提取外膜蛋白，制备甲硝唑人工抗原、甲硝唑多克隆抗体，并用Wester Blot方法比较了敏感、耐药菌株中能与甲硝唑结合的外膜蛋白。 实验方法 1．菌株来源 所有Hp菌株均分离自胃粘膜活检标本。Hp标准参考株为NCTC11637。病 浙江大学硕士学位论文人胃粘膜组织匀浆后接种于含10％羊血的哥伦比亚琼脂平板上，在微需氧环境中37oC培养3—sd。2．药敏试验 纸片扩散法初筛、二倍平皿稀释法确定Hp菌株对甲硝吧的耐药性。抑菌圈直径＜7 mm、最小抑菌浓度（MIC）38p旮1判为甲硝哩耐药株。3．DNA制备 采用常规的苯酚一氯仿法。4．PCR 引物序列：上游 了－GGGATTTTATTGTATGCTACAA－3’，下游5’－＊＊＊6＊口＊＊＊C＊＊＊＊＊＊＊＊＊丁3’。＊＊R反应总体积为100“ 内含：25＊。I几各dNTP、250 nmoffe各引物、15 mol［L MgCI。、2．SU T叫酶、100 ng DNA模板、IXPCR缓冲液（pHS．3）。PCR反应参数：94oC5min，XI 94OC305，50OC305，72t60s，X10；94OC305，50’C305，72t70s（以后每循环增加 10s），X20：72“C10min，XI。S．T－A克隆及测序 将扩增的目的片段克隆至 pUCm－T载体中，转化于 E．colt DH 5 a株并扩增，用碱变性法提取质粒，经限制性内切酶（BamH，EcoR）酶切鉴定后委托BBST公司测定插入片段的核苔酸序列。6．测序结果分析 采用 DNA Tool＊分子生物学软件分析上述临床菌株rdxA基因的核昔酸和氨基酸序列，并与国外报道的 Hp 26695株（NC－0009］5）及门 株Hp rdxA基因序列进行比较。7．茵株涉择 选用己行药敏试验及 rdx A基因克隆、测序，无 rdxA基因碱基插入、缺失的菌株。8．外膜蛋白提取 收集并洗涤培养后的 HP，用超声细胞破碎仪破碎，1000 X n 20min，去除没未破碎的细胞，加入蛋白酶抑制剂、RNA酶、DNA酶，超速离心 100000Xr for，去上清，沉淀用肌氨酸溶解、悬浮，再超速离心 40000 X r 0．shr，去上清，沉淀用去离子水 溶解、悬浮。9．甲硝陛多克隆抗体制备 9．1．全抗原MTZ－－BSA制各 用碳二亚胺法制备牛血清白蛋白与甲硝哇交联物。 9．2．抗体制备用昭个WZ免役家兔6周，心脏采血，分离血清。用田A预处理组做对 照，行双扩法鉴定是否有抗体产生，并初步判断抗体效价。 10．甲硝陛结合外膜蛋白比较：甲硝哇溶液甲硝陛溶液与敏感、耐药菌的外膜蛋白溶液均 匀混合，4℃过夜。以甲硝哇多克隆抗体为一抗，HRP标记的羊抗兔IgG 为二抗，用 Western blot检测能与甲硝哇结合的敏感、耐药幽门螺杆菌。 结 果 二药敏试验 临床分离的 ZI株 Hp（14株来自溃疡病患者，6株来自慢性胃炎患者，1株 来自胃癌患者）中，16株的MIC38Pg／ffil，耐药率为76．l％。 3 浙江大学硕士学位论文2 PCR 8株不同 MIC的 Hp临床菌株 rdxA基因扩增片段约为 880hP，HP标准参考株NCTC 637的rdxA基因扩增片段约为680hp。3 Hp临床苗株 rdxA基因序列分析 8株Hp临床菌株 rdxA基因扩增产物的核昔酸和氨基酸序列与 Hp 26695株比较，其核昔酸序列同源性为 90．IW95．l％。4甲硝哇耐药性与rd更多还原

【Abstract】 Helicobacter pylori is the main cause of chronic gastritis, peptic ulcer, gastric cancer and MALT. Now due to widely use of antibiotic in clinic, the phenomena of resistance become more and more prominent, which is the important factor leading to treatment failure. Metronidazole is the important factor for success treatment to Hpylori infection and is the main gradient of new triple therapy. But metronidazole combined therapy is also restricted due to the resistance.It’s difficult and complex task to clinically isolate and culture the Hpylori, which prevent susceptibility test being carried out clinically. It’s necessary to detect the resistance mechanism, only based on which can we carry out the susceptibility test using biochemistry method and instruct the clinical description. Besides these, detecting the resistance mechanism may be helpful in reverting the MTZ resistance.Up to now, the resistance mechanism of Hpylori to some medicines such as clarithromycin, amoxicillin is clear, but there are many viewpoints about metronidazole resistance of Hpylori. Of particular note is the mutation of rdxA gene, which encode an oxygen-insensitive NADPH nitroreductase. The cytotoxicity of MTZ is due to the unstable and less reduced intermediates that damage DNA, resulting in strand break, helix destabilization, unwinding and cell death. But up to now, no special nucleotide sequence related with Hpylori resistance phenotype was found.We have found membrane’s role in metronidazole resistance of Hpylori. In this study, rdxA genes of clinical isolates were sequenced and sequences were compared with Hp26695 sequence and the rdxA sequences reported by other researchers. Then we selected the clinical strains without rdxA gene frameshift mutation, prepared the outer membrane protein (OMP) and metronidazole polyclonal antibody. The main outer membrane proteins in sensitive and resistant strains were compared by Western Blot.Materials and methods1. Culture of Hpylori: All samples used in this study were clinical isolates from patients through endoscope biopsy. Each the sample was smeared on Hpylori selective Columbia agar plate containing 10% sheep blood. Hpylori cultures were incubated at 37℃ under microaorobic conditions. NCTC11637 was reference strain.2. Determination of minimal inhibition concentration (MIC): The MIC was determined by paper test and agar dilution method of NCCLS. Agar dilution plates were prepared with two fold serial dilution of metronidazole (sigma), ranging from 0.25 to 128μg/ml. The young exponentially growing Hpylori strains were prepared in sterile saline and adjusted to No.2 McFarland standard. About 1 to 5μl of adjusted inocula was delivered to agar dilution plate. Results were read after 72h incubation and the MIC determined as the lowest concentration of metronidazole in which no visible growth occurred. Strains the MIC value >8μg/ml were classified as resistant.3. Preparation of Hpylori Genomic DNA: Phenol-Chloroform and DNase-free RNase digestion were used for preparation and purification of Hpylori genomic DNA. The dried DNA was resuspended in 60μl TE buffer and stored at -20℃.4. PCR amplification of rdxA: The oligonucleotide primers rdxF(forward) GGGATTTTATTGTATGCTACAA and rdxR(backward) GCAGGAGCATCAGATAGTTCT were used to amplify PCR product (886bp long in total) that containing the entire 633bp rdxA gene. PCR was performed in an automated thermal cycler in a final volume of 100μl, containing 250nmol/L primers, 2.5mol/L dNTP, 15mol/L MgCl2, 2.5U Tag polymerase, 1× PCR buffer(pH8.3) and 100ng DNA template. The cycling conditions : 94℃5min, 41; 94℃30S, 50 ℃30S, 72℃60S, ×10: 94℃30S, 50℃30S, 72℃70S, ×20; 72℃10min, ×1. The result were identified by 1.5% agarose electrophoresis.5. T-A cloning and sequencing: The target DNA fragments amplified from rdxA genes were ligated to PUCm-T vector for 16h, and then transferred into E.Coli DH5a. Positive cloning containing the targe更多还原