【作者】 宝福凯；

【导师】 陈明清； 董坚；

【作者基本信息】 昆明医科大学 ， 外科学， 2012， 博士

【摘要】 研究目的：1.用实时荧光定量PCR确定伯氏疏螺旋体感染小鼠模型不同组织的病原体载量,探讨组织螺旋体载量与相应组织病理损伤的关系。2.建立伯氏疏螺旋体感染的小鼠模型,通过DECAL和微阵歹(microarray)等技术研究伯氏疏螺旋体在小鼠关节组织中的关节特异性基因表达谱,寻找伯氏螺旋体在关节中特异性高表达的基因。3.以伯氏疏螺旋体标准株B31株基因组DNA为模板,PCR扩增在关节中特异性高表达的bmpA全基因序列,定向克隆和高效表达重组蛋白BmpA(rBmpA)并纯化。对伯氏疏螺旋体BmpA蛋白进行亚细胞定位研究。4.通过用重组伯氏疏螺旋体膜蛋白A (rBmpA)定期局部注射昆明(KM)小鼠胫跗关节,诱导并建立莱姆关节炎(Lyme arthritis)昆明(KM)小鼠模型,在此基础上,从形态学、影像学、病理学水平探索关节炎病变,检测莱姆关节炎小鼠血清及关节液中Th-17细胞相关细胞因子IL-6、TGF-β及IL-17细胞因子含量,探讨Th-17细胞在莱姆关节炎致病机制中作用。5.从免疫细胞和分子水平初步研究rBmpA的致病机理。研究内容：1.培养低传代伯氏疏螺旋体至对数生长期,稀释为1×105/ml。为建立伯氏疏螺旋体感染小鼠模型,于每只小鼠皮内注射菌液100μl,在证实感染成功后,于第12d和18d分别取不同组织,提取总DNA,用实时荧光定量PCR分别测定组织中的螺旋体flaB基因拷贝数,并标准化为每106β-肌动蛋白所对应的flaB拷贝数(螺旋体数)。对不同组织的螺旋体载量进行统计学处理,确定不同组织螺旋体载量是否有显著性差异。2.建立伯氏疏螺旋体感染小鼠模型,在不同时间点处死小鼠,收集小鼠关节、心脏、皮肤和膀胱组织,从四种组织中分别提取总RNA；随后,用DECAL技术和微阵列技术分析不同时间点伯氏疏螺旋体在四种组织中的转录组；最后,将伯氏疏螺旋体在关节中的转录组依次与其他三种组织中的转录组进行比较,从而获得伯氏疏螺旋体不同时间点的关节特异性基因表达谱,找出仅在关节中表达的伯氏疏螺旋体基因。3.(1) BmpA基因的定向克隆与重组菌的产生：以伯氏疏螺旋体标准株B31株基因组DNA为模板,设计定向克隆引物,PCR扩增bmpA全基因序列,将bmpA基因定向克隆入表达载体pGEX-6p-1,酶切鉴定,转化大肠杆菌BL21菌株,获得bmpA重组菌。(2)重组bmpA高效表达与纯化：从重组菌培养温度,诱导时间,诱导剂的剂量,OD600等方面优化诱导条件,找到高效表达重组bmpA的最佳方案。用GSH柱纯化重组bmpA,探索纯化bmpA的最佳条件。(3)bmpA的亚细胞定位研究。4.(1)研究rBmpA在诱导莱姆关节炎致病中作用：优化rBmpA注射浓度、剂量、次数,探索rBmpA在诱导莱姆关节炎中的致病作用。(2)尝试在新的动物品系(KM小鼠)、新方法(局部关节注射)建立莱姆关节炎动物模型的可能性：探索采取局部注射KM小鼠胫跗关节,建立莱姆关节炎模型的方法。(3)从形态学、影像学、病理学水平探索关节炎病变。(4)探讨Th-17细胞相关细胞因子IL-6、IL-17及TGF-β细胞因子在莱姆关节炎致病机理中作用：检测莱姆关节炎小鼠血清及关节液中与Th-17细胞相关细胞因子IL-6、IL-17及TGF-β细胞因子含量,探讨Th-17细胞在莱姆关节炎致病机制中作用。5.研究rBmpA对小鼠腹腔巨噬细胞和脾脏淋巴细胞的活化作用。研究结果：1.在所检测的4种代表性组织中,膀胱伯氏疏螺旋体载量在两个典型时间点均最高,皮肤和关节次之,心脏最低。2.与其他组织相比,在感染后第15天,伯氏疏螺旋体在小鼠关节组织特异地表达21个基因,其中13个基因位于伯氏疏螺旋体染色体上,8个基因位于质粒上；在感染后的第105天,伯氏疏螺旋体在小鼠关节组织特异地表达24个基因,其中13个基因位于伯氏疏螺旋体染色体上,11个基因位于质粒上。在关节中表达最高的是bmpA、bmpB等基因。3. bmpA基因的克隆与表达研究。(1)在基因水平和蛋白水平上,都出现了目的条带和目标峰,确定表达载体bmpA-pGEX-6p-1构建成功,并表达重组bmpA。(2)通过分析比较,重组质粒在37℃, IPTG诱导浓度为0.1mmol/ml,诱导时间为6小时,菌液的0D值为0.5-1.0以及在LB培养基上培养GST-bmpA融合蛋白表达量达到最大。(3)在最佳表达条件下1L的重组菌能纯化到2.9-3.1mg的bmpA蛋白。(4)亚细胞定位研究表明,BmpA存在于螺旋体表面。4. BmpA诱导关节炎研究。(1)用rBmpA稀释液局部注射KM小鼠胫跗关节,成功建立了莱姆关节炎(Lyme arthritis)-昆明(KM)小鼠模型,拓展了新的莱姆关节炎动物造模方法。(2)从形态学、影像学、病理学水平证实rBmpA与莱姆关节炎发病密切相关。(3)检测莱姆关节炎小鼠血清及关节液中与Th-17细胞相关细胞因子IL-6、IL-17及TGF-β细胞因子含量与对照组及正常组比较无明显统计学意义,分析和探讨了相关原因,为未来进一步研究奠定了基础。5. rBmpA对小鼠腹腔巨噬细胞的作用不明显,但对小鼠脾淋巴细胞有明显的刺激增殖作用,并可以刺激脾淋巴细胞产生炎性细胞因子白细胞介素6。结论：1.伯氏疏螺旋体感染小鼠后,不同组织螺旋体载量有显著性差异,膀胱螺旋体载量在两个典型时间点均最高,皮肤和关节次之,心脏最低。组织螺旋体载量与组织损伤程度无密切关系。2.伯氏疏螺旋体在小鼠关节组织中存在独特的基因表达谱,其中螺旋体膜蛋白基因bmpA和bmpB在关节表达最高。这些在关节中特异表达的基因可能与莱姆关节炎的发生、发展有关。3.(1)成功的构建了表达重组蛋白bmpA的大肠杆菌原核表达系统,并且在基因水平和蛋白水平上得到鉴定。(2)找到了高效表达重组BmpA的最佳方案。用GSH柱纯化重组BmpA,探索出纯化重组BmpA的最适条件。(3)亚细胞定位研究表明,BmpA存在于螺旋体细胞外膜表面。4.(1)用rBmpA稀释液局部注射KM小鼠胫跗关节,成功建立了莱姆关节炎(Lyme arthritis)-昆明(KM)小鼠模型。(2)rBmpA与莱姆关节炎发病密切相关。5. rBmpA对小鼠脾淋巴细胞有明显的刺激增殖作用,并可以刺激脾淋巴细胞产生炎性细胞因子白细胞介素6。更多还原

【Abstract】 Objective:1. To quantify the burden of Borrelia burgdorferi in different representative tissues in the murine host.2. To identify the joint-specific expression profile of Borrelia burgdorfri in the murine hosts.3. To clone and highly express Borrelia recombinant bmpA, Genomic DNA of Borrelia burgdorferi reference strain B31is as template, using PCR to amplify bmpA gene sequences, directional cloning and high expression and purification of recombinant bmpA. To identify the subcellular location of bmpA.4. To establish Lyme arthritis model of KM mouse by injecting recombinant Borrelia burgdoferi membrance protein A(rBmpA) regularly and locally to the tibial tarsal joint and investigate the arthritic changes in X-ray and histopathological levels, on this basis, detect the contents of Th-17cell-associated IL-6, TGF-β and IL-17cytokines in the serum and synovial fluid of Lyme arthritis model of KM mouse and explore the role of Th-17cells in the pathogenesis of Lyme arthritis.5. To explore the roles of BmpA to activate the macrophages collected from peritoneal cavity, and murine lymphocytes from spleen, determine the effects of BmpA on the activation of macrophages and lymphocytes.Methods:1. To cultivate the low-passage Borrelia burgdorferi to logarithmic growth phase, and dilute bacterial culture to a concentration of1×105/ml for experimental use. To establish a Borrelia burgdorferi-infected murine model, mice were injected100μl diluted culture intradermally in the back. After artificial infection was confirmed, mice were sacrificed at day12and18postinfection in CO2box,skin, joints heart, and urine bladder samples were collected aseptically and frozen at-80℃, total DNA extracted, Borrelia burgdorferi flaB was quantified by real time quantitative PCR(Q-PCR). Data were analyzed statistically to determine if bacterial burdens in various tissues show a significant difference.2. Establishing the murine model of Lyme disease, sacrificing the infected mice at different time-points, collecting the joint, heart, skin, and urinary bladder samples of the infected mice, and extracting total RNA from the samples. DECAL technique and microarray were applied to btain the transcriptomes of Borrelia burgdorferi in the joints, heart, skin, and urinary bladder, the transcriptomes from different tissues were compared to identify the joint-specific expression profile of Borrelia burgdorferi.3.(1) BmpA gene cloning and the production of recombinant protein.Genomic DNA of Borrelia burgdorferi reference strain B31is as template, Designing primers, using PCR to amplify bmpA genome sequences, BmpA gene was cloned into the expression vector pGEX-6p-1, restriction enzyme digestion, transformed into E.coli strain BL21, get bmpA recombinant strain.(2) High level expression and purification of recombinant bmpA:From recombinant bacteria culture temperature, induction time, inducer dose, OD600optimal inducing conditions, etc, finding the best solution of High Expression Recombinant bmpA, Purified by GSH re bmpA, to explore the optimal conditions purified bmpA.(3) Identify the subcellular location of BmpA.4.(1) To validate the role of rBmpA in the induction of Lyme arthritis: Optimize the concentration, dose, frequency of rBmpA injected to verify the role of rBmpA in the induction of Lyme arthritis.(2) To establish animal models of Lyme arthritis in the new animal strains (KM mouse) and in the use of the new method (direct articular injection):Explore the new way of taking local tibial tarsal joint injection of KM mouse to establish the model of Lyme arthritis.(3)To explore the role of IL-6, IL-17, TGF-β-associated Th-17cells in the pathogenesis of Lyme arthritis. To detect the contents of IL-6, IL-17, TGF-β in the serum and synovial fluid of Lyme arthritis model of KM mouse to explore the role of Th-17cells in the pathogenesis of Lyme arthritis.5. To use BmpA to activate the macrophages collected from peritoneal cavity, and murine lymphocytes from spleen, explore the effects of BmpA on the activation of macrophages and lymphocytes.Result:1. Spirochete burden is highest in the urine bladder, medium in the skin and joint, lowest in the heart.2. Borrelia burgdorferi expresses21joint-specific genes, including13genes located in chromosome and9genes located in plasmids at day15after infection, and expresses24genes, including13genes in chromosome and11genes in plasmids at day105after infection. Borrelia burgdorferi shows specific gene expression profile in the murine joints. The sprochetal genes which express most highly in murine joints are bmpA and bmpB.3.(1) Target bands and peaks were appeared on level of gene and protein, showing the expression vector bmpA-pGEX-6p-1was successfully constructed and expressed recombinant bmpA.(2) through analysis and comparision, maximal expression of recombinant plasmid was induced by O.lmmol/ml IPTG at37℃for6h when OD value of bacterium was0.5-1.0.(3) the optimal expression conditions of recombinant1L can be purified to2.9-3.1mg of bmpA protein.(4) BmpA is located on the surface of Borrelia burgdorferi outer member.4.(1) rBmpA is closely related to the pathogenesis of Lyme Arthritis.(2)Successfully established Lyme arthritis model of KM mouse by using the way that directly injects rBmpA into tibial tarsal joint of KM mice locally and found the new way of establishing Lyme Arthritis animal model.(3) Detectd the contents of Th-17cell-associated IL-6, IL-17, TGF-β in the serum and synovial fluid of Lyme Arthritis model of KM mouse and compared it with those of the negative control group and normal group, results have no statistical difference.Finally, the relevant reasons were discussed and suggestions for further research were promoted. 5. Our preliminary results showed that Lyme spirochete BmpA can hardly activate the macrophages collected from peritoneal cavity, but can strongly activate murine lymphocytes from spleen.Conclusion:1. Difference of spirochete burden in various tissues is significant. Spirochete burden in the tissue is not positive related to the severity of this tissue.2. Borrelia burgdorferi expresses some specific genes different from those in the other tissues in infected murine joints. These joint-specific genes might be involved in the survival and arthritis pathogenesis of Borrelia burgdorferi.3.(1) As effort of our lab, prokaryotic expression system of E.coli of was successfully constructed, it was identified on gene and protein level.(2) A high expression of recombinant bmpA was accomplished, With purified recombinant GST bmpA, to explore the optimal conditions for purification of recombinant bmpA.(3) bmpA is located in the spirochetal cell surface.4.(1) Successfully established Lyme arthritis model of KM mouse by using the way that injecting rBmpA diluent into tibial tarsal joint of KM mice locally.(2) rBmpA is closely related with the attack of Lyme Arthritis.5. Our preliminary results showed that Lyme spirochete BmpA can hardly activate the macrophages collected from peritoneal cavity, but can strongly activate murine lymphocytes from spleen.更多还原