基于骨关节结核的TLR4信号传导通路中Tollip负反馈调控的机制研究.

Tuberculosis (TB) is one of the major diseases which cause serious damage to human health in the world,and epidemic situation is still very grim. Bone and joint TB mainly secondary to tuberculosis, it has higher morbidity. So to explore the pathogenesis of bone and joint TB, prevent and treat of tuberculosis has important significance .Research proves that in TLR pathway, Toll-like receptors and other regulatory genes and cytokines is closely.related to occur and develop of the pulmonary tuberculosis. Previous studies have found that IL-12and Tollip genes are abnormal expressed in bone and joint TB and is closely related to each other,thus speculated that Tollip genes abnormal expression is closely related to the negative feedback regulation and has important regulatory role in the development of bone and joint TB,but negative feedback regulation factors Tollip genes how to play a role of negative regulation and control mechanism has not seen research and reports.So this topic was proposed by constructing Tollip gene eukaryotic expression vector transfection to peripheral blood and tissue culture of TB lesions in vitro,using RNA interference (RNAi) silence technology research negative feedback control effect about Tollip negative feedback regulation TLR4 signaling pathways in vitro and animal body, reveal the development mechanism of Tollip negative feedback regulation TLR4 signaling pathways in the bone and joint TB and offer scientific theory foundation for looking new targets for prevention and control of joint and bone tuberculosis.

结核病是全世界严重危害人类健康的重大疾病之一,疫情仍十分严峻，骨关节结核主要继发于肺结核,有较高致残率。因此探讨骨关节结核的发病机制有重要意义。研究证明在TLR通路中，Toll样受体和负调控因子Tollip基因与肺结核发生发展密切相关。前期研究发现在骨关节结核病灶样本中TLR4及负反馈调控因子Tollip异常表达并与下游IL-12、CD14等细胞因子密切相关，由此推测Tollip异常表达可能与骨关节结核的发生发展有重要的负调控作用,而基于骨关节结核的TLR通路中Tollip的负调控作用及机制目前未见研究和报道。本课题拟通过构建人Tollip基因真核表达质粒载体转染人巨噬细胞株U937，利用RNA干扰沉默技术（RNAi）研究Tollip在TLR4通路在体外和动物体内的负反馈调控作用，揭示Tollip负反馈调控TLR4通路在骨关节结核发病的机理,为骨关节结核免疫防治寻找新靶点提供科学理论依据。

骨关节结核病仍严重危害人类健康的重大疾病之一,有较高致残率。前期研究发现在骨关节结核病灶样本中TLR4及负反馈调控因子Tollip异常表达并与下游IL-12、CD14等细胞因子相关，为此，本课题组对骨关节结核的TLR通路中Tollip的负调控作用及机制进行研究。研究内容有：1.百色地区原住壮族人群骨关节结核的基因特点可能为百色地区壮族人群脊柱结核患者和肺结核患者存在CD14/-159多态性,以TT为主,CD14基因-159TT和-1145AA基因型可能为百色地区原住壮族人群脊柱结核病的易感高风险因子；2.构建骨关节结核动物实验模型。 新西兰大白兔骨关节局部种植H37Rv标准菌株,可成功构建兔骨关节结核模型，本研究骨关节结核造模成功率为71.4%。3.利用Tollip 基因真核表达载体转染培养所得的巨噬细胞特异性沉默 Tollip 基因后，检测 TLR4 及其相关细胞因子 IL-12、TNF-α 的表达情况并在骨关节结核动物模型体内进行研究。经研究：成功构建pLV-U6-shRNA-Tollip-CMV-EGFP-T2A-Puro重组干扰慢病毒载体并为下一步动物实验做好准备。在骨关节结核动物模型腹膜接种带有沉默 Tollip 基因巨噬细胞后IL-12、TNF-α表达上调，而空质粒组和空白对照组 IL-12、TNF-α表达下调；TLR4变化相反。因此，本研究结论如下：新西兰大白兔胸腰椎体局部种植H37Rv标准菌株,可成功构建新西兰大白兔脊柱结核动物模型。本研究骨关节结核造模成功率为71.4%，与有关文献报道相似；TLR4及Tollip蛋白在结核病灶旁组织上呈微弱表达或不表达；在结核病灶组织中的表达明显增强，呈弥漫或颗粒状分布于细胞膜与细胞质上。Tollip蛋白表达与TLR4、IL-12、TNF-α的相关性呈负相关。Tollip在骨关节结核可能对TLR4信号通路有负反馈调控的作用。下一步可从TLR4通路上下游相关基因继续研究和探索，以期寻找骨关节结核早期诊断指标和可能治疗基因靶点。