肺炎支原体感染诱导猪 CYP3A29基因表达上调的分子机制

Mycoplasma Pneumonia of Swine (MPS) is pandemic in Chinese pig industry and incurs serious economic losses. Chinese native pig breeds, as represented by Meishan pig, are especially susceptable to this disease, compared with the introduced Western breeds. It provides us the reasons and feasibility to investigate the genetic basis and molecular mechanisms of Mycoplasma hyopneumonia (M. hyopneumonia) infection. We previously had conducted a series of studies on genetic differences of susceptability to MPS, and we found that mRNA expression of Cytochrome P450 3A29 (CYP3A29) was significantly up-regulated in the process of M. hyopneumonia infection. And high expression of CYP3A29 promotes expression of the major inflammatory cytokines in PAM cells caused by M. hyopneumoniae infection, via inhibition of the PPAR-γ signaling pathway. On this basis, artificial infection test for M. hyopneumonia in Meishan pigs and porcine alveolar macrophages (PAM) will be performed in this study, to investigate the relationship between TLR2/4, NF-kB, PXR, RXR-a and CYP3A29 at different onset stages after M. hyopneumonia infection. On the other hand, the expression level of TLR2/4, NF-kB, PXR, RXR-a will be over/interfere-regulated in cultured cells using the molecular tools of gene transinfection and RNAi, respectively, to identify the effect of TLR/NF-kB/PXR/RXR-a signal pathway on CYP3A29 expression. Then, gene mutation and chromatin immunopresipitaion (CHIP) will be used to investigate the binding site of PXR/RXR-a in CYP3A29 and NF-kB in PXR, and the effect of SNP in binding site of CYP3A29 on signal transduction and CYP3A29 expression after M. hyopneumoniae infection will be detected. Finally, the molecular mechanism of CYP3A29 expression up-regulation after M. hyopneumoniae infection in PAM cells will be analyzed. These results will help us to understand the genetic variance of suceptability to MPS, and to develop the molecular approaches to MPS.

猪支原体肺炎流行广、危害大，且在不同品种间存在敏感差异，有从遗传本质上探讨该病发生的分子基础和作用机制的依据与可能。项目前期研究发现猪CYP3A29基因在支原体肺炎感染过程中持续、显著表达上调，并能通过PPAR途径促进肺部炎症反应。本项目在此基础上，利用梅山猪及PAM细胞，开展肺炎支原体感染实验，检测不同发病阶段TLR2/4，NF-kB、PXR、RXR-α与CYP3A29的表达变化关系；进一步借助超表达、抑制表达实验，检测TLR/NF-kB/PXR/RXR-α信号通路在肺炎支原体感染PAM细胞诱导CYP3A29表达中的作用；最后，借助点突变和染色质免疫共沉淀确定RXR-α与CYP3A29启动子区转录因子结合位点，检测位点内SNP对肺炎支原体感染信号传导及CYP3A29表达的影响，揭示肺炎支原体感染后猪CYP3A29基因表达上调的分子机制，探索不同品种猪对肺炎支原体存在易感差异的遗传基础。

猪支原体肺炎具有发病率高和危害大等特点，在中国地方猪和西方品种猪之间具明显的敏感差异，有从遗传本质上揭示其发病分子基础和作用机制的依据与必要性。项目前期研究发现猪CYP3A29基因在支原体肺炎感染过程中持续、显著表达上调，并能通过PPAR途径促进肺部炎症反应，但肺炎支原体感染如何引起CYP3A29因表达上调目前尚不清楚。针于此，本项目利用梅山猪及PAM细胞，开展肺炎支原体感染实验，检测不同发病阶段TLR2/4，NF-kB、PXR、CYP3A29等目标基因的表达变化及相互关系，揭示肺炎支原体感染后猪CYP3A29基因表达上调的分子机制，探索不同品种猪对肺炎支原体存在易感差异的遗传基础。.研究结果表明：肺炎支原体入侵机体后，通过TLR2受体激活JNK及NF-kB/PXR信号通路协同上调CYP3A29基因表达，从而引起下游IL-1β、IL-6、IL-8、TNF-α等炎性因子表达，患猪表现肺部炎性症状；不同品种猪CYP3A29基因上游启动子区域-436 bp的A/G差异，导致位点结构蛋白差异及功能改变，形成肺炎支原体感染后炎性反应的差异。具体为：1、肺炎支原体感染后，TLR2受体在感染早期表达上调，随后逐渐下降；CYP3A29随支原体肺炎病程进展表达逐步上调，NF-kB、PXR及炎性因子IL-1β、IL-6、IL-8、TNF-α随炎症反应持续上调；2、肺炎支原体通过TLR2受体激活JNK及NF-kB/PXR通路，调节CYP3A29基因表达上调，引起下游炎症反应；3、不同品种猪CYP3A29启动子区-436 bp位置的A/G差异，导致相应位点蛋白结构与功能的改变，出现肺炎支原体感染后的炎性反应差异。项目结果揭示了猪肺炎支原体感染炎症反应的分子调控机制，及不同品种猪出现肺炎支原体易感差异的遗传本质，为猪支原体肺炎药物防治及抗性品系选育奠定了分子基础。