牛支原体脂质相关膜蛋白（LAMPs）诱导胎牛肺细胞IL-1β释放的分子机制研究

Since 2008, the first report about pneumonia caused by Mycoplasma bovis (M.bovis) in China, M.bovis has been an increasingly important factor that threatens the cattle industry in our country. Although great efforts have been made to understand the molecular basis of M.bovis infection, the cellular response to M.bovis infection is still largely unknown. Our previously study indicated that lipid-associated membrane proteins (LAMPs) of M.bovis induce embryonic bovine lung (EBL) cells to produce IL-1β by real-time PCR.. In this study, we further investigated the mechanisms underlying LAMPs inducing EBL cells to produce IL-1β by high throughput cDNA microarray. The differentially expressed (DE) genes were identified in the EBL cells by using the MAS 3.0 software which was based on the Kyoto Encyclopedia of Genes and Genomes database. The interactions between proteins encoded by DE genes were analyzed by STRING. Based on the analysis of the DE genes, the candidate genes which were involved in NF-κB and MAPK signaling pathway were selected for the next functional study. The EGFP, overexpression vector and RNA interference molecules targeting the candidate genes were constructed. The EBL cells were used as cell model to analysis the functions of the DE genes in the IL-1β production induced by M. bovis LAMPs.

自2008年在我国首次报到牛支原体肺炎以来，该病日益成为危害我国养牛业的重要因素。但是关于牛支原体的致病性及其致病机制尚不十分清楚。本实验室前期的研究证实牛支原体脂质相关膜蛋白（LAMPs）能够诱导胎牛肺细胞（EBL）产生IL-1β等细胞因子。. 为阐明牛支原体脂质相关膜蛋白（LAMPs）诱导EBL细胞产生IL-1β的分子机制。本研究拟采用高通量的基因芯片技术并结合MAS 3.0和STRING系统来鉴定牛支原体脂质相关膜蛋白（LAMPs）诱导EBL细胞产生的差异表达基因并对其所处的生物进程、信号通路和相互作用进行分析。同时，挑选出处于NF-κB和MAPK信号通路中差异表达的基因。通过构建差异表达基因的绿色荧光融合质粒，真核表达质粒和RNA干扰分子，研究其在牛支原体脂质相关膜蛋白（LAMPs）诱导EBL细胞IL-1β产生方面的作用。

养牛业是我国畜牧业的重要组成部分，疾病控制是保障养牛业健康发展的关键因素。牛支原体（Mycoplasma bovis，M.bovis）感染引起的犊牛的肺炎、关节炎、乳房炎、角膜结膜炎等与M.bovis相关的疾病被简称为MbAD。MbAD是引起我国养牛场中犊牛死亡的主要疾病之一。本实验室于2008年首次从患肺炎的犊牛肺脏中分离到牛支原体。我们完成了牛支原体Hubei-1株的全基因组测序。众所周知，感染性疾病的发生是病原体和宿主相互作用的结果。宿主细胞对病原菌膜蛋白免疫应答的分子机制对阐述病原菌的致病机理有着重要的意义。肺脏是牛支原体感染的靶器官，研究肺细胞对牛支原体重要膜蛋白的免疫应答机制对阐述牛支原体的致病机制和正确防控由牛支原体引起的疾病具有重要意义。.本研究以胎牛肺细胞（EBL）为研究对象，研究牛支原体LAMPs激活EBL细胞IL-1β释放的分子机制。证实，牛支原体LAMPs通过激活EBL细胞跨膜蛋白TLR2及接头分子MyD88并且激活转录因子P65的磷酸化及转核进而激活NF-κB信号通路激活EBL细胞IL-1β的产生。另一条激活通路为：TLR2、TLR1、MyD88及IRAK4介导的MAPK信号通路。完成对牛支原体LAMPs刺激EBL细胞的转录组测序。在FC≥1.5, p<0.05的筛选条件下，对差异表达基因进行筛选，共筛选鉴定到706个差异表达基因，其中上调差异基因364个、下调差异基因342个；对上述差异基因所处的生物进程和信号通路进行分析，结果显示，差异表达基因编码蛋白主要与细胞凋亡、免疫应答及细胞增殖等相关，并且其中40个差异表达基因与MAPK或NF-κB信号通路相关，包括23个上调表达基因和17个下调表达基因；利用荧光定量PCR对部分差异表达基因进行验证。鉴定到处于MAPK信号通路及NF-κB信号通路中的TRIM9、SEMA7A和PIM2基因，克隆并构建上述差异基因的荧光报告质粒、超表达质粒及干扰分子，对上述基因进行了亚细胞定位分析，证实，TRIM9、SEMA7A及PIM2基因定位于细胞浆中，PIM2能够抑制牛支原体LAMPs诱导的EBL细胞IL-1β的分泌。TRIM9和SEMA7A能够促进EBL细胞IL-1β的分泌。