蛇血清PLI-γ对sPLA2炎症通路的干预作用及相关机制研究

Secreted phospholipase A2 is a master switch in the upstream of arachidonic acid inflammatory pathway. Considered the severe side-effects of COX-2 inhibitor drugs, namely NSAIDs, which are the maximum prescription drug, drug discovery targeting to sPLA2 is of great significance both in anti-inflammatory pharmacology and clinical application. By the results of bioinformatics analysis and experimental trial in our preliminary study, we found an antidotal protein that isolated from Sinonatrix percarinata known as "γ type sPLA2 inhibitor (PLIγ)"，has potential inhibitive activity against human sPLA2. In this project, we are going to continue our research in three major aspects, 1) prepare the monoclonal antibody of PLIγ. With the mAb, co-immune precipitation will be carried out to screen human proteins (mostly, the PLA2s) that binding with PLIγ. These proteins are then separated by two dimensional electrophoresis(2DE) and identified by MS, and finally verified by western blot. 2) Develop a human monocyte cell line known as THP-1 that hosting PLIγ gene by transfection of recombinant plasmid of PLIγ-pIRES2-EGFP. After treatment with lipopolysaccharide(LPS), the recombined THP-1 cells and normal cells will exhibit different inflammatory response. Pro-inflammatory factors and cell factors in these cells are then assayed to assess the protective effect of PLIγ in vitro cell. 3) Replicate human severe acute pancreatitics (SAP) and rheumatoid arthritis（RA） on Wistar rats. Anti-inflammatory activities are valued in vivo on the two animal model. When the above studies on protein interactive, cell trial in vitro and animal experiment are finished, the anti-inflammatory effect and its related mechanism will be comprehensively illustrated.

sPLA2是花生四烯酸炎症通路的上游开关。由于现有的COX-2抑制剂类抗炎症药物（即NSAIDs）毒副作用强烈，因此寻找靶向sPLA2的药物具有重要的理论意义和应用价值。申请者前期通过生物信息学和相关实验，发现华游蛇解蛇毒蛋白"γ型sPLA2抑制剂（PLIγ）"具有潜在的抑制人sPLA2活性，是理想的抗炎症候选药。在此基础上，本项目拟开展如下研究：1）制备PLIγ单克隆抗体，通过免疫共沉淀、双向电泳和western blot技术，研究与PLIγ有相互作用的细胞蛋白质；2）构建PLIγ-pIRES2-EGFP真核表达载体，转染人THP-1细胞株，通过LPS诱导实验及炎症因子、细胞因子检测，研究PLIγ抗细胞炎症效果；3）复制人SAP和RA动物模型，评价PLIγ整体水平抗炎症作用。通过以上蛋白质相互作用，体外细胞实验和动物模型三个层次研究，系统地阐述华游蛇PLIγ抗炎症效果及作用机制。

本项目中，我们按照计划克隆了8种蛇PLIγ基因。构建了Pet28c-PLIγ重组质粒，通过正交设计法优化了表达条件，成功表达了His6-PLIγ融合蛋白。利用DNAstar软件，预测了华游蛇PLIγ的抗原表位肽，通过免疫小鼠和多轮筛选，最终获得了18株IgG型PLIγ单克隆抗体。该18株抗体均可以与重组表达的His6-PLIγ和天然华游蛇血清PLIγ结合。此外，利用其中的43号抗体，检测了华游蛇、银环蛇、金环蛇、眼镜蛇、五步蛇、颈棱蛇、王锦蛇、乌梢蛇、三线蛇、赤练蛇、红点锦蛇血清PLIγ表达情况。结果表明PLIγ在有毒蛇和无毒蛇中广泛存在。 本项目构建了pGC-FU -PLIγ-EGFP重组病毒载体，获得了具有感染性的慢病毒颗粒LV-PLIγ-EGFP。将该病毒颗粒感染人THP-1细胞株，获得了PLIγ阳性细胞株。THP-1阳性细胞株传代8代后EGFP仍然稳定表达，通过PCR和抗Flag标签western blot检测也表明PLIγ稳定表达。利用脂多糖对THP-1进行诱导，发现IIF和X 型sPLA2在LPS诱导后显著上调。除此之外，THP-1细胞中AA、TNF-α、IL-1β、IL-6、PGE2、PAF等炎症因子都显著表达上调。而在阳性THP-1组（ PLIγ组）中sPLA2、AA、TNF-α、IL-1β、IL-6、PGE2、PAF等因子的含量低于空病毒组（mock组），说明炎症时，PLIγ会对人sPLA2酶产生抑制，减少上述炎症因子释放。通过CoIP实验，发现PLIγ还可以与IB、IIA等PLA2相互结合，表明PLIγ对sPLA2亚型有广泛抑制效果。小鼠足趾肿胀实验表明，PLIγ具有良好的抗炎症作用，其效果显著优于地塞米松，并且呈剂量效应性。HE染色表明PLIγ组中嗜中性粒细胞数量远低于模型组。