人附红细胞体感染导致溶血与补体激活相关性研究

Hemoplasmas (or hemotrophic mycoplasmas, HM) infection is one kind of hematologic infectious zoonoses. The pathogen is a genus of blood-born epicellular parasitic organism found in different species of vertebrate. The prevalence of HM infection in our country is among the highest in the world, with over 35% in human and in house hold animals in the northwest parts of China during summer and autumn. HM from ovis aries，capra aegagrus hircus and bovine causes mild infection, but the pathogen from porcine causes severe infection. The main clinical manifestations include hemolysis, thrombosis, endothelial injuries and reduced immunity etc. HM infection has become a severe health problem in human and has a huge negative impact on livestock sector in our country, but the research has not been put in great effort in this area. .Our previous study revealed that C5b-9 abundant deposition was found on vascular endothelial cells in the HM infected mouse models. It is hypothesized that repeat or the infection from porcine HM may activate complements directly or indirectly, and causing damages to blood cells and endothelial cells, which promotes hemolysis, thrombosis, bleeding and immunosuppression. However, there is no report found regarding HM infection on the complement activation..Two key questions will be addressed in this project: is there any correlation between HM and complement activation? How does the HM infection affect complement regulators? To answer the questions, we will employ in vitro and in vivo studies. First, growing the blood and vascular endothelial cells isolated from wild type inbred mouse in tissue culture medium with HM pathogens in vitro , then quantifying membrane bound complement regulators and other CD molecules with flow cytometry and comparing gene expression patterns by RNA-Seq assay; second, inoculating murine models with HM isolated from infected human, analyzing MCP, CD55, CD59, C3b receptor, other adhesive molecules and C5b-9 deposition on different blood cells and vascular endothelium in vivo with flow cytometry, enzyme-linked immuno-sorbent assay (ELISA), enzyme-linked immuno-SPOT (ELISPOT) and immunohistology. If our hypothesis is correct, a new prevention and treatment approaches for HM infection will be proposed in the future.

附红细胞体（Hemoplasma,HM）病是人畜共患性传染病，内蒙古西部地区夏秋季人群感染率达35%以上，感染率可能居世界首位，具有明显的地区分布特点。已知源于牛羊的HM致病性弱，而源于猪的HM致病性强，可引起致命性溶血、血栓和流产等，给人群健康和畜牧业造成极大危害。HM导致溶血机制尚不明确，目前一些学者认为可能是因感染直接破坏了红细胞或使膜表面抗原暴露，产生抗自体红细胞抗体所致。我们前期实验发现，HM感染的人红细胞和动物模型血管内皮细胞有大量补体膜攻击复合物C5b-9沉积。本课题将采用HM感染小鼠模型及体外原代细胞培养，用流式细胞、酶联免疫、酶联斑点等技术检测HM感染对补体、补体调节蛋白、免疫复合物和炎性因子的影响，转录组测序技术（RAN-Seq）分析HM感染对血管内皮、血细胞基因表达谱的影响，揭示HM感染与补体活化的关系，从而探讨其溶血机制，为防治HM感染及其引发的并发症提供新的思路。

背景：附红细胞体（Eperythrozoon，Hemoplasma or hemotrophic mycoplasmas EP or HM）病是人畜共患性传染病，内蒙古地区夏秋季人群感染率达35%以上，感染率可能居世界首位。已知源于一些动物的EP致病性弱，如牛、羊、兔、鸡等，而源于猪的EP致病性强，可引起致命性溶血、血栓和免疫功能低下等，给畜牧业造成极大危害，由于人易感，所以对人健康也构成威胁。.内容及结果：我们应用PCR扩增技术确诊EP感染的人和饲养动物，PCR确诊EP感染的猪，其临床表现为食欲差或厌食、高热、皮肤出血、最终死亡。实验室应用流式细胞检测发现感染EP的猪红细胞膜表面沉积大量补体膜攻击复合物C5b-9、黏附分子降低；ELISA定量分析补体相关因子发现，C3活化产物C3b升高、血中循环免疫复合物升高等，而补体调节蛋白CD59显著降低；血管内皮细胞膜上也沉积大量C5b-9，炎性细胞聚集在血管内皮，产生大量炎性细胞因子IL1a、IFNgamma、IL2显著升高。.实验结果提示：EP感染可能诱发补体异常活化，导致红细胞溶解、血管内皮损伤。说明EP感染，特别是来源于猪的EP可以诱发补体过度活化，引发红细胞破裂、血管损伤，导致溶血、出血及血栓形成，同时发现EP感染的猪易继发其它细菌、病毒的感染，提示EP也可以导致免疫功能低下。此研究成果有助于我们从固有免疫异常活化的角度理解EP感染的致病机制，为防治EP感染提供新的研究思路。