印太江豚皮肤成纤维细胞识别革兰氏阴性菌的受体与分子机制研究

The recognition and prevention of Gram-negative bacteria infection by skin innate immune system is important for maintaining health of mammals. The mechanism of innate immunity in terrestrial mammals, such as human, has been well studied, however, the related research on cetaceans and other marine mammals, which living in the variable environment, is very lacking. Interestingly, the applicant recently discovered that the pattern recognition receptor from skin to recognize Gram-negative bacteria in Indo-Pacific Finless Porpoise is significantly different from the human beings (Toll-like receptor 4), which may be the C-type lectin receptor CLEC14A. However, the molecular mechanism of CLEC14A responding to Gram-negative bacteria is still unclear. Therefore, this project will analyze the recognition and resistance mechanism of Indo-Pacific Finless Porpoise to Gram-negative bacteria. Firstly, we will try to determine the potential receptor of the Indo-Pacific Finless Porpoise to recognize LPS by using DARTS. Second, the potential receptors will be transfected into the FP-LWH cell, then confirm the receptors which response to LPS stimulation. Finally, the resistance mechanism of Indo-Pacific Finless Porpoise to Gram-negative bacteria will be analyzed. With this project, we will be able to provide new insight for the in-depth study of the immune system of cetaceans, further improve the understanding of the mammalian immune system and provide new scientific support for the health risk assessment of the cetaceans.

通过皮肤先天免疫系统识别和抵御革兰氏阴性菌感染是哺乳动物维持机体健康的重要手段。目前，虽然对人类等陆生哺乳动物相关机制已有深入研究，但对于多变、多压力生存环境下的鲸豚类海洋哺乳动物的研究却非常缺乏。有趣的是，申请者发现印太江豚皮肤识别革兰氏阴性菌LPS的受体与人类皮肤受体（Toll样受体4）明显不同，其可能为C型凝集素受体CLEC14A，但尚需进一步确认，且具体分子机制还不清楚。为此，本项目拟以印太江豚皮肤成纤维细胞为研究对象，首先采取DARTS试验确定印太江豚识别LPS的潜在受体；再将不同的受体在FP-LWH细胞上进行鉴定；最后，在LPS刺激下，研究印太江豚皮肤成纤维细胞触发的信号通路及分子机制，阐明印太江豚皮肤先天免疫系统识别革兰氏阴性菌的受体和分子机制。本项目的研究成果将为了解鲸豚先天免疫系统提供新的思路，深化对哺乳动物免疫系统的认知，为鲸豚保护工作的开展提供科学技术支撑。

海洋中存在多种病原微生物严重威胁着鲸目动物的健康和生存，但人们目前对鲸目动物抵御病原微生物入侵的机制尚不清楚。研究表明，皮肤成纤维细胞对宿主识别和抵御病原入侵至关重要。为此，本项目以我国二级保护水生动物印太江豚的皮肤成纤维细胞为研究对象，采用生物信息学和分子生物学等技术深入探究印太江豚皮肤成纤维细胞识别和响应病原体入侵的机制。结果发现，印太江豚皮肤成纤维细胞不表达TLR4，其可能通过其他细胞表面受体识别革兰氏阴性菌。进一步研究发现，印太江豚皮肤成纤维细胞在欧文斯氏弧菌的刺激下激活NF-kB、MAPK和IL-17等信号通路并促使IL-6、IL-8、CXCL6、CCL2和CXCL5等免疫因子的表达。与此同时，我们通过成功构建的印太江豚的皮肤成纤维细胞系（FPT细胞）初步评估了重金属污染物（CuCl2、MeHg和CdCl2）以及持久性有机污染物（BDE47、PCB126和PCB153）对鲸目动物造成的健康风险，发现由于鲸目动物体内污染物浓度较高，其中MeHg和CdCl2对鲸目动物的健康造成较高风险，但CuCl2对鲸目动物的健康造成风险较低。此外，我们还鉴定并比较印太江豚消化道和皮肤伤口的微生物组成，发现印太江豚肠道微生物的优势门类为梭杆菌门、厚壁菌门和变形菌门，而在印太江豚皮肤伤口的微生物优势门类为变形菌门、厚壁菌门和拟杆菌门，并在印太江豚不同生理部位发现了多种潜在病原体，如发光细菌属、放线杆菌属、欧文斯氏弧菌属和丹毒丝菌属等。所获研究结果为深入了解印太江豚皮肤识别和响应病原入侵的机制奠定了良好基础，同时，环境污染物对鲸目动物的健康风险评估以及对印太江豚不同生理部位多种潜在病原体的研究为制定鲸目动物的保护策略提供了科学依据。