牛支原体体外肺精准切片感染模型的建立  

Establishment of Cattle Precision-Cut Lung Tissue Slices(PCLS)Models Infected with Mycoplasma bovis in vitro

作　　者：张慧 路豆昆 张怡秋 赵刚 陈颖钰[2] 陈曦[2] 胡长敏[2] 郭爱珍[2] ZHANG Hui;LU Doukun;ZHANG Yiqiu;ZHAO Gang;CHEN Yingyu;CHEN Xi;HU Changmin;GUO Aizhen(College of Animal and Veterinary Sciences,Southwest Minzu University,Chengdu 610041,China;College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China;School of Life Sciences,Ningxia University,Yinchuan 750021,China)

机构地区：[1]西南民族大学畜牧兽医学院,成都610041 [2]华中农业大学动物医学院,武汉430070 [3]宁夏大学生命科学学院,银川750021

出　　处：《畜牧兽医学报》2024年第10期4638-4645,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：西南民族大学中央高校基本科研业务费专项资金资助(ZYN2023045);西南民族大学引进高层次人才科研资助金资助(RQD2023031);宁夏回族自治区重点研发计划项目(2021BEF02028,2023BCF01038)。

摘　　要：本研究旨在建立一种牛支原体体外感染模型-牛肺组织精准切片(precision-cut lung tissue slices,PCLS)感染模型。采集2月龄犊牛新鲜肺脏,经低熔点琼脂糖灌注、固定后,使用振动切片机制成厚度为250μm的牛PCLS,利用牛支原体野毒株HB0801(P1)及其传代致弱株P150以108 CFU·孔^(-1)的剂量进行体外感染,分别经qRT-PCR、乳酸脱氢酶(LDH)细胞毒性试验、3D荧光显微镜全景扫描、免疫组化等技术评估牛支原体在PCLS中的感染效果。结果显示正常PCLS在体外培养72 h后,细胞活力可维持在60%以上,组织形态学观察PCLS边缘清晰,肺泡结构正常;qRT-PCR检测牛支原体强、弱菌株在PCLS中的基因拷贝数,发现二者均可存活和增殖,免疫组化观察牛支原体主要定位于PCLS的肺泡腔周围以及支气管间隙,LDH试验检测发现牛支原体强、弱菌株感染18 h出现细胞毒性反应,二者均可诱导PCLS产生促炎因子IL-1β和IL-8,且强毒株诱导水平显著高于弱毒株(P<0.05)。本研究初步建立了牛支原体PCLS体外感染模型,提供了一种可模拟牛支原体体内感染试验的离体模型,为今后研究牛支原体与宿主相互作用提供借鉴和参考。Mycoplasma bovis(M.bovis)is an important pathogen of bovine respiratory disease complex(BRDC),mainly causing pneumonia,mastitis,arthritis,keratoconjunctivitis,otitis,and genital disorders,leading to high economic losses in dairy and beef cattle production.Lack of small animals’models have greatly hindered the progress of effective vaccines and drugs for prevention and control in Mycoplasma bovis.In this study,we have established an in vitro infection model of M.bovis in Precision-cut lung tissue slices(PCLS).We collected bovine lungs from apparently healthy two-months cattle after slaughter,filled the lung airway with low melting point agarose,transferred it to the vibratome tissue slicer and then made the 250μm-thickness precision cut lung slices.After M.bovis wildtype strain HB0801(P1)and its attenuated strain P150 at a MOI of 108 CFU infected,quantitative real-time PCR,lactate dehydrogenase cytotoxicity as-say,3D fluorescence microscope panoramic scanning,immunohistochemistry analysis were per-formed.The results showed that cultured PCLS remained 60%cell viability for at least 72 h and maintained normal structural integrity including edge sharpness and alveolar structure well in un-infected PCLS.After infected with wildtype and attenuated M.bovis strains respectively,we found that both M.bovis could survive and proliferate in PCLS,tropism to alveolar and bronchi-al space,and showed cytotoxic reactions until 18 h post-infection.We also observed that after 36 h incubation with M.bovis wildtype strain P1 and its attenuated strain P150,cultured PCLS pro-duced pro-inflammatory factors IL-1βand IL-8 in PCLS,and M.bovis wildtype strain HB0801 could induce significantly higher level than P150(P<0.05).In conclusion,this study provides a model and method to investigate M.bovis infection in vitro which can mimic M.bovis infection in vivo.It is expected to provide reference for subsequent research on host-pathogen interaction in M.bovis.

关 键 词：牛支原体 肺组织精准切片模型 体外感染 炎性反应 

分 类 号：S852.62[农业科学—基础兽医学]