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自然感染绵羊肺腺瘤病毒羊肺组织线粒体自噬分子表达分析
梁浚哲,段续接,杜晓悦,张良,李慧萍,刘淑英*
0
(内蒙古农业大学 兽医学院/农业农村部动物临床诊疗技术重点实验室/内蒙古自治区基础兽医学重点实验室,呼和浩特 010018)
摘要:
为分析自然感染绵羊肺腺瘤病毒(Jaagsiekte sheep retrovirus,JSRV)羊肺组织中线粒体的质量及自噬相关蛋白表达的变化,本研究将自然感染 JSRV羊的肺组织作为研究对象,利用病理组织学、PCR和免疫组化法对自然感染JSRV羊肺组织进行鉴定。利用透射电镜技术观察病羊肺组织中线粒体形态结构变化,分别采用实时荧光定量PCR(RT-qPCR)和蛋白免疫印迹法(WB)检测感染组羊肺组织中线粒体相关因子线粒体外膜蛋白20(Outer membrane translocase 20,TOMM20)、线粒体内膜蛋白(Inner membrane translocase 23,TIMM23)、热休克蛋白 60(Heat shock proteins 60,HSP60)的mRNA和蛋白水平的相对表达量,利用 WB 检测病羊肺组织线粒体蛋白中自噬相关因子 Beclin1、LC3 和选择性自噬受体 p62 蛋白水平的相对表达量。结果表明:1)PCR扩增的目的条带与 JSRV env 预期扩增片段大小一致。光镜下可见肺组织中肺泡Ⅱ型上皮细胞呈乳头状生长,且伸入肺泡中,同时免疫组化结果显示在增生的肺泡Ⅱ型细胞中检测到JSRV Env大量表达,而阴性对照组未检出。2)与健康对照组相比,透射电镜观察到感染组羊组线粒体大量损伤,并且出现双层膜结构的线粒体自噬体包裹损伤的线粒体,TOMM20、TIMM23和HSP60的 mRNA 相对表达水平和蛋白相对表达水平(P<0.01)均极显著下降。3)自噬因子 Beclin1(P<0.05)和LC3(P<0.01)蛋白相对表达水平显著升高,p62 相对表达显著下降(P<0.001)。综上,本研究发现自然感染JSRV羊肺组织中线粒体损伤严重并出现线粒体自噬现象,为 JSRV 的致病机制提供新的研究方向。
关键词:  绵羊肺腺瘤病毒  绵羊肺组织  线粒体损伤  线粒体自噬
DOI:10.11841/j.issn.1007-4333.2024.08.12
投稿时间:2023-12-02
基金项目:国家自然科学基金项目(32072819、32360863);内蒙古科技重大专项(2021ZD0010);内蒙古草原英才创新团队项目(20151031);内蒙古教育厅创新团队项目(NMGIRT2412);牛羊疫病防控与发育工程创新团队资助项目(RZ2200001130);研究生科研创新资助项目(B202310722)
Expression analysis of mitochondrial autophagy factor in lung tissues of sheep naturally infected with Jaagsiekte sheep retrovirus
LIANG Junzhe, DUAN Xujie, DU Xiaoyue, ZHANG Liang, LI Huiping, LIU Shuying*
(Key Laboratory of Animal Clinical Diagnosis and Treatment of Ministry of Agriculture and Rural Affairs/Inner Mongolia Key Laboratory of Basic Veterinary Medicine/College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China)
Abstract:
The purpose of this study was to analyze the quality of mitochondria and the expression of autophagy-related proteins in the mitochondria in the lung tissues of sheep naturally infected with Jaagsiekte sheep retrovirus(JSRV). In this study, the lung tissues of sheep naturally infected with JSRV were taken as the object of study. Histopathology, PCR and immunohistochemistry were used to identify naturally infected JSRV lung tissues. The morphological and structural changes of mitochondria in diseased sheep lung tissues were observed by transmission electron microscopy (TEM). Real-time quantitative PCR (RT-qPCR) and Western blot (WB) were used to detect the relative expression of mitochondrial related factors outer membrane protein 20 (TOMM20),inner membrane protein 23 (TIMM23) and heat shock protein 60 (HSP60) in sheep lung tissues of healthy and naturally infected JSRV diseased sheep. WB was used to detect the relative expression of autophagy-related factors Beclin1, LC3 and selective autophagy receptor p62 in lung mitochondrial proteins of diseased sheep. The results showed that: 1) The PCR results showed that the target band was consistent with the expected fragments of JSRV env. Alveolar type II cells in the diseased tissues showed mastoid hyperplasia and extended into the alveolar cavity under the light microscope, and the immunohistochemical results showed a large amount of JSRV Env expression was detected in the proliferative alveolar type II cells, but not in the negative control group. 2) Compared with the healthy control group, the transmission electron microscope showed a large number of mitochondrial damage in the diseased sheep group, and the damaged mitochondria were wrapped by mitochondrial autophagosomes with double membrane structure. The relative expression levels of mRNA and protein of TOMM20, TIMM23 and HSP60 were extremely significantly decreased (P<0.01). 3) The relative expression of autophagy factors Beclin1 (P<0.05) and LC3 (P<0.01) were increased significantly, while the relative expression of p62 was significantly decreased (P<0.001). In summary, we found there was severe mitochondrial damage and mitochondrial autophagy in the lung tissues of diseased sheep, providing a new research direction for the pathogenesis of JSRV.
Key words:  Jaagsiekte sheep retrovirus  sheep lung tissues  mitochondrial damage  mitophagy