牦牛皮肤和肌肉成纤维细胞系的建立与比较分析  

Establishment and Comparative Analysis of Yak Skin and Muscle Fibroblast Cell Lines

作　　者：徐红梅 海欣怡 李悦悦 李巧 马梓峰 熊燕[1,2,3] 熊显荣[1,2,3] 兰道亮[1,2,3] 李键[1,2,3] 付伟 XU Hongmei;HAI Xinyi;LI Yueyue;LI Qiao;MA Zifeng;XIONG Yan;XIONG Xianrong;LAN Daoliang;LI Jian;FU Wei(Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization of Ministry of Education,Southwest Minzu University,Chengdu 610041,China;Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization Key Laboratory of Sichuan Province,Southwest Minzu University,Chengdu 610041,China;Key Laboratory for Animal Science of National Ethnic Affairs Commission of China,Southwest Minzu University,Chengdu 610041,China)

机构地区：[1]西南民族大学青藏高原动物遗传资源保护与利用教育部重点实验室,四川成都610041 [2]西南民族大学青藏高原动物遗传资源保护与利用四川省重点实验室,四川成都610041 [3]西南民族大学动物科学国家民委重点实验室,四川成都610041

出　　处：《华北农学报》2024年第S1期274-280,共7页Acta Agriculturae Boreali-Sinica

基　　金：国家重点研发计划项目(2021YFD1600200);四川省科技计划项目(2024YFTX0019);甘孜州“校州合作”项目(横20240054)。

摘　　要：旨在建立牦牛肌肉和皮肤成纤维细胞系,并分析比较两者的形态、生长、标志基因和蛋白的差异,为更好地了解和研究牦牛相关特性提供材料。首先,从妊娠3个月的牦牛胎儿分离皮肤和肌肉组织块,分别采用组织块法和酶解法培养其原代细胞,观察其生长状态和形态特点;之后,利用CCK8法检测皮肤成纤维细胞和肌肉成纤维细胞的增殖活力,并对2种细胞进行免疫荧光染色分析;最后,利用RT-qPCR分析2种细胞中成纤维细胞标志基因的表达。结果显示,酶解法比组织块法更容易获得胎牦牛皮肤和肌肉成纤维细胞,胎牦牛皮肤成纤维细胞的活力显著高于肌肉成纤维细胞(P<0.05),且皮肤成纤维细胞中标志蛋白VIMENTIN的荧光强度显著高于肌肉成纤维细胞,但二者组蛋白修饰H3K27me3的差异不显著;VIMENTIN和S100A4基因在皮肤成纤维细胞中显著高表达,而FAP和SMA基因在肌肉成纤维细胞中显著高表达,与免疫荧光染色结果一致。综上,建立了牦牛胎儿皮肤和肌肉成纤维细胞系,并通过CCK8、免疫荧光染色和RT-qPCR等方法比较了2种细胞的生理特性。The aim of this study was to establish yak muscle and skin fibroblast cell lines,analyze and compare the differences in morphology,growth,marker genes and proteins between two cell lines to provide materials for a better understanding of yak properties.Firstly,the skin and muscle tissue blocks were isolated from the yak fetus at 3 months of gestation,and the primary cells were cultured by tissue block method and enzymatic hydrolysis method respectively,and their growth status and morphological characteristics were observed.Following,the CCK8 method to detect the proliferation viability of skin and muscle fibroblasts,and immunofluorescence staining analysis was performed on the two cells.Finally,RT-qPCR was used to analyze the expression of fibroblast marker genes in the two kinds of cells.It was found that the enzymatic method was easier to obtain fibroblasts than the tissue method,and the viability of skin fibroblasts was significantly higher than that of muscle fibroblasts(P<0.05).The fluorescence intensity of the marker protein VIMENTIN in skin fibroblasts was significantly higher than that of muscle fibroblasts,but the histone-modified H3K27me3 was not significant between the two kinds of cells.Furthermore,VIMENTIN and S100A4 genes were significantly highly expressed in skin fibroblasts,while FAP and SMA genes were significantly high expressed in muscle fibroblasts,consisting with immunofluorescent staining results.In conclusion,it successfully established yak fetal skin and muscle fibroblast cell lines,and compared their physiological properties of two kinds of cells by using CCK8,immunofluorescence staining and RT-qPCR.

关 键 词：牦牛 胎儿成纤维细胞 分离培养 细胞活力 免疫荧光染色 

分 类 号：S823[农业科学—畜牧学]