树鼩原代耳缘成纤维细胞体外分离培养及鉴定的研究  

Research on isolation,culture and identification of primary tree shrew ear fibroblasts in Vitro

作　　者：冯梅 罗秋妮 郑思聪 彭碧燕 孙俊铭 张名媛 粟小平 欧阳轶强 李习艺[4] Feng Mei;Luo Qiuni;Zheng Sicong;Peng Biyan;Sun Junming;Zhang Mingyuan;Su Xiaoping;Ouyang Yiqiang;Li Xiyi(Laboratory Animal Center,Guangxi Medical University,Nanning 530021,China;School of Basic Medical Sciences,Guangxi Medical University,Nanning 530021,China;Guangxi Key Laboratory of Oral and Maxillofacial Rehabilitation and Reconstruction,Guangxi Medical University,Nanning 530021,China;Department of Nutrition and Food Hygiene,School of Public Health,Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学实验动物中心,南宁530021 [2]广西医科大学基础医学院,南宁530021 [3]广西口腔颌面修复与重建研究自治区级重点实验室,南宁530021 [4]广西医科大学公共卫生学院营养与食品卫生系,南宁530021

出　　处：《广西医科大学学报》2022年第11期1719-1725,共7页Journal of Guangxi Medical University

基　　金：国家自然科学基金资助项目(No.31560612);广西自然科学基金项目资助(No.2021GXNSFAA220005);南宁市青秀区科技局重点研发计划项目资助(No.2021011)。

摘　　要：目的:旨在建立树鼩耳缘成纤维细胞体外分离、培养纯化及鉴定的方法。方法:采用组织块贴壁法进行原代树鼩耳缘成纤维细胞(TSEF)的分离培养,将获得的细胞利用波形蛋白(Vimentin)进行免疫荧光鉴定,RT-qPCR法检测Vimentin基因mRNA的相对表达量;用苏木精—伊红(HE)染色,观察细胞形态;用细胞增殖试剂盒(CCK-8)检测TSEF的增殖情况;利用流式细胞仪检测第3代和第5代细胞周期和凋亡的情况。结果:通过组织块贴壁法可以成功分离出树鼩的耳缘成纤维细胞,在倒置荧光显微镜下原代细胞呈现出长梭形、多触角、鱼群状等形态;细胞免疫荧光Vimentin呈现强阳性;TSEFVimentin mRNA的相对表达量与小鼠3T3 Swiss albino细胞Vimentin mRNA的相对表达量无明显差异(P>0.05)。HE染色结果显示,该细胞形态呈典型的长梭形或星形,细胞质染为淡紫色,细胞核着深紫色,为规则的卵圆形;检测原代细胞增殖情况,细胞增殖曲线呈典型的S形,且第2至第3天是细胞的最佳增殖期。第3代和第5代细胞在G0/G1期所占的比例没有明显地改变(P>0.05),且处于G0/G1期的细胞占主导地位;两代细胞的凋亡率较低且没有明显变化(P>0.05)。结论:组织块贴壁法可成功提取TSEF,且细胞在第3至第5代内生长状况良好,可用于体细胞核移植等科学研究活动。Objective:To establish a method for isolation,culture,purification and identification of primary tree shrew ear fibroblasts in Vitro.Methods:The primary tree shrew ear fibroblasts(TSEF)were isolated and cultured by tissue block adherence method.The obtained cells were identified by immunofluorescence using vimentin,and the relative expression level of Vimentin mRNA was detected by RT-qPCR.Hematoxylin-eosin(HE)staining was used to observe cell morphology and the proliferation of TSEF was detected by cell counting kit-8(CCK-8)method.The cell cycle and apoptosis of the third and fifth passages were detected by flow cytometry.Results:The ear fibroblasts of tree shrew could be successfully isolated by the method of tissue block adherence.Under the inverted fluorescence microscope,the primary cells showed long spindle,multi-antennae and fish-like shapes and cell immunofluorescence Vimentin showed strong positive;there was no significant difference in the relative expression levels between TSEF Vimentin mRNA and mouse 3T3 Swiss albino cells(P>0.05).HE staining showed that the cell morphology was typical long spindle or star shape,the cytoplasm was light purple and the nucleus was dark purple with regular oval.The proliferation of primary cells was detected,the cell proliferation curve showed a typical S-shape and the optimal proliferation period was from the second to the third day.The proportion of cells in the third and fifth passages in the G0/G1 phase did not change significantly(P>0.05),and the cells in the G0/G1 phase were dominant;the apoptosis rate of the two passages of cells was lower and did not change significantly(P>0.05).Conclusion:TSEF can be successfully extracted by tissue block adhesion method,and the cells grow well from the third to fifth passage,which can be used for scientific research activities such as somatic cell nuclear transfer(SCNT).

关 键 词：树鼩 耳缘成纤维细胞 组织块贴壁法 体细胞核移植 波形蛋白 细胞增殖 

分 类 号：R-332[医药卫生]