树鼩视网膜微血管内皮细胞永生化细胞株的建立及鉴定  

Establishment and characterization of immortalized cell lines of retinal microvascular endothelial cells from tree shrew

作　　者：霍姝汭 邱敏 王文广[1] 陆彩霞[1] 罕园园[1] 代解杰[1] HUO Shurui;QIU Min;WANG Wenguang;LU Caixia;HAN Yuanyuan;DAI Jiejie(Institute of Medical Biology,Chinese Academy of Medical Sciences/Peking Union Medical College,Kunming 650118;China.2.Kunming Medical University,Kunming 650031)

机构地区：[1]中国医学科学院/北京协和医学院医学生物学研究所,昆明650118 [2]昆明医科大学,昆明650031

出　　处：《中国实验动物学报》2023年第4期471-477,共7页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家重点研发计划资助(2021YFF0702404);云南省基础研究计划面上项目(202101AT070291);云南省科技人才和平台计划项目(2017HC019);昆明市科技创新团队(2019-1-R-24483)。

摘　　要：目的建立树鼩视网膜来源的微血管内皮细胞的体外分离培养技术以及永生化细胞株,为体外利用树鼩视网膜微血管内皮细胞开展相关研究提供新的实验材料。方法利用Ⅱ型胶原酶、分散酶和DNaseⅠ酶消化法分离培养出原代视网膜微血管内皮细胞,利用差速消化法纯化内皮细胞,然后利用携带SV40T基因的慢病毒转染细胞,再挑取单克隆后进行传代培养。对传至50代以上的细胞进行形态学观察、免疫荧光鉴定以及核型鉴定。结果采用混合酶消化法能够分离获得微血管内皮细胞,纯化后的细胞呈不规则多角形和梭形。经慢病毒转染后的细胞传代培养后细胞形态一致,到第50代时细胞形态结构仍较好。细胞免疫荧光结果显示标志性蛋白VWF、CD34、Claudin1、ZO-1和永生化SV40T表达阳性。生长曲线结果显示:细胞生长旺盛,第2~4天时处于对数生长期,第4天进入平台期。细胞核型结果显示:染色体数与树鼩染色体相同,即所获取的细胞为永生化的树鼩视网膜微血管内皮细胞。结论成功建立的树鼩视网膜微血管内皮细胞永生化细胞株具有较好的形态结构与功能,为视网膜病变及眼科相关疾病的研究提供了新的实验材料。Objective To establish in vitro isolation and culture technology of tree shrew retina-derived microvascular endothelial cells and immortalize cell lines to provide new experimental materials for in vitro study of tree shrew retinal microvascular endothelial cells.Methods Primary retinal microvascular endothelial cells were isolated by treatment with collagenase typeⅡ,disperse and DNase I.The cells were purified by differential digestion.Morphological observation,immunofluorescence,and karyotyping were performed on cells passaged more than 50 times.Results Microvascular endothelial cells were isolated by enzyme digestion.Purified cells had an irregular polygonal shape and pavement pattern.The morphology of cells infected with a lentivirus was consistent after subculture,and cell morphology was intact at passage 50.Immunofluorescence showed that VWF,CD34,Claudin 1,ZO-1,and SV40T were positive.The growth curve showed that the cells grew vigorously and were in the logarithmic growth phase from day 2 to 4,which reached a plateau on day 4.Karyotyping showed that the chromosome number was consistent with the chromosome number of Sino-Burmese tree shrew.Therefore,the obtained cells were immortalized retinal microvascular endothelial cells.Conclusions The immortalized cell line of tree shrew retinal microvascular endothelial cells had an intact morphology,structure and functions,which provides new experimental material for the study of retinopathy and ophthalmology-related diseases.

关 键 词：树鼩 视网膜微血管内皮细胞 分离培养 永生化细胞株 

分 类 号：Q95-33[生物学—动物学]