树鼩脑微血管内皮永生化细胞系的建立及最适D-半乳糖浓度诱导BMECs衰老细胞模型的探讨  

Establishment of an immortalized tree shrew brain microvascular endothelial cell line and study of a senescence cell model of BMECs induced by the D-galactose concentration

作　　者：陈柳[1] 邱敏 丁相荣 霍姝汭 王文广[1] 陆彩霞[1] 代解杰[1] CHEN Liu;QU Min;DNG Xiangrong;HUO Shurui;WANG Wenguang;LU Caixia;DAJiejie(1nstitute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Kunming 650118,China;Kunming Medical University,Kunming 650500)

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650500

出　　处：《中国实验动物学报》2023年第3期337-345,共9页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家重点研发计划项目(2021YFF0702404);云南省基础研究计划面上项目(202101AT070291);云南省眼科疾病防治研究重点实验室(2017DG008);昆明市科技创新团队(2019-1-R-24483)。

摘　　要：目的建立树鼩永生化脑微血管内皮细胞(brain microvascular endothelial cells,BMECs),明确D-半乳糖对BMECs衰老的影响,探索其潜在机制。方法用携带SV40T基因的慢病毒转染BMECs,传至50代后进行形态观察、生长曲线测定以及免疫荧光和核型鉴定;再用不同浓度D-半乳糖(D-galactose,D-gal)诱导BMECs,通过细胞增殖实验确定最适诱导条件,用衰老相关β半乳糖苷酶染色分析细胞衰老情况,Western Blot检测衰老相关蛋白p53的表达水平,超氧化物歧化酶和脂质氧化检测试剂盒检测细胞内氧化应激水平。结果永生化的细胞生长状态较好,不同代次的细胞形态一致,单个细胞呈短梭形或多角形,整体呈典型的铺路石状;细胞生长曲线显示细胞数量在第2~5天时处于对数生长期,在第6天时达到高峰,之后缓慢增长进入平台期;免疫荧光显示该细胞特异蛋白vWF和CD31及永生化基因SV40T为阳性;细胞核型结果显示永生化细胞染色体数目与滇西亚种树鼩的染色体数量相符;D-gal诱导BMECs抑制细胞增殖,有时间和浓度依赖性;实验组,即浓度为10 g/L的D-gal诱导48 h的BMECs,β-半乳糖苷酶染色阳性明显增多、衰老相关蛋白p53显著增多、细胞内SOD酶活性降低和MDA含量增多。结论成功构建树鼩永生化脑微血管内皮细胞系,10 g/L的D-gal是建立永生化BMECs衰老模型的适合浓度,D-gal在体外促进细胞衰老可能是通过促进氧化应激水平、抑制细胞增殖来实现的。Objective Establish immortalized brain microvascular endothelial cells from tree shrews,clarify the effect of D-galactose on senescence of brain microvascular endothelial cells,and explore the potential mechanisms.Methods BMECs were transfected with a lentivirus carrying the SV40T gene,and immortalized cells were characterized by morphological observation,growth curve determination,immunofluorescence,and karyotype after expansion for>50 passages.Next,BMECs were treated with various concentrations of D-gal,and the optimal concentration was determined by cell proliferation assays.Senescence was analyzed by staining with senescence-associatedβ-galactosidase.Expression of senescence-associated p53 was measured by Western Blot.Intracellular oxidative stress levels were measured by superoxide dismutase and lipid oxidation assays.Results Immortalized cells grew with consistent morphology throughout passages.Single cells were shaped as short spindles or polygonal with a typical paving stone shape.The cell growth curve showed cells were in the logarithmic growth phase on days 2~5.Cell growth peaked at day 6 and then the cells grew slowly to a plateau.High levels of tree shrew vWF,CD31,and SV40T were detected by immunofluorescence.Karyotyping showed that the number of chromosomes in immortalized cells matched the number of chromosomes in tree shrew(Tupaia belangeri chinensises).D-galactose reduced BMEC proliferation in time-and dose-dependent manners.In the experimental group,i.e.BMECs treated by D-gal at a concentration of 10 g/L for 48 h,positiveβ-galactosidase staining was significantly increased,p53 was significantly increased,intracellular SOD activity was reduced,and MDA content was increased.Conclusions A tree shrew immortalized brain microvascular endothelial cell line was established successfully.The optimal concentration to establish an immortalized BMEC senescence model was 10 g/L D-gal.The mechanism by which D-gal promotes cellular senescence in vitro may be mediated through promotion of oxidative stress and

关 键 词：树鼩 脑微血管内皮细胞 永生化 D-半乳糖 衰老模型 

分 类 号：Q95-33[生物学—动物学]