摘要
为了建立蛙脑微血管内皮细胞原代培养方法,实验以黑斑侧褶蛙脑组织为实验材料,在无菌条件下分离出大脑灰质,利用0.1%Ⅱ型胶原酶和胶原酶-分散酶进行消化,经过Percoll密度梯度离心后,将获取的脑微血管段接种于鼠尾胶包被的培养瓶中,于28℃、5%CO_(2)条件下原代培养蛙脑微血管内皮细胞,并通过细胞形态学观察和Ⅷ因子相关抗原免疫荧光对细胞进行鉴定,最后用四唑蓝比色法(MTT)测定细胞的生长状态。结果显示,微血管段在体外培养1 d后开始贴壁生长,并逐渐扩大成团簇状,培养至7 d时,细胞呈“铺石路样”镶嵌式排列,表现为区域性单层生长;Ⅷ因子免疫荧光检测发现胞质呈红色,表达为阳性,阳性细胞率达97%以上;MTT实验显示,细胞在第3~5天时生长速率最快。本研究首次建立了蛙脑微血管内皮细胞的原代培养与鉴定方法,为体外研究蛙类神经性疾病的发病机理奠定基础,也为相关药物的研发和筛选提供了细胞模型。
In order to isolate the brain microvascular endothelial cells(BMECs)from Pelophylax nigromaculata,the brain tissue was used in this study.Under an asepic condition,the cerebral cortex was digested with 0.1% collagenase type Ⅱ and collagenase/dispase,then separated by Percoll density gradient centrifugation.BMECs were inoculated in culture flask coated with rat tail glue,and cultured at 28℃,5%CO_(2).Then,the cells were identified by morphologic observation and immunofluorescence staining with factor Ⅷ.Besides,the methyl thiazolyl tetrazolium(MTT)colorimetric assay was used to detect the cell viability.The results showed that after 1 day,the microvascular fragments grew adherently and proliferated in a clustered monolayer,then expanded in culture and formed confluent monolayers with the characteristic cobblestone shape on 7th day.Positive Ⅷ expression was frequently observed in P.nigromaculata BMECs cytoplasm.Besides,MTT assay showed the cell viability of P.nigromaculata BMECs grew the fastest on the 3rd day to the 5th.In this study,an available method of culturing P.nigromaculata BMECs was established for the first time,which is of great significance for studying the pathogenesis of frog neurological diseases in vitro,providing a cell model for the development and screening of related drugs.
作者
艾单寒
侯家昊
刘芳园
顾泽茂
胡瑞雪
AI Danhan;HOU Jiahao;LIU Fangyuan;GU Zemao;HU Ruixue(College of Fisheries,Huazhong Agricultural University,Wuhan 430070,China;Hubei Engineering Technology Research Center for Aquatic Animal Diseases Control and Prevention,Wuhan 430070,China;Shuangshui ShuanglüInstitute,Huazhong Agricultural University,Wuhan 430070,China;Engineering Research Center of Green development for Conventional Aquatic Biological Industry in the Yangtze River Economic Belt,Ministry of Education,Wuhan 430070,China)
出处
《水产学报》
CAS
CSCD
北大核心
2023年第8期87-94,共8页
Journal of Fisheries of China
基金
国家自然科学基金(32202990)
湖北省自然科学基金(2021CFB128)
中国博士后科学基金(2021M701351)。
关键词
黑斑侧褶蛙
脑组织
微血管内皮细胞
原代培养
Pelophylax nigromaculata
brain
microvascular endothelial cells
primary culture
