摘要
目的:探讨SD胎鼠海马神经干细胞(NSCs)源性外泌体(Exos)的提取、鉴定。方法:选取15 d胎鼠海马组织提取NSCs,首先光镜下观察细胞生长的形态变化,免疫荧光组织化学染色法检测巢蛋白(nestin)的表达;然后诱导分化培养基继续培养,行神经元特异性微管蛋白(β-tubulin-III)、胶质纤维酸性蛋白(GFAP)免疫荧光组织化学染色鉴定NSCs,最后收集细胞培养上清液,按照梯度离心方法提取、纯化Exos,电子显微镜下观察其形态特点,纳米颗粒分析检测其粒径大小,Western Blot检测其特异性标志物CD9、CD63、肿瘤易感基因101(TSG101)的表达。结果:成功分离、培养了SD胎鼠海马来源的NSCs,光镜下细胞呈典型神经球状生长,nestin表达阳性。NSCs经过诱导培养后成功分化为神经元和星形胶质细胞,β-tubulin-III与GFAP呈阳性表达。电子显微镜下Exos呈现双层膜的杯口状结构,平均粒径(171.8±67.6)nm,CD9、CD63与TSG101蛋白呈阳性表达。结论:分离并培养了15 d胎鼠海马来源的NSCs,成功提取NSCs来源的Exos。
Objective:To investigate the extraction and identification of exosomes(Exos)derived from hippocampal neural stem cells(NSCs)of SD fetal rat.Methods:NSCs were extracted from 15-day fetal rat hippocampus.First,the morphological changes of cell growth were observed under light microscope,and the expression of nestin was detected by immuno fluorescence chemical staining;Tubulin(β-tubulin-III)and glial fibrillary acidic protein(GFAP)immuno fluorescence chemical staining was used to identify NSCs.Finally,the cell culture supernatant was collected,and Exos was extracted and purified by gradient centrifugation.Its morphological characteristics were observed under an electron microscope.The particle size was detected by particle analysis,and the expression of specific markers CD9,CD63,and tumor susceptibility gene101(TSG101)was detected by Western Blot.Results:NSCs derived from SD fetal rat hippocampus were successfully isolated and cultured.The cells showed typical neurosphere growth under the light microscope,and the expression of nestin was positive.After induction and culture,the cells were successfully differentiated into neurons and astrocytes,andβ-tubulin-III and GFAP were positively expressed.Under the electron microscope,Exos showed a cup-shaped structure with a double-layered membrane,with an average particle size of(171.8±67.6)nm,and positive expressions of CD9,CD63,and TSG101 proteins.Conclusion:NSCs derived from the hippocampus of fetal rat were isolated and cultured for 15 days,Exos derived from NSCs were successfully extracted.
作者
陈珊
朱俊德
赵雪
龙婷婷
周璐
Chen Shan;Zhu Junde;Zhao Xue;Long Tingting;Zhou Lu(Department of Human Anatomy,Basic Medicine,Guizhou Medical University,Guiyang 550025,China)
出处
《神经解剖学杂志》
CAS
CSCD
2022年第6期657-663,共7页
Chinese Journal of Neuroanatomy
基金
国家自然科学基金(81660243)
贵州省科技厅自然科学基金(黔科合基础-ZK[2021]一般415)
贵州医科大学国家自然基金培育项目(20NSP006)。
关键词
神经干细胞
外泌体
细胞分化
提取及鉴定
大鼠
neural stem cell
exosomes
cell differentiation
extraction and identification
rat
