鼻黏膜间充质干细胞的培养、鉴定及诱导分化  

Culture, differentiation and characterization of nasal mucosa mesenchymal stem cells

作　　者：卓毅[1,2] 吴沛 段答 葛丽特[1,2] 王昊[1] 龙浪 胡莉[1] 卢明 Zhuo Yi;Wu Pei;Duan Da;Ge Lite;Wang Hao;Long Lang;Hu Li;Lu Ming(The Second Affiliated Hospital of Hunan Normal University, Department of Neurosurgery, the 163rd Hospital of Chinese PLA,Changsha 410003, China;Key laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, 410006, China)

机构地区：[1]湖南师范大学第二附属医院,解放军第163医院神经外科,长沙410003 [2]湖南师范大学生命科学学院,蛋白质化学与发育生物学教育部重点实验室,长沙410006

出　　处：《中国组织化学与细胞化学杂志》2018年第1期66-71,共6页Chinese Journal of Histochemistry and Cytochemistry

基　　金：国家自然科学基金(81371358);湖南教育厅科学研究项目(16B159)

摘　　要：目的建立Sprague Dawley(SD)大鼠鼻黏膜间充质干细胞(NM-MSCs)的获取、分离、培养方法,初步了解其生物学特性。方法通过SD大鼠鼻黏膜贴壁法体外分离、培养NM-MSCs,光镜下细胞形态学观察,用免疫荧光技术检测间充质干细胞和神经干细胞标记物,用流式细胞术检测细胞表面标记物,再诱导其向骨组织及脂肪组织方向分化,最后对其进行细胞周期检测及分析。结果分离培养的NM-MSCs光镜下以梭形与多角形细胞为主,呈放射状排列,且生长旺盛;免疫荧光染色显示NM-MSCs同时表达STRO-1和Nestin;第4代NM-MSCs不表达CD19、CD31、CD34、CD45及HLADR细胞表面标记物,但表达CD90、CD105基质细胞标记物;NM-MSCs经成骨、成脂诱导后,茜素红染色和油红O染色均呈阳性;细胞周期分析显示NM-MSCs符合干细胞生长的特性。结论 SD大鼠NM-MSCs组织块贴壁法获取容易,操作简单,且可大量扩增用于细胞实验研究,经体外诱导后具有多向分化潜能,具有间充质干细胞的一般生物学特性。SD大鼠鼻黏膜组织块贴壁法为组织细胞工程研究提供了充足的种子细胞来源。Objective To establish a method for isolating and culturing Sprague Dawley(SD)rats nasal mucosa mesenchymal stem cells(NM-MSCs)in vitro.Methods SD rat nasal mucosa was obtained through nasal mucosa adherent method.NM-MSCs were isolated from the nasal mucosa and cultured in vitro.The characterization of NM-MSCs included morphological observation under light microscope,cell immunofluorescence to examine mesenchymal stem cell and neural stem cell markers,flow cytometry to detect cell surface markers.NM-MSCs were then induced to differentiate towards bone and adipose tissue phenotypes in which cell cycle analysis were performed.Results NM-MSCs in culture were mostly spindle-like or polygonal in radial arrangement and highly proliferative under light microscope.Cell immunofluorescence showed NM-MSCs expressed STRO-1and nestin.At the4th passage,NM-MSCs were negative for CD19,CD31,CD34,CD45and HLA-DR,but positive for CD90and CD105.Following osteogenic or adipogenic induction,oil red O and alizarin red stained strong positive.Cell cycle analysis indicated NM-MSCs with the growth characteristics of stem cells.Conclusion Due to its simplicity and easy operation,SD rats nasal mucosa tissue adherence method can be used to isolate NM-MSCs for large scale in vitro experiment study,as well as for tissue and cell engineering.NM-MSCs which maintained general characteristics of mesenchymal stem cells were pluripotent and can be induced in vitro.

关 键 词：鼻黏膜间充质干细胞 原代培养 鉴定 诱导分化 

分 类 号：Q813[生物学—生物工程]