一种改良大鼠骨髓间充质干细胞培养方法  

Modified culture and identification of rat bone marrow mesenchymal stem cells

作　　者：王博荣[1] 鲁曦[1] 张敏龙[1] 李聪聪[1] 李鹏程[1] 王雅宁[2] 金发光[1] 

机构地区：[1]第四军医大学唐都医院呼吸内科,西安710038 [2]宝鸡市第二人民医院呼吸内科,宝鸡721000

出　　处：《中华肺部疾病杂志（电子版）》2017年第1期25-28,共4页Chinese Journal of Lung Diseases(Electronic Edition)

基　　金：国家自然科学基金资助项目(81570067)

摘　　要：目的建立大鼠骨髓间充质干细胞(BMSCs)的分离、改良培养、纯化方法,并进行细胞形态学观察、表面标志物鉴定及多向分化能力检测。方法通过改良全骨髓贴壁法对4周龄SD雄性大鼠脱颈处死,无菌条件下分离出骨髓进行原代培养、消化传代培养及纯化。对BMSCs进行形态学观察,收获第四代BMSCs进行流式细胞仪检测其细胞表面标记物CD90、CD29、CD34、CD45的表达率及向成脂方向诱导分化。结果 BMSCs的原代培养形态学观察可见骨髓细胞接种于培养皿后,细胞呈圆型,大小不一,悬浮于培养液中。24 h后部分细胞开始贴壁,呈圆形、梭形或多角形。通过换液去除未贴壁的杂质细胞,可见短梭形、星形细胞分散贴壁生长,四五天可见放射状排列的细胞集落,伸出长短不一、粗细不均的突起,梭形细胞为主,胞浆丰富,胞核大、核仁清晰。7~8 d细胞呈集落生长,融合80%~90%,呈漩涡状,同向排列,9~10 d细胞排列紧密,逐渐融合成片。传代培养可见消化传代后,传代细胞24 h完全贴壁生长。细胞形态均一,呈梭形生长,细胞生长旺盛。四至五天可传代1次。可稳定连续传代7代以上,细胞形态及生长速度未见明显变化。BMSCs表面标记物的表达通过流式细胞仪检测结果显示,培养的第4代大鼠BMSCs均一表达CD90,CD29,阳性率分别为96.9%,96.6%;而CD34,CD45,呈阴性,阳性率分别为0.395%,7.56%。BMSCs加入成脂诱导剂后18 d,诱导而成的脂肪细胞累积脂质,脂滴变大,合并呈串珠状,经油红O染色呈鲜红色。结论与传统全骨髓贴壁法相比,改良后的全骨髓贴壁法操作步骤简单,降低离心对细胞的损害,减少了污染机会,节省经费,且分离的BMSCs细胞活性高,可大量分离、纯化、扩增,所获细胞具有间充质干细胞的一般生物学特性,经诱导培养后具有多向分化潜能。可为组织器官缺损性疾病、恶性肿瘤等的治疗和组织工程提供充足的种Objective To establish the rat bone marrow mesenchymal stem cells（ BMSCs） were isolated and cultured,purified and modified optimization method in vitro,to observe cell morphology,and to assess surface markers and differentiation capacity detection. Mthods The bone marrows of 4-week-old,male Sprague Dawley rats were used to obtain m MSCs. Rats were euthanized via cervical dislocation. After 72 hours,the medium was replaced and fresh medium was provided every 3 days. BMSCs were identified via flow cytometry,multi-directional differentiation capacity,and morphology. Results Primary culture： Bone marrow cells were seeded in round culture dishes of different sizes and suspended in culture medium. After 24 hours,the part of the cells adhered to the culture dish were visible as round,fusiform,or polygonal. After removal of the non- adherent cells by media replacement,the adherent cells were found to be short,spindle or star shaped,and scattered on the plastic surface. After 4 or 5 days,radially arranged cell colonies were visible,which differed in length and thickness. Spindle cells comprised the main colonies, possessing abundant cytoplasm and a large nucleus. After a week,the cells showed colony growth,were fused at approximately 80%-90%,and were reminiscent of a whirlpool,all with the same directionality. After 10 days,the cells were arranged in close proximity to one another,gradually integrating into sheet form. Culture passage： After digestion and passage,the cells adhered to the plastic surface within 24 hours. The cells were homogeneous,spindle-shaped,and displayed strong cell growth. Each passaging was performed after 4 or 5 days of growth.Cell morphology and growth rate did not significantly differ after 10 generations of stable and continuous passage.The results of flow cytometry revealed a positivity rate of CD90 expression of 96.9%,and of 96.6% for CD29.Thus,the negativity rate of CD45 was 7.56%,and that of CD34 was 0.395% in fourth generation rat mM SCs.The mM SCs were added into an adipocytes-indu

关 键 词：骨髓间充质干细胞 原代培养 形态学 分化 鉴定 

分 类 号：R563[医药卫生—呼吸系统]