改良原代大鼠骨源间充质干细胞的体外培养方法及鉴定  

Improve the method of culture in vitro and identification of primary rat bone derived mesenchymal stem cells

作　　者：刘海琴 马华根 唐元瑜[3] Liu Haiqin;Ma Huagen;Tang Yuanyu(College of Integrated Traditional Chinese and Western Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China;College of Traditional Chinese Medicine,Beijing University of Chinese Medicine,Beijing 102488,China;College of Traditional Chinese Medicine,Fujian Unersity of Traditional Chinese Medicine,Fuzhou 350122,China)

机构地区：[1]福建中医药大学中西医结合学院,福州350122 [2]北京中医药大学中医学院,北京102488 [3]福建中医药大学中医学院,福州350122

出　　处：《中国组织化学与细胞化学杂志》2022年第1期61-67,共7页Chinese Journal of Histochemistry and Cytochemistry

基　　金：国家自然科学基金项目(81072714);国家大学生创新创业训练项目(202110393010)。

摘　　要：目的改良原代大鼠骨源间充质干细胞体外分离培养方法,以获得纯度高、增殖活力强、分化潜能大的理想工程细胞。方法基于“仿原位生态培养”方法及原理,剪取大鼠前后肢股骨、胫骨,剔除附着的肌肉、筋膜组织,完全不用冲洗骨髓腔,直接将其剪碎成约1 mm^(3)骨块后,移入0.1%Ⅱ型胶原酶中消化90 min;经洗涤、离心后,接种于培养皿中进行原代培养。当细胞融合度达80%~90%时,予0.25%胰蛋白酶消化液进行二次消化,按1:3比例进行传代培养。采用流式细胞仪检测第4代细胞表面CD分子标志物,并进行成脂、成骨诱导分化实验。结果接种48 h后,少量长梭形细胞从骨块周围爬出;96 h后,细胞逐渐融合成片;传至第4代时,细胞形态均一,以“成纤维样”细长梭形为主,融合后呈“涡旋式”生长,具有一定方向性;传至第10代,细胞形态仍基本保持不变。流式细胞仪检测结果提示CD44、CD90和CD29表达阳性,CD34、CD11b/c和CD45呈阴性表达;成脂、成骨诱导分化实验均为阳性。结论基于“仿原位生态培养”原理,采用组织块结合酶消化法,能够简单、高效分离培养出大鼠骨源间充质干细胞,值得推广。Objective To improve the in vitro isolation and culture method of primary rat bone-derived mesenchymal stem cells and obtain ideal cells with great differentiation ability.Methods The femurs and tibias of rats were taken out,then the muscle and fascia tissues were removed,and the bones were cut into about 1mm^(3) pieces.Then the bone fragments were transplanted into culture flasks containing 0.1%collagenase Ⅱ and digested for 90 min.After centrifugation,the fragments were inoculated in petri dishes for primary culture and subcultured at the ratio of 1:3 when the cell density reached 80%~90%.Cells of the fourth generation were detected by flow cytometry.The adipogenesis and osteogenesis experiments were also carried out.Results After 48 h of culture,a small number of short slender cells crawled out;after 96 h,the cells gradually fused into pieces.The cells were uniform in shape in the fourth generation,mainly long fusiform.Flow cytometry showed that CD44,CD90 and CD29 were positive,while CD34,CD11b/c and CD45 were negative.The adipogenesis and osteogenesis experiments showed positive results.Conclusion Based on the principle of“in situ ecological imitation cultivation”,BMSCs can be successfully isolated and cultured by bone fragments method combined with chemical enzyme digestion method.

关 键 词：骨 间充质干细胞 原代培养 仿原位生态培养 组织块结合酶消化法 免疫表型 细胞分化 

分 类 号：R-311[医药卫生]