改良全骨髓贴壁法分离及培养大鼠骨髓间充质干细胞  

Isolation and culture of rat bone marrow mesenchymal stem cells by modified whole bone marrow adherent method

作　　者：鲁燕妮 龙雯 常晓峰[1,2,3] 赵宁波 LU Yanni;LONG Wen;CHANG Xiaofeng;ZHAO Ningbo(Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research,College of Stomatology,Xi’an Jiaotong University,Xi’an 710004,China;Clinical Research Center of Shaanxi Province for Dental and Maxillofacial Diseases,College of Stomatology,Xi’an Jiaotong University;Department of Implant Dentistry,College of Stomatology,Xi’an Jiaotong University)

机构地区：[1]西安交通大学口腔医院陕西省颅颌面精准医学研究重点实验室,西安710004 [2]西安交通大学口腔医院陕西省牙颌疾病临床研究中心 [3]西安交通大学口腔医院种植科

出　　处：《山西医科大学学报》2023年第3期364-369,共6页Journal of Shanxi Medical University

基　　金：国家自然科学基金青年基金资助项目(82001085);陕西省科技厅社会发展领域一般项目(2018SF-117);西安交通大学口腔医院院级医疗新技术与新项目(xjkqxjs2021-14)。

摘　　要：目的建立简单、优化的分离及培养大鼠骨髓间充质干细胞(BMSCs)的方法,为组织工程支架研究提供细胞基础。方法分别采用常规和改良的全骨髓贴壁法分离和培养BMSCs。常规法按照传统全骨髓贴壁法,对骨髓冲洗液进行过筛和离心处理后加入完全培养基进行培养。在改良全骨髓贴壁法中,将骨髓冲出骨髓腔后直接加入完全培养基进行培养。倒置相差显微镜下观察两组细胞形态,采用细胞计数试剂盒(CCK-8)法比较两组BMSCs的增殖活性,流式细胞术检测表面标志物CD90、CD29、CD11b/c、CD45。对改良法培养的BMSCs进行定向诱导成骨及成脂分化,并通过茜素红染色检测其成骨分化潜能,通过油红O染色检测其成脂分化潜能。结果改良全骨髓贴壁法培养的BMSCs形态均一,细胞集簇排列呈“鱼群状”“漩涡状”。改良法比常规法培养的BMSCs增殖活性强(P<0.05)。改良法与常规法培养的BMSCs均高表达CD90和CD29,低表达CD11b/c和CD45。改良法培养的BMSCs成骨、成脂诱导分化后茜素红染色和油红O染色均为阳性。结论改良全骨髓贴壁法可成功分离并培养BMSCs,培养的BMSCs具有多向分化潜能,为组织工程支架的研究奠定了细胞基础。Objective To establish a simple and optimized method for isolating and culturing rat bone marrow mesenchymal stem cells(BMSCs),and provide a cellular basis for tissue engineering.Methods BMSCs were isolated and cultured using conventional and modified whole bone marrow adherent methods,respectively.In conventional method group,the bone marrow washings were filtered and centrifuged,and then added to the complete culture medium for culturing BMSCs.In modified whole bone marrow adherent method group,the marrow was flushed out of the marrow cavity and added directly to the complete culture medium for culturing BMSCs.The cell morphology in both two groups was observed under an inverted phase contrast microscope,and the proliferative activity of BMSCs was detected in two groups by cell counting kit(CCK-8)method,and the surface markers CD90,CD29,CD11b/c and CD45 were detected by flow cytometry.The osteogenic and lipogenic differentiation potential of BMSCs cultured by the modified method was detected by Alizarin red staining and oil red O staining.Results The BMSCs cultured by the modified method were homogeneous in morphology,with“fish swarm”and“swirl”arrangement of cell clusters.The proliferation activity of BMSCs cultured by the modified method was stronger than that of the conventional method(P<0.05).BMSCs cultured by both two methods highly expressed CD90 and CD29 and lowly expressed CD11b/c and CD45.The BMSCs cultured by the modified method were positive for Alizarin red staining and oil red O staining after osteogenic and lipogenic-induced differentiation.Conclusion BMSCs can be successfully isolated and cultured by the modified whole bone marrow adherent method,and have multi-directional differentiation potential,which lays a cellular foundation for tissue engineering.

关 键 词：改良全骨髓贴壁法 骨髓间充质干细胞 原代培养 细胞分化 

分 类 号：Q813.1[生物学—生物工程]