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幼兔髂骨穿刺抽取骨髓间充质干细胞分离培养鉴定：注意的细节与技术张聪;刘洪美;李庆伟;陈国武;梁啸;孟纯阳【摘要】背景：骨髓间充质干细胞被认为是构建组织工程骨修复骨与软骨缺损中较为常用的种子细胞，在其基本操作过程中注意常见问题并及时避免，对后期细胞学及组织工程学实验很有意义。
目的：通过作者实验操作过程中所遇问题的总结和分析，为初学者和科研人员提供可靠的骨髓间充质干细胞分离培养鉴定方法，减少操作过程中的人为失误和易犯问题。
方法：取16只幼年新西兰大白兔作为实验对象，进行髂骨穿刺抽取骨髓液。
采用密度梯度离心法联合贴壁培养法体外筛选纯化细胞，并且通过倒置相差显微镜观察其形态学特点、生长曲线和流式细胞术鉴定骨髓间充质干细胞表型。
结果与结论：实验过程中前5只兔骨髓抽吸、骨髓间充质干细胞分离过程中遇到不同的问题和困难，经认真总结和分析，后11只兔骨髓抽吸、骨髓间充质干细胞分离均获成功，在细胞培养过程中未发现细菌污染和细胞老化，第3代骨髓间充质干细胞高表达CD29、CD44抗原，而CD14、CD34抗原低表达，MTT测细胞生长曲线显示P3和P5增殖活性较高。
尽管骨髓间充质干细胞分离培养鉴定技术已较为成熟，但是如果操作过程中不注意细节问题，也将会导致实验困难重重或失败。
严格执行常规操作步骤可以得到纯度较高的骨髓间充质干细胞，提高成功率，为后续相关细胞实验和动物实验做好准备。
%BACKGROUND:Bone marrow mesenchymal stem cells are considered as commonly used seed cells to construct tissue-engineered for repair of bone and cartilage defects. It is of great significance for cytology and tissue engineering experiments to study the common problems existing in the basic operation and how to avoid these problems in a timely manner.OBJECTIVE:To summarize the common problems existing in the process of operation in order to provide reliable methods about separation, culture and identification of bone marrow mesenchymal stem cells for beginners and researchers. These can reduce or avoid some errors and problems during operation. METHODS:Sixteen New Zealand white rabbits were selected as experiment objects, and bone marrow mesenchymal stem cells were separated from rabbits by iliac puncture, purified and augmented by using density gradient centrifugation combined with adherent culture method. Then cellmorphology was observed by inverted phase contrast microscope, growth curve detected by MTT method and cellphenotype identified by flow cytometry. RESULTS AND CONCLUSION:We encountered some problems in the process of separation and culture, when we operated the first five rabbits. After careful y summarizing and analysis of the reasons, the operation was successful y completed on the rest 11 rabbits. Bacteria pol ution and cellaging were not found in the process of cellculture. What is more, the cells at passage 3 appeared with high-expression of CD29, and CD44, but low expression of CD14 and CD34. The cellgrowth curve showed that the proliferation activity of cells at passages 3 and 5 was higher than that at passage 10. Although the technology of separation, culture and identification of bone marrow mesenchymal stem cells is mature, the failure wil be happen if we do not pay attention to the details of operation. By strictly carrying out normal operations, we can get high purity of bone marrow mesenchymal stemcells, which lays a good foundation for celland animal experiments in the future.【期刊名称】《中国组织工程研究》【年(卷),期】2014(000)023【总页数】6页(P3639-3644)【关键词】干细胞;骨髓干细胞;骨髓间充质干细胞;种子细胞;细胞培养;表型鉴定;兔;山东省自然科学基金【作者】张聪;刘洪美;李庆伟;陈国武;梁啸;孟纯阳【作者单位】济宁医学院附属医院脊柱外科，山东省济宁市 272000;济宁医学院病理教研室，山东省济宁市 272000;济宁医学院附属医院脊柱外科，山东省济宁市 272000;济宁医学院附属医院脊柱外科，山东省济宁市 272000;济宁医学院附属医院脊柱外科，山东省济宁市 272000;济宁医学院附属医院脊柱外科，山东省济宁市 272000【正文语种】中文【中图分类】R394.20 引言 Introduction骨髓组织中除含有造血干细胞外，还含有少量的骨髓间充质干细胞[1]。
兔骨髓间充质干细胞体外成内皮细胞能力的研究何朝荣;张群林;黄从新【期刊名称】《中华老年多器官疾病杂志》【年(卷),期】2003(2)4【摘要】目的探讨骨髓间充质干细胞体外扩增及其向内皮细胞定向分化能力.方法无菌条件下采集兔骨髓,肝素抗凝,经淋巴细胞分层液密度梯度离心分离骨髓单个核细胞,通过贴壁培养法获得MSC,然后在内皮细胞生长环境中进行诱导分化,最后对扩增的细胞特征进行鉴定,同时初步研究其体外成血管特性.结果 MSC每扩增一代,细胞数量增加5～8倍,7.65×103个原代MSCs体外扩增3代即获得1.26×108个细胞.在70%～80%融合时呈现"铺路石"样形态,细胞免疫组化证实扩增细胞表达CD31和yon Willebrand factor(vWF),具有吞噬ac-LDL的功能,且扩增细胞在体外参与网状血管样结构形成.结论 MSC扩增能力强,在体外能诱导其向内皮细胞方向分化.【总页数】5页(P287-291)【作者】何朝荣;张群林;黄从新【作者单位】442000,十堰市,郧阳医学院附属太和医院心血管内科;442000,十堰市,郧阳医学院附属太和医院心血管内科;武汉大学附属第一医院湖北省人民医院【正文语种】中文【中图分类】R3【相关文献】1.PPARγ基因干扰体外对兔骨髓间充质干细胞成脂基因的影响 [J], 白祥;禹宝庆;黄建明;许大峰2.兔骨髓间充质干细胞的分离、体外培养、鉴定及成脂诱导 [J], 戚宗泽;罗云飞;侯勇;郭永章;朱洪3.三种细胞因子体外联合诱导兔骨髓间充质干细胞向血管内皮细胞的分化 [J], 陈毅;刘丹平4.兔骨髓间充质干细胞体外诱导血管内皮细胞 [J], 常青;程燕;高静媛;梁芳倩;于晓龙5.血管内皮细胞对骨髓间充质干细胞成骨能力影响的体外研究 [J], 曲哲;孙宏晨;郭英;张泽兵;杨儒壮;欧阳喈因版权原因，仅展示原文概要，查看原文内容请购买。
BMSCs的分离培养、纯化与鉴定作者：马民吕志刚杜以宽张桂娟马义【摘要】目的建立一种持续、稳定的可多向分化的骨髓间充质干细胞（BMSCs）体外分离培养体系。
方法运用密度梯度离心法从1月龄新西兰兔骨髓中分离培养BMSCs，利用相差显微镜观察其形态及生长情况，扫描电镜观察其细胞结构，运用流式细胞术分析分离细胞群所处细胞周期和细胞活力，用MTT比色法绘制细胞生长曲线，并用特定诱导液将分离的BMSCs向成骨细胞和成脂肪细胞定向诱导分化，利用ALP和油红O进行染色鉴定。
结果所分离的BMSCs细胞在形态学观察与生长动力学上均符合BMSCs特征，分离培养的BMSCs细胞在第3天进入对数生长期，第10天进入平台期；在成骨、成脂肪的诱导培养条件下，分别出现成骨、成脂肪表型特征，可进一步定向分化，结论所收获的细胞具有BMSCs的特异性。
【关键词】骨髓间充质干细胞；分离；成骨分化；成脂分化【Abstract】 Objective To establish a sustained and stable bone marrow derived mesenchymal stem cells (BMSCs) isolation and culture system which could be pluripotential in vitro. Methods BMSCs were isolated and cultured from the 1month old New Zealand white rabbits through density gradientcentrifugation. Growth and ultromicrostructure of the BMSCs were observed by light microscope and scan electron microscope. The cell activities and cell cycle were observed by flow cytometry. The grow curve of the BMSCs was described through MTT assay. BMSCs were induced and differentiate into osteoblasts and adipocytes by specific induction culture medium. Then the cells were stained with alkaline phosphatase (ALP) and Oil red O , and analyzed the result of all staining. Results The isolated BMSCs cells were in line with the characteristics of BMSCs in morphology and growth kinetics, and the isolated BMSCs cells would be into the logarithmic growth phase in the third day and into the platform phase in the tenth day. Conclusions In the osteogenic and adipogenic induced culture conditions, BMSCs have appeared the phenotypic characteristics of osteogenic and adipogenic respectively, and could be further directed differentiation. These indicated the harvested cells possessed BMSCs specificity.【Key words】 Bone marrow derived mesenchymal stem cells(BMSCs); Isolation; Osteogenic differentiation; Adipogenic differentiation软骨组织工程技术〔自体软骨细胞的移植、生长因子对软骨细胞的作用、骨髓间充质干细胞（又称骨髓基质干细胞，BMSCs）在软骨组织工程中的作用、转基因技术〕是当今的研究热点〔1，2〕。
