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世界最新医学信息文摘 2018年 第18卷 第61期91投稿邮箱：sjzxyx88@·基础研究·对骨髓间充质干细胞分化诱导的研究冯文钰1，王可新1，廉洁2，姚宏波2（通讯作者）（1.齐齐哈尔医学院基础医学院 卓越1班，黑龙江 齐齐哈尔 161006；2.齐齐哈尔医学院基础医学院 组胚教研室，黑龙江 齐齐哈尔 161006）0 引言髓间充质干细胞是由中胚层分化而来的干细胞，存在于骨髓基质内。
通过应用恰当的分化诱导方法及适宜的体外环境可以使其转化为神经细胞、肺泡上皮细胞、成骨细胞等其他各种组织细胞，更因为其具有独特的免疫学特征，可以使用免疫细胞化学的方法检测细胞的表面标志物来确定实验中诱导分化的细胞是否为我们所需的神经细胞[1]。
因其多向分化的生物学特性使骨髓间充质干细胞当之无愧的成为目前以及未来很长一段时间中组织工程的研究焦点。
1 实验方法1.1 细胞的分离：取健康4周龄大鼠，脱颈处死，浸泡于75%的乙醇溶液中20-30 min 进行消毒。
在无菌工作台上取大鼠双侧后肢股骨，用适量Hanks 液清洗以洗去血污。
用眼科剪剪开两端骨骺，暴露骨髓腔，用注射器抽取Hanks 液注入骨髓腔，冲出骨髓。
经过10 min 离心后，用吸管吸出多余的Hanks 液。
1.2 细胞的培养：将分离提取的细胞用培养液清洗1次，加入5 mL 20%培养液，放入培养瓶，置于37℃ 5%二氧化碳培养箱。
第二日换液，观察细胞贴壁情况。
以后每3-4 d 更换培养基一次，当细胞融合80%-90%时，按1:2进行传代培养。
1.3 细胞的分化诱导：取生长状态良好的第三代骨髓间充质干细胞以10^8/mL 的规格均匀接种于6孔板中，并分别向每孔中加入含体积分数为10%的无血清培养基血清替代物以及低糖DMEM 2 mL 。
随后将其放入37℃ 5%二氧化碳培养箱中进行培养。
当细胞达到60%-70%融合的状态时，改换为含3 mmol/L β-巯基乙醇的培养基预诱导24 h ，并用PH 为7.4的PBS 溶液洗涤，最终改换成含20 g/L 二甲基亚砜以及200 mmol/L 丁化羟基苯甲醚的无血清培养基进行诱导。
骨髓间充质干细胞诱导分化为肝样细胞的研究进展owen[1]首先将其命名为间充质干细胞（BM-MSCs)，最初认为它只能分化为同胚层的细胞，后来研究证实其分化潜能是超越胚层界限的。
BM-MSCs属于多能干细胞，来源于中胚层，具有很强的多向分化潜能，具有干细胞的共性[2-3]。
对BM-MSCs分化潜能的研究已成为国内外研究机构的热点。
目前BM-MSCs向肝细胞的分化已有研究，但仍处于初步阶段。
本文就BM-MSCs体外诱导分化为肝细胞的研究进展作一综述。
1 BM-MSCs的生物学特性1.1 BM-MSCs的来源及形态BM-MSCs从骨髓、脂肪组织、骨骼肌、成人外周血、血管周皮细胞、妊娠4-6个月羊水、胎儿的血和真皮组织、脐血中都可以分离得到间充质干细胞[4]，其中对BM-MSCs研究最充分最彻底。
BM-MSCs表现出早期细胞的特点，呈均一的成纤维细胞形态，染色质疏松，核仁明显，核浆比例大，辐射状或漩涡状排列，可聚集成均匀的集落。
1.2 BM-MSCs的鉴定鉴定BM-MSCs目前没有特异性的表型标志，研究从形态、表型及多分化功能来鉴定体外分离培养的BM-MSCs [5-6]，纯化的BM-MSCs表现为成纤维细胞梭形贴壁细胞，呈漩涡状、平行状或鱼群状生长；BM-MSCs的抗原表型，流式细胞仪检测不表达造血细胞CD14、CD34、CD45的标志，表达CD29、CD44、CD73、CD90、CDl06、CDl24、SH-2、SH-3、SH-3等；BM-MSCs具有跨胚层的多向分化，如成骨、成软骨、成脂肪，是BM-MSCs作为干细胞最基本特征[7]。
故BM-MSCs常通过负性筛选和细胞多向分化，鉴定BM-MSCs。
1.3 BM-MSCs的分离培养BM-MSCs在骨髓中含量很少，生理状态下20％为静止期细胞，所以BM-MSCs要应用于临床，其分离培养显得尤其重要。
目前分离获取BM-MSCs 的方法主要有4种[8-9]：全骨髓差异贴壁法、免疫磁珠法、密度梯度离心法、流式细胞仪分离法。
骨髓间质干细胞成软骨诱导分化实验原理
软骨的破坏、缺失是包括骨关节炎（osteoarthritis，OA）在内的多种骨科疾病的重要病理改变。
由于软骨特殊的解剖及组织特性，如缺乏血管和淋巴管的分布，使得其自我修复能力异常不足，因此通过人工方法修复软骨破坏具有重要临床意义。
近年来，随着间充质干细胞的发现和研究深入，其来源广泛、良好的增殖能力、多分化潜能、与各种3D支架材料具有良好的生物相容性等特性，使得其在软骨修复应用中具有令人期待的潜力。
MSC成软骨分化的过程：MSC首先收回其突起，聚集成团，这个团中间的细胞经分裂分化转变成一种大而圆的细胞，即成软骨细胞；成软骨细胞产生基质和纤维（主要为II型胶原蛋白），当基质的量增加到一定程度时，成软骨细胞被分隔在陷窝内，分化为成熟的软骨细胞。
体外诱导成软骨分化的鉴定主要围绕成软骨的标志物——II型胶原蛋白来进行，包括通过免疫组化、Westernblot检测II型胶原蛋白的表达和RT-PCR检测II型胶原蛋白的mRNA的表达，不同的角度进行鉴定；而甲苯胺蓝则主要检测成软骨细胞内酸性粘多糖。
下图是骨髓间充质干细胞的成软骨分化诱导图，可以看到软骨细胞埋藏在软骨间质内，它所存在的部位为一小腔，称为软骨陷窝（cartilage lacuna）。
中国组织工程研究与临床康复 第 12 卷 第 3 期 2008–01–15 出版Journal of Clinical Rehabilitative Tissue Engineering Research January 15, 2008 Vol.12, No.3综 述间充质干细胞的生物学特性及多向分化潜能*★张 凯1，王 毅1，邢国胜2Bio-characteristics and multi-differentiation potency of mesenchymal stem cellsZhang Kai1, Wang Yi1 Xing Guo-sheng2 AbstractBACKGROUND: Mesenchymal stem cells (MSCs) are an important cell type in hematopoietic microenvironment. They can differentiate to lots of tissues and have low immunogenicities. It has been verified by clinical tests that MSCs transplanting can be used for tissue repair and disease treatment of mesenchymal tissue genetic defects. OBJECTIVE: To understand bio-characteristics and multi-differentiations potency of MSCs. RETRIEVAL STRATEGY: A computerized search through the PubMed database was undertaken during January 1995 to December 2005. The key words were "mesenchymal stem cells, differentiation, biological character", and searching language was restricted in English. Meanwhile, a resemble search was conducted to the correlated articles basing on Chinese Journal Full-Text Database from January 1995 to December 2005, with the key words of "mesenchymal stem cells, differentiation, biological character", and the language was restricted in Chinese. Totally 78 related papers were searched out. Data were checked in the first trial. Inclusion criteria:①The literatures should be closely related with biological character and differentiation of MSCs.②The recent articles of the same field and that published in authority journals were preferred. Exclusion criteria: ①Repetitive study. ②Meta analysis. LITERATURE EVALUATION: The literature was compiled and analyzed about the biological character and multiple differentiation of MSCs. Thirty papers were selected, in which 4 were reviews and the others were clinical or fundamental researches. DATA SYNTHESIS: ①MSCs are especially rich in myeloid tissue and also present in placent, amniotic fluid, umbilical vein subendothelial layer, peripheral blood, liver, fat, muscles and skin. MSCs have potentials of fast proliferation, self renewing and multi-differentiation. Based on different inducing conditions, they can be differentiated into cartilage, bone, skeletal muscles, muscle tendon, fat, nerve and kidney parenchymal cell, etc.②Three isolation and culture methods are mainly used in vitro: Density gradient centrifugation and adherent culture methods: Basing on the density difference between MSCs and other cells, Percoll or Ficoll separation liquid can be used to separate cells and meanwhile separate the MSCs from the non-adherent cells depending on the adherent growing character of MSCs; Flow cytometer: It can be used for MSCs separation on the basis of its small volume and particle deficiency; Magnetic activated cell sorting: Positive or negative selection is carried out according to MSCs surface antigenic component is presented or deleted, and the relatively pure MSCs can be obtained by immobilizing with antibody. DMEM medium containing 5%－20% fetal bovine serum is commonly used for culturing MSCs. The identification of MSCs is mainly according to its morphous and functions. Furthermore, the surface marker is different from different sources.③Under specific physical and chemical environment or induced by cytokine, MSCs can differentiate into multiple types of cells, such as osteoblast, fat cell, nerve cell, cardiac myocyte, endothelium cell and so on. It can be affected by lots of factors and has low immunogenicity. So it is an ideal source of tissue engineering seed cells. Moreover, MSCs are easy to transfect and express the exogenous gene, so they will be widely used in cell therapy and genetic therapy. CONCLUSION: Because of the multi-differentiation properties, MSCs will be widely used in tissue engineering damage repair, cell replacement therapy, supporting effect of hemopoiesis and genetic therapy. But the monoclonal cell can not be obtained by separation and culture approaches currently used in vitro, and now the reliable identification standard also lacks. The problems, such as what are the differences between MSCs of different sources, should be solved. Zhang K, Wang Y, Xing GS.Bio-characteristics and multi-differentiation potency of mesenchymal stem cells.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(3):539-542(China) [/zglckf/ejournal/upfiles/08-3/3k-539(ps).pdf]1Department of Clinical Laboratory, Tianjin Hospital, Tianjin 300211, 2 China; Institute of Orthopaedics, Tianjin Hospital, Tianjin 300211, China Zhang Kai★, Master, Associate chief laboratorian, Department of Clinical Laboratory, Tianjin Hospital, Tianjin 300211, China zkkmail@yahoo. Supported by: General Program of Tianjin Science and Technology Committee, No. 05YFJMJC07600* Received:2007-09-13 Accepted:2007-11-02天 津 医 院 ,1 检 验 2 科， 骨科研究所， 天津市 300211 张 凯★，女， 1971 年生，天津 市人，汉族，2003 年南开大学毕业， 硕士， 副主任检验 师， 主要从事风湿 病、 骨代谢疾病实 验 室 检 测方 面 的 研究。
兔骨髓间充质干细胞定向诱导分化为成骨细胞的研究李雅娜;孙研;张玲;刘旭【期刊名称】《滨州医学院学报》【年(卷),期】2007(030)004【摘要】目的探讨兔骨髓间充质干细胞﹙bone marrow mesenchymal stem cells, BMSCs﹚体外分离培养、诱导分化为成骨细胞的方法,并研究其生物学特性.方法采用体外细胞培养技术自兔股骨、胫骨及肱骨中分离BMSCs进行纯化、培养.用地塞米松、β-甘油磷酸钠、维生素C定向诱导BMSCs分化为成骨细胞,改良MTT法测定其生长曲线,NBT/BCIP染色法、P-对硝基苯基质法及茜素红染色法检测BMSCs的成骨能力.结果成骨诱导1～5 d,培养的细胞增殖旺盛,3～5 d即可融合成单层,随着诱导时间的延长,细胞增殖速度减慢,出现细胞聚集的现象,培养细胞逐渐汇合呈铺路石状,细胞形态变为胞体较小的立方形,继续诱导,可出现多角形成骨样细胞,胞外基质分泌逐渐增多,胶原堆积、钙盐沉积,10～12 d形成不透光矿化结节,ALP活性增高,茜素红染色阳性,表示BMSCs向成骨细胞分化.结论兔BMSCs经体外分离、诱导培养,可以定向分化为成骨细胞.【总页数】5页(P252-256)【作者】李雅娜;孙研;张玲;刘旭【作者单位】滨州医学院组织学与胚胎学教研室,烟台市,264003;龙口市中医院骨科;云南省天然药物药理重点实验室;云南省天然药物药理重点实验室【正文语种】中文【中图分类】R329.2【相关文献】1.不同浓度地塞米松在体外定向诱导分化兔骨髓间充质干细胞为成骨细胞的实验研究 [J], 张建萍2.成人骨髓间充质干细胞向成骨细胞定向诱导分化的实验研究 [J], 刘晓静;任高宏;廖华;余磊;原林3.兔骨髓间充质干细胞诱导分化为成骨细胞的实验研究 [J], 笪虎;范卫民;崔维顶;高峰4.成人骨髓间充质干细胞向成骨细胞定向诱导分化的实验研究 [J], 朱伟;孙俊英;岑建龙;朱礼贤;顾联5.兔骨髓间充质干细胞定向诱导分化为脂肪细胞及成骨细胞的研究 [J], 李胜富;黄定强;卢晓风;刘瑾;孙明菡;李幼平;程惊秋;步宏;梁传余因版权原因，仅展示原文概要，查看原文内容请购买。
