1吉林大学第一医院神经外科，吉林省长春市 130021；2吉林大学药学院生物工程教研室，吉林省长春市 130021； 3吉林农业大学生命科学学院，吉林省长春市 130118

于 音☆，男，1977年生，吉林省长春市人，汉族, 吉林大学第一医院在读博士,主要从事颅底肿瘤的显微外科治疗及相关疾病的基因治疗研究。
fooish@sina.com Shaojj1982@163.
com

通讯作者：赵　　刚，教授, 吉林大学第一医院神经外科，吉林省长春市 130021

吉林省科委资助课题 （2003053
6-2）*

摘要
目的:骨髓间充质干细胞是存在于骨髓组织中多种干细胞的混和体，因其具有取材方便、扩增迅速、可自体移植并且具有多分化潜能等特点，是近年研究的热点。实验建立了体外分离、培养及鉴定兔骨髓间充质干细胞的方法，并通过此进一步验证其诱导成骨及成脂多向分化的潜能。
方法: 实验于2006-08/2007-06在吉林大学药学院生物工程实验室完成, 实验室属吉林大学重点实验室。①实验材料：四五月龄新西兰大白兔2只, 由吉林大学基础医学院动物培养中心提供, 体质量1.0~1.5 kg, 雌雄不限, 实验动物级别为二级, 实验过程中对动物处置符合动物伦理学标准。②实验方法：实验应用了密度梯度离心法与贴壁培养法相结合的方法从兔股骨、胫骨中分离、纯化骨髓间充质干细胞并在体外进行培养，以形态学及细胞表面标志的方法鉴定间充质干细胞，在倒置显微镜下观察细胞的形态特征，并利用成骨诱导剂包括0.1 μmol/L的地塞米松、50 mg/L的维生素C、10 mmol/Lβ-磷酸甘油钠和成脂诱导剂含10%FBS的L-DMEM、0.25 μmol/L地塞米松、50 μmol/L吲哚美辛、0.5 mmol/L IBMX、10 mg/L牛胰岛素诱导其向软骨细胞及脂肪细胞分化。
结果:①经原代及传代培养的骨髓间充质干细胞呈梭形，类似于成纤维细胞，骨髓间充质干细胞均一地表达CD29、CD44,而CD34、CD45均阴性表达。②成骨诱导14 d后细胞不明显，未形成钙结节，21 d后碱性磷酸酶钙钴法染色后大多数细胞的胞质呈棕黑色。③成脂诱导48 h后，细胞内有小脂滴出现，2周后脂滴数量增加并相互融合，细胞（有）由长梭形变为圆形或多边形，油红O染色显示有大量脂质沉积。
结论:密度梯度离心法与贴壁培养法相结合的方法是比较理想的骨髓间充质干细胞培养法，骨髓间充质干细胞经诱导培养后具有多向分化潜能。
关键词：骨髓；间充质干细胞；诱导；兔；多向分化

于音，赵刚，许侃，房学迅，邵佳甲.兔骨髓间充质干细胞体外培养向成骨和成脂方向的诱导分化[J].中国组织工程研究与临床康复，2008，12(8):1449-1452 [www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1449(ps).pdf]

中图分类号: R651.2
文献标识码: A
文章编号: 1673-8225
(2008)08-01449-04

收稿日期：2007-09-11
修回日期：2007-12-10
(07-50-11-6142/GW·Q)

Proliferation and differentiation of neural stem cells from neonatal rat basal forebrain of newborn rats into neurons in different culture conditions

Abstract

AIM:Bone marrow mesenchymal stem cells (BMSCs) are admixture of many kinds of stem cells in bone marrow, which have many characteristics including convenient use, prompt amplification, autotransplantation and multi-directional differentiation potency. It has been the hotspot in recent study. This study establishes the method of isolation and culture of rabbit BMSCs in vitro and confirms the potency of multi-directional differentiation by osteoblast and lipoblast formation induction.
METHODS: Experiments were performed at the Bioengineering Laboratory in Pharmacy School of Jilin University from August 2006 to June 2007. ①Two New Zealand rabbits (1.0-1.5 kg) aged 4-5 months after birth, of either sex, were bought from Animal Cultivation Center in Preclinical Medicine School of Jilin University. The hierarchy of animals was second grade. The disposition of the rabbits met the ethics standards. ② BMSCs were isolated from the femurs and tibias bones and purified and cultured in vitro by density gradient centrifugation and adherent culture. BMSCs were evaluated by morphology and surface marker. BMSCs culture was observed with an inverted microscope. BMSCs were induced to osteoblasts and lipoblasts by osteogenic inductor including 0.1 μmol/L dexamethasone, 50 mg/L vitamin C, 10 mmol/Lβ-phosphoglycerol and adipogenic inductor including 10% FBS-L-DMEM, 0.25 μmol/L dexamethasone, 50 μmol/L antifani, 0.5 mmol/L IBMX, 10 mg/L bovine insulin.
RESULTS: ①The BMCSs was fusiform, fibroblast-shape after cultivation of primary and subcultured. CD29 and CD44 were expressed, but CD34 and CD45 were not expressed. ②The cells did not form calcium nodus in the 14th day after osteogenic induction and the cytoplasm of the most cells presented brownish-black in the 21st day after alkaline phosphatase calcium-cobalt staining. ③Intra-cellular lipid droplet appeared after 48-hours adipogenic induction. The quantity of lipid droplet increased and were fused each other 2 weeks later. The morphous of fusiform shape became round or polygon, and a great quantity lipid deposited after oil red O staining.
CONCLUSION: Combination of density gradient centrifugation and adherent culture is an ideal method to culture BMSCs. BMSCs have the potency of multi-directional differentiation after induction.

Yu Y, Zhao G, Xu K, Fang XX, Shao JJ.Differentiation of mesenchymal stem cells from rabbit bone marrow into osteoblasts and lipobolasts in vitro.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(8):1449-1452(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1449(ps).pdf]

0 引言

骨髓间充质干细胞是存在于骨髓组织中多种干细胞的混和体，因其具有取材方便、扩增迅速、可自体移植并且具有多向分化潜能等特点[1-2]， 近年来越来越受到关注。兔骨髓间充质干细胞与神经组织修复、骨、软骨组织修复、肌组织、肌腱组织修复以及皮肤创面愈合等都有很密切的关系。
本实验从兔骨髓中取出骨髓基质细胞后进行体外原代及传代培养，经过多次传代以获得较纯的兔骨髓间充质干细胞，从形态学上观察和检测表面标志的方法鉴定兔骨髓间充质干细胞，对其进行成骨及成脂诱导，从而建立一套成熟的体外培养、鉴定和分化兔骨髓间充质干细胞的方法。

1 材料和方法

设计：形态学描述。
单位：吉林大学第一医院神经外科。
材料： 实验于2006-08/2007-06在吉林大学药学院生物工程实验室完成, 实验室属吉林大学重点实验室。四五月龄新西兰大白兔2只, 购自吉林大学基础医学院动物培养中心, 体质量1.0~1.5 kg, 雌雄不限, 实验动物级别为二级，实验过程中对动物处置符合动物伦理学标准。低糖DMEM培养基，胎牛血清FBS，0.25%胰蛋白酶，Percoll分离液，抗CD29、CD44、CD34、CD45单克隆抗体。
设计、实施、评估者：实验的设计由第二、三作者完成，由第一、三作者实施，由第二作者进行评估。
方法：
细胞的取材：取四五月龄的新西兰大白兔，3%戊巴比妥钠按50 mg/kg体质量的剂量行腹腔注射麻醉。无菌条件下，暴露左右两侧股骨及胫骨表面，用一只固定有18号针头内含0.1 mL肝素（3 000 U/mL）的5 mL注射器从骨髓中抽取2 mL骨髓液，抽吸物用PBS溶液冲洗3次后，用DMEM培养基悬浮，将单细胞悬液缓慢加在1.073 g/cm3 的Percoll分离液上部（细胞悬液与分离液之比为2∶1）， 350 g离心30 min，然后小心吸取位于分离液中间的乳白色细胞层，移入另一离心管中，用10 mL DMEM培养基重悬，180 g离心5 min，弃上清，再用5 mL DMEM培养基重悬后，用血细胞计数板计数后，以3×106密度接种于 50 mL塑料培养瓶中。
细胞的原代培养：将细胞置于37 ℃、体积分数为0.05的CO2、95%空气的培养箱内培养，接种后72 h首次换液，然后置换以5 mL新鲜的含10%胎牛血清的DMEM培养基，以后每3 d换液，第7天首次传代。
细胞的传代培养：将已经70%~80%长满的细胞用0.25%胰蛋白酶消化，再用含体积分数为0.10胎牛血清的DMEM培养基重悬，以1∶2比例接种于新培养瓶中，并置于37 ℃、体积分数为0.05的CO2、95%空气的培养箱内培养，以后每3 d换液，待细胞70%~80%长满时再次传代。
倒置显微镜观察：40及100倍倒差显微镜下逐日观察细胞的生长情况及形态特征。
细胞表面标记检测：用0.05%胰液酶消化收获细胞，PBS洗涤2次后，细胞计数，将细胞移入96孔板，每孔细胞数在5×105以上，后分别与抗CD29、CD44、CD34、CD45单克隆抗体室温避光反应30 min。PBS洗涤2次，1%多聚甲醛固定后，应用流式细胞仪检测标本。
成骨诱导分化及鉴定：取第3代细胞，6孔培养板每孔接种1×105个细胞，待80%~90%融合时加入成骨诱导剂包括0.1 μmol/L的地塞米松、50 mg/L的维生素C、10 mmol/L的β-磷酸甘油钠进行联合诱导培养，隔日换液，培养 14 d和21 d后，用钙钴法进行碱性磷酸酶染色。
成脂诱导分化及鉴定：取第3代细胞，6孔培养板每孔接种1×105个细胞，标准培养基培养24 h后加入成脂诱导剂含10%FBS的L-DMEM，0.25 μmol/L地塞米松，50 μmol/L吲哚美辛，0.5 mmol/L IBMX，10 mg/L牛胰岛素，每3 d换液，诱导2周后，细胞经PBS洗涤，10%甲醛固定30 min，进行油红染色检测脂肪沉积。
主要观察指标：①骨髓间充质干细胞的原代及传代培养形态。②骨髓间充质干细胞的诱导分化。

2 结果

2.1 骨髓间充质干细胞原代培养 细胞约24~48 h贴壁，细胞初为淋巴细胞样小圆细胞，

24~48 h后贴壁细胞明显增多，并开始分裂增殖，圆形细胞变形伸出伪足，见图1。约7 d可70%~80%长满培养瓶壁，此时细胞逐渐融合成单层，此时细胞形态为圆形、多角形、梭形或不规则形，呈集落状生长，并出现核分裂相。细胞常围绕集落中央呈岛状分布，呈多角形并可重叠生长，并分泌大量基质，见图2。
 

2.2 骨髓间充质干细胞（原代培养）传代培养 传代后细胞约24 h贴壁，逐渐伸展为梭形、纺锤形、多角形，胞体较原代略膨大，3~5 d即可长满培养瓶壁。连续传代后，随着传代次数增多，细胞变为形态更均一，排列更有序的成纤维细胞样，见图3。
2.3 骨髓间充质干细胞表面标记鉴定 流式细胞仪检测骨髓间充质干细胞均一地表达CD29、CD44,而CD34、CD45均阴性表达。
2.4 骨髓间充质干细胞成骨分化鉴定 成骨诱导14 d后细胞不明显，未形成钙结节，21 d后碱性磷酸酶钙钴法染色后大多数细胞的胞质呈棕黑色，见图4。
2.5 骨髓间充质干细胞成脂分化鉴定 成脂诱导48 h后，细胞内有小脂滴出现，2周后脂滴数量增加并相互融合，细胞（有）由长梭形变为圆形或多边形，油红O染色显示有大量脂质沉积，见图5。

3 讨论

一个多世纪前德国病理学家Cohnheim就提出在骨髓基质中存在有向非造血系统多向分化的干细胞，但直到20世纪60年代末才有了直接的证据[3]。1968年Friedenstein等首先证实骨髓间充质干细胞在骨髓组织中的存在。近年来，骨髓间充质干细胞的多向分化潜能越来越受到研究者的关注[4-5]，因其具有取材方便安全、来源丰富、损伤小、易于体外培养扩增，在体外可长时间保持未分化状态，体外基因转染率高，并能稳定高效表达多种治疗性外源基因，而且自体获取的骨髓间充质干细胞回植后不会发生免疫排斥反应等优点[6-7]，骨髓间充质干细胞已经成为重要的组织工程种子细胞，因此随着组织细胞工程学和基因工程学的兴起，若与其多向分化潜能相结合，通过导入目的基因，将细胞治疗和基因治疗结合在一起，必将为骨缺损、创伤愈合、神经组织功能修复等的治疗开辟广阔前 景[8-9]。
体外获得的骨髓间充质干细胞为各种细胞的混合体，至今尚未形成一套理想的分离纯化骨髓间充质干细胞的方法，如何纯化有待于对骨髓间充质干细胞的细胞学特性和分化各阶段细胞标志物作进一步研究。目前用于分离骨髓间充质干细胞常用的方法主要有贴壁筛选法、流式细胞仪分离法、免疫磁珠法和密度梯度离心法[10-11]，但流式细胞仪分离法和免疫磁珠法步骤繁琐且对细胞活性影响很大。本实验采用了密度梯度离心法与贴壁培养法相结合的方法，在取出兔的骨髓细胞后，用比重为1.073的Percoll密度梯度分离液分离骨髓细胞，经梯度离心后，由于比重差别能有效地将绝大部分红细胞、粒细胞、脂肪细胞和血小板除去，获得纯度较高的单个核细胞，然后根据骨髓间充质干细胞与血细胞贴壁性能差异，经体外贴壁培养，随换液弃除悬浮生长的血细胞。原代培养贴壁的细胞主要是骨髓间充质干细胞，也可能混有单核细胞、淋巴细胞，利用它们在培养瓶壁的贴壁性不同，每次传代用0.25%胰蛋白酶消化3~5 min，骨髓间充质干细胞迅速从培养瓶上脱落，而淋巴细胞、单核细胞由于贴附性强仍附于培养瓶壁，严格掌握消化酶的量和消化时间，保证骨髓间充质干细胞在短的作用时间内与培养瓶壁分开，从而使骨髓间充质干细胞得到进一步纯化。实验结果表明密度梯度离心法与贴壁培养法相结合的方法步骤相对简便且有效的避免了细胞活性的损伤。
本实验使用含有10%胎牛血清（FBS）的低糖DMEM培养基，通过本实验证实了低糖培养基较高糖培养基更利于骨髓间充质干细胞的生长。原代培养骨髓间充质干细胞约24~48 h贴壁，细胞初为淋巴细胞样小圆细胞，24~48 h后贴壁细胞明显增多，并开始分裂增殖，圆形细胞变形伸出伪足。约8~10 d可铺满培养瓶壁，细胞逐渐融合成单层。此时细胞为圆形、多角形、梭形或不规则形，呈集落状生长，并出现核分裂相。不传代细胞，可以持续生长，细胞常围绕集落中央呈岛状分布，呈多角形并可重叠生长，并分泌大量基质。传代培养细胞以0.25%胰酶消化，接种后24 h贴壁，逐渐伸展为梭形、纺锤形、多角形，胞体较原代略膨大，3~5 d即可长满。连续传代后，随着传代次数增多，细胞变为形态更均一、排列更有序的成纤维细胞样。通过流式细胞仪检测细胞表面标志，发现所培养干细胞均一表达其特异性表面标志CD29、CD44, 说明培养获得了较纯的骨髓间充质干细胞。通过加入成骨及成脂诱导剂成功的对骨髓间充质干细胞进行成骨及成脂诱导分化，磷酸酶钙钴法染色后细胞的胞质呈棕黑色，油红O染色后细胞内有大量脂质沉积，从而证实了骨髓间充质干细胞的多向分化潜能，为今后将其进一步应用于临床治疗颅骨缺损、神经功能缺损以及脑肿瘤奠定实验基础。

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