作者：刘斌钰，李宁毅，樊功为，金晓明，陈立强作者单位：（青岛大学医学院附属医院口腔科, 山东 青岛 266003） # k+ \7 s$ i# t7 O# U, O 8 p3 \( m+ u0 x
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【摘要】 目的 探讨大鼠骨髓间充质干细胞（BMSCs）的体外培养方法、生长规律及向成骨细胞分化的条件。方法 采用全骨髓培养法分离成年大鼠BMSCs，经条件培养液诱导培养后，进行形态学观察及碱性磷酸酶活性、细胞矿化作用、骨钙素(OCN)等的测定。结果 原代培养的BMSCs先形成细胞集落,14 d时集落融合; 传代细胞体积变大,约5～7 d传代1次。诱导条件下,细胞碱性磷酸酶活性明显增高,并出现了矿化结节。经过体外成骨诱导的BMSCs 表现出增殖、聚集、结节和矿化期的阶段性形态特征。在矿化期OCN 表达呈阳性。结论 BMSCs易于体外分离培养及扩增,体外成骨定向诱导的BMSCs 具有典型的成骨细胞的形态和功能性特征，可以作为骨组织工程的种子细胞。 , t5 @' ^0 j% J% @! ]. k
【关键词】骨髓间充质干细胞;诱导分化；细胞培养；碱性磷酸酶；骨钙素 ( B, }$ E4 n4 E$ {+ s3 F# X+ J: i EXPERIMENTAL STUDY OF CULTIVATION, IDENTIFICATION AND INDUCED DIFFERENTIATION OF BONE MARROW STROMAL STEM CELLS INTO OSTEOBLASTS IN RATSLIU BINYU， LI NINGYI， FAN GONGWEI，et al（Department of Stomatology, The Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, China） ［ABSTRACT］ObjectiveTo set up a protocol of culturing bone marrow stromal stem cells （BMSCs） of rats in vitro, and to study the condition for induced differentiation to osteoblasts.MethodsBMSCs were isolated from adult rats for anchoring culture. The passage cells were induced to differentiate into osteoblasts in a differentiation induction medium. The phenotype was determined by morphological method，and levels of alkaline phosphatase (ALP） and osteocalcin (OCN), and ability to form mineralized nodules were examined. ResultsColonies of BMSCs were formed in the primary culture and contacted to each other at day 14. In the passage culture, the cells became bigger than those in the primary culture and a new generation was produced in 5—7 days. Under induction, levels of ALP were elevated and the morphological changes of osteoblast，phases of proliferation，aggregation，nodulation and mineralization were observed. During the mineralization, expression of OCN was detected. ConclusionBMSCs can be easily isolated and cultured in vitro, with strong proliferation ability and osteogenic capacity. BMSCs can be used as seed cells for tissue engineering of bone.2 n4 \) R6 A" b* i0 `
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［KEY WORDS］mesenchymal stem cells； induction differentiation; cell culture; alkaline phosphatase; osteocalcin ' e$ _" Y9 d- [- ]6 I4 U1 Z- H1 z
随着科学技术的不断发展，现在人们已逐渐认识到，骨髓非造血干细胞有一类来源于中胚层的未分化的间充质干细胞，又称为骨髓间充质干细胞（BMSCs），该细胞具有多向分化潜能，易于分离培养，体外增殖能力强，可进行自体移植而不存在组织配型和免疫排斥的问题等特点，目前发现其具有能够分化形成至少7种组织细胞，如成骨细胞、成软骨细胞、成脂细胞、成肌细胞等组织细胞的潜能。为骨、软骨、肌肉等结缔组织的损伤修复提供了潜在的细胞来源，被认为是骨组织工程中最佳的种子细胞。BMSCs的成骨定向诱导是骨组织工程种子细胞研究的关注焦点。本实验通过全骨髓培养法分离BMSCs,并用诱导分化培养液做定向诱导培养, 观察BMSCs向成骨细胞分化过程中的细胞形态学变化，进行成骨细胞生物学表型鉴定以探讨BMSCs的培养方法、生长规律及向成骨细胞分化的条件。 I6 w# q7 r R$ M6 Y
4 i; `# _# o w2 n z! S) f1材料与方法8 W5 N1 a, c3 i2 |/ n6 }4 p
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