骨质疏松患者体外培养BMSCs的生物学特性.pdfVIP

医学临床研究 2007年l2月 第24卷 第 l2期 J Clin Res，Dec．2007，Vol 24，No12 · 2Ol9 · · 论著· 骨质疏松患者体外培养BMSCs的生物学特性 代平 詹瑞森 王卫国 (1．广东省深圳宝安区福永医院骨科，广东 深圳 518103；2．中南大学湘雅三医院骨科，湖南 长沙 41OO13) [摘要]【目的】探讨骨质疏松患者骨髓源骨髓基质干细胞(bone marrow stromal ceils，BMSCs)的体外培 养方法和作为种子细胞的可能性。【方法】从一骨质疏松患者髂骨中抽取骨髓 10ml，体外培养、传代和扩增， 对细胞形态进行观察及采用流式细胞仪鉴定 BMSCs；将第三代细胞分别接种在常规DMEM培养液及常规 DMEM+10ng／ml(PIX；F-BB)培养液，绘制两组的生长曲线、倍增时间及用 MTT法分析其存活和增殖能力； 将第三代细胞分别用常规DMEM培养液、常规矿化液和常规矿化液+PDGF-BB(10ng／m1)培养液培养，向成 骨细胞定向诱导分化后测定三组细胞内碱性磷酸酶(ALP)含量及茜素红法(ARS)钙化结节染色。【结果】倒 置相差显微镜下观察显示原代细胞具有成纤维细胞的生长特性；生长曲线显示常规DMEM+PDGF-BB培养 液组较常规DMEM培养液组细胞增殖快且细胞倍增时间短；流式细胞仪鉴定细胞群为 BMSCs，纯度在95 以上；MTT法显示三组两两之间统计学分析均有显著性差异(P0．01)；ALP定量测定显示三组之问统计 学分析有显著性差异(P0．01)；茜素红法(ARS)钙化结节染色结果显示常规矿化液组和常规矿化液+ PDGF-BB培养液两组均为阳性。【结论】从骨质疏松患者骨髓源中获得的BMSCs，经体外培养、纯化、扩增后 可达到组织工程骨所要求的10 ～10 细胞数量级，且其可诱导分化为成骨细胞；PDGF-BB能促进 BMSCs的 增殖，但不影响其分化成骨能力。 [关键词] 骨质疏松； 骨髓细胞 Biological Characteristics of Bone Marrow StromaI Cells 0btained from 0steoporotic Patients in vitro f Ping(Department of Orthopaedics．Fuyong Hospital of Baoan District．Shenzhen 5181O3，( ina) [Abstrdct][Objective]To study the proliferation and osteogenic differentiation of bone marrow stromal ceils (BMSCs)isolated from osteoporotic patients in vitro and to explore the feasibility of which they were as the seed cells for bone tissue engineering．[Methods]BMSCs obtained from osteoporotic patientsilium were cultured and prolifera— ted in DMEM medium in vitro．The biological characteristics of BMSCs were observed under inverted phase contrast microscopes and examined by flow cytometry．The cells of third passage were inoculated into DMEM medium and DMEM were added to lOng／ml PDGF—BB medium．and then proliferative activity of cells was detected by methythia— zolyl tetrazolium (MTT)．Growth curve was drawed and the cell diploproliferatio