| 改良人骨髓间充质干细胞分离方法提高细胞定向分化能力 |
| 投稿时间：2010-04-14 点此下载全文 |
| 引用本文：张卉,董学君,邵健忠,项黎新.改良人骨髓间充质干细胞分离方法提高细胞定向分化能力[J].医学研究杂志,2010,39(9):57-62 |
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| 基金项目:浙江省省部共建项目（WKJ2007-2-037）；浙江省自然科学基金项目（Y2090337） |
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| 中文摘要:目的改良人骨髓间充质干细胞（hBM-MSCs）分离方法，提高细胞定向分化能力。方法用密度梯度离心法从少量人骨髓中分离得到单个核细胞，贴壁培养3天后换液，实验组换液后72h内每隔12h更换新鲜培养液，传代时胰蛋白酶37℃作用2min后终止消化；对照组未经频繁换液，胰蛋白酶消化3min。镜下观察分离细胞的形态变化。用成骨、脂肪和软骨细胞诱导液诱导两组第3代（P3）细胞分化，碱性磷酸酶钙钴染色法、茜素红染色法和Von Kossa银染色法检测成骨细胞分化；油红O染色法检测脂肪细胞分化；阿尔新蓝染色法检测软骨细胞分化。结果首次换液72h后，实验组与对照组相比，贴壁细胞数量较少但形态均一性高。不同染色结果显示：诱导分化成骨细胞14天时，实验组95%以上细胞分化，对照组为60%~70%；诱导分化脂肪细胞21天时，实验组50%~60%细胞分化，对照组为40%；诱导分化软骨细胞21天时，实验组90%以上细胞分化，对照组为60%~70%。结论首次换液72h内频繁更换新鲜培养液和传代时缩短胰蛋白酶消化时间的方法能够提高hBM-MSCs的定向分化能力。 |
| 中文关键词:人骨髓间充质干细胞 成骨细胞 脂肪细胞 软骨细胞 |
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| An Improved Protocol for Isolating and increasing the Multiple Differentiation Potentials of Mesenchymal Stem Cells from Human Bone Marrow |
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| Abstract:ObjectiveTo explore an improved protocol for isolating and increasing the multiple differentiation potentials of human bone marrow mesenchymal stem cells (hBM-MSCs). MethodsMononuclear cells were isolated from human bone marrow by density gradient centrifugation and the medium was changed after 3 days. In the following 72 hours, the medium was changed every 12 hours in experimental group but not in control group. hBM-MSCs were digested with trypsin for 2 minutes in experimental group, while the time was 3 minutes in control group. The morphological changes of the separated cells were observed by phase-contrast microscope. Three induction media induced oriented differentiation of hBM-MSCs at passages 3 into osteoblasts, adipocytes and chondrocytes. Osteoblasts were identified by ALP stain, Alizarin Bordeaux stain and Von Kossa stain, and adipocytes by morphological change and Oil Red-O stain, as well as chondrocytes by Alcian Blue stain. ResultsAfter 72 hours when medium was exchanged for the first time, the number of adherent cells in experimental group was less than that in control group, while the uniform of the cells was higher. Different staining methods showed that when the cells were induced in the osteoblast induction media for about 14 days, the differentiation rate of experimental group was more than 95%, the control group was 60%~70%; when the cells were induced in the adipocyte induction media for about 21 days, the differentiation rate of experimental group was 50%~60%, the control group was 40%; when the cells were induced in the chondrocyte induction media for about 21 days, the differentiation rate of experimental group was more than 90%, the control group was 60%~70%. ConclusionFrequent medium change during 72 hours after first-time exchange of medium and diminishing the trypsinization time could increase the multiple differentiation potentials of hBM-MSCs. |
| keywords:Human bone marrow mesenchymal stem cells Osteoblasts Adipocytes Chondrocytes |
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