| 引用本文： |
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张月双,卫华,马健.人脐血间充质干细胞分离培养方法的改良[J].同济大学学报（医学版）,2010,31(1):27-31. [点击复制]
- ZHANG Yue-shuang,WEI Hua,MA Jian.Improved culture method of mesenchymal stem cells from human umbilical cord blood[J].同济大学学报（医学版）,2010,31(1):27-31. [点击复制]
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| 摘要: |
| 目的探讨一种新的能提高脐血间充质干细胞体外分离培养成功率的方法。方法利用Ficoil分离脐血，采用改良的密度梯度两步离心法分离脐血单个核细胞，接种于含20％胎牛血清和5％人脐血血清的原代培养液中，在倒置相差显微镜下进行形态观察，直接免疫荧光法检测人脐血间充质干细胞相关抗原CD29的表达。加入成骨诱导剂体外诱导脐血间充质干细胞10d碱性磷酸酶染色鉴定其成骨分化能力。结果本实验方法成功分离培养出人脐血间充质干细胞。原代培养第4天即可见到有成纤维样细胞贴壁，10d左右集落形成，约25d进行传代。传代后生长加速，一周左右即可长满，免疫荧光检测显示脐血间充质干细胞相关抗原CD29呈阳性表达，成骨诱导10d碱性磷酸酶染色阳性。结论利用改良的密度梯度离心法可获得人脐血间充质干细胞． |
| 关键词: 脐血 间充质干细胞 细胞分离 培养 |
| DOI：10.3969/j.issn1008-0392.2010.0l.006 |
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| 基金项目:上海浦江人才计划资助项目 |
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| Improved culture method of mesenchymal stem cells from human umbilical cord blood |
| ZHANG Yue-shuang,WEI Hua,MA Jian |
| (Dept.of Preclinical Stomatology,Stomatology Hospital,Tongji University,Shanghai 200072,China) |
| Abstract: |
| Objective An effective method w as explored for i sol ation and cultiv ation of mesenchymal stem cells from human umbilical cord blood (HUMS Cs) in vitro . Methods Umbilical cord blood mononuclear cells were separated by modifying two steps of Ficoll density gradient centrifugation with different low velocities. They were planted into the medium containing 20% fetal bovine serum and 5% human serum. Cell adherence and growth were observed under the inverted microscope, the cell surface antigen phenotype CD29 were examined through direct immunofluorenscence, the osteogenic differention were examined by the staining of alkaline in vitro successfully isolated from cord blood by this modified method. HUMSCs appeared to be fibroblastlike cells on the fourth day of the primary culture and were harvested at the subconfluence about 25 days later. Specifities of antigen CD29 positive by related with these mesenchymal stem cells. The alkaline phosphatases were positive in HUMSCs after 10 days of osteogenic induction. Conclusion HUMSCs can be effectively acquired from the improved method of density gradient centrifugation. |
| Key words: umbilical cord blood mesenchymal stem cells cell isolation culture |
