| 改良组织块混合酶消化法成骨样细胞培养 |
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Received:January 24, 2003
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| 作者 | Author | 单位 | Unit | E-Mail |
| 李靖 |
LI ling |
第四军医大学西京医院全军骨科研究所,陕西 西安 710033 |
Institute of Orthopaedics of Chinese PLA, Xijing Hospital, The Fourth Military Medical University Shanxi Xi' an ,710033 , China |
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| 黄鲁豫 |
HUANG Luyu |
第四军医大学西京医院全军骨科研究所,陕西 西安 710033 |
Institute of Orthopaedics of Chinese PLA, Xijing Hospital, The Fourth Military Medical University Shanxi Xi' an ,710033 , China |
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| 王全平 |
WANG Quanping |
第四军医大学西京医院全军骨科研究所,陕西 西安 710033 |
Institute of Orthopaedics of Chinese PLA, Xijing Hospital, The Fourth Military Medical University Shanxi Xi' an ,710033 , China |
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| 吕荣 |
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第四军医大学西京医院全军骨科研究所,陕西 西安 710033 |
Institute of Orthopaedics of Chinese PLA, Xijing Hospital, The Fourth Military Medical University Shanxi Xi' an ,710033 , China |
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| 白建萍 |
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第四军医大学西京医院全军骨科研究所,陕西 西安 710033 |
Institute of Orthopaedics of Chinese PLA, Xijing Hospital, The Fourth Military Medical University Shanxi Xi' an ,710033 , China |
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| 倪国华 |
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解放军160医院骨科 |
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| 期刊信息：《中国骨伤》2003年16卷，第11期，第661-663页 |
| DOI：doi:10.3969/j.issn.1003-0034.yyyy.nn.zzz |
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目的:应用组织块与混合酶消化联合使用进行大鼠颅骨成骨细胞体外培养。
方法:取新生2～3d的SD大鼠颅骨剪碎依次用不同浓度的胰蛋白酶和Ⅱ型胶原酶混合分2次对颅骨进行消化。纯化、接种后进行原代和传代培养并对细胞进行鉴定。
结果: 原代培养的细胞ALP染色阳性区域可以达90%以上,传代培养后细胞阳性率为100%。18d左右有钙结节形成。电镜观察示细胞表面有微绒毛,粗面内质网丰富。原代培养细胞BMP兔疫组化呈弱阳性表达,传代培养后细胞BMP表达逐渐增强,以第5代左右细胞表达强度最高。
结论:改良组织块混合酶消化法成骨细胞培养具有细胞成分单一、表型稳定的特点。可为成骨细胞的代谢和生理的体外研究提供高纯度的细胞。 |
| [关键词]:细胞,培养的 成骨细胞 表型 |
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| Osteoblastic-like cell culture with modified method of mixed enzymatic digestion |
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Abstract:
Objective:To investigate possibility of culture of osteoblast from rat cranial bone in vitro with modified method which based on the combination of tissue block and mixed enzyme digestion
Methods:The newly born SD rats were sacrificed and the cranial bones of the rats were obtained and cut to blocks of 1 mm× 1 mm.Then the blocks were digested by collagenase combined with trypsogen two times with 10 minutes for the first time and 30 minutes for the second time. The ratio of collagenase to trypsogen is 3 to 2 for the first time,3 to 1 for the second time, respectively. ALP stains for the cultured cells and TEM investigation were performed to determine the activity of osteoblast.
Results:The area positive for ALP staining is no less than 90% in the primary culture and almost 100% in the subculture.Calcium nodule appeared on the 18th days.Ultrastructural investigations showed that there are microvillus on the surface and plenty of rough endo-plasmic reticulum in cells. BMP-immunoeactivity was detected in the primary culture cells and increased gradually and peaked in fifth subculture using immunocytochemical technique.
Conclusion:The modified culture of osteoblastic-like cells has the advantages of single component and stable phenotype, therefore can be used for the study of metabolism and physiology of osteoblast in vitro. |
| KEYWORDS:Cell, culture Osteoblast Phenotype |
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| 引用本文，请按以下格式著录参考文献： |
| 中文格式: | 李靖,黄鲁豫,王全平,吕荣,白建萍,倪国华.改良组织块混合酶消化法成骨样细胞培养[J].中国骨伤,2003,16(11):661~663 |
| 英文格式: | LI ling,HUANG Luyu,WANG Quanping.Osteoblastic-like cell culture with modified method of mixed enzymatic digestion[J].zhongguo gu shang / China J Orthop Trauma ,2003,16(11):661~663 |
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