中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (15): 2661-2666.doi: 10.3969/j.issn.1673-8225.2010.15.001

• 骨组织构建 bone tissue construction •    下一篇

正常人成骨细胞系的建立及鉴定

马文辉1，张学敏2，时述山3，李亚非3   

  1. 1解放军白求恩国际和平医院骨科，河北省石家庄市  050082；2河北国防建医院，河北省石家庄市050081；3解放军北京军区总医院骨科，北京市
    100072
  • 出版日期:2010-04-09 发布日期:2010-04-09
  • 作者简介:马文辉★，男，1973年生，河北省石家庄市人，汉族，2000年解放军军医进修学院毕业，硕士，主治医师，主要从事关节外科与骨修复研究。hmq318@hotmail.com

Establishment and identification of a normal human osteoblast system

Ma Wen-hui1, Zhang Xue-min2, Shi Shu-shan3, Li Ya-fei3   

  1. 1 Department of Orthopaedics, Bethune International Peace Hospital of Chinese PLA, Shijiazhuang   050082, Hebei Province, China; 2 Hebei National Defence Construction Hospital, Shijiazhuang   050081, Hebei Province, China; 3 Department of Orthopaedics, General Hospital of Beijing Military Area Command of Chinese PLA, Beijing   100072, China
  • Online:2010-04-09 Published:2010-04-09
  • About author:Ma Wen-hui★, Master, Attending physician, Department of Orthopaedics, Bethune International Peace Hospital of Chinese PLA, Shijiazhuang 050082, Hebei Province, China hmq318@hotmail.com

PDF

367

被引次数

7

摘要：

背景：骨细胞的体内研究由于受许多因素的影响而不便于结果分析。分离的骨细胞培养则可获得单一的细胞系，以此来观察各种不同物质或实验手段对细胞的直接效应，可以避免许多干扰因素的影响。
目的：探讨建立可行的、能大量生产人正常成骨细胞培养方法和保存方式。
方法：取引产胎儿四肢皮质骨，彻底刮除皮质骨表面的骨膜及内部的骨髓，并反复冲洗去除残余组织，胰酶、胶原酶消化后，将骨粒平铺于尼龙网上培养，建立正常人成骨细胞系。观察细胞形态及组织学变化；以免疫组织化学方法测定其生成Ⅰ型胶原情况。利用液氮冻存细胞以建立适当的保存方法。选取对数生长良好的细胞进行遗传学特性-染色体的制备。
结果与结论：成骨细胞呈梭形，多突起；细胞核呈卵圆形，偏于一侧；胞体大，胞浆丰富；碱性磷酸酶染色可见胞浆中含有大量的灰黑色颗粒，某些部位染成黑色，定量分析细胞内的碱性磷酸酶、骨钙素水平明显高于成纤维细胞；细胞免疫组织化学染色表明其主要合成Ⅰ型胶原。染色体分析表明，其具有23对染色体，未发现异常染色体，从而证实了其为正常人体来源的细胞，经培养与多次传代后仍保持正常，未发生变异。通过使用对新生骨有特异性的荧光染料四环素进行染色标记，证实了此培养细胞具有成骨能力。证实建立了可靠的正常人成骨细胞系，并能采用液氮冷冻的方式长期保存。经多次传代其遗传性质及生物学特征均未发生变异，复苏后的冻存细胞仍具有稳定的生物学特性。

关键词: 成骨细胞, 培养, 生物学特征, 人, 骨组织工程

Abstract:

BACKGROUND: In vitro research of bone cells were difficult for result analysis due to various factors. The separated bone cells could obtain single cell line. Therefore, the single cell line was used to observe direct effect of various materials or methods and avoid some interfering factors.
OBJECTIVE: To investigate a feasible method to mass-produce, culture, and reserve normal osteoblast of people. 
METHODS: Diaphysis of extremities from normal fetus aged four months were harvested to digest in collagenase and pancreatin to generate single cell suspensions, the cells and tissue segments were cultured for establishing normal osteoblast system of people. Morphology and histology were observed; type Ⅰ collagen produce was measured using immunohistochemistry; cells were reserved using liquid nitrogen frozen method; cells in good logarithmic growth phase were collected for preparation of genetics-chromosome.
RESULTS AND CONCLUSION: Osteoblast was fusiformed-shaped and had plentiful processes. Nucleus was orbicular-ovate and leaning to lateral side. Soma was large, and plasma was abundant. Alkaline phosphatase staining suggested that a great number of gray-black particles were observed in plasma, and some region was darkly stained. Quantitative analysis demonstrated that levels of alkaline phosphatase and osteocalcin in the plasma were significantly higher than fibroblast; immunohistochemical staining suggested that the type Ⅰ collagen was mainly produced. Chromosome analysis indicated that there were 23 pairs of chromosomes, and abnormal chromosome was not detected, suggesting that the obtained samples were normal human cells which were still normal following various cultures. Fluorochrome-tetracycline staining demonstrated that the cultured cells in this study had the osteogenesis ability. In this study, a normal osteoblast system of people was established and could be reserved with liquid nitrogen for a long time. The cells had stable biological characteristics following both various passages and frozen reservation.

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