中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (20): 3620-3625.doi: 10.3969/j.issn.1673-8225.2012.20.003

• 骨组织构建 bone tissue construction • 上一篇    下一篇

重组人骨形成蛋白2诱导成肌细胞成骨分化中的矿化反应***☆

张  力1, 2，王  伟2   

  1. 1辽宁中医药大学，辽宁省沈阳市  110032；2锦州市中心医院骨科，辽宁省锦州市  121000
  • 收稿日期:2011-11-03 修回日期:2011-11-14 出版日期:2012-05-13 发布日期:2012-05-13
  • 通讯作者: 王伟，博士生导师，教授，主任医师，锦州市中心医院骨科，辽宁省锦州市 121000
  • 作者简介:张力☆，男，1980年生，湖北省黄冈市人，汉族，辽宁中医药大学在读博士，主治医师，主要从事骨与周围神经再生修复研究。
  • 基金资助:

    国家自然基金资助项目(30772190)，转分泌性CNTF基因的肌源性干细胞复合支架修复周围神经缺损的实验研究；中国博士后科学基金(2005038055)，转CNTF基因的肌源性干细胞修复周围神经缺损的相关研究；辽宁省科技计划项目(2005225003- 14)，新型人工生物复合材料修复骨缺损的研究。

Mineralization reaction during osteogenic differentiation of myoblasts stimulated by bone morphogenetic protein 2***☆

Zhang Li1, 2, Wang Wei2   

  1. 1Liaoning University of Traditional Chinese Medicine, Shenyang  110032, Liaoning Province, China; 2Department of Orthopedics, Jinzhou Central Hospital, Jinzhou  121000, Liaoning Province, China
  • Received:2011-11-03 Revised:2011-11-14 Online:2012-05-13 Published:2012-05-13
  • Contact: Wang Wei, Doctoral supervisor, Professor, Chief physician, Department of Orthopedics, Jinzhou Central Hospital, Jinzhou 121000, Liaoning Province, China weiwang_ly@yahoo.com.cn
  • About author:Zhang Li☆, Studying for doctorate, Attending physician, Liaoning University of Traditional Chinese Medicine, Shenyang 110032, Liaoning Province, China; Department of Orthopedics, Jinzhou Central Hospital, Jinzhou 121000, Liaoning Province, China zhangli2004jz@yahoo.com.cn
  • Supported by:

     the National Natural Science Foundation of China, No.30772190*; Postdoctorate Foundation Program of China, No. 2005038055*; Science and Technology Development Program of Liaoning Province, No. 2005225003-14*

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摘要：

背景：近年来成肌细胞在重组人骨形态发生蛋白2诱导下向成骨细胞分化已经通过多种方式得到了证实。
目的：探讨成肌细胞在重组人骨形态发生蛋白2诱导下向成骨分化过程的矿化反应及其体外诱导表达成骨表型的可行性。
方法：采用差速贴壁法和酶消化法获取Wistar乳鼠成肌细胞并体外培养，纯化鉴定后取第3代培养成肌细胞加入含重组人骨形成蛋白2的培养液进行诱导，对照组不加入重组人骨形成蛋白2，体外培养21 d。
结果与结论：成肌细胞经重组人骨形成蛋白2诱导后，细胞增殖减缓，相邻细胞聚集呈层状排列，诱导8 d可见胞浆有少量不透光结节，诱导14 d结节增多，21 d时胞浆内可见大量不透光结节，未经重组人骨形成蛋白2诱导的成肌细胞融合成有收缩性的肌管。诱导后的成肌细胞碱性磷酸酶活性随时间延长而增强，诱导21 d时细胞经碱性磷酸酶染色、Ⅰ型胶原免疫组化染色和钙结节染色均呈阳性反应。表明大鼠成肌细胞经重组人骨形成蛋白2诱导可以出现矿化反应，可在体外一定条件下诱导能够转化为成骨细胞。

关键词: 成肌细胞, 成骨细胞, 骨形态发生蛋白, 分化, 矿化

Abstract:

BACKGROUND: In recent years, it has been confirmed by a variety of ways that myoblasts can differentiate into osteoblasts under the induction of recombinant human bone morphogenetic protein 2 (rhBMP-2).
OBJECTIVE: To explore the mineralization reaction during the osteogenic differentiation of myoblasts under the induction of recombinant rhBMP-2 and the feasibility of osteogenic phenotype expression by in vitro induction.
METHODS: Myoblasts were isolated and harvested from neonatal Wistar rats using differential velocity adherent technique and trypsinization method. After in vitro culture, purification and identification, myoblasts at passage 3 were induced by a medium containing rhBMP-2 for 21 days. Myoblasts in the control group were cultured in vitro in complete medium without rhBMP-2 for 21 days.
RESULTS AND CONCLUSION: After rhBMP-2 induction, myoblast proliferation gradually slowed down. A small quantity of opaque secretory granules were found in the cytoplasm on day 8 after induction; the number of opaque secretory granules increased on day 14 after induction; and a great quantity of opaque secretory granules were found in the cytoplasm on day 21 after induction while the myoblasts without induction fused into contractile myotubes. The alkaline phosphatase activity of the induced myoblasts increased as time extended; myoblasts reacted positively in the alkaline phosphatase staining, immunochemical staining for type Ⅰ collagen and calcium node staining on day 21 after induction. These findings suggest that mineralization reaction is found in rat myoblasts by rhBMP-2 induction and myoblasts can differentiate into osteoblasts under certain inducing conditions in vitro.

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