中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (19): 3433-3437.doi: 10.3969/j.issn.1673-8225.2011.19.003

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

人LIM矿化蛋白1基因腺病毒重组表达载体构建及在犬骨髓间充质干细胞中的表达

蒲  超，倪卫东，高仕长，邱  宇   

  1. 重庆医科大学附属第一医院骨科，重庆市神经病学重点实验室，重庆市  400016
  • 收稿日期:2011-02-14 修回日期:2011-03-20 出版日期:2011-05-07 发布日期:2011-05-07
  • 通讯作者: 倪卫东，主任医师，重庆医科大学附属第一医院骨科，重庆市 400016 niweidong18@163.com
  • 作者简介:蒲超★，男，1984年生，四川省南充市人，汉族，重庆医科大学在读硕士，主要从事骨折愈合研究。 puchao3310@sina.com
  • 基金资助:

    重庆市医学科技计划项目。

Construction of human LIM mineralization protein-1 gene recombinant adenovirus and its expression in dog bone marrow mesenchymal stem cells

Pu Chao, Ni Wei-dong, Gao Shi-chang, Qiu Yu   

  1. Department of Orthopedics, First Hospital Affiliated to Chongqing Medical University, Chongqing Key Laboratory of Neurology, Chongqing  400016, China
  • Received:2011-02-14 Revised:2011-03-20 Online:2011-05-07 Published:2011-05-07
  • Contact: Ni Wei-dong, Chief physician, Department of Orthopedics, First Hospital Affiliated to Chongqing Medical University, Chongqing Key Laboratory of Neurology, Chongqing 400016, China niweidong18@163.com
  • About author:Pu Chao★, Studying for master’s degree, Department of Orthopedics, First Hospital Affiliated to Chongqing Medical University, Chongqing Key Laboratory of Neurology, Chongqing 400016, China puchao3310@sina.com
  • Supported by:

    Medical Science and Technology Plan of Chongqing City*

PDF

363

被引次数

11

摘要：

背景：研究表明LIM矿化蛋白1在体内和体外都可促使骨发生。
目的：应用Adeasy-1腺病毒系统构建含人LIM矿化蛋白1基因的腺病毒重组体，并感染犬骨髓间充质干细胞，检测其体外表达及诱导成骨作用。
方法：构建重组腺病毒质粒pAd-LMP-1，经人胚胎肾293细胞包装、扩增后得到复制缺陷重组腺病毒Ad-LMP-1，以最佳感染复数值体外感染犬骨髓间充质干细胞，行RT-PCR及 Western Blot检测LIM矿化蛋白1、骨形态发生蛋白7基因的表达。重组Ad-LMP-1和(或)外源性重组人骨形态发生蛋白7处理犬骨髓间充质干细胞，21 d后行矿化(钙)结节茜素红染色分析LIM矿化蛋白1基因的诱导成骨作用。
结果与结论：①将Ad-LMP-1以感染复数值100感染犬骨髓间充质干细胞可获得最佳感染效率，感染后骨髓间充质干细胞能在基因和蛋白水平表达LIM矿化蛋白1，未引起骨形态发生蛋白7基因的表达。②Ad-LMP-1或重组人骨形态发生蛋白7均不能单独促使骨髓间充质干细胞向成骨细胞转化，两者联合可促使骨髓间充质干细胞向成骨细胞转化。③实验成功构建重组腺病毒载体Ad-LMP-1，并实现其在犬骨髓间充质干细胞中表达，证实了LIM矿化蛋白1在诱导成骨作用中表现为与外源性重组人骨形态发生蛋白7的协同效应。

关键词: 重组人骨形态发生蛋白7, 骨髓间充质干细胞, LIM矿化蛋白, 腺病毒, 骨组织工程

Abstract:

BACKGROUND: Studies shows that the LIM mineralization protein 1 (LMP-1) can stimulate bone formation in vivo and in vitro.
OBJECTIVE: To construct the recombinant adenovirus vector containing LMP-1 gene by using the Ad-Easy system, then to detect the gene expression and study the mechanism of bone formation induced by LMP-1 in infected dog bone marrow mesenchymal stem cells (BMSCs).
METHODS: The recombinant plasmid pAd-LMP-1 was constructed. The replication-defective recombinant adenovirus Ad-LMP-1 was packaged and amplified in the human embryonic kidney 293 cells (HEK293). Dog BMSCs were infected by Ad-LMP-1 in the best value of MOI in vitro. The gene expression of LMP-1and BMP-7 was detected by RT-PCR and Western Blot. Dogs BMSCs were treated with Ad-LMP-1 and recombinant human bone morphogenetic protein 7 (rhBMP-7) to study the mechanism of bone formation of LMP-1 undergoing mineralization (Ca) nodules at 21 days by alizarin red staining.
RESULTS AND CONCLUSION: ①Ad-LMP-1 was successfully constructed and obtained titers in the packaging about 1×1011 efu/mL after amplified in HEK293. ②The best efficiency could be obtained at the most appropriate MOI 100 after Ad-LMP-1 infected BMSCs. Infected BMSCs can express mRNA and protein of LMP-1, but not caused expression of BMP-7. ③ Dog BMSCs were treated with Ad-LMP-1 and (or) rhBMP-7 for 21 days. We found that Ad-LMP-1 with exogenous rhBMP-7 promote transformation into osteoblasts together. Neither Ad-LMP-1 nor rhBMP-7 alone induced any transformation on day 21. The recombinant adenoviral vector containing hLMP-1 gene was successfully constructed and expressed effectively in dogs BMSCs. It is confirmed that the mechanism of bone formation induced by LMP-1 is synergistic effect with the performance of exogenous rhBMP-7.

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