中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (19): 3451-3457.doi: 10.3969/j.issn.1673-8225.2010.19.007

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

重组腺病毒Ad-LMP-1感染大鼠骨髓间充质干细胞

赵忠海1，朱 悦2，林 乐3，阿 良1，李洪秋1   

  1. 1沈阳医学院奉天医院骨科，辽宁省沈阳市  110024；
    2中国医科大学附属第一医院骨外科，辽宁省沈阳市  110021；
    3大连大学附属中山医院，辽宁省大连市  116001
  • 出版日期:2010-05-07 发布日期:2010-05-07
  • 通讯作者: 朱 悦，博士，主任医师，中国医科大学附属第一医院骨外科，辽宁省沈阳市 110021
  • 作者简介:赵忠海，男，1969年生，辽宁省葫芦岛市人，汉族，1992年锦州医学院毕业，副主任医师，主要从事骨外科工作，特别是创伤外科的临床实践。 zzh-hy@126.com

Recombinant adenovirus Ad-LMP-1 infects rat bone marrow mesenchymal stem cells

Zhao Zhong-hai1, Zhu Yue2, Lin Le3, A Liang1, Li Hong-qiu1   

  1. 1Department of Orthopaedics, Fengtian Hospital, Shenyang Medical College, Shenyang   110024, Liaoning Province, China;
    2Department of Bone Surgery, First Affiliated Hospital, China Medical University, Shenyang   110021, Liaoning Province, China;
    3Zhongshan Hospital Affiliated to Dalian University, Dalian   116001, Liaoning Province, China
  • Online:2010-05-07 Published:2010-05-07
  • Contact: Zhu Yue, Doctor, Chief physician, Department of Bone Surgery, First Affiliated Hospital, China Medical University, Shenyang 110021, Liaoning Province, China
  • About author:Zhao Zhong-hai, Associate chief physician, Department of Orthopaedics, Fengtian Hospital, Shenyang Medical College, Shenyang 110024, Liaoning Province, China zzh-hy@126.com

被引次数

4

摘要：

背景：LIM矿化蛋白1 (LIM Mineralization protein, LMP-1)是一种细胞内非分泌蛋白，可以通过招募众多的成骨因子共同参与成骨细胞的分化。LMP-1在成骨上游水平发挥的强大作用提示了其在骨骼重建方面具有巨大的应用潜力。
目的：观察重组腺病毒Ad-LMP-1感染骨髓间充质干细胞的效果，及对其分化的影响。
方法：大鼠骨髓间充质干细胞分离培养，传代后，检测特异性标记物CD44的表达情况，矿化液诱导细胞成骨分化后用茜素红染色鉴定。感染重组腺病毒Ad-LMP-1后通过观察绿色荧光蛋白表达情况计算转染效率，RT-PCR及Western blot验证感染效果，观察感染Ad-LMP-1 2，7，14 d后骨髓间充质干细胞的形态变化和对细胞增殖活力的影响。
结果与结论：传代细胞形态一致，排列紧密。矿化液诱导后，细胞形态明显改变。茜素红染色结果表明，诱导后出现钙化结节。观察绿色荧光蛋白信号表明重组病毒Ad-LMP-1感染骨髓间充质干细胞效率在85%左右。感染重组腺病毒Ad-LMP-1后第7天出现了细胞形态改变，14 d出现类钙化结节，但不伴有细胞增殖活力改变。结果提示重组腺病毒Ad-LMP-1可以有效地感染原代培养的骨髓间充质干细胞，并诱导其向成骨细胞分化。

关键词: 腺病毒, 骨髓间充质干细胞, 细胞分化, LIM矿化蛋白1, 干细胞

Abstract:

BACKGROUND: LIM mineralization protein 1 (LMP-1), an intracellular non-secreted protein, can recruit a large number of osteogenic factors to participate in osteoblast differentiation. The role of LMP-1 in the upper levels of osteogenic differentiation suggested the powerful roles in bone reconstruction.
OBJECTIVE: To observe the effect of recombinant adenoviruson Ad-LMP-1 infection on bone marrow mesenchymal stem cells (BMSCs) and the effect on its differentiation.
METHODS: Rat BMSCs were isolated and cultured. The expression of specific marker of BMSCs, CD44, was successfully detected. Mineralized solution was added into the study group medium to induce osteogenic differentiation and the calcified nodules were detected by alizarin red staining. After infecting with recombinant adnovirus Ad-LMP-1, the infection efficiency was evaluated by observing the green fluorescent protein (GFP) signals. The upregulated expression of LMP-1 was confirmed by RT-PCR and Western blot, while the effects of Ad-LMP-1 on morphological and proliferative changes were observed at 2, 7 and 14 days.
RESULTS AND CONCLUSION: The morphology of subcultured BMSCs was consistent and arranged closely. After induction by mineralized solution, osteogenous changes were observed in cell morphology. Alizarin red staining results showed that the calcified nodules were produced after induction. Observation of GFP signal showed that the infection efficiency of recombinant adenovirus Ad-LMP-1 was about 85%. After infection with Ad-LMP-1, the morphological changes appeared at day 7, and calcified nodules were observed at day 14, but not accompanied with any change in proliferative ability. Results indicated that recombinant adenovirus Ad-LMP-1 can effectively infect primary cultured BMSCs, and induce osteogenic differentiation.

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