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中华老年骨科与康复电子杂志

中华老年骨科与康复电子杂志 ›› 2018, Vol. 04 ›› Issue (06) : 341 -345. doi: 10.3877/cma.j.issn.2096-0263.2018.06.005

所属专题： 文献；

基础研究

转染音猬因子对骨髓间充质干细胞成骨分化的影响
贾祎佳1, 孙吉平1, 刘强1, 郝海虎1, 朱剑1, 吴斗1,†()   
  1. 1. 030032　太原，山西医学科学院　山西大医院骨科
  • 收稿日期:2018-06-19 出版日期:2018-12-05
  • 通信作者: 吴斗
  • 基金资助:
    山西省自然科学基金(2013011057-4)

The effects of bone marrow stromal cells transferred with lentivirat—mediated Sonic Hedgehog (Shh) gene in osteoporosis

Yijia Jia1, Jiping Sun1, Qiang Liu1, Haihu Hao1, Jian Zhu1, Dou Wu1,†()   

  1. 1. Department of Orthopaedics, Shanxi Academy of Medical Sciences, Shanxi Dayi Hospital, Taiyuan 030032, China
  • Received:2018-06-19 Published:2018-12-05
  • Corresponding author: Dou Wu
  • About author:
    Corresponding author: Wu Dou, Email:
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引用本文：

贾祎佳, 孙吉平, 刘强, 郝海虎, 朱剑, 吴斗. 转染音猬因子对骨髓间充质干细胞成骨分化的影响[J]. 中华老年骨科与康复电子杂志, 2018, 04(06): 341-345.

Yijia Jia, Jiping Sun, Qiang Liu, Haihu Hao, Jian Zhu, Dou Wu. The effects of bone marrow stromal cells transferred with lentivirat—mediated Sonic Hedgehog (Shh) gene in osteoporosis[J]. Chinese Journal of Geriatric Orthopaedics and Rehabilitation(Electronic Edition), 2018, 04(06): 341-345.

目的

建立能够持续稳定表达音猬因子（Shh）的骨髓基质干细胞（BMSCs），观察其成骨分化能力，为体内治疗骨质疏松提供可行性依据。

方法

使用Gateway Technology构建pDown-DsRed-Shh，贴壁培养法获取SD大鼠BMSCs，慢病毒转染法将pDown-DsRed-Shh、pDown-DsRed报告质粒转染进BMSCs，分为DsRed-BMSCs组（A组）和Shh-DsRed-BMSCs组（B组），荧光显微镜下观察DsRed表达，判断转染效率。48 h后使用RT-PCR法和Western印迹法检测Shh基因的表达情况，7 d后检测碱性磷酸酶（ALP）活性，28 d后茜素红染色检测骨髓间充质干细胞的成骨情况。

结果

慢病毒转染24 h后Shh-DsRed-BMSCs的转染率约为90%。同A组相比，B组能持续稳定高水平表达Shh mRNA和蛋白，同A组相比，B组的ALP活性更强，差异具有统计学意义（t=17.665，P<0.05），B组的茜素红染色表达情况明显高于A组。

结论

慢病毒转染音猬因子的骨髓间充值干细胞可以导致Shh的持续稳定高水平表达，并具有很高的成骨细胞分化能力，为骨质疏松的体内治疗提供了理论基础。

关键词: 间充质干细胞, 骨质疏松症, 转染, 音猬因子
Objective

To observe osteogenous differentiation of modified bone marrow stromal cells (BMSCs) which can express Sonic Hedgehog (Shh) stably and provide the treatment of osteoporosis in vivo feasibility.

Methods

BMSCs were infected with lentiviruses using a lentiviral vector containing the DsRed or the Shh-DsRed gene and then divided into Shh-DsRed- BMSCs group (group A) and DsRed- BMSCs group (group B). The transfection efficiency were evaluated by fluorescence microscopy using DsRed expression. The expression of Shh was tested by RT-PCR and Western-blot analysis after 48 hours. The osteogenous differentiation of BMSCs were analysed by ALP testing after 7 days, and tested by alizarin red staining after 28 days.

Results

The transfeetion efficiency was about 90% after 24 hours in Shh-DsRed-BMSCs. Compared with the group B, the Shh-DsRed-BMSCs can stably secrete Shh mRNA and protein. The ALP activity in Shh-DsRed-BMSCs group was significantly greater than those in DsRed-BMSCs. The alizarin red staining expression in group A was markedly higher than those in group B.

Conclusion

Shh-DsRed-BMSCs can long-term and stably secrete high levels of Shh, has a high ability of osteoblast differentiation and was then the basis for the subsequent treatment for osteoporosis in vivo.

Key words: Mesenchymal stromal cells, Osteoporosis, Transfection, Sonic hedgehog
图4~5 慢病毒载体转染BMSCs 5 d后。图4 DsRed-BMSCs（×100）；图5 Shh-DsRed-BMSCs（×100）
图6 Shh基因的mRNA的表达
图7 Shh-BMSCs中Shh蛋白的表达情况
图8~9 两组成骨诱导分化28 d后茜素红染色情况。图8 A组茜素红染色情况（×100）；图9 B组素红染色情况（×100）
图10 两组成骨诱导分化28 d后茜素红染色吸光度值的比较
图11 两组ALP活性定量检测比较
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