[1]贾祎佳,孙吉平,刘强,等.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,(06):341-345.[doi:10.3877/cma.j.issn.2096-0263.2018.06.005]
　Jia Yijia,Sun Jiping,Liu Qiang,et al.The effects of bone marrow stromal cells transferred with lentivirat—mediated Sonic Hedgehog (Shh) gene in osteoporosis[J].Chin J Geriatr Orthop Rehabil(Electronic Edition),2018,(06):341-345.[doi:10.3877/cma.j.issn.2096-0263.2018.06.005]
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转染音猬因子对骨髓间充质干细胞成骨分化的影响()
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中华老年骨科与康复电子杂志[ISSN:1674-3911/CN:11-9292/R]

卷:
期数:
2018年06期
页码:
341-345
栏目:
基础研究
出版日期:
2018-11-23

文章信息/Info

Title:
The effects of bone marrow stromal cells transferred with lentivirat—mediated Sonic Hedgehog (Shh) gene in osteoporosis
作者:
贾祎佳; 孙吉平; 刘强; 郝海虎; 朱剑; 吴斗
030032 太原，山西医学科学院 山西大医院骨科
Author(s):
Jia Yijia;  Sun Jiping;  Liu Qiang;  Hao Haihu;  Zhu Jian;  Wu Dou
Department of Orthopaedics, Shanxi Academy of Medical Sciences, Shanxi Dayi Hospital, Taiyuan 030032, China
关键词:
间充质干细胞;  骨质疏松症;  转染;  音猬因子
Keywords:
Mesenchymal stromal cells;  Osteoporosis;  Transfection;  Sonic hedgehog
DOI:
10.3877/cma.j.issn.2096-0263.2018.06.005
文献标志码:
A
摘要:
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。
Abstract:
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.

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备注/Memo

备注/Memo:
基金项目：山西省自然科学基金（2013011057-4）
更新日期/Last Update: 2018-11-23