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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 ›› 2021, Vol. 07 ›› Issue (02) : 65 -72. doi: 10.3877/cma.j.issn.2096-0263.2021.02.001

所属专题： 文献；

基础研究

BMP-2/Smads信号通路促进骨代谢失衡大鼠骨髓间充质干细胞的成骨分化
张伟丽1, 罗敏2, 彭江3, 赵斌4,†()   
  1. 1. 100039　北京，解放军总医院第三中心住院与病案管理科
    2. 100039　北京，解放军总医院泌尿外科
    3. 100853　北京，解放军总医院骨研所
    4. 100853　北京，解放军总医院第三中心骨科
  • 收稿日期:2020-03-21 出版日期:2021-04-05
  • 通信作者: 赵斌
  • 基金资助:
    国家自然科学基金项目(NSFC81472092)

The BMP-2/Smads signaling pathway promote the osteogenic differentiation of mesenchymal stem cells in rat with bone metabolism imbalance

Weili Zhang1, Min Luo2, Jiang Peng3, Bin Zhao4,†()   

  1. 1. Inpatient and Medical Record Management Division of 3rd Medical Center, Chinese PLA General Hospital, 69 Yong ding Road, Hai dian District, Beijing 100039, China
    2. Urology Department of 3rd Medical Center, Chinese PLA General Hospital, 69 Yong ding Road, Hai dian District, Beijing 100039, China
    3. Institute of Orthopaedics, Chinese PLA General Hospital, 28 Fuxing Road, Haidian District, Beijing 100853, China
    4. Orthopaedic Department of 3rd Medical Center, Chinese PLA General Hospital, 69 Yong ding Road, Hai dian District, Beijing 100039, China
  • Received:2020-03-21 Published:2021-04-05
  • Corresponding author: Bin Zhao
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张伟丽, 罗敏, 彭江, 赵斌. BMP-2/Smads信号通路促进骨代谢失衡大鼠骨髓间充质干细胞的成骨分化[J]. 中华老年骨科与康复电子杂志, 2021, 07(02): 65-72.

Weili Zhang, Min Luo, Jiang Peng, Bin Zhao. The BMP-2/Smads signaling pathway promote the osteogenic differentiation of mesenchymal stem cells in rat with bone metabolism imbalance[J]. Chinese Journal of Geriatric Orthopaedics and Rehabilitation(Electronic Edition), 2021, 07(02): 65-72.

目的

本研究旨在探讨BMP-2/Smads信号通路对骨代谢失衡大鼠骨髓间充质干细胞（rBMSCs）成骨分化的影响。

方法

切除雌性SD大鼠双侧卵巢，建立大鼠骨代谢失衡模型，即骨质疏松（osteoporosis，OP）模型。通过成骨诱导鉴定rBMSCs的成骨分化。采用脂肪诱导和流式细胞术（FCM）检测脂肪分化和rBMSC表面标志物的表达。将rBMSCs分为空白组、NC组（转染大鼠rBMSCs的阴性对照质粒）、SI-BMP2组（转染大鼠rBMSCs具有低表达BMP-2质粒）和OE-BMP2组（转染大鼠rBMSCs具有高表达BMP-2质粒）。Western blot和定量实时聚合酶链反应（qRT-PCR）检测关键因子和成骨相关因子的表达水平。茜素红染色观察钙化结节的形成。碱性磷酸酶染色测定ALP活性。

结果

OP组大鼠体重增加，骨密度下降（均P<0.01）。OP大鼠rBMSCs具有成骨分化和成脂分化能力，CD44（91.3±2.9%）和CD105（94.8±2.1%）高表达。与空白组相比，OE-BMP2组BMP-2和SMAD1水平升高，钙化结节和ALP阳性染色面积增加（均P<0.05）。OE-BMP2组RUNX2、OC、OPN表达水平均高于空白组（均P<0.05）。SI-BMP2组的结果与OE-BMP2组相反。

结论

BMP-2激活的BMP信号通路可促进骨代谢失衡大鼠rBMSCs的成骨分化。

关键词: BMP信号通路, 骨髓间充质干细胞, 骨质疏松, 成骨分化
Objective

The purpose of this study was to investigate the effect of BMP-2/Smads signaling pathway on osteogenic differentiation of bone marrow mesenchymal stem cells (rBMSCs) in rat with bone metabolism imbalance.

Methods

Ovaries of female SD rats were excised and OP model was established. The osteogenic differentiation of rBMSCs was identified by osteogenic induction. Fat induction and flow cytometry (FCM) were used to detect fat differentiation and the expression of rBMSC surface markers. rBMSCs were divided into blank group, negative control (NC) group (transfected with NC BMP-2 plasmid), SI-BMP2 group (transfected with low expressing BMP-2 plasmid) and OE-BMP2 group (transfected with high expressing BMP-2 plasmid). Western blot and quantitative real-time PCR were used to detect the expression of key factors and osteogenic related factors. The formation of calcified nodules was observed by alizarin red staining. ALP activity was determined by alkaline phosphatase staining.

Results

In OP group, the weight and bone mineral density of rats increased (P<0.01). rBMSCs of OP rats have the ability of osteogenic differentiation and lipogenic differentiation. CD44 (91.3±2.9%) and CD105 (94.8±2.1%) are highly expressed. Compared with the blank group, the level of BMP-2 and Smad1 in OE-BMP2 group increased, and the area of calcified nodule and ALP positive staining increased (P<0.05). The expression level of Runx2, OC and OPN in OE-BMP2 group was higher than that in blank group (P<0.05). The results of SI-BMP2 group were opposite to those of OE-BMP2 group.

Conclusion

The BMP signaling pathway activated by BMP-2 can promote the osteogenic differentiation of rBMSCs in OP rats.

Key words: BMP signaling pathway, Bone marrow mesenchymal stem cells, Osteoporosis, Osteogenic differentiation
图1~2 OP模型组与正常组大鼠体重及骨密度的比较。图1　OP模型组和正常组大鼠体重；图2　OP模型组和正常组大鼠骨密度
图5~8 OP大鼠rBMSCs表型特征的鉴定
图9~11 OP大鼠rBMSCs成骨和成脂分化的鉴定。图9　成骨诱导21 d后茜素红染色；图10　光镜观察7 d后成脂；图11　成脂诱导14 d后油红O染色
图12~14 各组BMP相关蛋白表达的比较。图12　BMP相关蛋白mRNA表达；图13　BMP相关蛋白的蛋白表达；图14　BMP相关蛋白表达的统计分析
图25~27 各组成骨相关基因Runx2、OC和OPN的表达。图25　Runx2、OC、OPN的mRNA表达；图26　Runx2、OCT、OC、OPN的蛋白质；图27　Runx2、OC和OPN蛋白质表达的统计分析
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