[1]李锐冬,邓忠良,张亚梅,等.不同剂量TGF-β1在BMP9诱导间充质干细胞成骨分化中的作用[J].第三军医大学学报,2014,36(02):98-104.
 Li Ruidong,Deng Zhongliang,Zhang Yamei,et al.Effects of different concentrations of TGF-β1 on BMP9-induced osteogenesis in mesenchymal stem cells[J].J Third Mil Med Univ,2014,36(02):98-104.
点击复制

不同剂量TGF-β1在BMP9诱导间充质干细胞成骨分化中的作用(/HTML )
分享到:

《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
36卷
期数:
2014年第02期
页码:
98-104
栏目:
论著
出版日期:
2014-01-30

文章信息/Info

Title:
Effects of different concentrations of TGF-β1 on BMP9-induced osteogenesis in mesenchymal stem cells
作者:
李锐冬邓忠良张亚梅汪洋尹良军陈亮
重庆医科大学附属第二医院:骨科,麻醉科
Author(s):
Li Ruidong Deng Zhongliang Zhang Yamei Wang Yang Yin Liangjun Chen Liang
Department of Orthopaedics, Department of Anesthesiology, Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400010, China
关键词:
骨形成蛋白9转化成因子β1间充质干细胞成骨分化
Keywords:
bone morphogenetic proteins transforming growth factor-β1 mesenchymal stem cells osteogenic differentiation
分类号:
R322.71;R329.21;R341
文献标志码:
A
摘要:
目的      分析联合应用不同剂量TGF-β1对BMP9诱导间充质干细胞成骨分化过程的影响,寻找最佳的促进BMP9成骨的TGF-β1剂量,为联合不同细胞因子促进组织工程骨再生寻找有效途径。      方法      应用鼠间充质干细胞株C3H10T1/2,以重组腺病毒法将其导入BMP9,再分别施加0、5、10、20 ng/mL TGF-β1蛋白处理,建立BMP9联合TGF-β1诱导间充质干细胞成骨分化模型。条件培养基法获取含活性良好的TGF-β1蛋白上清液;改良SEAP化学发光法和组织化学染色法检测碱性磷酸酶(alkaline phosphatase, ALP)表达及活性;茜素红S染色检测钙盐沉积;实时荧光定量PCR和Western blot法分别检测成骨相关基因Ⅰ型胶原(collagenⅠ,COLⅠ)、骨桥素(osteopontin, OPN)、骨钙素(osteocalcin, OCN)的mRNA转录表达水平和蛋白表达水平改变;流式细胞仪检测细胞周期;台盼蓝拒染法计数活细胞监测细胞增殖。      结果      5 ng/mL TGF-β1与BMP9联合处理较BMP9单独处理能更早、更多地提高ALP活性和引起钙盐沉积,持续处理17 d后钙盐沉积结果不再显示明显差异,TGF-β1单独处理与对照组间无明显差异。随TGF-β1剂量增加,ALP活性和钙盐沉积效应受到抑制。5 ng/mL TGF-β1与BMP9联合处理较BMP9单独处理使COLⅠ、OPN、OCN的mRNA转录水平和蛋白表达水平明显增高,COLⅠ增高变化发生较早和显著;TGF-β1应用剂量达到20 ng/mL出现基因转录和蛋白表达受到抑制,但COLⅠ升高趋势仍能维持。BMP9促进细胞增殖,TGF-β1对该效应起抑制作用,细胞周期转化表现G0/G1期阻滞。      结论      在BMP9诱导间充质干细胞成骨分化中,适量的TGF-β1联合应用具有促进作用;BMP9与TGF-β1在诱导成骨分化中可能存在协同作用关系。
Abstract:
Objective      The effectiveness of bone morphogenetic protein 9 (BMP9)-induced osteogenic differentiation of mesenchymal stem cells (MSCs) in combination with transforming growth factor-β1 (TGF-β1) is investigated to find the optimal concentration of TGF-β1 for bone regeneration of tissue engineering.       Methods      Mouse MSC C3H10T1/2 cell line was used to construct the osteogenic differentiation cell model by the steps of transfection of recombinant adenovirus with BMP9 and stimulation with different concentrations of TGF-β1 protein(0, 5, 10, and 20 ng/mL). Alkaline phosphatase (ALP) activity was assayed by the modified SEAP chemoluminescence assay and histochemical staining assay. Alizarin Red S staining was used for matrix mineralization. Real-time PCR and Western blotting were used to detect the mRNA and protein expression of collagen type Ⅰ (COL Ⅰ), osteopontin (OPN) and osteocalcin (OCN). Viable cell counting with trypan blue staining was used to detect cell proliferation and flow cytometry to test cell cycle.       Results      It was found that BMP9 combined with 5 ng/mL TGF-β1 produced higher ALP activity and more matrix mineralization at earlier period as compared to single BMP9 treatment group. Matrix mineralization had no significant difference after 17 days’s treatment. Meanwhile, TGF-β1 alone had no difference with control group. BMP9 combined with higher concentration TGF-β1 inhibited ALP activity and matrix mineralization in a dose-dependent manner. In real-time PCR and Western blot results, BMP9 combined with 5 ng/mL TGF-β1 increased mRNA transcription and protein expression of COL Ⅰ, OPN and OCN. COL Ⅰ expression increase was observed earlier and more significant.  TGF-β1 of 20 ng/mL combined with BMP9 decreased the expression of OPN and OCN but COL Ⅰ. TGF-β1 inhibited the proliferation of MSCs during the BMP9-induced osteogenic differentiation and arrested the cells in G0/G1 phase.       Conclusion      Suitable concentration of TGF-β1 can promote BMP9-induced osteogenic differentiation of MSCs. TGF-β1 and BMP9 may have synergistic effect.

参考文献/References:

李锐冬, 邓忠良, 张亚梅, 等. 不同剂量TGF-β1在BMP9诱导间充质干细胞成骨分化中的作用[J].第三军医大学学报,2014,36(2):98-104.

相似文献/References:

[1]任格,聂利,杨霞,等.Smad信号通路在骨形成蛋白9促进牙囊干细胞成骨向分化中的研究[J].第三军医大学学报,2018,40(12):1066.
 REN Ge,NIE Li,YANG Xia,et al.Smad signaling pathway regulates bone morphogenetic protein 9induced osteogenic differentiation of rat dental follicle stem cells in vitro[J].J Third Mil Med Univ,2018,40(02):1066.

更新日期/Last Update: 2014-01-21