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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2017, Vol. 37 Issue (10): 8-15    DOI: 10.13523/j.cb.20171002
研究报告     
DLX1对BMP9诱导的间充质干细胞C3H10T1/2成骨分化的影响
徐丽, 吉彩霞, 刘晓骅, 喻婷婷, 罗进勇
重庆医科大学检验医学院 重庆医科大学检验医学教育部重点实验室 重庆 400016
Effects of DLX1 on BMP9-induced Osteogenic Differentiation of C3H10T1/2 Mesenchymal Stem Cells
XU Li, JI Cai-xia, LIU Xiao-hua, YU Ting-ting, LUO Jin-yong
Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Chongqing Medical University, Chongqing 400016, China
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摘要： 目的：分析和确认转录因子DLX1在骨形态发生蛋白9（bone morphogenetic protein 9，BMP9）诱导的间充质干细胞C3H10T1/2成骨分化中的作用。方法：首先，BMP9腺病毒感染C3H10T1/2细胞，RT-PCR和Western blot检测DLX1表达变化；随后，利用重组腺病毒技术分别过表达DLX1和RNA干扰（RNA Intenference，RNAi）抑制DLX1的表达，并利用碱性磷酸酶（Alkaline Phosphatase，ALP）染色、钙盐沉积实验（茜素红染色），免疫细胞化学检测骨钙素（osteocalcin，OCN）表达和裸鼠皮下异位成骨实验分析DLX1对于BMP9诱导的C3H10T1/2细胞成骨分化的影响。荧光素酶报告基因实验和Western blot分析DLX1对于BMP9诱导的C3H10T1/2细胞Smad1/5/8信号途径的影响。结果：BMP9可以促进C3H10T1/2细胞中DLX1基因和蛋白表达水平；过表达DLX1在体外可进一步促进BMP9诱导的C3H10T1/2细胞的ALP活性、钙盐沉积以及OCN的表达，过表达DLX1亦可促进BMP9诱导的裸鼠皮下异位成骨；反之，RNAi抑制DLX1表达后，由BMP9诱导的C3H10T1/2细胞的ALP活性、钙盐沉积、OCN表达和裸鼠皮下异位成骨均相应受到抑制。过表达DLX1可进一步增强BMP9诱导的C3H10T1/2细胞中Smad/1/5/8的转录调控活性，RNAi降低DLX1表达则可抑制BMP9诱导Smad/1/5/8的转录调控活性。但是，无论过表达DLX1和RNAi降低DLX1表达均不会对BMP9诱导的Smad/1/5/8磷酸化造成影响。结论：DLX1可以调节BMP9诱导的间充质干细胞C3H10T1/2细胞成骨分化，其调节作用可能是通过影响Smad1/5/8信号的转录调控活性而实现的。
关键词： 间充质干细胞;  同源异形盒基因;  DLX1;  骨形态发生蛋白;  成骨分化    
Abstract: Objective:To confirm the effects of DLX1 on BMP9-induced osteogenic differentiation of C3H10T1/2 mesenchymal stem cells.Methods:C3H10T1/2 cells were infected with recombinant adenoviruses expressing BMP9.Expression level of DLX1 upon BMP9 stimulation was measured by RT-PCR and Western blot. Then, the effects of DLX1 on BMP9-induced osteogenic differentiation of C3H10T1/2 cells were determined by ALP staining, Alizarin red S staining, immunocytochemical and entopic bone formation assay. Luciferase reporter assay and Western blot were conducted to analyze the activation level of Smad1/5/8 signal. Results:BMP9 increased the DLX1 expression of C3H10T1/2 cells. BMP9-induced ALP activity, calcium deposition and OCN expression of C3H10T1/2 cells in vitro was enhanced by overexpression of DLX1, but was inhibited by RNAi of DLX1. Overexpression of DLX1 led to an increase in new bone formation compared with the BMP9 group, however, RNAi of DLX1 resulted in a decrease in new bone formation in vivo. Moreover, BMP9-induced activation of Samd1/5/8 were accordingly increased along with overexpression of DLX1, and yet decreased along with DLX1 RNAi. Conclusions:Collectively, these above results implied us that DLX1 may play a private role in regulating BMP9-induced osteogenic differentiation of MSCs at last partly through Smad1/5/8 signal activation.
Key words: MSCs    Bone morphogenetic proteins    DLX    Osteogenic differentiation
收稿日期: 2016-12-18 出版日期: 2017-10-25
ZTFLH:  Q291  
基金资助: 国家自然科学基金（31071304，81272006）资助项目
通讯作者: 罗进勇,luojinyong@sina.com     E-mail: luojinyong@sina.com
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引用本文:

徐丽, 吉彩霞, 刘晓骅, 喻婷婷, 罗进勇. DLX1对BMP9诱导的间充质干细胞C3H10T1/2成骨分化的影响[J]. 中国生物工程杂志, 2017, 37(10): 8-15.

XU Li, JI Cai-xia, LIU Xiao-hua, YU Ting-ting, LUO Jin-yong. Effects of DLX1 on BMP9-induced Osteogenic Differentiation of C3H10T1/2 Mesenchymal Stem Cells. China Biotechnology, 2017, 37(10): 8-15.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20171002    或    https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I10/8

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