• 1. Department of Pharmacology, West China School of Pharmacy, Sichuan University, Chengdu Sichuan, 610041, P. R. China;
  • 2. Department of Orthopaedics and Traumatology, Li Ka Shing Faculty of Medicine, the University of Hong Kong;
  • 3. Regenerative Medicine Research Center, Chengdu Sichuan, 610041, P. R. China;
  • 4. Division of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University;
DENGLi, Email: dengli2000@21cn.com
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Objective To analyze the expression profile changes of osteogenic-related genes during spontaneous calcification of rat bone marrow mesenchymal stem cells (BMSCs). Methods BMSCs were isolated from 3-day-old healthy Sprague Dawley rats;cells at the 4th generation were used to establish the spontaneous calcification model in vitro. Spontaneous calcification process was recorded by inverted phase contrast microscope observation and alizarin red staining after 7 and 14 days of culture. For gene microarray analysis, cell samples were collected at 0, 7, and 14 days after culture; the differentially expressed genes were analyzed by bioinformatics methods and validated by real-time quantitative PCR (RT-qPCR) assay. Results Rat BMSCs calcified spontaneously in vitro. When cultured for 7 days, the cells began to aggregate and were weakly positive for alizarin red staining. After 14 days of culture, obvious cellular aggregation and typical mineralized nodules were observed, the mineralized nodules were brightly positive for alizarin red staining. A total of 576 gene probe-sets expressed differentially during spontaneous calcification, corresponding 378 rat genes. Among them, 359 gene probe-sets expressed differentially between at 0 and 7 days, while only 13 gene probe-sets expressed differentially between at 7 and 14 days. The 378 differentially expressed genes were divided into 6 modes according to their expression profiles. Moreover, according to their biological functions, differentially expressed genes related to bone cell biology could be classified into 7 major groups:angiogenesis, apoptosis, bone-related genes, cell cycle, development, cell communication, and signal pathways related to osteogenic differentiation. In cell cycle group, 12 down-regulated genes were linked with each other functionally. Matrix metalloproteinase 13 (Mmp13), secreted phosphoprotein 1 (Spp1), Cxcl12, Mmp2, Mmp3, Apoe, and Itga7 had more functional connections with other genes. The results of genes Spp1, Mgp, Mmp13, Wnt inhibitory factor 1, Cxcl12, and cyclin A2 by RT-qPCR were consistent with that of gene microarray. Conclusion The first 7 days after rat BMSCs were seeded are a key phase determining the fate of spontaneous calcification. Multiple genes related with cell communication, bone-related genes, cell cycle, transforming growth factor-β signaling pathway, mitogen-activated protein kinase signaling pathway, and Wnt signaling pathway are involved during spontaneous calcification.

Citation: JIAOXuefeng, HUANGYongcan, HUANGYizhou, WUChengguang, DENGLi. EXPRESSION PROFILE OF OSTEOGENIC-RELATED GENES DURING SPONTANEOUS CALCIFICATION OF RAT BONE MARROW MESENCHYMAL STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(2): 133-141. doi: 10.7507/1002-1892.20140032 Copy

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