基于iTRAQ技术的脱氢表雄酮(DHEA)促进骨髓间充质干细胞向成骨细胞分化机制研究

Incidence of osteoporosis has increased year by year in a aging population. Aging causes reduced ability to differentiation of bone marrow mesenchymal stem cells into osteoblasts and the incidence of osteoporosis is closely related. Studies have shown that DHEA can improve the ability of differentiation of bone marrow mesenchymal stem cells into osteoblasts. So far, however, protein molecules and DHEA to promote research on osteogenic differentiation of mesenchymal stem cells, have not been reported. HMSCs osteogenic induction system proposed for this project ± DHEA, collected at different time points of drug treatment group and a control group of g using iTRAQ quantitative Proteomics technology to create drug treatment group and control group differences in protein expression profiles, based on differences of translation and transcription level of validation of protein molecules with two-dimensional gel electrophoresis of semi-quantitative results corroborate. Select target 2-3 potential molecular markers using gene transfer technology or RNAi technology, to filter out the important differences between protein molecules function verification analysis of the molecules in the DHEA promote the role of differentiation of mesenchymal stem cells into osteoblasts, to further explore the protein value in increasing stem cells in osteogenic differentiation potential provides a theoretical and experimental basis.

伴随人口老龄化，骨质疏松发病率有逐年增高趋势。老龄化引起骨髓间充质干细胞成骨分化能力下降与骨质疏松发病存在密切关系。研究表明DHEA能够提高骨髓间充质干细胞成骨分化能力。然而目前为止，对蛋白质分子与DHEA促进间充质干细胞成骨分化能力的研究，未见报道。本项目拟采用hMSCs成骨诱导体系±DHEA，采集不同时间点的药物处理组与对照组蛋白，运用定量蛋白质组学iTRAQ技术创建药物处理组和对照组的差异蛋白表达谱，通过对差异性蛋白质分子翻译水平和转录水平的验证与双向凝胶电泳的半定量结果相印证。选择2-3个潜在分子标志物为目标，运用基因转染技术或RNAi技术，对筛选出的重要差异分子进行蛋白功能验证，分析该分子在DHEA促进间充质干细胞成骨分化过程中的作用，为进一步探索该蛋白在提高干细胞成骨分化能力潜在的应用价值提供理论依据和实验基础。

研究表明，骨质疏松症(OP)是由于成骨和破骨细胞平衡受破坏引起，而成骨又与骨髓间充质干细胞(BMSCs)的功能存在密切联系。脱氢表雄酮(DHEA)是可由肾上腺分泌的一种物质，OP患者通过口服DHEA能有效改善症状。由此我们推测，DHEA在促进BMSCs向成骨分化中起到重要作用。本研究以人骨髓间充质干细胞(hBMSCs)为研究对象，验证DHEA对hBMSCs作用后相关成骨基因表达情况；以同位素标记相对和绝对定量技术(iTRAQ)检测药物处理组和对照组的差异蛋白表达谱，并以RT-PCR对筛选出的重要差异分子进行验证。我们的研究结果表明：(1) DHEA+成骨诱导体系对促进hBMSCs成骨分化效果最佳；成骨基因在DHEA+诱导组中也明显上调。(2)质谱分析结果显示：在DHEA+成骨诱导体系中，蛋白表达数量较空白对照组和实验对照组明显上升；通过质谱分析及PCR验证，我们发现DHEA很可能通过CHAD/JAK/STAT信号通路干扰下游关键基因干扰素调节因子9（IRF9）的表达，从而参与到诱导成骨形成的过程。我们的研究为进一步探索CHAD在促进hBMSCs成骨分化上提供了理论依据和实验基础，同时也为DHEA抗OP治疗靶点的研究提供了新的思路。