USP22调控骨髓间充质干细胞成骨分化及其差异蛋白质组学研究

Elucidating the molecular mechanisms that regulate human mesenchymal stem cells(hMSCs) differentiating into osteogenic lineage is important for the development of anabolic therapies for treatment of osteoporosis. Recently, we found that USP22 was up-regulated during early stage (day7) of osteogenic differentiation in hMSCs. Furthermore, down-regulation of USP22 attenuated osteogenic differentiation of hMSCs. Therefore, we propose that USP22 is a crucial modulator for osteogenic differentiation. In this study, we plan to up-regulate or down-regulate USP22 expression in hMSCs by lentivirus system. After differentiating hMSCs into osteoblasts in vitro, we will detect the changes of ALP, matrix mineralization and other osteoblastic functions, thus gain the direct evidences that USP22 is involved in osteogenic differentiation. Furthermore, we will use USP22-knockdown hMSCs and control group (7 days post osteogenic differentiation) for comparative proteomic analysis to reveal the differential proteins. Lastly, we will perform western blot analysis to confirm the mass spectrometry data. We expect that these results will elucidate the effects and molecular mechanisms of USP22 on regulating osteogenic differentiation of hMSCs, and understand the mechanisms of osteogenic differentiation and provide novel targets for osteoporosis therapy.

阐明间充质干细胞(hMSCs)定向分化为成骨细胞的分子机制对于防治骨质疏松症具有至关重要的意义。近期，我们发现原癌基因泛素特异性水解酶22（USP22）的转录在hMSCs向成骨细胞分化早期(第7天)显著激活，进而观察到下调该基因导致hMSCs成骨能力下降，据此提出USP22是调控成骨分化关键分子的新观点。本课题首先利用慢病毒载体分别介导USP22在hMSCs中过表达和下调，经成骨诱导培养，鉴定成骨细胞表型，以获得USP22调控成骨分化的直接证据；基于USP22的转录调控和蛋白质修饰的双重功能，利用蛋白质组学技术分析USP22敲除后hMSCs中蛋白质的差异表达，寻找并验证USP22作用的靶点。实验结果将揭示USP22在抗骨质疏松症中的作用，并有助于大规模发掘未知的USP22调控分子，对于深入理解hMSCs定向分化的调控网络并确立治疗骨质疏松新的新靶点具有积极意义。

阐明间充质干细胞(hMSCs)定向分化为成骨细胞的分子机制对于防治骨质疏松症具有至关重要的意义。根据研究计划，本课题利用慢病毒载体分别介导 USP22 在 hMSCs 中过表达和下调，经成骨诱导培养，鉴定成骨细胞表型，以获得 USP22调控成骨分化的直接证据；在此基础上，利用ChIP-seq技术寻找细胞中USP22潜在的靶基因，结合mRNA-seq结果，筛选出多个靶基因并予以验证，在转录水平锚定五个重要的USP22靶基因；进一步，利用蛋白质组学技术分析 USP22 敲除后 hMSCs 中蛋白质的差异表达，寻找USP22 去泛素修饰作用的靶蛋白，应用Western Blot对质谱结果进行验证。我们将转录组数据与蛋白组学数据进行联合分析，发现成骨细胞特异性转录因子Osterix可能是USP22的潜在靶点。研究结果初步阐明USP22调控对人类成骨细胞分化的影响，对于深入理解 hMSCs 定向分化的调控网络并确立治疗骨质疏松新的新靶点具有积极意义。