组蛋白去甲基化酶LSD1在人脂肪基质细胞成骨向分化中的作用及机制

Human adipose-derived stromal cells (hASCs) have become a highly attractive source of adult mesenchymal stem cells in bone tissue engineering because they can be obtained from adipose tissues that offer a more abundant and accessible pool of mesenchymal stem cells with a less invasive and less expensive procedures. How to effectively promote osteogenic differentiation of hASCs then has become a core issue in bone tissue engineering fields. Histone methylation has been regarded as a major mechanism of epigenetic modulation, especially in the lineage determination and differentiation of stem cells. A better understanding of how histone methyaltion contributes to osteogenic differentiation of hASCs will improve the efficiency of their osteoblastic lineage differentiation. In our previous work published on Stem Cells journal, we demonstrated that histone H3K4 trimethylation (H3K4me3) elimination governs the ability of osteogenic differentiation of hASCs. However, whether histone H3 lysine 4 monomethyl or dimethyl (H3K4me1 or H3K4me2) plays a role in this process has not been examined. Lysine specific demethylase 1 （LSD1） is a major histone demethylase that specifically catalyzes demethylation of H3K4me1 or H3K4me2. To characterize the role of LSD1 in osteogenic differentiation of hASCs, we will manipulate LSD1 expression in hASCs by using lentiviral vectors expressing siRNAs against LSD1 as well as lentiviral vetors carrying FLAG-LSD1. The difference of osteogenic differentiation between the knockdown /overexpression group and control group will be examined both in vitro and in vivo. In order to establish the internal link between LSD1 and osteogenic differentiation of hASCs, we will check the expression of osteogenesis-associated genes, such as ALP, OC and OSX between the LSD1 knockdown/overexpression hASCs and control hASCs. To further explore the underlying mechanism of LSD1-mediated regulation of osteogenic differentiation of hASCs, we will investigate the physical association between LSD1 and RUNX2, a master regulator of osteogenesis. Our Stem Cells paper reported that histone demethylase RBP2, which mainly take H3K4me3 as its substrate, regulates osteogenic differentiation of hASCs. In this study, we will further explore whether and how LSD1 and RBP2 coordinately regulate osteogenic differentiation of hASCs. Understanding the functional role of LSD1 will not only improve our knowledge of the epigenetic mechanisms governing the osteoblastic lineage differentiation of hASCs, but also provide valuable information on how to promote osteogenic differentiation of hASCs for the bone tissue engineering field. It may be possible to develop highly specific LSD1 inhibitors that can impact on epigenetic profiles of hASCs, thereby benefit the therapeutic pursuits for many human diseases.

人脂肪基质细胞(hASCs)具有多潜能分化、易扩增、易获得等优点，是骨组织工程较理想的干细胞来源。本课题组发现组蛋白H3K4三甲基去甲基化酶RBP2在hASCs成骨向分化中发挥着重要作用,初步证实以组蛋白甲基化为代表的表观遗传学调控与hASCs成骨向分化密切相关。基于此发现，本研究继续探讨具有H3K4一甲基和二甲基去甲基化酶活性的LSD1在hASCs成骨分化中的作用和机制，为构建hASCs成骨向分化的表观遗传学调控网络提供依据。本研究拟敲低以及过表达LSD1，分别从基因和蛋白水平检测其在hASCs成骨向分化中的作用和机制，探讨其与成骨相关基因及重要转录因子Runx2的关系，明确其与去甲基化酶RBP2是否协同调节成骨向分化。探讨hASCs成骨分化的表观遗传调控机制（即以染色质为基础的基因表达调控），将为今后利用小分子药物或相应酶抑制剂进行表观治疗或干预从而为促进hASCs成骨向分化奠定基础。

人脂肪基质细胞(hASCs)具有多潜能分化、易扩增、易获得等优点，是骨组织工程较理想的干细胞来源。近年来表观遗传学的兴起使我们想到探索hASCs成骨向分化的表观遗传学机制，从而通过改变hASCs的表观遗传学特性来促进其成骨向分化。本课题组发现组蛋白H3K4三甲基去甲基化酶RBP2在hASCs成骨向分化中发挥着重要作用,初步证实以组蛋白甲基化为代表的表观遗传学调控与hASCs成骨向分化密切相关。基于此发现，本研究继续探讨具有H3K4一甲基和二甲基去甲基化酶活性的LSD1在hASCs成骨分化中的作用和机制，为构建hASCs成骨向分化的表观遗传学调控网络提供依据。.. 本研究成功构建LSD1 RNAi及过表达质粒，包装慢病毒感染hASCs从而实现LSD1的敲低及过表达。通过体外实验（碱性磷酸酶，茜素红的染色及定量实验）和体内实验（裸鼠异位成骨模型）证明了LSD1抑制hASCs的成骨向分化。比较实验及对照组成骨相关基因的表达可见LSD1抑制了成骨相关基因的表达，ChIP实验表明LSD1能直接调节OC及Osx的表达，并且该调节依赖于其去甲基化活性。使用LSD1的小分子抑制剂来修饰细胞的表观遗传状态从而影响基因表达是近来的研究热点。我们通过实验证明LSD1非特异性抑制剂帕吉林及特异性抑制剂CBB1007均能够促进hASCs细胞的成骨向分化。并通过体内外实验证明了LSD1抑制剂帕吉林对老年鼠的骨质疏松状态有一定的挽救作用，对去势鼠的骨质疏松形成有一定的预防作用。.. 本项目将在本课题组以往研究的基础上，进一步探讨组蛋白H3K4位点的甲基化水平对hASCs成骨向分化的影响。阐明其作用及机制对于建立hASCs成骨向分化的表观遗传学调控网络，从而深入认识hASCs成骨向分化的表观遗传学机制(即以染色质为基础的基因表达调控)具有重要作用。本项目的完成也将进一步丰富表观遗传学中组蛋白密码学说的内容。我们通过LSD1的抑制剂促进hASCs的成骨向分化，并通过抑制剂来治疗和预防骨质疏松，即通过小分子药物修饰细胞的表观遗传状态从而影响基因表达，将表观干预引入骨组织工程学，也是有着非常重要的意义。表观治疗在肿瘤等疾病的治疗中已初显价值，该方法也必将在口腔颌面部组织工程化骨的应用或相关干细胞治疗技术方面展示良好的前景。