KDM和KMT调控老年骨质疏松BMSC成骨-成脂分化失衡的表观遗传机制研究

Osteoporosis has a close relationship with the transition of aged BMSC from osteogenesis to adipogenesis. It was reported that overexpression of histone demethylases KDM4B or KDM6B significantly enhanced osteogenic differentiation while knockdown of KDM4B or KDM6B increased adipogenic differentiation. However, the mechanism of how histone demethylases (KDM) and histone transmethylases (KMT) regulate the imbalance of osteogenic and adipogenic differentiation of aged BMSC has not been studied. We previously found that UTX increased significantly while EZH2 decreased significantly in the young BMSC after osteogenic stimulation, however, EZH2 and UTX changed slightly after osteogenic differentiation of aged BMSC. We hypothesize that UTX and EZH2 are the key factors regulating the imbalance of osteogenesis and adipogenesis of aged BMSC. We will further identify the target transcription factors inducing MSC cell lineage commitment of UTX and EZH2 by ChIP and prove how UTX and EZH2 regulate the imbalance of osteogenic and adipogenic differentiation of aged BMSC. Secondly, we will try to promote the osteogenic differentiation of aged BMSC through overexpression and knockdown of UTX and EZH2.Finally, the epigenetically regulated BMSC will be injected into the bone marrow cavity of OVX mice to treat osteoporosis. We hope to find a new way to treat osteoporosis through epigenetic modifications.

骨质疏松骨中骨与脂肪组织"此消彼长"的现象与老化骨髓间充质干细胞（BMSCs）的成骨-成脂分化失衡有关。已证实组蛋白去甲基化酶KDM4B和KDM6B能使BMSC成骨分化，而低表达则诱导成脂分化，但尚不明确组蛋白甲基化转移酶（KMT）和KDM与衰老BMSC成骨-成脂分化失衡现象之间的联系。课题组前期实验中发现年轻BMSC在成骨刺激后UTX显著升高、EZH2显著降低，而衰老BMSC的变化没有那么明显。本研究假设UTX和EZH2是调控老年BMSC成骨-成脂分化失衡的关键因子，拟通过敲减或过表达UTX和EZH2,明确UTX和EZH2与RUNX2、BMP等成骨转录因子的关系，并尝试通过调控UTX和EZH2的表达来扭转衰老BMSC成骨-成脂分化失衡现象，最后验证经表观调控的衰老BMSC能否改善裸鼠骨质疏松症状。如获成功，本研究将为今后利用小分子药物或蛋白酶抑制剂进行骨质疏松症的表观治疗或干预打下基础。

本研究的目的是要阐明EZH2在骨性关节炎（OA）发病中的作用并研究调控EZH2活性对OA的疗效。我们用western blotting法分别测定了正常和OA病人膝关节软骨中EZH2的表达量，用PCR法测定软骨细胞过表达或敲减EZH2表达后软骨细胞肥大相关基因的表达量变化，还用染色质免疫沉淀（ChIP）PCR法分析了软骨细胞中Wnt抑制因子SFRP1启动子上的组蛋白甲基化水平。我们还对OA小鼠的关节软骨进行了组织学检测以评估EZH2抑制剂EPZ005687对软骨细胞的保护作用。通过分析我们发现正常和OA病人膝关节软骨中EZH2的表达量明显不同，OA病人软骨中EZH2表达量明显较正常人的高。将正常软骨细胞过表达EZH2表达后软骨细胞肥大相关基因 Indian Hedgehog, MMP-13, ADAMTS-5 and COLX 的表达量明显升高，而敲减EZH2表达后以上基因表达量则明显下降。此外，软骨细胞过表达EZH2后，Wnt抑制因子SFRP1的活性明显下降，相应的，ChIP PCR检测的结果也证实SFRP1启动子上的组蛋白出现了过甲基化。最后，我们发现将EZH2抑制剂EPZ005687注射入OA小鼠膝关节后明显延缓了OA的发展。以上结果验证了EZH2在OA发病过程中的作用，药物抑制EZH2表达也许会成为治疗OA的一种有效途径。