衰老微环境中HDAC9调控自噬影响骨髓间充质干细胞再生能力的机制研究

Plenty researches have proved that reduced regeneration capacity of mesenchymal stem cells (MSCs) caused by aging microenvironment is the main cause of bone mass loss in age-related osteoporosis. Our previous work proved that the inhibited regeneration of aged-MSCs is probably caused by decreased autophagy. However, the mechanism of how aging microenvironment regulates the autophagy in MSCs is still unclear. Some researchers suggest that epigenetics regulated the autophagy, which made the cells adapt the changed environment during the aging process. Protein acetylation, in particular acetylation autophagy-related (ATG) proteins, has emerged as a regulator of autophagy. Our preliminary experiments showed that the expression pattern of histone deacetylase 9 (HDAC9) significantly increased in senescence tissues. In addition, HDAC inhibitor could up-regulate the autophagy and rescue the function of age-related MSCs. Therefore, we infer that HDAC9 could affect the function of age-related MSCs via modulating the autophagy directly. In this study, we design three part experiments to demonstrated the intrinsic relationship among microenvironment, epigenetic and bio-characteristic of MSCs. The experiments include the investigation of HDAC9 on modulating the acetylation of specific histone target, the mechanism of HDAC9 regulates the autophagy via CHIP-seq and GRO-seq analyses. Finally, we testify the effect of regulating HDAC9 on age-related osteoporosis in mice model. This study will not only elucidate the mechanism of the age-related osteoporosis from a new perspective, but also provide a new therapeutic target for curing it.

衰老微环境抑制骨髓间充质干细胞（BMMSCs）再生是导致衰老相关骨质疏松的重要原因。我们前期研究证实BMMSCs自噬下降导致其再生能力降低并引发骨质疏松，然而衰老微环境如何影响BMMSCs自噬的机制仍不明确。表观遗传可调控自噬使细胞在衰老过程中发生功能改变，其中自噬相关蛋白的乙酰化被认为是其重要调节因素。我们预实验发现组蛋白去乙酰化酶9（HDAC9）在衰老骨、骨髓及BMMSCs中显著升高，而其抑制剂能有效恢复衰老BMMSCs自噬及其再生能力。由此，我们推测衰老微环境下，HDAC9可能发挥调控BMMSCs自噬的重要作用。本课题拟从HDAC9修饰关键组蛋白乙酰化位点的确定，HDAC9调控自噬的分子机制及对BMMSCs再生能力的影响，体内改变HDAC9治疗衰老骨质疏松三方面探讨微环境、表观调控及干细胞再生能力之间的关系。我们期望从新的角度阐明衰老骨质疏松的分子机制，并为其预防及治疗提供新的思路。

本课题组围绕主要研究内容及预期目标，开展了以下几方面的研究工作：① 发现衰老骨组织及骨髓间充质干细胞（Bone marrow-derived mesenchymal stem cell, BMMSCs）中组蛋白去乙酰化9（histone deacetylase 9，HDAC9）表达均显著升高，且BMMSCs在连续传代后仍不影响HDAC9的表达变化：② 使用HDAC广谱抑制剂或者沉默HDAC9均能有效恢复衰老BMMSCs的功能；③ 发现衰老骨组织及BMMSCs中自噬及自噬潮均显著下降，且沉默HDAC9后，能有效恢复BMMSCs自噬水平；④ 通过 CHIP-seq 和 GRO-seq 高通量测序结合染色质免疫共沉淀，证明HDCA9/H3K9直接与自噬相关基因Beclin1、LC3a及LC3b的启动子结合，从而发挥抑制自噬基因表达的作用；⑤ 将HDAC9 siRNA注射入16月龄衰老小鼠骨髓腔，结果表明抑制HDAC9表达能有效增加衰老小鼠骨量抑制其骨髓脂肪含量，进一步分离BMMSCs，发现其自噬水平升高，成骨分化能力增强而成脂分化能力下降，证明HDAC9 siRNA能有效逆转骨衰老；⑥ 以第一作者和通讯作者发表SCI9篇，其中IF>5.0 SCI 6篇, 培养硕士研究生1名，博士研究生3名；参加国际特邀学术报告2次，国内特邀学术报告4次。