线粒体去乙酰化酶SIRT3参与脂肪源性干细胞成骨分化调控的机制研究

Adipose derived stem cells can be isolated from fat tissues and have potentials to differentiate into a variety of cells. Recent studies suggest promising applications of ADSCs in dental implant osseointegration, periodontal regeneration. Mitochondria are tightly involved within regulations of cell proliferation, differentiation, senescence and apoptosis. Besides as a power supplier, mitochondria also plays vital roles in regulating cell signal transduction pathway. SIRT3 within mitochondria, as a member of Sirtuins family, is NAD+-dependent deacetylase. It can regulate cell metabolism and signaling transduction by mediating deacetylation of various proteins. However, there are few studies concerning on the potential role of SIRT3 involved during osteogenesis of mesenchymal stem cells. Based upon previous studies, knockdown of SIRT3 expression by siRNA technique leads to significant decrease in osteogenic potential of ADSCs, along with increased oxidative stress within cells. Our study aims to explore the potential roles of mitochondria and FOXO3/β-catenin/Runx2 in SIRT3-mediated regulation of ADSCs osteogenic differentiation. This may provide novel evidence for SIRT3 to act as a new target to be modulated during applications of ADSCs in bone tissue engineering.

脂肪源性干细胞在促进口腔种植体骨整合、牙周组织重建及颅颌面骨缺损修复中有着广阔的应用前景。细胞增殖、分化、衰老及凋亡等过程与线粒体功能活性调节关系密切。位于线粒体内的SIRT3，可介导线粒体内蛋白去乙酰化修饰，从而参与物质的代谢、能量合成及信号转导。但至今鲜有关于SIRT3参与调控间充质干细胞分化机制的研究报道。基于本课题的前期研究发现脂肪源性干细胞在成骨分化过程中SIRT3表达量明显升高，而使用siRNA技术抑制SIRT3表达后，成骨能力显著减弱，并伴随细胞内氧化应激的改变。本课题拟从“线粒体”角度出发，探讨SIRT3可能通过介导MnSOD及PGC-1a等与线粒体稳态相关因子的表达，参与脂肪源性干细胞成骨分化过程中线粒体数目、形态及功能活性等相关调控；同时研究SIRT3是否通过FOXO3/β-catenin/Runx2等通路，介导成骨分化的调控。

脂肪源性干细胞在促进口腔种植体骨整合、牙周组织重建及颅颌面骨缺损修复中有着广阔的应用前景。位于线粒体内的去乙酰化酶Sirtuin 3(SIRT3)，可介导线粒体内蛋白去乙酰化修饰，从而参与物质的代谢、能量合成及信号转导。本项目体外提取大鼠脂肪源性干细胞（adipose-derived stem cells, ASCs）并诱导成骨分化，通过以慢病毒为载体调控SIRT3表达，探索及验证SIRT3通过调节细胞内氧化应激状态从而影响ASCs成骨分化。同时在糖尿病状态下，骨组织内会有大量糖基化终末产物（advanced glycation end products,AGEs）聚集，而大量AGEs会抑制干细胞的成骨分化能力。鸢尾素irisin作为一种骨骼肌分泌的肌肉因子，可介导运动给多种靶器官所致的效应，因此本项目在前期基础上，使用一定浓度的AGEs体外模拟糖尿病微环境，探究irisin在AGEs条件下对ASCs成骨能力的影响及SIRT3所介导的调控机制。实验结果显示：1、SIRT3介导细胞内氧化应激状态参与调控ASCs成骨分化能力。2、AGEs条件下ASCs成骨分化能力明显减弱，而irisin处理可缓解由AGEs所致的ASCs成骨能力降低，此过程由SIRT3所介导。3、进一步探寻SIRT3所介导的分子机制，发现irisin对AGEs条件下ASCs增殖和成骨能力的改善，由SIRT3调控细胞、线粒体内的氧化应激状态、线粒体功能、细胞内多种抗氧化物酶活性、线粒体生物合成及线粒体自噬等多方面所介导。4、构建2型糖尿病大鼠模型，在其颅骨上建立临界性骨缺损，局部移植包裹ASCs和irisin的凝胶会明显促进骨缺损愈合。上述结果阐述了SIRT3介导irisin改善AGEs条件下ASCs增殖或成骨分化的分子机制，揭示了在糖尿病状态下irisin对骨组织再生的调控作用，并为SIRT3作为干预靶标以促进糖尿病状态下骨组织再生提供理论依据。