MAPK信号参与钙磷基纳米粒子诱导成骨系细胞自噬调控成骨分化的机制研究

Nano hydroxyapatite (NanoHAP) could promote cell attachment, proliferation and differentiation in bone regeneration, however, the underline molecular mechanisms is still not very clear. Recently, nanoparticles were defined as a novel class of autophagy activators and autophagy plays a pivotal role in skeletal cell homeostasis. It reported that an important autophagy-related gene leads to osteoblast differentiation through a mechanism involving elevated and prolonged MAPK activity. Thus, we hypothesize that "osteoblast autophagy induced by NanoHAP and Ca2+ could promote osteoblast differentiation through MAPK signaling pathway which could regulate the expression of osterix." To test this hypothesis, the nano material science, cytology and molecular biology will be used to confirm the hypothesis comprehensively. The effect of NanoHAP on osteoblast autophagy will be studied. The key biological signal pathway MAPK of osteoblast autophagy regulates osteoblast differentiation will also be studied. Then elucidate the mechanisms that autophagy and MAPK regulate osteoblast differentiation. The successful implementation of the project will help to evaluate the effects of nanomaterial for bone repair and regeneration.

纳米羟基磷灰石（NanoHAP）在骨再生中促进成骨系细胞的黏附、增殖和分化，但NanoHAP粒子调控成骨系细胞功能的分子机制尚缺乏深入的研究。依据纳米粒子具有诱导细胞自噬的共性、细胞自噬关键基因通过MAPK信号通路调控成骨分化等研究成果以及我们的研究基础，提出以下假说“NanoHAP诱导成骨系细胞发生自噬进而通过MAPK信号通路调控成骨特异转录因子Osterix的表达来促进成骨分化”。为验证这一假说，本项目拟采用纳米材料学、细胞学以及分子生物学的先进技术方法，研究NanoHAP粒子对成骨系细胞自噬的影响，探讨细胞自噬影响成骨系细胞分化的关键信号通路，从而探明NanoHAP粒子与细胞相互作用过程中细胞自噬及MAPK信号通路的调控机制，此项目如能成功实施将对钙磷基纳米材料在骨修复及再生中的生物学功效提供更深入的理论依据。

羟基磷灰石（HAP）是骨骼的主要无机成分，在骨骼形成和骨重塑中起着重要作用。合成的HAP结构类似天然HAP，已广泛应用于骨科和口腔科材料植入。已知纳米羟基磷灰石（Nano-HAP）在高浓度下容易团聚，Nano-HAP的分散可以很好地解决这个问题，但分散后Nano-HAP在成骨细胞分化过程中的生物学效应和作用机制仍未阐明。在本研究中，我们首先用化学沉淀法制备了Nano-HAP，通过高压灭菌方法将针状Nano-HAP转变为短棒状，并添加六偏磷酸钠来控制纳米颗粒的团聚。分散的纳米HAP与成骨细胞MC3T3E1共培养，发现会促进ALP活性、COL I分泌以及成骨相关marker基因表达，从而以剂量依赖的方式促进成骨分化。进一步的研究结果表明，Nano-HAP粒子在细胞内位于典型的自噬小体中，并促进了细胞自噬marker基因LC3II/LC3I的比率，提示Nano-HAP诱导了细胞自噬。此外，纳米HAP诱导自噬还依赖于MTOR信号通路的抑制。总之，本项目研究结果显示，Nano-HAP通过调控自噬和mTOR信号通路以剂量依赖性的方式调节成骨细胞分化。我们的研究为深入理解Nano-HAP对成骨细胞的生物学效应提供了新的视角，并为Nano-HAP的临床应用提供了参考依据。