血小板因子4对骨髓间充质干细胞增殖和骨向分化潜能的负性调控作用和分子机制的研究

Osteoporosis (OP) is related to oral bone loss and the pathogenesis is still obscure. Many evidences exhibited that the impaired osteogenic differentiation on BMMSCs played a key role in osteoporosis. However, the cytokines in bone marrow microenvironment are important to osteogenic differentiation of BMMSCs. Platelet factor 4 (PF4) is one kind of cytokines in bone marrow. Previous research showed that PF4 can directly down-regulate the proliferation of hematopoietic stem cell. Moreover, PF4 down-regulated the ability of osteogenesis in mice model of radiation injury. Both aforementioned studies implied that PF4 maybe induce BMMSCs silencing and down-regulate ability on osteogenic differentiation during osteoporosis. The further preliminary experiment showed that PF4 was increased in the bone marrow accompanied with the processing of OP. The study in vitro exhibited that PF4 decreased the relative gene expression of RUNX2, SP7, and SPP1 through ERK1/2-MAPKs pathways and inhibited osteogenesis. Moreover, the galectin-1 can also inhibite the biological activity of PF4 except heparin. The aforementioned studies on PF4 and bone metabolism have not been reported. Therefore, we plan to study the relationship of PF4 and the process of OP firstly. Next, we focus on the PF4’s effects on the proliferation and osteogenic differentiation of BMMSCs in and ex vivo. And then, the mechanisms on down-regulating the osteogenic differentiation of BMMSCs by PF4 will be explored using western blot, real time RT-PCR, immunoprecipitation, or gene chip. Finally, the therapeutic target aimed at PF4 will be designed and experimented for osteoporosis. Thus, the study will shed new light on the mechanisms of the osteogenic differentiation of BMMSCs and prevention or treatment of osteoporosis.

骨质疏松与口腔骨丢失关系密切，其中骨髓间充质干细胞（BMMSCs）骨向分化能力受损是骨质疏松发生的关键因素之一。文献报道血小板因子4（PF4）可直接抑制造血干细胞增殖或放射损伤时下调BMMSCs成骨潜能，这提示PF4高表达可能是造成BMMSCs成骨潜能降低的原因之一。预实验表明，小鼠去势后髓内PF4的表达持续增高，可能与骨质疏松病程相关。进一步研究表明PF4通过ERK1/2-MAPKs信号通路下调RUNX2等基因表达来抑制BMMSCs骨向分化；而半乳凝集素1能抑制PF4的上述效应。以上PF4在骨代谢中的作用均未见文献报道。因此，本研究拟探索PF4在小鼠骨质疏松病程中的表达时相规律；进一步研究PF4在BMMSCs增殖和骨向分化中的负性调控作用及机制；最后评估半乳凝集素1防治骨质疏松和口腔骨丢失的效果。综合上述方面来解析PF4对BMMSCs的调控规律，为骨质疏松和口腔骨丢失的治疗奠定理论基础。

项目背景：血小板因子4是一种包含多种生理功能的趋化因子，是由存在于骨髓中的巨核细胞分泌而成。最近的研究表明在骨髓微环境中，血小板因子4可直接调节造血干细胞静默。但是血小板因子4对骨髓间充质干细胞和骨质疏松发挥何种作用仍不清楚。因此，本研究的目的是评估血小板因子4引发小鼠骨损伤的效果及潜在机制。.主要研究内容：通过卵巢切除法构建小鼠去势骨质疏松模型，并利用ELISA和qPCR探索血清和骨髓上清中血小板因子4的表达情况。同时采用microCT和组织学切片评估小鼠去势或外源给予血小板因子4后骨丢失的效果。进一步，采用CCK8实验和流式细胞术评估血小板因子4对骨髓间充质干细胞增殖和细胞周期的作用。同时，采用碱性磷酸酶和茜素红染色和定量的方法评估血小板因子4对骨髓间充质干细胞骨向分化潜能的抑制情况。最后，采用qPCR和WB评估血小板因子4抑制了何种骨髓间充质干细胞骨向分化基因以及分子通路。.重要结果和关键数据：小鼠去势后，整体上血清和骨髓上清中的血小板因子4水平显著高于假手术组。而且，小鼠腹腔注射外源性血小板因子4后股骨出现剂量依赖性的骨丢失，并且这种骨丢失是与成骨能力受损相关的。接下来，研究发现在骨髓间充质干细胞中加入血小板因子4后其细胞增殖、迁移和骨向分化能力出现显著下降，进一步研究发现细胞增殖下降是由于血小板因子4干扰了骨髓间充质干细胞的细胞周期有关。而且研究表明血小板因子4导致骨髓间充质干细胞多种成骨基因下调并且这是与抑制了ITGA5-FAK-ERK细胞通路有关。.科学意义：血小板因子4通过抑制去势小鼠骨形成从而导致骨丢失。血小板因子4通过抑制ITGA5-FAK-ERK细胞通路来减弱骨髓间充质干细胞骨向分化潜能。上述发现有利于深入了解骨质疏松的发生发展机制并为研究临床骨质疏松新疗法提供借鉴。