随着社会的飞速发展，外伤引起骨折的发生率显著增高，由于年龄及疾病因素等的影响，患者经常面临骨折不易愈合的状况以及随之而来的肢体废用性萎缩等并发症。间充质干细胞（MSCs）的增殖、迁移及成骨向分化在骨折愈合中具有重要作用，但如何在骨折愈合过程中对其进行有效调控仍需进一步研究。申请人在前期工作中发现神经肽Y对MSCs成骨向分化的调控可能是脑损伤合并骨折患者具有更快骨折愈合速度的原因； 褪黑素处理能够上调神经细胞中锌吸收转运体Zip1的表达。结合此前口服锌上调NPY表达的报道，提示褪黑素可能通过对Zip1及NPY水平的调控参与骨折愈合作用。本项目拟采用大鼠股骨骨折模型及原代骨髓间充质干细胞(BMSCs)作为研究对象，从体内及体外两个方面探讨褪黑素对Zip1及NPY水平的影响及其在促进MSCs成骨分化及骨折愈合中的作用及其可能机制，为更有效的骨折愈合药物及治疗手段的开发提供实验及理论依据。

With the rapid social development, the incidence of fracture caused by traumatic injuries is higher. Patients offen face the status of nonunion fractures and the subsequent complications such as disuse atrophy because of the effects as age, disease, etc. The proliferation, migration and osteoblast differentiation of mesenchymal stem cells are essential for bone healing, but the effective regulation needs to be studied further during bone healing. Our previous research has found that the regulation of MSCs osteoblast differentiation by neuropeptide Y may be the cause that patients with brain injury and fracture heal at a quicker rate than patients with simple fracture. Also, our studies have shown that melatonin treatment can increase the expression of zinc transporter, Zip1 in neurocyte. Combined with the earlier report that oral administration of zinc can up regulate the expression of NPY, we have a sufficient reason to speculate that melatonin may control the level of NPY and Zip1, and then play an important role in fracture healing. This project intends to use femoral fracture rats and bone marrow mesenchymal stem cells as research models to investigate the effects and mechanism of Zip1 and NPY on the promotion effect of MSCs osteoblast differentiation and fracture healing by melatonin in vitro and in vivo. This study is designed to provide further experimental bases and theoretical foundation for the research of fracture healing.

神经肽Y（neuropeptide Y，NPY）是人体内含量丰富的神经肽之一，其水平与骨折愈合过程具有相关性，能够通过对NPY Y1受体的调控影响骨折愈合，但其机制尚不明确；褪黑素（melatonin，MLT）在骨折愈合中具有重要作用，但其具体调控机制仍不明确，因此本课题通过体内实验明确褪黑素对NPY水平的影响及NPY在褪黑素促进骨折愈合过程中的作用，并在细胞水平上验证褪黑素及NPY对MSCs成骨向分化的调控，明确褪黑素促进骨折愈合的分子调控机制。本实验通过体内实验和体外实验进行验证：（1）建立大鼠股骨骨折模型，分别应用褪黑素（MLT）及NPY拮抗剂干预模型，对大鼠骨折愈合情况进行检测，结果显示MLT可促进成骨细胞和胶原纤维的生成，抑制破骨细胞生成，增强血清中ALP及NPY活性，并上调NPY及NPY1R基因和蛋白的表达，而NPY拮抗剂可抑制成骨细胞和胶原纤维的生成，促进破骨细胞形成，逆转MLT引起的ALP与NPY活性的增强及MLT引起的NPY及NPY1R基因和蛋白表达的上调。以上结果说明MLT可促进大鼠股骨骨折的愈合，加入NPY拮抗剂后可抑制大鼠股骨骨折的愈合，提示MLT通过促进NPY及NPY1R的表达进而促进大鼠骨折的愈合。（2）诱导大鼠骨髓间充质干细胞成骨向分化，验证MLT对大鼠骨髓间充质干细胞成骨向分化的影响，结果表明MLT可上调成骨标志因子ALP、COL1A1、OCN及Runx2基因的表达，上调COL1A1及Runx2蛋白的表达，增强ALP活性，促进MLT诱导的细胞成骨向分化，并上调NPY及其受体NPY1R蛋白的表达；NPY拮抗剂可抑制MLT诱导的细胞成骨向分化，并下调NPY及其受体NPY1R蛋白的表达。以上结果表明MLT通过促进NPY及其受体NPY1R的表达进而促进骨髓间充质干细胞的成骨向分化。本项目的完成为骨折愈合的临床治疗及更有效的临床药物的开发提供新的思路及研究靶点，具有重要的理论意义和应用价值。