自然铜（pyritum）是我国中医骨伤科的接骨要药,有"非煅淬不能用"的传统认识。前期研究发现，自然铜的矿相特征、Fe元素形态及溶出量随煅制温度、时间的不同呈现规律性的变化，自然铜煅淬品能促进成骨细胞增殖，缩短骨折愈合时间。项目将基于前期实验基础，制备代表性的自然铜煅淬样品。采用扫描电子显微镜（SEM）、电子探针X射线显微分析（EPMA）及ICP-MS联用等现代分析技术对自然铜不同样品的矿相特征，Fe、Zn、Cu、Mn、Pb、As等元素的形态进行研究；建立成骨细胞和骨折动物模型，检测ALP活性、Ca、P、BMP-2和IGFmRNA等相关效应指标，研究自然铜在煅淬过程中其矿相特征、元素形态及成骨效应三个方面的变化规律，并分析三者的内在联系，寻找自然铜治疗骨折的最佳元素配比。通过项目的研究阐明自然铜"非煅淬不能用"的炮制机理，为临床合理使用自然铜及其制剂提供指导，为中药矿物药的研究提供思路。

Pyritum is a very important crude mineral drug, which was usually been prescribed to set broken bone in traditional Chinese medicine (TCM). It is commonly believed that "pyritum cannot to be used to set bones before it was prepared by tempering process first". Former research of our laboratory revealed that the mineralography characters along with the element speciation of ferrum and its quantity of release undertook regularly changing with the temperature and time utilized in tempering process. Tempering processed pyritum can promote osteoblast proliferation and shortening the union time of broken bones. This project will be based upon the previous research. Representative tempering pyritum samples will be prepared. Those sophisticated techniques such as scanning electronic microscope (SEM), electron probe x-ray micro analyzer (EPMA) and ICP-MS will be utilized to explore the mineralography characters of tempering pyritum and examine the element speciation of ferrum (Fe), zinc (Zn), copper(Cu), manganese(Mn), lead(Pb) and arsenium(As) in those samples. Osteoblast cell model and bone fractured animal model will also be built up to examine the activity of ALP and effect related indicators such as Ca、P、BMP-2和IGFmRNA etc. The changing regularity of mineralography characters of pyritum, element speciation and osteogenetic effect will be investigated and the inner relationship between them will be revealed in order to find the optimized element ratio for catagmatic effect. This project will elaborate the processing mechanism of "pyritum cannot to be used to set bones before it was prepared by tempering process first", provide useful information and guideline for clinical use related to pyritum. Besides, we also try to pave a road for the research of crude mineral drugs in TCM.

自然铜（pyritum）为硫化物类矿物黄铁矿族黄铁矿（FeS2），具有疗折伤、续筋骨、散瘀排脓、止痛定惊之功效。自然铜临床自古就有“非煅淬不能用”的传统认识，而对其煅制后促进骨折愈合的作用机制和元素与药效的相关性等则缺乏研究。课题运用矿物学知识和现代仪器分析技术开展：（1）自然铜古今文献研究；（2）在不同煅制温度、时间和醋淬次数下制备自然铜样品，33种样品总铁含量为43.92%~56.97%；（3）采用X射线衍射（XRD）、傅里叶变换红外光谱（FTIR）、扫描电镜分析（SEM）以及电子探针X射线显微分析（EPMA）等技术探求自然铜炮制过程中物相及矿相显微结构的变化规律。自然铜生品为FeS2，400 ℃时少部分被氧化为Fe2O3；高于500 ℃，FeS2首先转化为Fe7S8，再逐渐被氧化为Fe3O4和Fe2O3，遵循从黄铁矿逐渐向磁黄铁矿、磁铁矿、赤铁矿转化的相变规律。FTIR和EPMA显示与XRD一致的结果；SEM电镜结果显示自然铜生品粉末颗粒较大，棱角明显，煅淬后则棱角和粒径大小显著下降；（4）采用电感耦合等离子体发射光谱法（ICP-OES）测定自然铜不同炮制品粉末及水煎液中微量元素含量，结果自然铜粉末中Fe，Zn，Mn等元素的含量随着煅制温度升高呈上升趋势，有害元素As的含量在高于600 ℃时显著降低，而Pb的含量则保持不变；随着煅制温度升高，水煎液中总铁含量和亚铁含量均呈升高的趋势；磁性测定结果显示，随着煅制温度升高、煅制时间延长和醋淬次数增加，自然铜炮制品的磁性逐渐增大。（5）以大鼠骨缺损模型比较其促进骨折愈合功效，自然铜能够促进骨折愈合，且煅淬品优于生品；以成骨细胞模型比较自然铜不同炮制品含药血清对成骨细胞活性的影响，结果自然铜含药血清能够促进成骨细胞的增殖、分化和矿化，且煅淬品的活性均优于生品；自然铜通过TGF-β1/Smad 信号通路调节成骨细胞矿化，其含药血清能提高ALP、Col I、OC 基因的表达和上调TGF-β1、p-Smad2/3、Smad4蛋白的表达，且煅淬品优于生品；（6）自然铜中Fe元素与其促进成骨细胞增殖有良好的相关性。课题为以自然铜为代表的矿物药的研究提供了思路和方法。