生物活性玻璃陶瓷涂层介导的Hedgehog信号通路调控成骨细胞分化的研究

The bioactive glass ceramics has been successfully applied as Ti alloy coatings in the clinical therapy of bone. However, the effects of Hh signaling pathway regulating in the process of osteoblasts differentiation over bioactive glass ceramics have not been studied yet. In this research we will focus on the key molecules in the Hh signaling pathway which is regulating osteoblasts differentiation, and establish in vitro cell models for expression or inhibition Hh signaling pathway Gli-2 gene, that will also be treated by Hh signaling Ihh gene, the pathway antagonist cyclopamine, BMP-2 and BMP-2 antagonist. The key factors of Hh signaling pathway in the regulating of osteoblast adhesion, proliferation, differentiation, apoptosis and matrix calcification will be studied by using MTT, flow cytometric detection, immunofluorescence stain and immunohistochemical stains, RT-PCR, Western blot, scanning electron microscopy, the alkaline phosphatase (ALP) qualitative and quantitative analysis and alizarin red vital staining service (ARS) etc. In this work we will design and prepare the bone defect model and transplant into animal for further clarifying the effects of BGC coatings in the Hh signaling pathway regulating and will provide strong theoretical basis for the BGC coatings as the clinical application of Ti alloy coatings.

生物活性玻璃陶瓷（BGC）作为钛合金涂层的应用，已在临床上获得成功。但目前为止还没有研究BGC涂层介导成骨过程中Hh信号通路调控作用的文献报道。本课题以骨髓间充质干细胞（BMSCs）在BGC涂层成骨分化时起调控作用的Hedgehog(Hh)信号通路关键分子为研究的切入点，构建表达或干扰Hh信号通路中Gli-2基因的体外细胞模型，分别处理于Hh信号通路的Ihh基因、该通路抑制剂、BMP-2及其抑制剂。用MTT检测、流式细胞仪检测、免疫荧光染色、免疫组化染色、RT-PCR、Western blot、扫描电镜观察、碱性磷酸酶分析和茜素红染色（ARS）等方法，研究Hh信号通路关键因子对成骨细胞粘附、增殖、凋亡和分化及基质钙化的作用。制作骨缺损的动物模型，将BGC涂层植入体内，进一步明确在体内成骨过程中BGC涂层对Hh信号通路调控作用，将为BGC作为钛合金涂层的临床应用，提供有力的理论依据。

背景：生物活性玻璃陶瓷作为钛合金涂层的应用，已在临床上获得成功。而Hedgehog(Hh)信号通路与成骨关系密切，但目前为止还没有生物活性玻璃陶瓷涂层与Hh信号通路调控成骨过程的关系的研究。探讨生物活性玻璃陶瓷涂层与Hh信号通路调控成骨过程的关系。.主要研究内容：①制备生物活性玻璃陶瓷涂层材料，应用扫描及透射电子显微镜观察涂层结构；②将原代培养大鼠骨髓间充质干细胞接种在生物活性玻璃陶瓷涂层上，应用荧光定量RT-PCR、Western blot、免疫荧光染色等方法检测成骨标志物骨形态发生蛋白2和Hh信号通路关键因子Gli1(Gliloma-association oncogene homoglog)的表达，并观察细胞的迁移能力。.结果与结论：①电镜下生物活性玻璃陶瓷涂层表面无裂纹，表面光滑，具有致密的介孔结构,涂层厚度约350 nm；②生物活性玻璃陶瓷组中骨形态发生蛋白2和Gli1 mRNA和蛋白表达水平显著高于对照组，且Gli1和骨形态发生蛋白2蛋白在成骨过程中相互联系；与对照组相比，生物活性玻璃陶瓷组的骨髓间充质干细胞的迁移能力明显增强。③结果提示生物活性玻璃陶瓷涂层通过增加Hh信号通路的转录因子Gli1的表达，进而促进激活Hh信号通路，增强Hh信号通路活性，协同增加骨形态发生蛋白2，共同促进成骨细胞的生长增殖。