TGF-β1/TβR-Ⅰ、TβR-Ⅱ信号轴与骨形成改建的相关性分析及PNS 对牵张成骨骨愈合影响机制

Distraction Osteogenesis (DO) technique has broad prospects in the fields of craniomaxillofacial plastic surgery, postoperative reconstruction after tumor resection, etc. However, this technique has long treatment course, and complications such as fibrous healing or bone nonunion were often found after the treatment. The application of traditional chinese medicine Panax notogensing on promoting bone fracture healing enjoys a very long history, the effective component of Panax notogensing in promoting bone regeneration during distraction osteogenesis is Panax notogensing saponins(PNS). This research firstly adopts targeted gene knocked out technology. By knocking out TGF-beta 1, we study on the mandible morphological structure, mechanical properties and the influence for morphology and function of bone marrow mesenchymal stem cells. Then overexpression and gene silencing methods will be adopted, key receptor TβR-Ⅱ of TβR-Ⅰand TβR-Ⅱ signal axis silenced/overexpressed, which activates Smads signal pathways. The effect of osteogenic differentiation and prompting osteogenesis for bone marrow mesenchymal stem cells of rabbits and rabbits distraction osteogeneis model is obeserved after using PNS. Meanwhile, the effect of PNS for downstream signaling molecules regulatory protein RanBP3 and key control protein phosphatase PPM1A in TGFβ 1/ Smads signaling pathways is detected. All research further clarify the function of TβR-Ⅰand TβR-Ⅱ signal axis in the process of PNS promoting osteogenesis, and target of drug action, which will provide scientific basis of prompting bone healing in the process of distraction osteogenesis for PNS.

牵张成骨技术在颅颌面整形、肿瘤术后重建等方面应用前景广阔，但疗程过长且常出现纤维性愈合或骨不连等并发症。中药三七应用于促进骨伤的愈合已有悠久的历史，其促进牵张成骨新骨形成的有效成分为三七总皂苷(PNS)。本课题拟采用基因靶向敲除TGF-β1基因，研究TGF-β1对下颌骨形态结构、力学性能的影响和对骨髓间充质干细胞(BMSCs)形态和功能的影响，并采用基因沉默和过表达，沉默/过表达参与Smads信号通路激活的TGFβ1与TβR-Ⅰ、TβR-Ⅱ信号轴的关键受体TβR-Ⅱ，并转染兔BMSCs，观察三七主要成分PNS作用于BMSCs成骨分化、兔牵张成骨模型和TGF-β1/Smads细胞信号通路下游信号分子及调控蛋白RanBP3、关键调控蛋白磷酸酶PPM1A的影响，阐明TGFβ1与TβR-Ⅰ、TβR-Ⅱ信号轴在PNS促进成骨过程中的作用，及其作用基因靶点，为PNS用于促进牵张成骨骨愈合提供科学依据。

背景：牵张成骨（DO）新骨形成的速度明显领先于生长发育高峰期的婴幼儿，甚至超越了部分恶性肿瘤的生长速度。前期研究中，证实了中药三七的主要活性提取物三七总皂苷（PNS）可以上调兔骨髓间充质干细胞（rBMSCs）以及DO牵张间隙新生骨组织转化生长因子β1（TGF-β1)表达，促进rBMSCs的成骨功能及DO新生骨组织质量。探索PNS调控TGF-β1信号传导机制将为临床中应用PNS促进DO治疗提供理论基础。.内容：1.PNS通过TGF-β1/Smads信号传导调控rBMSCs成骨功能的机制研究；2.过表达rBMSCs中TβR-Ⅱ，进一步探索PNS通过TGF-β1/Smads信号传导调控rBMSCs成骨功能的机制；3.沉默rBMSCs中TβR-Ⅱ，进一步探索PNS通过TGF-β1/Smads信号传导调控rBMSCs成骨功能的机制；4.PNS通过TGF-β1/Smads信号传导促进兔DO新骨形成的作用机制研究。.结果：1.PNS可上调rBMSCs中TβR-Ⅱ的表达，激活TGF-β1/Smads信号通道，上调Smad2、Smad3的表达促进TGF-β1/Smads信号传导，抑制PPM1A、RanBP3的表达降低TGF-β1/Smads信号传导的终止作用，从而促进rBMSCs的成骨分化，提升rBMSCs的成骨功能；2.过表达TβR-Ⅱ在不影响rBMSCs功能的情况下，上调TβR-Ⅱ表达，进一步激活TGF-β1/Smads信号通道，上调Smad2、Smad3并下调PPM1A、RanBP3的表达，进一步促进TGF-β1/Smads的信号传导，提升PNS对rBMSCs成骨分化作用，增强成骨功能；3.沉默TβR-Ⅱ在不影响rBMSCs功能的情况下，下调TβR-Ⅱ表达，抑制TGF-β1/Smads信号通道，下调Smad2、Smad3并上调PPM1A、RanBP3的表达，抑制TGF-β1/Smads的信号传导，降低PNS对rBMSCs成骨分化作用，抑制成骨功能；4.PNS可上调DO牵张间隙内TβR-Ⅱ及Smad2、Smad3的表达、下调PPM1A、RanBP3的表达，促进TGF-β1/Smads信号传导，通过调控TGF-β1/Smads信号通道促进DO过程中的新骨形成速度，提高新骨生成质量。