BMP-9 基因修饰的ADSCs复合淫羊藿苷/纳米晶胶原基骨修复兔大段骨缺损的实验研究

With the rapid development of tissue engineering,it supports a new idea and method for treating bone defects in clinic. As for the seed cells,some reports and our previous studies had shown that adipose-derived stem cells (ADSCs) have the ability of multiple differentiation potential, especially in osteogenic differentiation,and they are easily obtained in large quantities. Therefore,they are expected to take place of Embryonic stem cells and Bone Marrow Stem cells (MSCs ). However, in vivo and in vitro ,the efficacy of bone formation of ADSCs should be enhanced further. The bionic artificial Nano-hydroxyapatide/collagen(nHAC) which was independently developed by our nation has demonstrated good performance in bone scaffold. A few of Reports have shown that ADSCs can be induced osteogenic differentiation by bone morphogenetic protein 9 (BMP-9) and promoted the bone formation. It did the same to MSCs for Chinese medicine Icarii .By transfecting with BMP-9 gene in ADSCs, the BMP-9 could be continuously expressed in the tissue. It will be the first time for ASCs combined with Icariin and nHAC that maded into the three-dimensional scaffold. Then the composite scaffold will be used to treat a large segment bone defect of radius in rabbit. Our experimentation is aim to study on the efficacy of bone regeneration of ASCs promoted by BMP-9 combinded with Icariin ,and the biocompatible of ASCs mixed with nHAC. To explore the possible mechanism of action of icariin and BMP-9 by detecting the expression of the related genes of JNK signaling pathway. Futhermore, This study may provide the evidences of theory and experimentation for the future study of treating bone defects.

组织工程学的迅速发展，为骨缺损这一临床难题的解决提供了新的思路和方法。 在种子细胞方面，本课题组的前期研究及文献都显示脂肪干细胞（ADSCs）具有多向分化潜能，尤其是成骨分化，且其来源丰富，有望替代骨髓干细胞(MSCs)，但目前ADSCs的成骨效能有待增强。支架方面，我国自行研制的纳米晶胶原基人工骨(nHAC)已经展现了良好的性能。文献显示骨形态发生蛋白-9（BMP-9）可诱导ADSCs成骨分化，而中药淫羊藿苷（Icariin）能诱导并增强MSCs的成骨分化。本研究拟在前期研究基础上，将BMP-9基因转染ADSCs，使局部持续表达BMP-9，复合Icariin与nHAC组装成三维缓释支架，并将此支架作为ASCs的载体，修复兔大段桡骨缺损。观察BMP-9 及Icariin的促成骨效能；通过检测JNK信号通路相关基因的表达，探索其可能的分子作用机制。为骨缺损治疗的进一步研究提供理论和实验依据。

本项目为选用淫羊藿苷、BMP-9诱导脂肪干细胞的成骨分化，并结合nHAC支架修复兔大段骨缺损的实验研究。重点研究了：淫羊藿苷协调BMP-9诱导脂肪干细胞成骨分化的影响；淫羊藿苷及BMP-9诱导脂肪干细胞成骨通路的分子机制；淫羊藿苷对ADSCs的毒性影响、IC50浓度；淫羊藿苷不同浓度促ADSCs的成骨效能。同时研究了：Ad-BMP-9感染脂肪干细胞合适的MOI值；植骨材料复合体的构建（BMP-9修饰的脂肪干细胞+ nHAC支架、淫羊藿苷），及其在动物体内成骨效能研究；ADSCs分离、培养、传代的方法，及证实其为干细胞的鉴定。 取得了一些重要进展结果，体内、外实验证实：传统中药单体淫羊藿苷可促进脂肪干细胞成骨，并且能协同、增强BMP-9诱导ADSCs的成骨作用；明确了JNK位点是淫羊藿苷及BMP-9诱导脂肪干细胞成骨通路关键交叉靶点。而且JNK位点对经典BMPs-Smad 成骨信号通路确实有影响，也即证实经典BMPs-Smad 信号途径经过JNK位点。取得了一些关键数据：在本实验室条件下①明确了淫羊藿苷对脂肪干细胞的毒性浓度，IC50值为33.67µg/ml。②摸索出了病毒（Ad-BMP-9）感染ADSCs细胞合适的MOI值为100。③明确了体外实验中协调增强BMP-9 诱导脂肪干细胞成骨、淫羊藿苷的最佳浓度是25µg/ml。 科学意义：为进一步利用ADSCs作为种子细胞的骨组织工程的研究提供了一定的理论、实验依据。