NAC-PCCs载骨形态发生蛋白2基因纳米微球促大鼠骨髓间充质干细胞成骨分化

Osteogenic differentiation of rat bone marrow mesenchymal stem cells induced by bone morphogenetic protein gene 2 vector

目的构建半胱氨酸及磷酸胆碱共接枝的仿生壳聚糖载体（NAC-PCCs）,并包封骨形态发生蛋白2（BMP2）基因进行诱导骨髓间充质干细胞成骨分化的研究。方法制备p BMP2/NAC-PCCs材料,检测其粒径、形态,绘制DNA缓释曲线,研究微球抗DNA酶降解能力。在骨髓间充质干细胞与材料共培养过程中,检测材料的转染效能、ROS清除能力、BMP2蛋白分泌水平、成骨相关基因RUNX2、OC表达、碱性磷酸酶活性等指标,以研究其对骨髓间充质干细胞成骨分化的影响。结果微球材料能有效避免DNA被生物酶降解,其转染效率为23.1%,ROS清除率为（36.13±0.47）%。同时实验结果显示与NACPCCs共培养的细胞,其细胞内BMP2表达水平高于其余各组且成骨分化效果最佳。结论 NAC-PCCs包封BMP2基因形成的纳米微球材料具有促进BMSC成骨分化作用。

Objective To study the effect of acetylcysteine/phosphorylcholine-modified chitosan（NAC-PCCs） encapsulation of bone morphogenetic protein gene 2 nanoparticle materials in promoting the osteogenic differentiation of SD rat bone marrow mesenchymal stem cells（BMSC） in vitro. Methods The p BMP2/NAC-PCCs nanoparticles were prepared, and its characterization, in vitro release and the ability to protect bone morphogenetic protein gene 2 against DNaseⅠwere examined. BMSC cells, nanoparticles in the process of cultivation, and transfection efficiency were determined by fluorescence microscopy and flow cytometry. The secretion of BMP2 protein levels was determined by ELISA experiment and the expression of RUNX2 and OC gene was determined by RT-PCR. The calcium deposition analysis was performed using alizarin red staining. Results Gel electrophoresis showed that nanoparticles could effectively protect DNA from being digested by DNaseⅠ. Fluorescent microscopy and flow cytometry showed that the gene delivery nanoparticles had better transfection efficiency. The results of ROS activity test showed that the material had the ability to scavenge ROS, and the ELISA experiment showed that the expression level of BMP2 in the group of p BMP2/NAC-PCCs was the highest within 14 days. QRT-PCR, AKP activity detection and alizarin red staining showed that the osteogenic differentiation of p BMP2/NAC-PCC was the best. Conclusion p BMP2/NAC-PCCs nanoparticles can promote the osteogenic differentiation of BMSC.