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

doi:10.13418/j.issn.1001-165x.2018.02.013

中国临床解剖学杂志 ›› 2018, Vol. 36 ›› Issue (2): 187-191.doi: 10.13418/j.issn.1001-165x.2018.02.013

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

高浚淮¹，　邱素均¹，　殷杰²，　史本超¹，　闵少雄¹

1.南方医科大学珠江医院脊柱外科，广州 510280；　2.宁波市第六医院，宁波 315040

收稿日期:2018-02-28 出版日期:2018-03-25 发布日期:2018-05-04
通讯作者: 闵少雄，博士，主任医师，Tel: 020-62782579，E-mail：msxbear24@163.com
作者简介:高浚淮（1992-），硕士，主要从事骨组织工程方向的研究，E-mail：1440358630@qq.com
基金资助:
广州市科技计划（201607010266）

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

GAO Jun-huai¹, QIU Su-jun¹, YIN Jie², SHI Ben-chao¹, MIN Shao-xiong¹

1. Department of Spinal Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, China; 2. Ningbo No.6 Hospital, Ningbo 315040, China

Received:2018-02-28 Online:2018-03-25 Published:2018-05-04

摘要/Abstract

摘要：

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pBMP2/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NAC-PCCs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。

关键词: 骨损伤修复, 　非病毒载体, 　成骨分化, 　骨形态发生蛋白2

Abstract:

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 pBMP2/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 pBMP2/NAC-PCCs was the highest within 14 days. QRT-PCR, AKP activity detection and alizarin red staining showed that the osteogenic differentiation of pBMP2/NAC-PCC was the best.　Conclusion pBMP2/NAC-PCCs nanoparticles can promote the osteogenic differentiation of BMSC.

Key words: Repair of bone injury, 　Non-viral vector, 　Osteogenic differentiation, 　Bone morphogenetic protein gene 2

高浚淮,邱素均,殷杰,史本超,闵少雄. 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[J]. 中国临床解剖学杂志, 2018, 36(2): 187-191.

GAO Jun-huai, QIU Su-jun, YIN Jie, SHI Ben-chao, MIN Shao-xiong. Osteogenic differentiation of rat bone marrow mesenchymal stem cells induced by bone morphogenetic protein gene 2 vector[J]. Chinese Journal Of Clinical Anatomy, 2018, 36(2): 187-191.

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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

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