超声内空化调控rhBMP-2基因转染干细胞可视化治疗骨质疏松性骨折的研究

骨质疏松性骨折因骨组织微结构退化易出现愈合不良，移植重组人骨形态发生蛋白2(rhBMP-2)基因转染的骨髓间充质干细胞(BMSCs)、改善内环境为骨质疏松性骨折治疗带来了希望。然而此基因疗法仍停留于基础研究，要真正应用于临床须解决安全高效、可视化、可调控问题。超声靶向微泡破坏技术(UTMD)是一种安全的由空化核介导空化效应的基因转染新方法。但因空化核位于胞外，仅能提高细胞膜通透性，不能开放细胞核膜，故转染效率偏低。基于BMSCs的内吞特性，我们提出设想并体外验证了，借助天然生物泡(GVs)作为空化核，负载rhBMP-2基因后先经BMSCs内吞引入细胞质，再联合UTMD激发细胞内空化效应，直接开放细胞核膜，提高基因转染效率。同时基于GVs可超声成像的特性，有望实现BMSCs体内实时示踪，及超声可视化定点调控BMSCs基因转染，为实现安全高效、可视化、可调控的骨质疏松性骨折基因疗法提供新思路。

Osteoporosis fracture tends to poor fracture healing because of a deteriorated bone microstructure. Recombinant human BMP-2 (rhBMP-2) gene transfection to bone mesenchymal stem cells (BMSCs), improving the internal environment of fracture, is hot in osteoporosis fracture treatment. However, this therapy is still basic study because that it is unsafe, inefficient, unvisualized and incontrollable. Ultrasound-targeted microbubble destruction (UTMD) is a new method which uses ultrasound induced cavitation effect enhancing membrane permeability to promote gene transfection. It can only affect cell membrane but not nuclear membrane because of the extracellular cavitation nucleus, which leads to low transfection efficiency. Herein, we formulate hypothesis and confirm in vitro that the rhBMP-2 gene loaded gas vesicles (GVs) can be phagocytosed by BMSCs. After treated with UTMD, rhBMP-2 gene loaded GVs (cavitation nucleus) enhance nuclear membrane permeability by intracellular cavitation effect and improve transfection efficiency. Besides, GVs is natural acoustic contrast agent for in vitro and in vivo ultrasound imaging, which enable BMSCs to be tracked by ultrasound and realize controllable gene transfection by ultrasound. In summary, this study will improve the safety, efficiency, visualization and controllability of gene transfection, which provides a new approach to visualized precision therapy for osteoporosis fracture.