缓释核壳结构利用基因传递用于骨缺损的研究

Gene delivery based on sustained releasing core-shell structure for therapy against bone defect

目的利用巯基烷基化壳聚糖(TACS)与质粒形成纳米粒子,在其外围包裹羟丁基壳聚糖(HBC)用以增加纳米粒子稳定性,并赋予其缓释的功能。方法利用骨形态发生蛋白4(BMP4)-绿色荧光蛋白(EGFP)融合基因的负电性与TACS的正电性相互作用形成核结构(TACS-pBMP4-EGFP),再与HBC相互作用形成核壳结构(TACS/HBC-pBMP4-EGFP)。利用动态光散射,透射电镜,凝胶电泳,体外转染,Western blot及动物成骨实验验证TACS、HBC对pBMP4-EGFP的包裹及缓释。结果随着氮磷比增加,TACS-pBMP4-EGFP直径逐渐减小当氮磷比大于8时,TACS-pBMP4-EGFP直径小于200 nm,包裹HBC后,TACS/HBC-pBMP4-EGFP粒径小幅度增加且电势趋于中性,易于被细胞转染表达出BMP4。流式细胞仪和Western blot结果均显示随着转染时间增加,核壳结构转染率逐渐增高兔桡骨缺损修复过程中,X线片可见携载核壳结构的缺损处骨密度明显增高,皮质骨连续性较好,呈骨性愈合。结论

Objective To explore the effects of core-shell structure in releaseing plasmid and repairing of bone defect. Methods Thiolated N-alkylated chitason and hydroxylbutyl chitason were taken to interact with eGFP-BMP plasmid to form TACS/HBC-pBMP4-EGFP. Then dynamic light scattering, Transmission Electron Microscope, agarose gel electrophoresis, vitro transfection, western blot and vivo experiment to test whether they could delivery plasmid into cell and their ability of sustained release. Results With addition of thiolated N-alkylated chitason, the diameter of formed nanoparticle became smaller. When the N/P ratio was 8, the diameter of nanoaprticle was less than 200 nm. After forming a shell out of nanoparticle by addition of hydroxylbutyl chitason, the diameter became a litter larger and the zeta potential came to more neutral. We took advantage of some plasmid which contains both the base pair sequence of eGFP and BMP plasmid. The expression of eGFP was tested by flow cytometry and the ex- pression of BMP was examined by Western blot. X-ray showed that more woven bone-like tissue were visible and trabecular-like structure was formed in the erperiment group. Conclusion TACS/HBC-pBMP4-EGFP owns the a- bility of sustained release and may be used to repaire bone defect.