摘要目的 构建一种缓释FGF2基因的“壳核”结构的壳聚糖缓释微球.“核”是包埋FGF2基因质粒的巯基烷基化壳聚糖(TACS)、“壳”是羟丁基壳聚糖(HBC),探讨该壳聚糖壳核微球对颗粒脂肪移植存活率的影响.方法 制备HBC@TACS-pFGF2-EGFP壳核缓释微球,检测核壳结构包载基因缓释基因的释放规律.Western-blot检测该缓释微球体外转染293T细胞后表达FGF2蛋白的情况.细胞增殖实验证明10μg/ml的pFGF2质粒可促进293T细胞生长.取18只新西兰白兔用于颗粒脂肪移植实验.兔左耳作为实验组,移植2 ml脂肪颗粒和HBC@TACS-pFGF2-EGFP;兔右耳作为对照组,移植2 ml脂肪颗粒和HBC@TACS-空载质粒.分别于颗粒脂肪移植术后第4、8、12周切取标本,行大体观察、HE染色、免疫组化染色,观察移植物的生物学特性、脂肪移植成活率及新生血管密度.结果 HBC@TACS-pFGF2-EGFP在体外可缓慢释放pFGF2-EGFP基因,而且在转染293T细胞后可成功表达FGF2蛋白.颗粒脂肪移植术后不同时间点取材,发现移植后的脂肪组织体积随时间推移逐渐减小,整个实验过程中实验组的脂肪体积、脂肪移植成活率均大于对照组(P＜0.05),HE染色显示实验组的新生脂肪组织细胞排列较对照组更为规则,免疫组化染色显示实验组脂肪组织的微血管密度和FGF2蛋白表达水平均高于对照组(P＜0.05).结论 缓释FGF2基因的HBC@TACS-pFGF2-EGFP微球可提高颗粒脂肪移植存活率.

abstractsObjective To construct a novel carrier with core-shell structure-inner core of pFGF2-EGFP-loaded TACS coated by hydroxybutyl chitosan (HBC),and to explore its effects on granular fat graft survival.Methods The core-structured particles (TACS-pFGF2-EGFP) and core-shell-structured particles (HBC@TACS-pFGF2-EGFP) were prepared to explore the release pattern of pFGF2-EGFP of these particles.The expression of FGF2 protein was detected by Western-Blot in 293T cells transfected with the sustained-release microspheres in vitro.Cell proliferation assay demonstrated that 10μg/ml pFGF2 plasmid could promote 293T cells growth.Eighteen New Zealand white rabbits were used for adipose tissue transplantation experiment.Rabbit left ear was treated as experimental group,2 ml fat granules and HBC@TACS-pFGF2-EGFP were implanted;rabbit right ear was used as control group,2 ml fat granules and HBC @TACS-empty plasmids were transplanted.The specimens were harvested at 4,8,12 weeks separately after fat transplantation.Gross view,HE staining,and immunohistochemical staining were performed to observe graft survival,biological characteristics,and neovascular density.Results HBC@TACS-pFGF2-EGFP particles could sustained release Pfgf2 gene in vitro,and successfully express FGF2 protein after transfecting 293T cells.At different time points after transplantation,the volume of adipose tissues was gradually reduced with time.The fat volume and survival rate of adipose tissues in the experimental group were significantly higher than that in the control group (P ＜ 0.05).HE staining showed that the arrangement of new adipocytes in the experimental group was more regular than that in the control group.Immunohistochemistry staining showed that the micro vessel density and FGF2 protein expression level in the adipose tissue of the experimental group were higher than those in the control group (P ＜ 0.05).Conclusions HBC@TACS-pFGF2-EGFP particles with sustained release of FGF2 can improve the survival of granule fat transplantation.