缓释FGF2基因壳聚糖核壳微球提高颗粒脂肪移植存活率的研究

摘要

目的 构建一种缓释FGF2基因的"核壳"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).探讨该壳聚糖核壳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,检测该壳核结构包载基因相较于核结构包载基因缓释的释放规律. 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.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周、第8周、第12周切取标本,并行大体观察、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,实验组脂肪组织的新生血管密度高于对照组(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.%Objective To construct a novel core-shell-structured chitosan particles with sustained-release FGF2 plasmids (the core was pFGF2-EGFP-loaded TACS and the shell was HBC), and to explore the effect of the particles on the survival rate of adipose tissue transplantation. Methods The core-structured particles (TACS-pFGF2-EGFP) and core-shell-structured particles (HBC@TACS-pFGF2-EGFP) were prepared to explore the release regulation of pFGF2-EGFP of these particles. The expression of FGF2 protein was detected by Western Blot after in vitro transfection of 293T cells. Eighteen New Zealand white rabbits were used for adipose tissue transplantation experiment. The left ears were treated as experimental group and the right ears were treated as control group. The ears in experimental group were implanted with 2 mL fat particles and HBC@TACS-pFGF2 particles while the ears in control group were implanted with 2 mL fat particles and HBC@TACS-empty plasmids. The new adipose tissues were harvested to analyze the survival rate at 4, 8, 12 weeks after transplantation. Besides, the biological characteristics of grafts and the density of new blood vessels were observed at the same time by HE staining and immunohistochemistry. Results The FGF2 protein can be slowly released by HBC@TACS-pFGF2 particles in vitro and be successfully expressed after transfection of 293T cells. At different time point, new fat tissues were observed. The volume of adipose tissues were gradually reduced after transplantation. The survival rates of adipose tissues in the experimental group were significant higher than in control group at any time point (P<0.05). HE staining showed the new fat cells arranged more regularly in experimental group than the cells in control group. Immunohistochemistry staining showed the density of new blood vessels in adipose tissue in experimental group was higher than that in control group (P<0.05). Conclusion HBC@TACS-pFGF2-EGFP particles can improve the survival rate of fat transplantation.