【作者】 董晓庆；

【导师】 常津；

【作者基本信息】 天津大学 ， 材料学， 2007， 硕士

【摘要】 本文采用W/O/W方法制备聚乳酸-羟基乙酸共聚物（PLGA）载表皮生长因子（EGF）缓释微球。采用模型药物BSA研究内水相乳化剂浓度、外水相乳化剂浓度、有机溶剂配比、外水相pH值、盐离子浓度五个制备条件对药物包封率和体外释放行为的影响,得到最优制备条件。通过透射电镜观察微球形貌,动态光散射粒径分析仪测定微球粒径及粒径分布;ELISA法检测微球体外释药行为。结果显示,微球呈规则圆形,平均粒径193.5nm,多分散系数为0.176,生长因子包封率达85.6%,体外释放实验表明微球在PBS缓冲溶液中具有较好的缓释效果,释药规律符合Higuchi释放动力学模型Q=46.80738+10.09451t^1/2（R=0.94341）,累积释放时间达24小时。采用MTT法进行EGF活性和缓释微球安全性的细胞实验,结果表明不同浓度的缓释微球对小鼠成纤维细胞都有促进增殖的能力,缓释微球促进细胞增殖的能力与微球浓度相关,10μg/L为其促进细胞生长的最适浓度。对比10μg/L EGF缓释微球和等量EGF原液对细胞增殖作用的影响发现,缓释微球促进细胞增殖效果优于EGF原液,具有统计学意义（P<0.01）。研究不同浓度空白微球对细胞增殖能力的影响发现,从0.01μg/L到100μg/L这个浓度梯度内,缓释微球材料对细胞均没有毒性作用。采用糖尿病溃疡模型大鼠研究微球对糖尿病溃疡的修复作用。通过计算溃疡面积变化可以直观看出微球组治疗效果优于生长因子原液组和空白对照组,并且具有显著性差异,治疗21天时微球组愈合率已达96.61%。观察组织病理切片、EGFR表达、PCNA表达、电镜照片发现,空白微球未表现出生物毒性和对溃疡愈合的抑制作用,进一步说明此种技术的可行性。另外还发现缓释微球能很好地保持生长因子活性,同时能够持续释放生长因子,保持对细胞的连续刺激,促进糖尿病皮肤溃疡愈合效果较传统生长因子原液要好,体现了缓释微球技术优势。该研究为缓释微球的在糖尿病皮肤溃疡方面临床应用提供了重要的试验依据。更多还原

【Abstract】 The modified w/o/w extraction–evaporation technique was chosen to prepare epidermal growth factor （EGF） loaded microspheres using poly （lactic-co-glycolic） acid （PLGA）. Bovine serum albumin（BSA） was used as molding drug. The emulsification concentration of inner aqueous phase, the mixture ratio of solvent, the emulsification concentration of outer aqueous phase, pH value, salt concentration was researched for the highest encapsulation efficiency. The microspheres were characterized for morphology by transmission electronic microscopy （TEM）. The particle sizes of microspheres were determined by Particle Size Analyzer. The release performances of EGF loaded PLGA microspheres in vitro were studied, and EGF was measured by enzyme-linked immunosorbant assay （ELISA）. The result showed that microspheres were smooth and spheriacal with a mean particle size 193.5nm, the index of polydispersity was 0.176, and the encapsulation efficiency was 85.6%..There was a linear relationship between the amount of accumulative release Q and time square root t^1/2 Q=46.80738+10.09451t1/2（R=0.94341）. The release of EGF from PLGA microspheres can persist for 24h.The proliferation effects of microspheres on fibroblast were evaluated by MTT assay. We found that all of concentration of EGF loaded microspheres can accelerate the proliferation of fibroblast, however, there were some relationships between the concentration of microspheres and the proliferation effects. The result showed that 10μg/L was the optimal concentration for the proliferation. The proliferation effects of 10μg/L EGF loaded microspheres were much better compared with equal EGF. The proliferation effects of different concentration of blank microspheres on fibroblast were investigated. There was no toxicity on fibroblast with blank microspheres concentration range of 0.01μg/L to 100μg/L. All values were treated by statistical analysisThe diabetic ulcer model rats were used for researching the healing effects of EGF loaded microspheres. The change of ulcer areas visual displayed that there was significantly enhanced wound closure with EGF loaded microspheres from the 14th to 21th day compared with the control （EGF solution, the blank microspheres and PBS treatment） groups. Histological examination, the expression of the proliferative cell nuclear antigen（PCNA） and EGF receptor（EGFR）, observation with electron microscope were used to observe the theraputic effects in microscopic view. Firstly, the results showed that the blank microspheres was non-toxic and no inhibited action. Secondly, EGF activity was well maintained by EGF loaded microspheres, which can release EGF slowly and stimulate fibroblast continuously. The healing effects of EGF loaded microspheres were more effective than traditional EGF solution treatment.This work offers evidences for treating the diabetic ulcer using EGF loaded microspheres in clinic.更多还原