【作者】 李明；

【导师】 谢红炬；

【作者基本信息】 南华大学 ， 外科学， 2010， 硕士

【摘要】 目的应用W/O/W复乳溶剂蒸发法制备碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）-聚乳酸-聚羟基乙酸共聚物（Polylactic-co-glycolic acid,bFGF-PLGA）缓释微球,考察其对新西兰大白兔静脉皮瓣成活的影响。方法本文首先建立了BCA法测定缓释微球中牛血清白蛋白（ BSA ）含量,以及ELISA法测定bFGF-PLGA缓释微球模拟体外释放bFGF的含量,研究了以BSA作为模型药物制备注射用PLGA含药微球,并应用正交设计试验进一步优化bFGF微球的制备工艺,采用优化处方制备bFGF-PLGA微球,并考察其外观形态和粒径分布,检测其载药量和包封率,研究其体外释药的性质;以新西兰大白兔为实验模型制作侧腹壁静脉皮瓣,并术前5天分别皮内注入bFGF-PLGA（Ⅰ组）,bFGF+空微球悬浊液组（Ⅱ组）和生理盐水（Ⅲ组）。术后,①计算术后7天皮瓣的成活面积;②取兔皮肤标本行CD34+免疫组化染色,检测CD34+表达情况及平均血管数。结果所制微球表面光滑圆整,球体均匀度好,无黏连现象。微球粒径的98%分布在12.50～43.49μm之间,平均粒径为26.93μm,径距0.611±6.60。载药量为[（23.11±0.44）×10^-3]%,包封率为（86.51±0.83） %。突释期内微球的体外释放度为27.78% , 30d后体外累积释放度高达81.56% ,微球的体外释药规律符合Higuichi方程（ r =0.997）。缓释微球组、空白微球组和生理盐水组术后7天的皮瓣平均面积分别为72.03±1.81、51.03±1.77、50.49±2.36%（ P < 0.05） ,平均血管数目分别为34.01±2.30、23.81±3.03、22.56±2.26个/cm2 （ P < 0.05）。结论1.所制备的bFGF-PLGA缓释微球表征良好,载药量和包封率高;微球通过较长时间地持续释放活性bFGF。2.术前5天局部皮下注射bFGF-PLGA缓释微球持续释放bFGF,有效促进新生血管的形成,增加皮瓣的血运,提高缺缺血皮瓣的成活面积,是一种简单有效的细胞因子治疗方法。更多还原

【Abstract】 Objective To prepare the basic fibroblast growth factor （bFGF）-Polylactic-co- glycolic acid（PLGA）sustained release microspheres （MS） by W/O/W multiple emulsion evaporation method, and investigate their effects on the survival of venous flaps.Methods In this study, BCA and ELISA were established in the determination of sustained-release microspheres content of bovine serum albumin BSA and bFGF-PLGA microspheres slow-release of bFGF in vitro release of simulated.BSA were used as a model protein drug and fabricated protein loaded PLGA microspheres were prepared for injectable cell delivery used as microspheres.The formulation of bFGF-PLGA [poly（ lactic-co- glycolic -acid）]-MS was optimized by orthogonal design. The morphology,size distribution, drug loading volume encapsulation efficiency and in vitro drug release behavior of bFGF-PLGA-MS being measured. In 24 New Zealand white rabbits , lateral abdominal wall skin flap supplied by the epigastric vein was created, and five days before operation, respectively, intradermal injection of bFGF-PLGA microspheres （Ⅰgroup） , bFGF solution or bFGF- impregnated PLGA （Ⅱgroup） and normal saline （Ⅲgroup）. After seven days,①calculation of the survival area of skin;②Rabbit skin samples from CD34+ immunohistochemical staining to detect the expression of CD34+ blood vessels, and the average number of vascular.Results The bFGF MS prepared based on optimized formulation exhibited well-defind properties,with the even and uniform sphere in appearance, regular particles without adhesion, about 98% of particles with a size distribution between 12.50 to 43.49μm,with a mean particle size of 26.93μm and size span of 0.611±6.60. The drug loading volume and encap sulation efficiency of bFGF MS reached [（23.11±0.44 ）×10^-3]% and （86.51±0.83） % , respectively. In the burst release phase, the rate of in vitro drug release amounted to 27.78% , but rose to 81.56% accumulatively 30 days later.The in vitro drug release of bFGF MS corresponded with Higuichi equation （ r =0.997）. after 7 days, The sustained-release microspheres, blank microspheres and normal saline group, the average survival of the flap were 72.03±1.81、51.03±1.77、50.49±2.36% （P <0.05）, the average number of blood vessels were 34.01±2.30、23.81±3.03、22.56±2.26 /cm2 （P <0.05）.Conclusion1.bFGF MS with good morphology, high drug loading volume and encapsulation efficiency can be obtained using optimized formulation.2. 5 preoperative days,local subcutaneous injection of the bFGF MS can promote the survival of venous flaps through a long period due to sustained release of bFGF.更多还原