聚乳酸聚乙醇酸微球冷冻切片方法的研究  

Study on cryo-sectioning technique of poly (lactic-co-glycolic acid) microspheres

作　　者：黎呐[1] 麦海燕[2] 罗宇燕[2] 钟晨[1] 张永明[2] 

机构地区：[1]中山大学药学院,广东广州510006 [2]中山大学附属第三医院,广东广州510630

出　　处：《广东药学院学报》2014年第1期1-5,共5页Academic Journal of Guangdong College of Pharmacy

基　　金：广东省重大专项科技计划项目(2011A080504003)

摘　　要：目的研究聚乳酸聚乙醇酸(PLGA)微球切片的制备方法,以便使用扫描电镜获得孔洞结构完整清晰的微球截面图片,为后期使用图像处理软件计算微球的孔隙率提供基础。方法使用冷冻包埋剂包埋载有牛血清白蛋白(BSA)的PLGA微球,冷冻切片获得微球截面,并用扫描电子显微镜观察切片形态,分别考察包埋剂的种类、浸泡时间、液氮冷冻等对切片质量的影响。结果优化方法为:将微球浸泡在自制的质量浓度10%明胶+5%甘油混合冷冻包埋剂中,于37℃浸泡3 h,然后转移至质量浓度20%明胶+5%甘油混合冷冻包埋剂中,于37℃浸泡1 h,再转移至-20℃的冰箱中使包埋剂凝固;切片前将包埋块浸入液氮中急速冷冻5 min。结论使用优化方法所得的PLGA微球切片,截面孔洞结构完整清晰,且操作方法简单、快速和有效。Objective To develop an experimental method to obtain complete cryo-sections, which show the structure of poly （ lactic-eo-glycolic acid） （PLGA） microspheres clearly with the internal pores retaining their shape.Methods The microspheres were suspended in two different embedding agents, OCT （optimal cutting temperature） compound and an aqua solution containing gelatin and glycerin, and the fixed samples were embedded,frozen in liquid nitrogen and eryo-sectioned. Then the sections were dried and photographed by scanning electron microscopy.The technical parameters of eryo-seetioning such as the type of embedding agents, the immersion time and the procedure of quick freezing before cryo-sectioning were investigated, and the optimized method was established. Results The optimized cryo-seetioning method was as follows：microspheres were soaked in an aqueous solution containing 10% gelatin-5% glycerin for 3 h at 37 ℃ , then soaked in 20% gelatin-5% glycerin for 1 h at 37℃ , after solidification by stored in - 20℃ , the microspheres containing gelatin-glycerin jelly were frozen in liquid nitrogen for 5 min and cryo-sectioned. Conclusions The porous structure without distortion of the sections could be clearly observed by scanning electron microscopy. The optimized method was easy, fast and effective for the observation of internal structure of PLGA microspheres.

关 键 词：聚乳酸聚乙醇酸 微球 微观结构 冷冻切片技术 

分 类 号：R943[医药卫生—药剂学]