碱性成纤维细胞生长因子缓释微球促进神经干细胞生长的体外实验研究  

Promotion role of basic fibroblast growth factor slow-release microsphere in neural stem cells proliferation in vitro

作　　者：薛杉[1] 吴钢[2] 祝爱萍[3] 郭燕舞[1] 汪求精[1] 周振军[1] 姜晓丹[1] 柯以铨[1] 全大萍[3] 

机构地区：[1]南方医科大学珠江医院神经外科、广东神经外科研究所、广东省脑功能修复与再生重点实验室,广州510282 [2]南方医科大学珠江医院肿瘤中心,广州510282 [3]中山大学高分子化学与物理研究所,广州510275

出　　处：《中华神经医学杂志》2013年第8期769-773,共5页Chinese Journal of Neuromedicine

基　　金：广东省医学科研基金（B2011204）;广东省自然科学基金（粤科基办字[201113号）;广东省科技计划项目（20118061200046）;国家临床重点建设专科资助项目

摘　　要：目的 制备一种能够负载足量碱性成纤维细胞生长因子（bFGF）并且能够以理想的速度缓慢释放的载体. 方法 构建负载bFGF的缓释微球,并检测其体外释放过程.将bFGF缓释微球与神经干细胞共培养,以常规多次添加bFGF的神经干细胞培养组作为对照,用AlamarBlue法检测培养细胞的存活情况,ELISA法检测培养上清bFGF含量,定期观察细胞形态学变化,并进行巢蛋白荧光免疫细胞化学染色,检测神经干细胞标记物. 结果 AlamarBlue法检测结果显示,培养3d、5d时bFGF缓释微球组和常规培养组之间细胞增殖率差异有统计学意义（P＜0.05）.ELISA法检测结果显示,仅负载bFGF缓释微球组能测量到培养上清中bFGF含量,大致维持在200 pg/mL水平.细胞形态学观察发现bFGF缓释微球组能够在培养第3天即形成神经干细胞球,并且在培养第7天仍保持较好透光度,保持较好的细胞活性.荧光免疫细胞化学染色发现bFGF缓释微球组培养的神经干细胞球巢蛋白染色阳性. 结论 本课题组制作的bFGF缓释微球材料具有较好的生物相容性.缓慢释放的bFGF能够满足神经干细胞生长需要,并且保持神经干细胞自身的细胞特性.该bFGF缓释微球安全有效,可应用于中枢神经系统损伤的在体实验研究.Objective To prepare a kind ofmicrosphere being able to load and slowly release basic fibroblast growth factor （bFGF）.Methods The bFGF-sustained release microspheres were established and their in vitro release process was detected.The bFGF sustained release microspheres and neural stem cells were co-cultured as experimental group; neural stem cells added bFGF for several times were chosen as control group.AlamarBlue assay was employed to detect the cell survival; ELISA was used to observe the supematant bFGF content; morphological changes of the cells were observed regularly and nidogen fluorescence immunocytochemical staining was employed to detect the neural stem cell marker.Results AlamarBlue assay showed that the cell survival of the experimental group and control group had significant difference on the 3rd,5th d of culture （P〈0.05）.ELISA showed that supematant bFGF could only be detected in the experimental group,reaching 200 pg/mL.Cell morphology observation found formation of neural stem cell spheres in the experimental group on the 3rd d of culture,maintaining good transparency and cell activity on the 7th d of culture.Fluorescence immunocytochemistry staining revealed nidogen positive expression in the experimental group.Conclusion The bFGF sustained release microsphere material has no biological toxicity,enjoying good biological compatibility; the slow release ofbFGF can meet the need of neural stem cell growth and keep their cell characteristics of neural stem cells; the bFGF sustained release microspheres is safe and effective,and can be applied to clinical experimental study of central nervous system injury.

关 键 词：碱性成纤维细胞生长因子 缓释 神经干细胞 

分 类 号：R363[医药卫生—病理学]