脊髓组织工程的体外构建  

In vitro construction of spinal cord by tissue engineering

作　　者：谢青松[1] 李立新[1] 黄保胜[1] 付震[1] 胡卫星[1] 鲁艾林[1] 

机构地区：[1]南京医科大学第一附属医院神经外科,江苏省南京市210029

出　　处：《中国组织工程研究与临床康复》2007年第10期1850-1853,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：江苏省"135工程"医学重点人才基金资助项目(135-57)~~

摘　　要：目的:观察神经干细胞在聚乳酸-羟基乙酸支架上的生长和分化行为,探讨组织工程体外构建人工脊髓修复脊髓损伤。方法:实验于2005-10/2006-06在南京医科大学第一附属医院中心实验室完成。①在聚乳酸-羟基乙酸支架(孔径200～300μm,孔隙率90%,聚乳酸/聚羟基乙酸=75∶25;山东医疗器械研究所)上种植孕14d的胚胎大鼠脑细胞,培养7d。②将培养7d的聚乳酸-羟基乙酸支架置入含体积分数为0.05的胎牛血清和含脑源性神经营养因子20μg/L的DMEM/F12培养基中继续培养5d,诱导其向神经元分化。③用倒置相差显微镜和扫描电镜观察细胞在聚乳酸-羟基乙酸支架上的生长和分化行为;应用免疫组织化学鉴定聚乳酸-羟基乙酸支架上培养的细胞。结果:①神经干细胞生长形态:神经干细胞克隆球充满聚乳酸-羟基乙酸支架孔隙,其形状近似孔隙的不规则几何形状。②神经干细胞超微结构:诱导分化后,大量神经干细胞长出的神经突触跨越支架孔隙的三维空间结构,布满支架的表面和孔隙,并彼此建立了突触联系。③诱导分化前后的聚乳酸-羟基乙酸石蜡切片免疫组织化学鉴定结果:诱导分化前后免疫组织化学鉴定分别为巢蛋白和微管相关蛋白2抗体阳性,说明诱导分化前是神经干细胞,诱导分化后有神经元形成。结论:聚乳酸-羟基乙酸适合神经干细胞的生长和分化,其孔隙结构规范调节了神经干细胞生长增殖的空间排列,而脑源性神经营养因子调控了神经干细胞的分化方向,可以利用该支架的空间结构和细胞因子适度调控神经干细胞在聚乳酸-羟基乙酸上的生长和分化构建脊髓组织。AIM： To observe the growth and differentiation of neural stem celis cultured on poly lactic-co-glycolic acid （PLGA）, and explore the fabrication of spinal cord by tissue engineering in vitro in treating spinal cord injury. METHODS： The experiment was conducted in the central laboratory of First Hospital affiliated to Nanjing Medical University between October 2005 and June 2006. ①Cells isolated from the brain of 14-day embryonic rat ware inoculated onto the PLGA for 7 days （200-300 p.m pore diameter, 90% interval porosity, the proportion of polylactic acid and polyglycolic acid was 75：25; provided by Shandong Research Institute of Medical Instrument）. ②Then the scaffold was put into DMEM/F12 medium containing 0.05 volume fraction fetal bovine serum and 20μg/L brain-derived neurotrophic factor （BDNF） for 5 days to induce cell differentiation. ③The cells growth and differentiation implanted on PLGA ware observed by phase contrast microscope and scanning electron microscope, and immunohistochemistry was applied to identify the cells on PLGA. RESULTS： ①Neural stem cells appearance： PLGA pores ware filled with neural stem cells, whose shape resembled the irregular geometry of pores. ②Ultrastructure of neural stem cells： After cell differentiation induced by BDNF, formation of the three-dimensional network poly was covered with neurite contacted each other. ③Paraffin section identification by immunohistochemistry： Nestin-positive cells and microtubule associated protein two-positive cells ware found in the PLGA before and after differentiation induced by BDNE, indicating that neural stem cells cultured on PLGA ware induced differentiation into neuron cells by BDNF. CONCLUSION： PLGA supports the growth and differentiation of neural stem cells. The organization and differentiation direction of neural stem cells can be regulated by three-dimensional formation of PLGA scaffold and directed by BDNF. Spinal cord tissue engineering can be fabricated in vitro by 3D formation of PL

关 键 词：干细胞 脑源性神经营养因子 细胞分化 组织工程 脊髓损伤 

分 类 号：R318[医药卫生—生物医学工程]