胶质细胞源性神经营养因子联合转化生长因子β1体外诱导大鼠脊髓源性神经干细胞的分化(英文)  

Glial cell-derived neurotrophic factors combined with transforming growth factor-beta 1 for in vitro differentiation of neural stem cells from rat spinal cord

作　　者：高明勇[1] 肖建德[1] 李振宇[1] 闫洪印[1] 白润涛[2] 韩漫夫[2] 

机构地区：[1]南方医科大学附属深圳医院脊柱外科,广东省深圳市518035 [2]南方医科大学附属深圳医院神经内科,广东省深圳市518035

出　　处：《中国组织工程研究与临床康复》2007年第24期4856-4860,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：广东省自然科学基金项目(5009437);深圳市科技计划项目~~

摘　　要：背景:胶质细胞源性神经营养因子与转化生长因子β1同属于转化生长因子β超家族,两者在哺乳动物中枢与外周神经系统发育、分化及细胞周期的调节中扮演重要角色。目的:观察胶质细胞源性神经营养因子联合转化生长因子β1体外诱导脊髓源性神经干细胞的分化,并与单一因子诱导培养液比较。设计:对照观察。单位:南方医科大学附属深圳医院中心实验室。材料:选用10只清洁级孕16d的SD大鼠,由华中科大同济医学院动物中心提供。主要试剂及材料:DMEM/F12,B27(GIBCO);碱性成纤维细胞生长因子,EGF,胶质细胞源性神经营养因子,转化生长因子-β1(PeproTech);胎牛血清(Hyclone);神经巢蛋白(nestin)多抗(武汉博士德);胶质纤维酸性蛋白(GFAP)多抗,神经丝蛋白(NF-200)单抗(Sigma)。方法:实验于2005-10/2006-09在南方医科大学附属深圳医院中心实验室完成。无菌条件下分离大鼠胚胎,进行脊髓源性神经干细胞的分离培养。实验分①基础培养基组:含青霉素、链霉素、两性霉素B及体积分数为0.02 B27添加液的DMEM/F12。原代培养1周后,获取体外稳定增殖的鼠脊髓源性神经干细胞克隆。②对照组:在基础培养基中加入体积分数为0.1的胎牛血清。③碱性成纤维细胞生长因子组。④转化生长因子β1组。⑤胶质细胞源性神经营养因子+转化生长因子β1组。换用诱导培养基,即分别在基础培养基中加入20μg/L碱性成纤维细胞生长因子、2μg/L转化生长因子β1、10μg/L胶质细胞源性神经营养因子+2μg/L转化生长因子β1。①光镜观察在不同因子诱导情况下脊髓源性神经干细胞的分化情况。②免疫细胞化学染色标记检测神经元与星状胶质细胞的表达。③通过荧光激发流式细胞仪分选技术对分化细胞计数,检测不同诱导环境下神经干细胞分化为神经元及星状胶质细胞的阳性百分率。主要观察指标:①各组细胞分化�BACKGROUND： Glial cell-derived neurotrophic factor （GDNF） and transforming growth factor-beta 1 （TGF-β1）co-subordinate to TGF-β family. Both of them play very important roles in the development and differentiation of central and peripheral nervous system, and regulation of cell cycle in mammals.OBJECTIVE： To observe the differentiation of spinal cord-derived neural stem cells（NSCs） induced by GDNF combined with TGF-β1, and make a comparison of differentiation results with GDNF or TGF-β1 culture fluid.DESIGN： Controlled observation.SETTING： Central Laboratory, Shenzhen Hospital Affiliated to Southern Medical University.MATERIALS： Ten SD rats of clean grade, which were at conception for 16 days, were provided by the Experimental Animal Center, Tongji Medical College, Huazhong University of Science and Technplogy. Main reagents and materials：DMEM/F12,B27（GIBCO）; basic fibroblast growth factor （bFGF）, GDNF; TGF-β1（PeproTech）;fetal bovine serum （FBS,Hyclone）; nestin multiple antibody （Boster, Wuhan）; glial fibrillary acidic protein （GFAP） multiple antibody; neurofilament protein （NF-200） monoclonal antibody （Sigma）.METHODS： This experiment was carried out in the Central Laboratory, Shenzhen Hospital Affiliated to Southern Medcial University between October 2005 and September 2006. Under the aseptic condition, rat fetus was isolated for isolation and culture of spinal cord-derived neural stem cells. In this study, five groups were divided： basal medium group, control group, bFGF group, TGF-β1 group, GDNF＋ TGF-β1 group. In the basal medium group, DMEM/F12 containing penicillin,streptomycin, amphotericin （AMPH） B and 0.02 volume fraction of B27 annex solution. At 1 week after primary culture, rat spinal cord-derived NSC clones proliferated in vitro stably were harvested. In the control group, 0.1 volume fraction of FBS was added into basal medium. In the later three groups, induced medium was exchanged, i.e. 20 μg/L bFGF, 2 μg/L TGF-β1, and 10 μ

关 键 词：神经干细胞 分化 神经元 胶质细胞源性神经营养因子 转化生长因子Β1 

分 类 号：R394.2[医药卫生—医学遗传学]