胶质细胞源性神经营养因子与转化生长因子β1联合诱导大鼠脊髓源性神经干细胞的分化特点  

Differentiation traits of rat spinal cord derived neural stem cells following combined inducement of glial cell line-derived neurotrophic factor and transforming growth factor beta 1

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

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15期2842-2845,2860,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：2005年深圳市科技计划面上项目;广东省自然科学基金自由申请项目(5009437)~~

摘　　要：目的:观察胶质细胞源性神经营养因子与转化生长因子β1体外联合诱导对脊髓源性神经干细胞分化的影响,为了解神经干细胞在体内多因素环境下的分化表现提供实验依据。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。①碱性成纤维细胞生长因子,表皮生长因子,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);神经巢蛋白多抗(武汉博士德);胶质纤维酸性蛋白多抗,神经丝蛋白单抗(Sigma)。②选用清洁级孕16d的SD大鼠10只,无菌条件下取出胚胎,进行脊髓源性神经干细胞的分离培养。基础培养基组成:含青霉素、链霉素、两性霉素B及体积分数为0.02B27添加液的DMEM/F12。③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,在基础培养基中加入体积分数为0.1的胎牛血清作为对照。同时设立碱性成纤维细胞生长因子组、转化生长因子β1组、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。观察在不同因子诱导情况下脊髓源性神经干细胞的分化行为,免疫细胞化学染色标记神经元与星状胶质细胞的表达。④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。结果:①细胞分化的形态学观察:分化早期可见神经球贴壁,大量细胞向四周爬出,1周后迁徙的大部分细胞完全贴壁,完成分化过程。在细胞密度较低区域可辨认出3种神经细胞类型:神经元样细胞、星状胶质样细胞、寡突胶质样细胞。②神经干细胞免疫组织化学检测:血清对照组、转化生长因子β1组存在大量胶质纤维酸性蛋白�AIM： To observe the in vitro combined inducement effect of glial cell line-derivecl neurotrophic factor （GDNF） and transforming growth factor （TGF） beta 1 on the differentiation of spinal cord derived neural stem cells （NSCs）, and provide experimental evidences for differentiation of NSCs under multiple factor invivo niche. METHODS： The experiment was performed at the Central Laboratory of Shenzhen Hospital Affiliated to Southem Medical University from October 2005 to September 2006. ①There were basic fibroblast growth factor （bFGF）, epidermal growth factor （EGF）, GDNF, TGF β 1 （PeproTech）, fetal calf serum （Hyclone）, polyclonal antibody of nestin （Wuhan Boster）, polyclonal antibody of glial flbrillary acidic protein, monoclonal antibody of neurofllament protein （Sigma）. ② Totally 10 SD rats conceived 16 days of clean grade were selected. Embryon was removed under sterile condition for isolation and culture of spinal cord derived NSCs. Basal medium was composed of penicillinum, phytomycin, amphotericin B and DMEM/F12 containing B27 annex solution of 0.02 volume fraction. ③1 week after primary culture, in vitro spinal cord derived NSCs clone was obtained. Feta calf serum of 0.1 volume fraction was added in the basal medium. At the same time, bFGF group, TGF β 1 group, GDNF plus TGF β 1 group were set up, and then 20 μg/L bFGF, 2 μg/L TGF β 1, 10μg/L GDNF plus 2 μg/L TGF β 1 were added in basal medium, respectively. Differentiation of spinal cord derived NSCs was observed under different inducement. Expressions of neuron and astrocyte ware labeled by immunocytochemical stain. ④Differentiated cells were counted by fluorescence activated cell sorting （FACS） of flow cytometry. Positive percentage of NSCs differentiation into neuron and astrocyte was detected under different inducement condition. RESULTS： ①morphological observation of cell differentiation： At early phase, large quantity of cells crept following the neurosphere attachment, and the co

关 键 词：神经系统 干细胞 细胞分化 神经元 神经生长因子类 转化生长因子Β 

分 类 号：R394.2[医药卫生—医学遗传学]