神经生长因子对神经干细胞分化及神经元轴突形成的影响  

Effects of nerve growth factor on the differentiation of neural stem cells and the formation of neuronal axons

作　　者：乌优图[1] 王运杰[1] 

机构地区：[1]中国医科大学附属第一医院神经外科,辽宁省沈阳市110001

出　　处：《中国组织工程研究与临床康复》2008年第29期5631-5635,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景：将大量的神经干细胞定向诱导分化后的神经细胞能否与其他神经细胞建立功能联系是目前解决神经干细胞应用于临床的重要问题之一。目的；观察神经生长因子对体外培养的神经干细胞生长和分化的影响，以及神经生长因子对神经轴突形成和生长的作用。设计、时间及地点：细胞学体外观察，于2007—06／2008—12在中国医科大学设备处完成。材料：清洁级二三天龄新生SD雄性大鼠3只，神经生长因子为peprotech公司产品。方法：酶消化和机械分离法相结合体外分离培养新生鼠神经干细胞，传至第4代的细胞克隆团行巢蛋白免疫细胞化学染色观察，剩余细胞团用机械法分散，采用有限稀释法进行单克隆神经干细胞培养，加入完全培养基制成10^8L^-1的单细胞悬液，分为2组滴入培养板，对照组加入10％FBS，诱导组加入10％FBS＋50μg／L神经生长因子，培养5～7d。连续观察5个神经元特异烯醇化酶染色阳性且未与其他神经元发生连接的孤立神经元，求助于以神经元为圆心的同心圆，分别计数内径为37．5μm和75μm圆环内的突触数量，将两者均值视为神经元轴突数量，通过此同心圆测量最长轴突的长度。主要观察指标：通过巢蛋白、神经元特异烯醇化酶、胶质原纤维酸性蛋白免疫组化染色对培养细胞进行鉴定。检测神经元数量及轴突数量、长度。结果：所培养出的细胞团均为巢蛋白阳性，诱导分化后均可产生神经元特异烯醇化酶、胶质原纤维酸性蛋白阳性细胞。诱导分化第6天，诱导组神经元数量、单个神经元轴突数量、最长轴突长度均明显高于对照组（t=3．301，2．982，4．012，P均〈0．01）。结论：神经生长因子可促进神经干细胞向神经元的分化，还可以增加由神经干细胞分化而来的神经元突起数量及长度。BACKGROUND： The differentiation of neural stem cells （NSCs） is an important for NSCs to be applied in clinical treatment. And whether the neurons differentiated from NSCs can be connected with other neurons or not comes into being a critical problem. OBJECTIVE： To observe the effect of nerve growth factor （NGF） on the growth and differentiation of in vitro cultured NSCs, and on the formation and growth of axons. DESIGN, TIME AND SETTING： The cytology in vitro experiment was performed at the Equipment Center of China Medical University from June 2007 to December 2008. MATERIALS： Three 2-3 day male Sprague Dawley rats were used in this study. NGF was purchased from Peprotech. METHODS： NSCs were isolated from neonatal rats by enzyme digestion and mechanical separation. At the fourth passage, cell clone masses received nestin immunocytochemistry. Remaining cells were dispersed by mechanical separation. Monoclone NSCs were incubated by limiting dilution assay, and made into 10^8 L^-1 monoplast suspension in complete medium. NSCs were assigned into 2 groups. NSCs in the control group were incubated in 10% fetal bovine serum （FBS）. NSCs in the induction group were incubated in the 10% FBS＋50 μ g/L NGF for 5-7 days. The five isolated neurons with positive expression of neuron specific enolase （NSE） were observed. The number of axons was measured through concentric circles （37.5 μ m and 75 μ m diameter） circling neurons to detect the length of long axon. MAIN OUTCOME MEASURES： Cultured cells were identified by nestin, NSE and glial fibrillary acidic protein （GFAP） immunohistochemistry to test the number of neurons, number and length of axons. RESULTS： Neurospheres were Nestin-positive and could differentiate into the NSE-positive or GFAP-positive cells. At day 6, the numbers of neurons and axons were significantly more, and the length of longest axons was significantly longer in the induction group than in the control group （t=3.301,2.982, 4.012, P 〈 0.01）. CONCLU

关 键 词：神经干细胞 神经生长因子 神经元 轴突 

分 类 号：R394.2[医药卫生—医学遗传学]