体外神经干细胞培养特性观察

Characteristics of neural stem cells in in Vitro cell culture

目的探讨神经干细胞（NSCs）体外分离培养和增殖的特性。方法从新生24h内的SD大鼠脑组织分离NSCs，采用无血清悬浮培养法进行NSCs体外扩增培养。倒置相差显微镜观察细胞形态，通过绘制细胞生长曲线观察NSCs的自我更新和增殖能力，采用免疫细胞化学法检测NSCs标志物神经上皮干细胞蛋白（Nestin）的表达及分化后细胞神经元特异性烯醇化酶（NSE）、胶质纤维酸性蛋白（GFAP）和2，3-环核甘酸磷酸二脂酶（CNP）的表达。结果从新生SD大鼠脑组织分离的细胞在无血清的培养基中形成悬浮的神经球。神经球具有自我更新和表达Nestin的能力，分化后的细胞能表达神经元、星型胶质细胞及少突胶质细胞的特异性抗原。结论从新生大鼠的脑组织中成功分离出NSCs，其具有在体外自我更新和增殖、多向分化潜能及表达Nestin的能力。

Objective To investigate the characteristics of neural stem cells （NSCs） including isolation,culture and expansion in vitro. Methods Primary NSCs were isolated from whole brain of neonatal Sprague Dawley rats （within 24h after birth）. The cells were suspended and cultured in serum--free medium in vitro. The cell morphology was observed with inverted phase--contrast microscope. Plotted growth curve was used to mornitor the capacity of self--renewing and proliferation of NSCs. Immunocy- tochemistry was performed to detect the expression of neuroepithelial stem cell protein （Nestin）, neuron specific enolase （NSE）, glial fibrillary acidic protein （GFAP）, and cyclic nucleotide phosphohydrolase （CNP）. Results The proliferating cells isolated from neonatal Sprague Dawley rats grew into floating neurospheres in the serum--free medium. The neurospheres had the capacities of self--renewing and expressing Nestin. The cells differentiated from these neurospheres could express the specific antigens of neurons,astrocytes,and oligodendrocytes. Conclusions The NSCs were isolated from neonatal rats successfully. In vitro, they showed the capacities of self--renewing and proliferation, pluripotentiality of differentiation,and expressed the Nestin antigen.