不同胎龄人胎脑室下区神经干细胞生长特征及增殖分化实验研究

Experimental study on growth, proliferation and differentiation of neural stem cell from subventricular zone of human fetal brain at different gestational age

目的探讨不同胎龄人胎脑室下区神经干细胞(NSCs)的生长特征。方法收集胎龄16～36周的胎儿90例,每例研究对象取室下区脑组织作为实验材料。所有研究对象产前经B超诊断患有先天性心脏病或消化道畸形,均无脑发育异常,孕妇及其丈夫要求终止妊娠。采用免疫组化技术对人胎脑室下区NSCs的形态、存在方式以及数量进行检测,采用细胞培养技术对其进行培养、传代、分化观察,并应用免疫组化技术对培养及分化的细胞进行鉴定。结果人胎脑室下区NSCs具有星形、圆形、椭圆形及梭形等形态,以星形最多。细胞胞浆相对较少;核呈圆形,染色质疏松,1～4个核仁不等。可见NSCs对称分裂和不对称分裂现象,NSCs多数单个散在分布,有的NSCs与别的NSCs形成突触联系。不同胎龄人胎脑室下区NSCs随着胎龄的增加而减少(X2=4644．602,P<0．01)。不同胎龄以及同一胎龄不同个体的NSCs在上述方面存在一定的差别。从人胎脑室下区分离的细胞在无血清培养时呈悬浮状态生长,形成神经球。该细胞可传代培养,表达神经巢蛋白抗原(Nestin);而在含血清培养时能分化,分化细胞表达神经元细胞、少突及星形胶质细胞的特异性抗原。结论不同胎龄人胎脑室下区均存在NSCs,且在形态、存在方式及数量上存在一定的差异。不同胎龄人胎脑室下区均能在体外培养出NSCs。

Objective To study growth characteristics of neural stem cells （NSCs） from subventricular zone （SVZ） of the different human fetal brain at different gestational age and to provide experimental and theoretical evidences for clinical application of NSCs for treatment of certain diseases. Methods Ninety human embryos at gestational age 16-36 weeks were collected and were divided into six groups according to gestational age： 16 w, 20 w, 24 w, 28 w, 32 w and 36 w. Each group had 15 embryos and brain tissues were taken from each embryo＇s SVZ. All subjects had congenital heart disease or digestive tract abnormity diagnosed with B ultrasound at antepartum, but none had abnormal developmemt of brain. Pregnant mother and her husband desire termination of pregnancy. The morphology, existing mode and the number of neural stem cells in subventricular zone were examined with immunohistochemical method. The NSCs in subventricular zone were cultured, passaged and differentiated with cell culture technique, then were identified with immunohistochemical method. Results NSCs in SVZ from the different human fetal brain existed in a scattered manner in the network formed by stellate cells, NSCs had round, ellipse and fusiform shape, especially in stellate shape. NSCs had larger and smaller size and distributed in dense or scattered forms, each having zero to two enations, most had one or two. NSCs had less cytoplasm. The nucli of the NSCs had a round shape with loose chromatin and 1-4 nucleoli. Most of NSCs existed in singular scattered form, some of them showed symmetral or asymmetral division, some of them showed synaptic connection with other NSCs. The number of NSCs in SVZ from groups with different fetal age decreased with increasing gestational age （x^2 = 4644. 602, P 〈 0. 01 ）. NSCs in SVZ from the different human fetal brain cultured with serum-free medium formed typical neurospheres in suspension. The cells could be passaged continuously, and could express nestin antigen. Serum-contained medium induced neural stem cells to differentiate and express specific antigens of neuron, astrocyte and oligodendrocyte. Conclusions NSCs existed in SVZ of human embryos at different gestational age. There are differences in morphology, existing pattern and the number of NSCs in SVZ at different gestational age. NSCs in SVZ at different gestational age may be cultured in vitro.