成人脑神经干细胞体内外增殖、分化和迁移研究

目的探索从成人脑组织获取的神经干细胞（成人-hNSCs）在体外的增殖能力、分化特性、以及在裸鼠颅内的存活、迁移及分化情况。方法分别留取癫痫患者手术切除的颞叶脑组织和10W左右人类自然流产胎儿纹状体组织，体外分离成单细胞悬液，无血清培养基培养、传代并诱导分化。软琼脂糖集落形成实验检测NSCs的增殖能力。免疫荧光法检测NSCs标志物神经上皮巢蛋白（Nestin）和诱导分化后神经元标志物13．tubllin以及神经胶质细胞标志物胶质纤维酸性蛋白（GFAP）的表达；利用动物立体定向仪将体外悬浮培养2W的人NSCs移植入裸鼠颅内，检测NSCs在裸鼠脑组织局部的存活、迁移和分化状况。结果成人-hNSCs集落形成能力较胚胎脑组织来源的NSCs（胎儿-hNSCs）明显减弱，免疫荧光染色显示分离的NSCs呈Nestin阳性，诱导分化后可见（β-tubllin和GFAP阳性的神经细胞，其中80％的细胞为GFAP阳性的星形胶质细胞，20％左右为β-tubllin阳性细胞。分别将成人-hNSCs和胎儿．hNSCs移植入裸鼠纹状体，1个月后，冰冻切片，荧光显微镜观察到来源于成人脑组织来源的NSCs仅见沿针道的近距离迁移，免疫荧光染色在成人-hNSCs移植裸鼠颅内只检测到GFAP阳性的星形胶质细胞。而胎儿-hNSCs可穿过针道，沿大脑廉向脑实质广泛迁移，免疫荧光染色能检测到GFAP和少量（β-tubllin阳性细胞。结论成人脑组织和胚胎纹状体组织中均能成功分离到神经前体细胞，而与胎儿．hNSCs相比，成人-hNSCs体外增殖能力、多向分化潜能和体内迁移能力都明显减弱。

Study on the proliferation, differentiation and migration of neural stem cells derived from adult brain in vitro and in vivo

Objective To investigate the proliferation and differentiation of human adult neural stem cells （adult-hNSCs） in vitro and in vivo. Methods Central nervous system-derived human neural stem cells （CNS- hNSCs） were obtained from adult human brain tissues and human aborted fetus brain （ 10 w） （fetus-hNSCs）, and were suspension cultured, passaged and induced to differentiate in vitro. NSCs marker-Nestin, neuron marker β- tublin and glial cell marker-glial fibrilary acidic protein （GFAP） were detected by method. Furthermore, the properties of NSCs in vivo was studied in nude mice rr~el. Results Compared with fetus-hNSCs, the ability of colony formation of the adult-NSCs was decreased significantly. Using the iinmunofluorescence method, nestin could be identified in adult-hNSCs and the differentiated cells expressed β-tublin and GFAP. After being transplanted into the corpus striatum of nude mice, adult-hNSCs could be detected to survive at least for 6 w, and differentiate into astrocytes. Conclusion CNS-hNSCs can be derived from adult brain and fetus brain. They could be cultured, passaged and induced to differentiate in vitro successfully. Compared with fetus-hNSCs, adult-hNSCs have limited abilities in proliferation, differentiation and migration in vivo.