摘要
目的探讨新生小鼠室管膜下区(subventricular zone,SVZ)神经干细胞(neural stem cells,NSCs)体外培养方法,为治疗神经系统疾病寻找合适的种子细胞。方法取SPF级新生ICR小鼠SVZ组织,采用机械法和酶消化法分离培养NSCs并传代,倒置显微镜下观察细胞形态。取第3代细胞行抗SOX-2和抗巢蛋白(Nestin)免疫荧光染色鉴定,Brd U标记法及MTT法检测比较培养3、7 d细胞增殖能力。以未添加b FGF和EGF的无血清培养基诱导第3代NSCs分化,抗β-微管蛋白Ⅲ(Tuj-1)、GFAP免疫荧光染色检测比较诱导3、7 d NSCs向神经元及星形胶质细胞分化的能力。结果成功分离培养新生小鼠SVZ组织中细胞,倒置显微镜下见原代培养3 d即有神经球形成,至7 d时见大量悬浮生长神经球,且体积较前增大,部分神经球出现融合及贴壁分化现象。细胞呈典型NSCs形态。免疫荧光染色鉴定获得细胞为NSCs。细胞增殖能力检测显示,体外培养3 d的NSCs中Brd U阳性细胞数为(75.817±2.961)个、吸光度(A)值为0.478±0.025,均显著高于培养7 d的(56.600±4.881)个、0.366±0.032(t=3.366,P=0.028;t=2.752,P=0.011)。经诱导培养后,NSCs能分化为神经元、星形胶质细胞;细胞分化能力检测显示,诱导分化3 d时Tuj-1、GFAP阳性细胞百分比分别为23.1%±3.7%、23.7%±3.8%,显著低于诱导分化7 d(40.1%±3.6%、37.1%±4.5%)(t=3.285,P=0.030;t=3.930,P=0.017)。结论体外培养的新生小鼠SVZ处NSCs时具有自我增殖和多分化潜能,且体外培养时间不同,细胞增殖、分化能力亦不同。
Objective To establish the system of isolation, cultivation, and identification of the neural stem cells （NSCs） from subventricular zone （SVZ） of neonatal mice so as to seek for the appropriate seed cells for potential therapeutic interventions of neurological disorders. Methods NSCs were isolated enzymatically and mechanically from SVZ of neonatal mice and cultured. The cellular morphology was observed by inverted microscopy. Immunocytochemical stainings of anti-Nestin and anti-SOX-2 were used to identify NSCs of passage 3. To study the differentiation of NSCs, NSCs were plated into 24-weUs in the medium supplemented without epidermal growth factor （EGF） and basic fibroblastic growth factor （bFGF）＇ for 3 or 7 days. To compare the differentiation and proliferation potential of NSCs with different cultivation time, the BrdU pulse-labeling method and MTT test were used. To identify neurons and astrocytes, the anti-β-tubulin Ill （Tuj-1） and anti-glial fibrillary acidic protein （GFAP） staining were used. Results The ceils of the SVZ can be isolated and cultured in vitro, and these cells began to form neurospheres after cultured for 3 days at primary passage. While cultured for 7 days, these cells formed more neurospheres, and the volume of the neurospheres became bigger than neurospheres cultured for 3 days. In addition, after cultured for 7 days, the phenomena of fusion of neurospheres and adherent differentiation of neurospheres were observed under inverted microscope. These cells were provided with the typical phenotype of NSCs. The immunofluorescence staining results revealed that these cells showed positive immunoreactivity to Nestin and SOX-2. During the 4 hours BrdU pulse, the number of proliferated NSCs cultured for 3 days （75.817±2.961） was significantly higher than that of NSCs cultured for 7 days （56.600±4.881） （t=3.366, P=0.028）. The results of MTT assay revealed that the absorbance （A） value of NSCs cultured for 3 days （0.478±0.025） was significantly high
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2015年第6期766-771,共6页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金资助项目(81271723)~~
关键词
室管膜下区
神经干细胞
细胞分化
细胞增殖
小鼠
Subventricular zone
Neural stem cells
Differentiation
Proliferation
Mouse
