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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 investigate the in vitro culture methods of neural stem cells (NSCs) in subventricular zone (SVZ) of newborn mice and find appropriate seed cells for the treatment of neurological diseases. Methods SVF tissues from SPF newborn ICR mice were isolated and cultured by mechanical method and enzymatic digestion method. The morphology of the cells was observed under inverted microscope. The third generation cells were identified by immunofluorescence staining of SOX-2 and Nestin. BrdU labeling and MTT assay were used to compare the proliferation of cells cultured for 3 and 7 days. The third generation of NSCs were induced to differentiate in the third generation of NSCs without bFGF and EGF, and the anti-β-tubulin Ⅲ (Tuj-1) and GFAP immunofluorescence staining were used to detect neuronal and astrocytes The ability of glial cells to differentiate. Results The cells in SVZ tissue of neonatal mice were successfully isolated and cultured. Under inverted microscope, neurospheres were formed 3 days after primary culture, and large numbers of neurons were suspended in suspension at 7 days. And adherent differentiation phenomenon. Cells showed typical morphology of NSCs. Immunofluorescence staining identified cells as NSCs. The results of cell proliferation assay showed that the number of BrdU positive cells in vitro cultured in vitro for 3 d was (75.817 ± 2.961) and the absorbance (A) was 0.478 ± 0.025, which were significantly higher than that of the control group (56.600 ± 4.881) , 0.366 ± 0.032 (t = 3.366, P = 0.028; t = 2.752, P = 0.011). After induction, NSCs could differentiate into neurons and astrocytes. The results of cell differentiation assay showed that the percentage of Tuj-1 and GFAP positive cells were 23.1% ± 3.7% and 23.7% ± 3.8% (40.1% ± 3.6%, 37.1% ± 4.5%) (t = 3.285, P = 0.030; t = 3.930, P = 0.017). Conclusions The NSCs of SVZ in vitro cultured in vitro have the potential of self-proliferation and differentiation, and have different culture time in vitro, and their proliferation and differentiation ability are also different.