人胚胎纹状体来源神经干细胞体外培养生物学特性
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摘要
神经干细胞(neural stem cells, NSCs)是对存在于哺乳动物及鸟类等中枢神经系统内的一类具有多向分化潜能和自我更新能力的细胞群的统称。神经干细胞具有广阔的应用前景,是神经系统替代治疗可供选择的最理想细胞。通过对神经干细胞的基础研究不仅可以为临床神经干细胞移植提供直接的资料同时还可以对人类神经系统的发育机制进行研究。本文旨在探讨人类胚胎来源神经干细胞体外培养的部分生物学特性。
我们取8-16周自然流产的人胚胎纹状体区域神经干细胞在体外含有生长因子的无血清培养基中进行增殖。用Accutase酶消化结合巴斯德管机械吹打对神经球进行传代操作。通过Nestin化学免疫荧光检测,显示体外培养的神经干细胞中约有90%为Nestin阳性。分别使用Brdu掺入实验和克隆形成率来检测神经干细胞体外培养增殖能力。神经干细胞体外培养平均Brdu掺入率为39.5±5%,克隆形成率平均为7±0.5%。使用去bFGF和EGF含10%胎牛血清的基础培养基作为分化培养基对神经干细胞进行血清诱导分化。分别采用Tuj-1、GFAP、MBP、nestin抗体对10%血清条件下分化6-8天的神经干细胞进行免疫荧光染色。Tuj-1阳性细胞体积较小,形态多较规整,边缘光滑,呈梭形、椭圆形或近圆形,突起较少而长,多数有1-2个突起。细胞核较小,圆形或椭圆形,核浆致密,居于胞体一侧,DAPI染色较深,胞浆体积与核的体积相近,也较致密,着色较深。多呈团簇样聚集生长,突起相互交织成网,多分布在距神经球较近的地方。Tuj-1阳性细胞约占分化出细胞总数的56.8%。GFAP阳性细胞体积较大,边缘不规则,多边体形,类似于星形,胞浆丰富,胞核体积较大,DAPI染色较浅。位于分化细胞底层,由神经球至分化冠缘较均匀分布,细胞间多较重叠,占分化出细胞总数的43.2%。在实验中,我们没有发现MBP阳性细胞。对分化出的细胞进行nestin抗体免疫荧光反应,我们看到神经球中多数细胞为nestin阳性。
通过本实验我们证实人类胚胎神经干细胞可以在体外无血清条件下长期培养,并保持神经干细胞的自我增殖和多向分化等基本特性。
Neural stem cells are defined as cells which possess the capacity about self-renewing and multi-directional differentiation potentiality like neurons, astrocytesin and oligodendrocytes which exist in various regions of the central nervous system throughout the mammalian lifespan. The present study was initiated to observed the growth characteristic and the features of serum induced differentiation of hfNSCs, and search a high-performance method that can be used for procuring and propagating NSCs in vitro. To explored a feasibility source of suitable cell for NSCs-based therapy.
Brain tissue of aborted human fetal aged 8-16 weeks gestation, Neural stem cells were cultured in serum-free growth medium, the proliferation capability of the cells was observed. Brdu incorporation assay the percentage of Proliferation was about 37.9% and the Cloning efficiency was about 6%-7%. Neural stem cells were induced to differentiate in DMEM/F12 with 10% fetal bovine serum but without the growth factors. Anti-Nestin, anti-MBP, anti-Tuj-1 and anti-GFAP were used in Immunofluorescence labeling. Tuj-1-positive cells were smaller and had a clear fringe. The proportion of Tuj-1-positive in differentiated NSCs from striatum was about 56.8%. The GFAP-positive cells were biger than the other kinds cells, and the appearance of them was irregular. The proportion of GFAP-positive in differentiated NSCs from striatum was about 43.2%. The MBP-positive cell was never seen in the differentiated NSCs from striatum in our study. The nestion-positive cells were located interior the neurospheres. The Tuj-1-positive cells and GFAP-positive cells were all nestion-positive but the fluorescence of differentiated was dimmish compared with the cells before differentiation.
Neural stem cells can be harvested from human fetal brain, and the cells maintain their undifferentiated features and have the capacity of self-renewal and multi-directional differentiation potentiality in vitro.
我们取8-16周自然流产的人胚胎纹状体区域神经干细胞在体外含有生长因子的无血清培养基中进行增殖。用Accutase酶消化结合巴斯德管机械吹打对神经球进行传代操作。通过Nestin化学免疫荧光检测,显示体外培养的神经干细胞中约有90%为Nestin阳性。分别使用Brdu掺入实验和克隆形成率来检测神经干细胞体外培养增殖能力。神经干细胞体外培养平均Brdu掺入率为39.5±5%,克隆形成率平均为7±0.5%。使用去bFGF和EGF含10%胎牛血清的基础培养基作为分化培养基对神经干细胞进行血清诱导分化。分别采用Tuj-1、GFAP、MBP、nestin抗体对10%血清条件下分化6-8天的神经干细胞进行免疫荧光染色。Tuj-1阳性细胞体积较小,形态多较规整,边缘光滑,呈梭形、椭圆形或近圆形,突起较少而长,多数有1-2个突起。细胞核较小,圆形或椭圆形,核浆致密,居于胞体一侧,DAPI染色较深,胞浆体积与核的体积相近,也较致密,着色较深。多呈团簇样聚集生长,突起相互交织成网,多分布在距神经球较近的地方。Tuj-1阳性细胞约占分化出细胞总数的56.8%。GFAP阳性细胞体积较大,边缘不规则,多边体形,类似于星形,胞浆丰富,胞核体积较大,DAPI染色较浅。位于分化细胞底层,由神经球至分化冠缘较均匀分布,细胞间多较重叠,占分化出细胞总数的43.2%。在实验中,我们没有发现MBP阳性细胞。对分化出的细胞进行nestin抗体免疫荧光反应,我们看到神经球中多数细胞为nestin阳性。
通过本实验我们证实人类胚胎神经干细胞可以在体外无血清条件下长期培养,并保持神经干细胞的自我增殖和多向分化等基本特性。
Neural stem cells are defined as cells which possess the capacity about self-renewing and multi-directional differentiation potentiality like neurons, astrocytesin and oligodendrocytes which exist in various regions of the central nervous system throughout the mammalian lifespan. The present study was initiated to observed the growth characteristic and the features of serum induced differentiation of hfNSCs, and search a high-performance method that can be used for procuring and propagating NSCs in vitro. To explored a feasibility source of suitable cell for NSCs-based therapy.
Brain tissue of aborted human fetal aged 8-16 weeks gestation, Neural stem cells were cultured in serum-free growth medium, the proliferation capability of the cells was observed. Brdu incorporation assay the percentage of Proliferation was about 37.9% and the Cloning efficiency was about 6%-7%. Neural stem cells were induced to differentiate in DMEM/F12 with 10% fetal bovine serum but without the growth factors. Anti-Nestin, anti-MBP, anti-Tuj-1 and anti-GFAP were used in Immunofluorescence labeling. Tuj-1-positive cells were smaller and had a clear fringe. The proportion of Tuj-1-positive in differentiated NSCs from striatum was about 56.8%. The GFAP-positive cells were biger than the other kinds cells, and the appearance of them was irregular. The proportion of GFAP-positive in differentiated NSCs from striatum was about 43.2%. The MBP-positive cell was never seen in the differentiated NSCs from striatum in our study. The nestion-positive cells were located interior the neurospheres. The Tuj-1-positive cells and GFAP-positive cells were all nestion-positive but the fluorescence of differentiated was dimmish compared with the cells before differentiation.
Neural stem cells can be harvested from human fetal brain, and the cells maintain their undifferentiated features and have the capacity of self-renewal and multi-directional differentiation potentiality in vitro.
引文
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3、Reynolds B A, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. [J]. Science.1992,255:1707-1710
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8、Xu R, Wu C, Tao Y, et al. Nestin-positive cells in the spinal cord:a potential source of neural stem cells. [J]. Int J Dev Neurosci.2008,26(7):813-820
9、Walton RM, Wolfe JH. In vitro growth and differentiation of canine olfactory bulb-derived neural progenitor cells under variable culture conditions. [J]. J Neurosci Methods.2008,169(1):158-167
10、Cameron HA, Hazel TG, Mckay RD. Regulation of neurogenesis by growth factors and neurotransmitters. [J]. J Neurobiol.1998,36(2):287-306
11、Dictus C, Tronnier V, Unterberg A, et al. Comparative analysis of in vitro conditions for rat adult neural progenitor cells. [J]. J Neurosci Methods.2007,161(2): 250-258
12、Hulspas R, Tiarks C, Reilly J et al. In vitro cell density-dependent clonal growth of EGF-responsive murine neural progenitor cells under serum-free conditions. [J]. Exp Neurol.1997,148(1):147-156
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14、Thilippe T, Jasodhara R, Wolfgang H, et al. FGF-2-Responsive neural stem cell proliferation requires CCg, a novel autocrine/paracrine cofactor. [J]. Neuron.2000, 28(2):385-397
15、Vsecovi AL, Parati EA, Gritti A, et al. Isolation and cloning of mutipotential stem cells from the embryonic human CNS and establishment of transplantable neural stem cells lines by epigenetic stimulation. [J]. Exp Neurol.1999,156(1):71-83
16、Alvarez-B A, Garcia-V JM. Neurogenesis in adult subventricular zone. [J]. Neurosci.2002,22(3):629-634
17、Uchida N, Buck DW, He D, et al. Direct isolation of human central nervous system stem cells. [J]. Proc Natl Acad Sci U S A.2000,97(26):14720-14725
18、Gritti A, Parati EA, Cova L, et al. Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor. [J]. 1996,16(3):1091-1100
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