神经干细胞在人胎脑及缺血缺氧性脑病中变化规律研究
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摘要
第一部分人胎脑神经干细胞发育规律及其克隆实验研究
目的：探讨人胎脑发育中NSCs的分布、形态、生长方式以及数量等特点，并对不同胎龄不同部位的脑组织在体外分别进行NSCs培养，同时鉴定其增殖及分化潜能，最终探明胎脑发育中NSCs的变化规律，为进一步在体内外进行调控NSCs的增殖分化机制研究以及临床应用NSCs治疗神经系统退行性疾病提供实验及理论依据。
方法：收集胎龄16～36w的自愿水囊引产胎儿90例作为研究对象，按胎龄16w，20w，24w，28w，32w，36w分成6组，每组15例；每例研究对象分别取海马、纹状体、室下区、额叶、颞叶、枕叶、顶叶等7个部位的脑组织作为实验材料。采用原位杂交、免疫组织化学以及光镜技术分别对6组7个部位胎脑组织NSCs的分布、形态、生长方式以及数量进行检测。采用包含bFGF及EGF的无血清培养基和单细胞克隆技术，分别从6组7个部位胎脑组织中分离出NSCs，并在体外进行培养、传代、分化，同时应用免疫荧光染色技术对培养的NSCs及其分化的细胞进行鉴定。
结果：6组7个部位胎脑组织均存在NSCs，NSCs包括大小圆形、大小椭圆形、梭形、星形、三角形、多角形等细胞形态。可观察到两两对称和不对称分裂的NSCs、2-8个NSCs形成的集落、群状、簇状等NSCs生长方式，群状、簇状分布的NSCs好似群状迁移的NSCs群也好似NSCs发生中心，有的NSCs突起伸向别的细胞，形成突触联系。不同胎龄不同部位NSCs在分布部位、形态、生长方式以及数量等方面存在一定差异。不同胎龄同一部位的NSCs数量随着胎龄的增加而减少，同一胎龄
不同部位的NSCs数量自海马、室下区、纹状体、额叶、颖叶、顶叶以
及枕叶依次减少，且相互之间存在显著性差异。星形NSCs呈Nestin和
CFAP双表达。在28w胎龄组的海马及室下区、32w和36w胎龄组的海
马、室下区及纹状体等部位均存在Nestin和CD34蛋白双表达阳性NSCs。
在16w，20w，24W，28w和32w胎龄组的7个部位均存在Nestin和CD133
蛋白双表达阳性NSCs。6组7个部位的脑组织在无血清培养基中培养时
细胞呈悬浮状态生长，形成神经球，该细胞具有连续克隆能力，可传代
培养，表达神经巢蛋白抗原;在含血清培养基中培养时NSCs可被诱导
分化，分化后的细胞分别表达神经元细胞、星形胶质细胞和少突胶质细
胞的特异性抗原。各胎龄组、各部位分别培养的NSCs在细胞形态、增
殖和多分化潜能方面无明显差别。
结论:胎龄16~36w人胎脑海马、室下区、纹状体、额叶、颜叶、
顶叶等部位均存在NSCs。冻存细胞和水囊引产后48h内分离的原代细胞
均能培养出NSCs，其原代NSCs与死亡时间呈负相关;bFGF和EGF是
NSCs存活和增殖的关键生长因子。Nestin和CD133是人胎脑NSCs的特
征性标记物，CD34可能是人胎脑晚期NSCs的特征性标记物。不同胎龄
同一部位人胎脑NSCs在分布部位、形态、生长方式以及数量等方面存
在差异，同一胎龄不同部位人胎脑NSCs在分布部位、形态、生长方式
以及数量等方面同样存在差异。人胎脑室下区及海马齿状回是NSCs的
发源地，但人胎脑可能存在室下区及海马齿状回以外的NSCs生发中心。
人胎脑NSCs仍然是移植的重要材料来源。人胎脑在体NSCs的形态多样
性与体外培养NSCs的形态单一性不一致的现象提示，在体调控自身
Nscs的增殖和分化可能是治疗疾病另一条更有前景的道路。
Section One
A study on neural stem cells about developing laws and experimental
cloning in human fetal brain
Objectives To investigate the features of neural stem cells(NSCs) from the developing fetal brain of human,the features including distribution, shape, growth mode and amount. To culture NSCs of fetal brain tissue from different gestational age and different location in vitro respectively and determine potency of proliferation and differentiation.Eventually,to find developing laws of NSCs, so as to provide experimental and theoretic evidences for further studying mechanism about controlling proliferation and differentiation in vivo and in vitro and treating regressional diseases of nervous system.
Methods 90 cases of fetuses at gestational ages 16~36w and by induction of labor with water bag were collected and divided into six groups according to gestational age 16w,20w, 24w,28w,32w and 36w,each including 15 cases. Experimental materials including hippocampus, striatum, subventricular zone (SVZ),the frontal lobe,the temporal lobe,the parietal lobe and, the occipital lobe were cutted from brains of cases. Distribution,shape,growth mode and the number of NSCs in six groups
including seven locations were examined with hybridization in situ and immunohistochemical method under light microscope.NSCs in six groups including seven locations were isolated, cultured,passed and differentiated respectively with serum free medium containing basic fibroblast growth factor(bFGF) and epidermal growth factor(EGF) and single cell clone tecnique,then the cells including cultured NSCs and differentiated cells were identified with immunofluorescence staining.
Results NSCs that included large or small round and elliptic,fusiform, cajal,triangle and multiangle cells were existed in fetal brain tissues in six groups including seven locations.NSCs had several growth modes including unsymmetral or symmetral cleavage,clony with two or eight cells,cluster and crowd,as if the clusters and crowds of NSCs settled down on the migration way,or they were cell generative centres.Enations of some NSCs extended to others,there,they forming synapse.There were some different items including distribution,shape,and growth mode at different location in different gestational age.The number of NSCs in the same region were decreasing with the increase of gestational age.NSCs of the same gestational age in hippocampus,striatum,SVZ,the frontal lobe,the temporal lobe,the parietal lobe and the occipital lobe decreased by returns,there was a significant differences among them.Astrocytes of NSCs expressed not only nestin but also GFAP.NSCs expressed nestin and CD34 existed in hippocampus and SVZ at gestational age 28w,they also existed in hippocampus,striatum and SVZ at gestational age 32w and 3 6w.There were NSCs expressed nestin and CD34 at gestational age 16w, 20w, 24w, 28w and 32w.The less the gestational age,the more the NSCs.The NSCs from brain tissues in six groups including seven locations were success fully isolated and cultured with serum free midium,they formed typical neuraospheres in suspension.These NSCs could be cloned and passed continuously,expressing nestin antigen.Serum midium induced these NSCs to differentiate and express specific antigens of neuron,astrocyte and oligodendrocyte.In cultured NSCs respectively,There
were no significant differences on shape,proliferation and differentiation.
Conclusions Our results suggest following conclusions:First,NSCs exist in locations of human fetal brain at gestational age 16-36w,the locations including hippocampus,striatum,SVZ,the frontal lobe,the temporal lobe,the parietal lobe and the occipital lobe.The second, Freezed and primary cells from fetal brain by induction of labor with water bag have be cultured with serum free midium,but there is an inverse relationship between the number of spheres with the time course of postmortem.The third,EGF and bFGF are the key growth factor for p NSCs' proliferation. The fourth,Nestin and CD 133 are the characteristic markerof NSCs,CD34 may be the c
目的：探讨人胎脑发育中NSCs的分布、形态、生长方式以及数量等特点，并对不同胎龄不同部位的脑组织在体外分别进行NSCs培养，同时鉴定其增殖及分化潜能，最终探明胎脑发育中NSCs的变化规律，为进一步在体内外进行调控NSCs的增殖分化机制研究以及临床应用NSCs治疗神经系统退行性疾病提供实验及理论依据。
方法：收集胎龄16～36w的自愿水囊引产胎儿90例作为研究对象，按胎龄16w，20w，24w，28w，32w，36w分成6组，每组15例；每例研究对象分别取海马、纹状体、室下区、额叶、颞叶、枕叶、顶叶等7个部位的脑组织作为实验材料。采用原位杂交、免疫组织化学以及光镜技术分别对6组7个部位胎脑组织NSCs的分布、形态、生长方式以及数量进行检测。采用包含bFGF及EGF的无血清培养基和单细胞克隆技术，分别从6组7个部位胎脑组织中分离出NSCs，并在体外进行培养、传代、分化，同时应用免疫荧光染色技术对培养的NSCs及其分化的细胞进行鉴定。
结果：6组7个部位胎脑组织均存在NSCs，NSCs包括大小圆形、大小椭圆形、梭形、星形、三角形、多角形等细胞形态。可观察到两两对称和不对称分裂的NSCs、2-8个NSCs形成的集落、群状、簇状等NSCs生长方式，群状、簇状分布的NSCs好似群状迁移的NSCs群也好似NSCs发生中心，有的NSCs突起伸向别的细胞，形成突触联系。不同胎龄不同部位NSCs在分布部位、形态、生长方式以及数量等方面存在一定差异。不同胎龄同一部位的NSCs数量随着胎龄的增加而减少，同一胎龄
不同部位的NSCs数量自海马、室下区、纹状体、额叶、颖叶、顶叶以
及枕叶依次减少，且相互之间存在显著性差异。星形NSCs呈Nestin和
CFAP双表达。在28w胎龄组的海马及室下区、32w和36w胎龄组的海
马、室下区及纹状体等部位均存在Nestin和CD34蛋白双表达阳性NSCs。
在16w，20w，24W，28w和32w胎龄组的7个部位均存在Nestin和CD133
蛋白双表达阳性NSCs。6组7个部位的脑组织在无血清培养基中培养时
细胞呈悬浮状态生长，形成神经球，该细胞具有连续克隆能力，可传代
培养，表达神经巢蛋白抗原;在含血清培养基中培养时NSCs可被诱导
分化，分化后的细胞分别表达神经元细胞、星形胶质细胞和少突胶质细
胞的特异性抗原。各胎龄组、各部位分别培养的NSCs在细胞形态、增
殖和多分化潜能方面无明显差别。
结论:胎龄16~36w人胎脑海马、室下区、纹状体、额叶、颜叶、
顶叶等部位均存在NSCs。冻存细胞和水囊引产后48h内分离的原代细胞
均能培养出NSCs，其原代NSCs与死亡时间呈负相关;bFGF和EGF是
NSCs存活和增殖的关键生长因子。Nestin和CD133是人胎脑NSCs的特
征性标记物，CD34可能是人胎脑晚期NSCs的特征性标记物。不同胎龄
同一部位人胎脑NSCs在分布部位、形态、生长方式以及数量等方面存
在差异，同一胎龄不同部位人胎脑NSCs在分布部位、形态、生长方式
以及数量等方面同样存在差异。人胎脑室下区及海马齿状回是NSCs的
发源地，但人胎脑可能存在室下区及海马齿状回以外的NSCs生发中心。
人胎脑NSCs仍然是移植的重要材料来源。人胎脑在体NSCs的形态多样
性与体外培养NSCs的形态单一性不一致的现象提示，在体调控自身
Nscs的增殖和分化可能是治疗疾病另一条更有前景的道路。
Section One
A study on neural stem cells about developing laws and experimental
cloning in human fetal brain
Objectives To investigate the features of neural stem cells(NSCs) from the developing fetal brain of human,the features including distribution, shape, growth mode and amount. To culture NSCs of fetal brain tissue from different gestational age and different location in vitro respectively and determine potency of proliferation and differentiation.Eventually,to find developing laws of NSCs, so as to provide experimental and theoretic evidences for further studying mechanism about controlling proliferation and differentiation in vivo and in vitro and treating regressional diseases of nervous system.
Methods 90 cases of fetuses at gestational ages 16~36w and by induction of labor with water bag were collected and divided into six groups according to gestational age 16w,20w, 24w,28w,32w and 36w,each including 15 cases. Experimental materials including hippocampus, striatum, subventricular zone (SVZ),the frontal lobe,the temporal lobe,the parietal lobe and, the occipital lobe were cutted from brains of cases. Distribution,shape,growth mode and the number of NSCs in six groups
including seven locations were examined with hybridization in situ and immunohistochemical method under light microscope.NSCs in six groups including seven locations were isolated, cultured,passed and differentiated respectively with serum free medium containing basic fibroblast growth factor(bFGF) and epidermal growth factor(EGF) and single cell clone tecnique,then the cells including cultured NSCs and differentiated cells were identified with immunofluorescence staining.
Results NSCs that included large or small round and elliptic,fusiform, cajal,triangle and multiangle cells were existed in fetal brain tissues in six groups including seven locations.NSCs had several growth modes including unsymmetral or symmetral cleavage,clony with two or eight cells,cluster and crowd,as if the clusters and crowds of NSCs settled down on the migration way,or they were cell generative centres.Enations of some NSCs extended to others,there,they forming synapse.There were some different items including distribution,shape,and growth mode at different location in different gestational age.The number of NSCs in the same region were decreasing with the increase of gestational age.NSCs of the same gestational age in hippocampus,striatum,SVZ,the frontal lobe,the temporal lobe,the parietal lobe and the occipital lobe decreased by returns,there was a significant differences among them.Astrocytes of NSCs expressed not only nestin but also GFAP.NSCs expressed nestin and CD34 existed in hippocampus and SVZ at gestational age 28w,they also existed in hippocampus,striatum and SVZ at gestational age 32w and 3 6w.There were NSCs expressed nestin and CD34 at gestational age 16w, 20w, 24w, 28w and 32w.The less the gestational age,the more the NSCs.The NSCs from brain tissues in six groups including seven locations were success fully isolated and cultured with serum free midium,they formed typical neuraospheres in suspension.These NSCs could be cloned and passed continuously,expressing nestin antigen.Serum midium induced these NSCs to differentiate and express specific antigens of neuron,astrocyte and oligodendrocyte.In cultured NSCs respectively,There
were no significant differences on shape,proliferation and differentiation.
Conclusions Our results suggest following conclusions:First,NSCs exist in locations of human fetal brain at gestational age 16-36w,the locations including hippocampus,striatum,SVZ,the frontal lobe,the temporal lobe,the parietal lobe and the occipital lobe.The second, Freezed and primary cells from fetal brain by induction of labor with water bag have be cultured with serum free midium,but there is an inverse relationship between the number of spheres with the time course of postmortem.The third,EGF and bFGF are the key growth factor for p NSCs' proliferation. The fourth,Nestin and CD 133 are the characteristic markerof NSCs,CD34 may be the c
引文
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45. Noctor S C,Fint A C,Weissman T A,et al.Neurons derived from radial glial cells establish radial units in neocortex.Nature, 2001, 409 (6821):714-720.
46. Flax J,Aurora S.Engraftable human stem cells respond to cues, replace, and express foreign genes.Nature Biotech, 1998,16(2): 1033-1099.
47. Santa-Olalla J.Basic fibroblast growth factor promotes epidermal growth factor responsiveness and survival of mesencephalic neural precuror cells. J.neurobiol, 1999, 40(1): 14-27.
48. Kirino T,Tsujita Y,Tamum A.Induced toderance to ischemia in gerbil hippocampal neurons.J Cereb Blood Flow Metab, 1991,11(2):299-307.
49．安沂华，瞿晶，历俊华，等．离体培养神经干细胞的超微结构学研究．中国康复理论与实践．2004，10(1)：11-12．
50. Calli R,Regulation of neuronal differentiation in human CNS stem cell progeny by leukemia inhibitory factor.Dev Neurosci, 2000,22(3):86-95.
51. Cataudella T,Conti L,Cattaneo E.Neural stem and progenitor cells: choosing the fight Shc.Prog Brain Res.2004,146(2): 127-133.
52. An YH,Wan H,Zhang ZS,et al.Effect of rat Schwann cell secretion on proliferation of human neural stem cells.Biomed Environ Sci,2003, 16(1):90-94.
53. Riess P,Zhang C,Saat man KE,et al.Tranplanted neural stem cells survive, differentiate,and improve neurological motor function after experimental traumatic brain injury. Neurosurgery. 2002, 51(4):1043-1052.
54. Li XK, Guo AC,Zuo PP.Survival and differentiation of transplanted neural stem cells in mice brain with MPTP-induced Parkinson disease.Acta, Pharmacol Sin,2003,24(12):1192-1198.
55. Galli R, Gritti A,Bonfanti L,et al.Neural stem cells.Circ Res, 2003, 92(6):598-608.
56．陆华，蒋云召，苗宗宁，等．人胚脑皮层神经干细胞的分离培养及鉴定．中国微循环，2002，6(1)：9-10．
57. Johansson CB,Momma S,Dianal T,et al.Identification of a neural stem cell in the adult mammalian central nervous system.Cell, 1999,96(1):25-34.
58．程飙，付小兵，盛志勇．干细胞：未来疾病治疗的新希望[J]．中国临床康复，2003，7(2)：178-180．
59. Fuchs E,Sergre J.Stem cell:A New Lease on Life.Cell ,2000,100(1): 143-155.
60. Clarke D,Johansson C,Wilbertz J,et al.Generalized potential of adult neural stem cells.Science, 2000;288(5471): 1660-1663.
61. Wachs FP,Couillard-Despres S,Engelhardt M,et al.High efficacy of clonal growth and expansion of adult neural stem cells.Lab Invest 2003, 83(7):949-962.
62. Poltavtseva RA, Revishchin AV, Aleksandrova MA,et al.Neural stem and progenitor cells of human embryos and fetuses as a basis of biomedical new technologies Ontogenez 2003;34(3):211-215.
63. Wang L,Hu H,Zhang C,et al.Isolation, cultivation and identification of neural stem cell from human embryonic CNS Sheng Wu Yi Xue Gong Cheng Xue Za Zhi 2002; 19(2):264-267.
64．张朝佑．人体解剖学．第二版．北京：人民卫生出版社，1998．1311-1325．
65. Potter E D,Ling Z D,Carvey P M.Cytokine-duced conversion of mesencepalic-derived progenitor cells into dopamine neurons.Cell Tissue Res, 1999,296(2) :235-246.
66．余小强，阮怀珍，蔡文琴，等．PD大鼠纹状体提取液诱导中脑神经干细胞分化为多巴胺能神经元的研究．第三军医大学学报，2004，26(1)：18-21．
67．王忠成．神经干细胞在中枢神经系统损伤修复中的应用前景．中国康复理论与实践．2004，10(1)：1．
68．胡志兵，陆雪芬，郑得枢，等．神经节苷脂GM1对缺血缺氧后脑中HSP70表达的影响．卒中与神经疾病，2001，8(5)：278-281．
69．王培福，田成林，蒲传强．大鼠神经干细胞的培养和鉴定．军医进修学院学报同，2003，24(5)：30-32．
70. JLindvall O,Hagell P.Cell therapy and transplantation inParkinson's disease.Clin Chem Lab Med,2001,39(3):356-361.
71. Reynolds BA,Tetzlaff W,Weiss S.A multipotent EGF-re-sponsive striatal embryonic progenitor cell produces neuronsand astrocytes. J Neurosci, 1992,12 (1):4565-4574.
72. Whittemore SR,Morassutti DJ,Walters WM etal.Mitogenand substrate differentially affect the lineage restriction ofadultratsubventricularzone neural precursor cell populations.Exp Cell Res,1999,252(3):75-95.
73. Ozawa K,Urtmo T,Miyakawa K et al.Expression of the fi-broblast growth factor family and their receptor family genesduring mouse brain development.Brain Res Mol Brain Res, 1996,41 (1):279-288.
74. Gritti A,Parati EA,Cova Let al.Multipotential stem cells from the adult mouse brain proliferate and self-renew in re-sponse to basic fibroblast growth factor.J Neurosci, 1996,16(3): 1091-1100.
75．尹晓娟，封志纯，杜江．分离培养不同胚龄人神经干细胞的方法比较．中国临床康复，2004，8(9)：1726-1727．
76．尹晓娟，封志纯．神经干细胞及其应用前景．中国临床康复，2004，8(4)：714-715．
77. Kilpatrick TJ,Bartlett PF.Cloned multipotential precursorsfrom the mouse cerebrum require FGF-2,whereas glial re-stricted precursors are stimulated with either FGF-2or EGF.J Neurosci, 1995,15(4):3653-3661.
78. Svendsen CN, Fawcett JW,Bentlage C.Increased survival of rat EGF-generated CNS precursor cells using B27 supplemented medium.Exp Brain Res, 1995,102(3):407-414.
79. Morshead CM,Reynolds BA,Craig CG. Neural stem cells in the adult mammalian forebrain:A relatively quiescent subpopulation of subependymal cells.Neuron, 1994,13(5): 1071-1082.
80. Studer L,Csete M,Lee SH,et al.Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen. J Neurosci,2000,20(5):7377-7383.
81. Hoshida S,Kuzuyu T,Fuji H. Sublethal ischemia alters myocardial antioxidant activity in canine heart .Am J Phisiol, 1993,264(1Pt2):33-39.
82. Rordorf G,Koroshetz WJ,Bonveutre JV.Heat shock protects cultured neurons from glutamate toxicity.Neuron, 1991,7(6): 1043-1051.
83. Burmester T,Weich B,Reinhardt S,et al.A vertebrate globin expressed in the brain.Nature,2000,407(6803):52-523.
84．张莉莉，蔡玲玲，张伟，等．缺氧缺血性脑病动物模型实验治疗的病理观察．中华病理学杂志，1996，25(2)：102-104．