纹状体神经干细胞的分离培养及其鉴定

胎脑内存在有能分化为神经元和神经胶质细胞的神经干细胞 .在表皮生长因子 (EGF)或碱性成纤维细胞生长因子 (b FGF)存在的条件下 ,从 14d胎鼠纹状体分离培养神经干细胞 ,并通过间接免疫荧光细胞化学法对其鉴定 ,发现大量呈 Nestin抗原阳性的干细胞团的形成 ,EGF和 b FGF对神经干细胞的增殖及分化能产生一定的影响 ,但它们对增殖及分化的影响是有差别的 .     

大鼠胎脑神经干细胞的分离和培养

胚龄14．5～16．5d(E14．5～16．5)的大鼠胎脑组织获得大鼠胎脑神经干细胞(rat fetal neural stem cells，rFNSCs)，培养于含有碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)和表皮生长因子(epidermal growth factor，EGF)的无血清培养液DMEM／F12中，用^3[H]胸苷掺入试验检测EGF和bFGF对大鼠胚胎神经干细胞分裂和增殖的影响．BrdU结合反应和nestin免疫组化检测显示培养细胞在早期时代约90％以上具有分裂增殖能力并显示nestln阳性，而且这些细胞在培养过程中可以分化神经元、星形胶质细胞和少突胶质细胞，证明分离培养的是神经干细胞，可用于移植、定向分化和基因转移的研究．     

成年神经干细胞研究及应用展望

成年神经干细胞(adult neural stem cells ANSCs)起源于人脑室管膜下层、海马的齿状回和嗅回。它具有增殖、多向分化的潜能，能分化成神经元和神经胶质细胞，并迁移到相应的脑区整合在神经网络中。这为利用神经干细胞治疗脑的损伤、退行性病变、帕金森氏综合症(Parkinson’s)、老年性痴呆、多发性硬化症等带来了十分诱人的前景。     

马钱子苷对神经干细胞的增殖、存活和分化的调节作用

为探讨不同浓度的马钱子苷对神经干细胞的增殖、存活和分化的调节作用及其相关分子机制，本实验从成年小鼠大脑中分离培养了神经干细胞，用不同浓度的马钱子苷进行干预，观察马钱子苷对神经干细胞增殖、存活和分化的影响。结果显示：成年小鼠神经干细胞在含有中、高浓度马钱子苷的增殖培养基中培养5 d和7 d后，神经球数量和直径与对照相比显著增加（P<0.05）；中、高剂量的马钱子苷能够促进神经干细胞的有丝分裂，低剂量马钱子苷处理显著促进神经干细胞发生分化（P<0.01），并增加神经元和星形胶质细胞的数量及比例（P<0.01）；中、高剂量马钱子苷抑制神经干细胞分化（P<0.05），高剂量的马钱子苷使得神经元的数量减少（P<0.05）。研究结果表明，高浓度的马钱子苷能够促进神经干细胞存活，并通过促进神经干细胞有丝分裂来提高其增殖能力；低浓度的马钱子苷促进神经干细胞分化，有利于神经再生和少突胶质细胞再生。研究结果为神经干细胞治疗中枢神经系统疾病的研究奠定了理论和实验基础。     

人和小鼠神经干细胞的体外培养的分化研究

首次克隆了小鼠神经元标志性微管蛋白βⅢ基因，从核苷酸序列推导出小鼠与人两者之间在其羧基端有相同的EAQGPK六肽，进一步证实用抗人微管蛋白βⅢ单抗可检测小鼠神经干细胞分化成的神经元细胞，免疫组化鉴定显示小鼠神经干细胞在体积分数为1％胎牛血清（FBS）诱导下，可分化成神经元，星形胶质细胞，少突胶质细胞，同时培养了13周龄胎儿脑来源的人类神经干细胞，用特异性的抗人nestin抗体鉴定，全部为阳性细胞，但它们经诱导分化产生较不同寻常的细胞分化细胞和分化程度，在生长因子减半和1％FBS诱导条件下可分化为神经元和星形胶质细胞，而无少突胶质细胞分化，NF单抗检测证实为早期分化的神经元。     

新生大鼠海马神经干细胞的体外培养及鉴定

的探讨新生大鼠海马神经干细胞（NSC）的体外培养和诱导分化的条件和特点。方法分离出生1d大鼠海马,在表皮生长因子、碱性成纤维生长因子和B27联合作用下使其稳定增殖,用5-溴脱氧尿苷（BMU）标记处于增殖状态的神经干细胞,应用免疫荧光染色方法行巢蛋白（Nestin）、5-溴脱氧尿苷（BrdU）、β-Ⅲ型微管蛋白（Tuj-1）、波形蛋白（Vimenfin）和Galc-C免疫荧光染色,对NSC的增殖及其分化的细胞进行鉴定。结果体外培养的NSC增殖成神经干细胞球并传代,鉴定为Nestin染色阳性细胞和5-溴脱氧尿苷（BrdU）标记染色阳性细胞,并可诱导分化为神经元细胞（Tuj-1染色阳性细胞）、神经胶质细胞（Vimentin染色阳性细胞）和少突胶质细胞（GMc-C染色阳性细胞）。结论采用无血清培养基中加入特定生长因子的培养技术,可培养出在体外稳定增殖并有多向分化潜能的新生大鼠海马神经干细胞。     

新生大鼠海马神经干细胞的体外培养及鉴定

目的 探讨新生大鼠海马神经干细胞(NSC)的体外培养和诱导分化的条件和特点.方法 分离出生1d大鼠海马,在表皮生长因子、碱性成纤维生长因子和B27,联合作用下使其稳定增殖,用5-溴脱氧尿苷(BrdU)标记处于增殖状态的神经干细胞,应用免疫荧光染色方法 行巢蛋白(Nestin)、5-溴脱氧尿苷(BrdU)、β-Ⅲ型微管蛋白(Tuj-1)、波形蛋白(Vimentin)和Galc-C免疫荧光染色,对NSC的增殖及其分化的细胞进行鉴定.结果 体外培养的NSC增殖成神经干细胞球并传代.鉴定为Nestin染色阳性细胞和5-溴脱氧尿苷(BrdU)标记染色阳性细胞,并可诱导分化为神经元细胞(Tuj-1染色阳性细胞)、神经胶质细胞(Vimentin染色阳性细胞)和少突胶质细胞(Calc-C染色阳性细胞).结论 采用无血清培养基中加入特定生长因子的培养技术,可培养出在体外稳定增殖并有多向分化潜能的新生大鼠海马神经干细胞.     

神经生长因子对神经干细胞向少突胶质细胞分化的诱导作用研究

目的体外分离和培养大鼠海马神经前体细胞,在此基础上探索在体外环境下使神经干细胞定向分化为少突胶质细胞,为后续的神经干细胞移植提供实验基础.方法取孕10 d的Wistar大鼠胚胎脑的海马,分离神经前体细胞,将单克隆的神经干细胞球贴壁,并使用生长因子(NGF),分析不同浓度的NGF对神经干细胞分化的影响.在含NGF的NSC培养基中进行体外培养,以GalC免疫组化标记分化后的少突胶质细胞,对神经干细胞的分化特性进行鉴定.结果NGF可促进神经前体细胞的增殖及神经球的克隆形成,并获得了Nestin阳性的神经前体细胞,其可分化为分别表达GaIC的阳性细胞.结论体外分离和培养的大鼠海马神经前体细胞,在NGF生长因子的作用下,神经干细胞可定向分化为少突胶质细胞,并与培养基中NGF的浓度有一定的关系,有望应用于脱髓鞘神经系统疾病的细胞移植治疗.     

人骨髓间充质干细胞对小鼠神经干细胞增殖与分化培养的影响

通过在体外培养、鉴定人的骨髓间充质干细胞与小鼠神经干细胞,用骨髓间充质干细胞条件培养基分别在增殖与分化条件下对神经干细胞进行培养.发现,间充质干细胞条件培养基在增殖条件下能加快神经球内神经干细胞的迁移,使神经球解聚,对神经干细胞增殖没有影响;而间充质干细胞条件培养基在分化条件下,能增加神经干细胞向少突胶质细胞分化的能力,降低向星型胶质细胞的分化能力,对向神经元分化能力没有影响,间充质干细胞可能是通过促进神经干细胞迁移、分化而加快神经损伤的修复的.     

人胚神经干细胞的体外培养

目的　探讨人胚神经干细胞体外培养的条件和分化情况 ,以摸索出一种切实可行的能获得较纯且多潜能人胚神经干细胞的方法 .方法　采用取 3月龄人胎脑 ,用胰蛋白酶消化法分离单个细胞 ,部分冻存 ,另一部分进行细胞培养 ,加EGF ,bFGF刺激生长 ,有限稀释法获得单细胞克隆 ,血清诱导分化 ,并用免疫组化方法进行鉴定 .结果　EGF和bFGF同时存在于无血清培养基中 ,有大量神经干细胞团生成 ,含血清培养基则诱导神经干细胞分化成为神经元、星型胶质细胞、少突胶质细胞 .结论　神经干细胞的存活和分裂有赖于EGF和bFGF的共同作用 .经冻存后的胎脑细胞同样能分离培养出有活性的神经干细胞 .     

Proliferation and differentiation of neuronal stem cells regulated by nerve growth factor

Nerve growth factor plays an important part in neuron-target interactions in the late embryonic and adult brain. We now report that this growth factor controls the proliferation of neuronal precursors in a defined culture system of cells derived from the early embryonic brain. Neuronal precursor cells were identified by expression of the intermediate filament protein nestin. These cells proliferate in response to nerve growth factor but only after they have been exposed to basic fibroblast growth factor. On withdrawal of nerve growth factor, the proliferative cells differentiate into neurons. Thus, in combination with other growth factors, nerve growth factor regulates the proliferation and terminal differentiation of neuroepithelial stem cells.     

Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides

Murine embryonic stem (ES) cells are pluripotent cell lines established directly from the early embryo which can contribute differentiated progeny to all adult tissues, including the germ-cell lineage, after re-incorporation into the normal embryo. They provide both a cellular vector for the generation of transgenic animals and a useful system for the identification of polypeptide factors controlling differentiation processes in early development. In particular, medium conditioned by Buffalo rat liver cells contains a polypeptide factor, ES cell differentiation inhibitory activity (DIA), which specifically suppresses the spontaneous differentiation of ES cells in vitro, thereby permitting their growth as homogeneous stem cell populations in the absence of heterologous feeder cells. ES cell pluripotentiality, including the ability to give rise to functional gametes, is preserved after prolonged culture in Buffalo rat liver media as a source of DIA. Here, we report that purified DIA is related in structure and function to the recently identified hematopoietic regulatory factors human interleukin for DA cells and leukaemia inhibitory factor. DIA and human interleukin DA/leukaemia inhibitory factor have thus been identified as related multifunctional regulatory factors with distinct biological activities in both early embryonic and hematopoietic stem cell systems.     

Adult neural stem cells-Functional potential and therapeutic applications

The adult brain has been thought traditionally as a structure with a very limited regenerative capacity. It is now evident that neurogenesis in adult mammalian brain is a prevailing phenomenon. Neural stem cells with the ability to self-renew, differentiate into neurons, astrocytes and oligodendrocytes reside in some regions of the adult brain. Adult neurogenesis can be stimulated by many physiological factors including pregnancy. More strikingly, newborn neurons in hippocampus integrally function with local neurons, thus neural stem cells might play important roles in memory and learning function. It seems that neural stem cells could transdifferentiate into other tissues, such as blood cells and muscles. Although there are some impediments in this field, some attempts have been made to employ adult neural stem cells in the cell replacement therapy for traumatic and ischemic brain injuries.     

神经干细胞的体外克隆

为建立神经干细胞体外稳定克隆的培养方法并对克隆进行鉴定,从胚胎大鼠脑组织中分离神经干细胞,采用无血清培养技术,在生长因子的作用下使其稳定增殖克隆。同时利用免疫荧光染色对神经干细胞进行鉴定。结果培养的神经干细胞增殖成神经干细胞球并传代,鉴定为nestin染色阳性细胞。说明利用无血清技术和特定生长因子,可使神经干细胞在体外稳定增殖克隆。     

A role for adult TLX-positive neural stem cells in learning and behaviour

Neurogenesis persists in the adult brain and can be regulated by a plethora of external stimuli, such as learning, memory, exercise, environment and stress. Although newly generated neurons are able to migrate and preferentially incorporate into the neural network, how these cells are molecularly regulated and whether they are required for any normal brain function are unresolved questions. The adult neural stem cell pool is composed of orphan nuclear receptor TLX-positive cells. Here, using genetic approaches in mice, we demonstrate that TLX (also called NR2E1) regulates adult neural stem cell proliferation in a cell-autonomous manner by controlling a defined genetic network implicated in cell proliferation and growth. Consequently, specific removal of TLX from the adult mouse brain through inducible recombination results in a significant reduction of stem cell proliferation and a marked decrement in spatial learning. In contrast, the resulting suppression of adult neurogenesis does not affect contextual fear conditioning, locomotion or diurnal rhythmic activities, indicating a more selective contribution of newly generated neurons to specific cognitive functions.