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.     

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。     

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 .     

全反式维甲酸对小鼠胚胎不同阶段神经干细胞诱导作用研究

目的:探讨全反式维甲酸对小鼠胚胎不同阶段神经干细胞(NSCs)的诱导分化情况.方法:分离孕12.5d及15.5d的胚胎小鼠脑皮质.取第3代NSCs,用含1μmol·L-1的全反式维甲酸在体外诱导小鼠胚胎不同阶段的NSCs.诱导5d后,通过神经元微管相关蛋白2(MAP2)免疫荧光染色和Westernblots检测NSCs分化为神经元的比例.结果:与对照组相比,全反式维甲酸可明显提高神经元分化的比例.E12.5干细胞和E15.5胚胎干细胞分化为神经元的比例分别为30%±1.47%和16.21%±1.36%.结论全反式维甲酸具有显著的促神经干细胞分化成神经元的效应,并对胚胎不同阶段神经干细胞的诱导作用有所不同.     

神经干细胞移植的研究近况

神经干细胞能够再生和自我更新,并可以分化为神经元、星形胶质细胞和少突胶质细胞,因而神经干细胞移植可用于神经变性、脊髓损伤和神经脱髓鞘等疾病的治疗.本文主要总结了神经干细胞移植在帕金森氏病、脱髓鞘症、脊髓损伤、阿耳茨海默氏病和脑中风治疗中的应用.     

人胚胎脑神经干细胞分离纯化及体外培养的研究

为了探讨人胚神经干细胞体外培养条件下的生物学特性,为其应用于临床治疗奠定基础.取胎龄16周的人流产胚胎,胰酶消化结合机械法分离成单细胞悬液,以2×106个细胞/mL接种到含hEGF和h-bFGF的DMEM/F12、N2培养基进行体外培养;观察细胞生长情况,用10% FBS诱导神经干细胞球分化,免疫细胞化学鉴定. 结果显示从人胚大脑分离出的细胞经悬浮培养可以形成细胞球,表达Nestin蛋白.经诱导分化后具有表达神经元,神经胶质细胞的特异性抗原. 说明人胚神经干细胞在体外可以稳定生长,并能分化成为神经原及胶质细胞.     

大鼠胚胎神经干细胞体外培养及诱导分化

神经干细胞在理论研究和临床应用上有着广泛的前景.本文主要在体外分离培养SD大鼠胚胎前脑的神经干细胞,并分别用去除生长因子或添加全反式维甲酸(ATRA)两种方法诱导分化.免疫荧光染色技术分别检测细胞巢蛋白(Nestin)的表达及分化后β微管蛋白Ⅲ(β-Ⅲtubulin)、胶质纤维酸性蛋白(GFAP)的表达,计算分化率.结果显示:细胞生长状态良好,呈Nestin表达阳性.分化后可获得β-Ⅲtubulin及GFAP表达阳性的细胞,其中ATRA诱导方法获得β-Ⅲtubulin阳性细胞较多.     

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.     

大鼠胚胎神经干细胞的培养和鉴定

目的　体外培养和鉴定神经干细胞 .方法　从胎鼠前脑取出脑组织 ,采用酶消化、机械吹打、对倍稀释成单个细胞 ,经无血清培养获得单细胞克隆 ,由免疫细胞化学方法鉴定分离的神经干细胞 .结果　从胎鼠脑中分离的细胞具有连续传代形成克隆的能力 ,表达神经干细胞蛋白 (nestin) ,并能诱导分化成神经元和神经胶质细胞 .结论　分离的细胞是神经干细胞 .     

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。     

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．     

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.     

神经干细胞及其临床应用潜能

近年来神经干细胞已在成年哺乳动物中的中枢神经系统中分离成功。神经干细胞的最基本特征是具有分化为神经元、星状胶质细胞和少突胶质细胞的潜能，具有自我更新能力，并足以维持整个大脑所需。神经干细胞在修复受伤神经组织及治疗神经系统退行性疾病，如帕金森病、阿尔茨海默病、和亨庭顿病等方面有很好的应用前景。但在达到临床实际应用之前仍有一系列问题需要解决，最首要的是搞清神经干细胞的分化机制。     

Neural stem cell transplantation in the repair of spinal cord injury

Neural stem cells are a pronising candidate for neural transplantation aimed at neural cell replacement and repair of the damaged host central nervous system (CNS). Recent studies using neural stem cells have shown that implanted neural stem cells can effectively incorporate into the damaged CNS and differentiate into neurons, astrocytes, and oligodendrocytes. The recent explosion in the field of neural stem cell research has provided insight into the inductive factors influencing neural stem cell differentiation and may yield potential therapies for several neurological disorders, including spinal cord injury. In this review, we summarize recent studies involving neural stem cell biology in both rodents and humans. We also discuss unique advantages and possible mechanisms of using neural stem cell trans plantation in the repair of spinal cord injury.     

Differential Notch signalling distinguishes neural stem cells from intermediate progenitors

During brain development, neurons and glia are generated from a germinal zone containing both neural stem cells (NSCs) and more limited intermediate neural progenitors (INPs). The signalling events that distinguish between these two proliferative neural cell types remain poorly understood. The Notch signalling pathway is known to maintain NSC character and to inhibit neurogenesis, although little is known about the role of Notch signalling in INPs. Here we show that both NSCs and INPs respond to Notch receptor activation, but that NSCs signal through the canonical Notch effector C-promoter binding factor 1 (CBF1), whereas INPs have attenuated CBF1 signalling. Furthermore, whereas knockdown of CBF1 promotes the conversion of NSCs to INPs, activation of CBF1 is insufficient to convert INPs back to NSCs. Using both transgenic and transient in vivo reporter assays we show that NSCs and INPs coexist in the telencephalic ventricular zone and that they can be prospectively separated on the basis of CBF1 activity. Furthermore, using in vivo transplantation we show that whereas NSCs generate neurons, astrocytes and oligodendrocytes at similar frequencies, INPs are predominantly neurogenic. Together with previous work on haematopoietic stem cells, this study suggests that the use or blockade of the CBF1 cascade downstream of Notch is a general feature distinguishing stem cells from more limited progenitors in a variety of tissues.     

Platelet-derived growth factor from astrocytes drives the clock that times oligodendrocyte development in culture

The various cell types in a multicellular animal differentiate on a predictable schedule but the mechanisms responsible for timing cell differentiation are largely unknown. We have studied a population of bipotential glial (O-2A) progenitor cells in the developing rat optic nerve that gives rise to oligodendrocytes beginning at birth and to type-2 astrocytes beginning in the second postnatal week. Whereas, in vivo, these O-2A progenitor cells proliferate and give rise to postimitotic oligodendrocytes over several weeks, in serum-free (or low-serum) culture they stop dividing prematurely and differentiate into oligodendrocytes within two or three days. The normal timing of oligodendrocyte development can be restored if embryonic optic-nerve cells are cultured in medium conditioned by type-1 astrocytes, the first glial cells to differentiate in the nerve: in this case the progenitor cells continue to proliferate, the first oligodendrocytes appear on the equivalent of the day of birth, and new oligodendrocytes continue to develop over several weeks, just as in vivo. Here we show that platelet-derived growth factor (PDGF) can replace type-1-astrocyte-conditioned medium in restoring the normal timing of oligodendrocyte differentiation in vitro and that anti-PDGF antibodies inhibit this property of the appropriately conditioned medium. We also show that PDGF is present in the developing optic nerve. These findings suggest that type-1-astrocyte-derived PDGF drives the clock that times oligodendrocyte development.     

<Emphasis Type="Italic">In vitro</Emphasis> induced dopaminergic differentiation of expanded rat mesencephalic neural stem cell

Recent progress in stem cell biology and recognition of the unique biological properties of stem cell have made it possible to treat the neurodegenerative diseases includ- ing Parkinson抯 disease (PD) by the approach of cell-re- placement and nutritional support with NSCs. Tissue re-construction based on the stem cells endowed us some unpredicted application and market opportunities. Studies in a variety of systems have highlighted perfect prospects for the application of stem cells in the t…     

Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease

Parkinson's disease is a widespread condition caused by the loss of midbrain neurons that synthesize the neurotransmitter dopamine. Cells derived from the fetal midbrain can modify the course of the disease, but they are an inadequate source of dopamine-synthesizing neurons because their ability to generate these neurons is unstable. In contrast, embryonic stem (ES) cells proliferate extensively and can generate dopamine neurons. If ES cells are to become the basis for cell therapies, we must develop methods of enriching for the cell of interest and demonstrate that these cells show functions that will assist in treating the disease. Here we show that a highly enriched population of midbrain neural stem cells can be derived from mouse ES cells. The dopamine neurons generated by these stem cells show electrophysiological and behavioural properties expected of neurons from the midbrain. Our results encourage the use of ES cells in cell-replacement therapy for Parkinson's disease.     

Degeneration in vitro of post-mitotic neurons overexpressing the Alzheimer amyloid protein precursor.

A pathological hallmark of Alzheimer's disease is the deposition of amyloid fibrils in the brain. The principal component of amyloid fibrils is beta/A4 amyloid protein, which can be generated by the aberrant processing of a large membrane-bound glycoprotein, the beta/A4 amyloid protein precursor (APP)3. To test whether overexpression of APP generates abnormally processed derivatives that affect the viability of neurons, we stably transfected full-length human APP complementary DNA into murine embryonal carcinoma P19 cells. These cells differentiate into post-mitotic neurons and astrocytes after exposure to retinoic acid. When differentiation of the APP cDNA-transfected P19 cells was induced, all neurons showed severe degenerative changes and disappeared within a few days. The degenerating neurons contained large amounts of APP derivatives that were truncated at the amino terminus and encompassed the entire beta/A4 domain. These results suggest that post-mitotic neurons are vulnerable to overexpressed APP, which undergoes aberrant processing to generate potentially amyloidogenic fragments.