兔胚胎神经干细胞的分离、培养和鉴别

目的：研究兔胎脑神经干细胞体外生长特性,为探讨神经干细胞的临床应用及神经系统的发育奠定基础。方法：采用含碱性成纤维细胞生长因子（bFGF）和表皮细胞生长因子（EGF）的N2无血清培养技术,取18天龄兔胚胎脑组织,分离神经干细胞,并观察分离的细胞体外培养、增殖、分化潜能,免疫组化鉴定。结果：从18天龄兔胎脑皮质和纹状体中成功分离出具有自我更新和多分化潜能的神经干细胞,在无血清培养时细胞呈半贴壁状态生长,形成神经球,可传代。细胞呈Nestin免疫反应阳性;在含血清培养基中培养时则分化,分化后的细胞表达神经元细胞、星形胶质细胞和少突胶质细胞的特异性抗原。结论：来自兔胎脑神经干细胞能在体外培养、增殖并保持传代能力。无血清N2EGF、bFGF培养基有利于兔胎脑神经干细胞的存活和增殖,含血清培养基能诱导兔胎脑神经干细胞分化。     

维甲酸和EGF对大鼠脑胚胎神经干细胞增殖和分化的影响

目的 观察全反式维甲酸(RA)和表皮生长因子(EGF)对大鼠胚胎神经干细胞增殖和分化的影响。方法 从大鼠胚胎脑中分离神经干细胞,经RA和EGF处理后,用台盼蓝确定细胞数量,BrdU标记分析细胞生长能力,采用免疫细胞化学法鉴定神经干细胞和分化的神经细胞。结果 20ng／ml EGF和1μmol／LRA处理的培养细胞均显示增殖效应,但EGF处理组增殖速度明显高于RA组,悬浮细胞中有大量nestin和BrdU阳性细胞。用EGF和EGE／RA诱导的神经元分化率分别为17％和31％,而RA处理的神经元分化率显升高至89％。由EGF、EGF／RA和RA诱导的星形胶质细胞分化率分别为83％、69％和11％。结论 EGF主要促进神经干细胞增殖并主要诱导星形胶质细胞的生成,RA主要诱导神经干细胞向神经元分化,二无明显协同效应。     

bFGF和BMP-2联合应用对体外培养兔骨髓间充质干细胞增殖与骨向分化的影响

目的:研究碱性成纤维细胞生长因子(bFGF)和骨形成蛋白-2(BMP-2)联合应用对体外培养兔骨髓间充质干细胞(BMSCs)增殖与骨向分化的影响.方法:体外培养兔骨髓间充质干细胞,在第2代细胞培养液中加入不同浓度的bFGF和BMP-2,依据加入bFGF和BMP-2浓度的不同分为5个实验组(组1:80 ng/ml bFGF;组2:80 ng/ml BMP-2;组3:30 ng/ml bFGF 30 ng/ml BMP-2;组4:50ng/ml bFGF 50ng/ml BMP-2;组5:80ng/ml bFGF 80ng/ml BMP-2)和对照组(不加任何生长因子),采用绘制生长曲线,四唑盐比色法(MTT),碱性磷酸酶(ALP)活性检测法和碱性磷酸酶(ALP)免疫组化染色法比较各组间差异,观察不同浓度的bFGF和BMP-2联合应用对兔骨髓间充质干细胞增殖与骨向分化的影响.结果:与对照组相比,单独应用80 ng/ml bFGF可显著促进BMSCs的增殖,但对BMSCs的骨向分化显著抑制;单独应用80 ng/ml BMP-2对BMSCs的增殖和骨向分化均有促进作用;30ng/ml bFGF 30 ng/ml BMP-2、50 ng/ml bFGF 50 ng/ml BMP-2和80 ng/ml bFGF 80 ng/ml BMP-2可显著地促进BMSCs增殖和促进BMSCs的骨向分化,且呈正性剂量-效应关系,联合应用两种生长因子较二者单独应用促细胞增殖及骨向分化的效果更为显著.结论:一定浓度范围内,bFGF和BMP-2的联合应用促进BMSCs增殖的同时也促进其骨向分化,两者对BMSCs有明显的协同增强的生物学效应.     

体外神经干细胞克隆球的超微结构-透射电镜观察

为观察培养的神经干细胞克隆球内部的超微结构特征,采用无血清培养技术,在体外进行小鼠纹状体神经干细胞克隆球的培养传代,经过免疫细胞化学鉴定后,对单一的神经干细胞克隆球进行固定,常规透射电镜观察。结果表明,神经干细胞可以在bFGF等生长因子存在的情况下,在无血清培养液内增殖生成悬浮状态的神经干细胞克隆球,这种克隆可被诱导生成神经细胞和神经胶质细胞,电镜下,神经干细胞克隆球内部细胞相互间可形成特化的膜性结构,细胞内可有小泡出现,部分细胞有凋亡的形态。     

APP5肽模拟物P165对体外培养大鼠海马神经干细胞增殖和分化的影响

目的研究一种小分子多肽─APP5肽的模拟物P165对体外培养的大鼠胚胎海马神经干细胞（neuralstem cells,NSCs）增殖和分化的影响,以期能找到一种可代替神经营养因子的小分子物质,能够促进NSCs的增殖或分化,为将来的临床应用提供理论依据。方法（1）原代培养SD大鼠胚胎脑海马NSCs;（2）利用5-溴脱氧尿嘧啶核苷（BrdU）和神经元、星型胶质细胞、少突胶质细胞的特异性标记物微管相关蛋白2（MAP2）、胶质纤维酸性蛋白（GFAP）、2,3-环核苷酸-3磷酸二酯酶（CNPase）对培养的NSCs进行鉴定;（3）将培养的NSCs分为对照组、血清组、APP5肽反序列组和P165组,观察各组细胞形态的变化;（4）将培养的NSCs分为对照组、APP5肽反序列组和P165组,利用细胞计数,测定干细胞克隆形成率、干细胞克隆形成大小的方法分析P165对海马NSCs增殖的影响。结果（1）海马神经干细胞呈神经球聚集生长,BrdU染色阳性;加入血清后神经球周围有细胞呈放射状向四周生长,并带有突起。染色呈MAP2、GFAP或CNPase阳性;（2）海马NSCs加入P165及其反序列后细胞形态上与对照组相比没有明显改变;（3）与对照组相比,加P165后海马NSCs数量明显增加,克隆形成率和克隆形成的直径均有明显的增加,并有统计学差异。结论P165能够促进海马NSCs的增殖,但并不促进其分化。     

胚胎大鼠脑和脊髓神经干细胞的分离和培养

研究采用显微解剖、无血清细胞培养和免疫荧光细胞化学染色等实验技术 ,成功地建立了胚胎大鼠脑和脊髓神经干细胞 (NSCs)的分离和培养方法。结果显示 ,( 1)在含成纤维细胞生长因子 2 (FGF 2 )和表皮生长因子(EGF)的无血清培养液中 ,两种来源的NSCs经体外培养 8- 10代后 ,其细胞数呈指数级增加 ,其中脑来源的NSCs数由原代培养时的 1× 10 6 增加至 1× 10 12 ,脊髓来源的NSCs数从 1× 10 6 增加至 1× 10 11。增殖的细胞表达神经上皮干细胞蛋白 (nestin) ;( 2 )在含 1%胎牛血清 (FBS)的培养条件下 ,它们都能被诱导分化为神经元、少突胶质细胞和星型胶质细胞。但其分化比例可随细胞传代次数的增加而改变 ,其中 ,大脑来源的NSCs分化为神经元的比例从第二代 (P2 )的 11 95± 2 5 %下降至第五代 (P5)的 1 97± 1 16% (P <0 0 1) ,而少突胶质细胞的分化比例则基本保持不变 ,这一分化格局同样可在脊髓来源的NSCs中发现。结果表明 ,我们所分离和培养的细胞在体外经多次传代后仍具有很强的增殖能力和多向分化潜能 ,它们都表达nestin ,属于中枢神经系统的干细胞     

L-型钙通道对大鼠胚胎海马神经干细胞增殖和分化的调控

为鉴定大鼠胚胎海马神经干细胞(NSCs)是否表达功能性的L-型钙通道,L-型钙通道是否参与了对大鼠胚胎NSCs增殖和分化调控.分离孕15天Wistar大鼠胚胎海马组织,制成单细胞悬液,利用无血清培养技术,在添加bFGF、EGF、N-2和B27 supplement的DMEM/F12培养液中进行培养.采用细胞免疫荧光法对原代至第5代细胞进行鉴定,均有巢蛋白(nestin)的表达,第3代nestin阳性细胞比例达97%.把培养的细胞诱导分化5天后,这些细胞表现为神经元和星形胶质细胞的形态,且分别呈Ⅲ型β-微管蛋白(Tuj1)阳性和胶质纤维酸性蛋白(GFAP)阳性;细胞免疫印迹结果显示,NSCs表达L-型钙通道的Cav1.2α1C亚单位,而无Cav1.3α1D亚单位的表达;利用全细胞膜片钳技术在NSCs上记录到了L-型钙电流,证明了NSCs所表达的L-型钙通道具有功能.进一步对细胞进行药理学干预,发现L-型钙通道的激活不仅可以促进胚胎NSCs的增殖,而且使增殖的NSCs向神经元分化的比例显著增加.以上结果表明,Wistar大鼠胚胎海马NSCs表达功能性的L-型钙通道;L-型钙通道参与了胚胎NSCs增殖和分化的调控.     

单细胞因子支持人胚胎干细胞生长的培养体系

[目的]为2株中国人胚胎干细胞系建立培养液中仅添加1种细胞因子的培养体系。[方法]两株人胚胎干细胞h ES-846XX和h ES-18 46XY分别使用含有成纤维细胞生长因子(b FGF)160ng/ml、转化生长因子(TGFβ1)20ng/ml和noggin 200ng/ml的培养液进行无饲养层培养,观察细胞的生长情况并对所得细胞进行鉴定。[结果]培养液中添加b FGF 160ng/ml可以有效地支持人胚胎干细胞的长期增殖,传代8次的细胞仍然保持胚胎干细胞的特性。添加TGFβ1或noggin的培养基无法维持人胚胎干细胞的长期增殖,3代以后细胞完全分化。[结论]证明培养液单独添加高浓度b FGF 160ng/ml支持2株中国人胚胎干细胞长期保持未分化状态增殖,实际未分化率为68.2±1.08%。     

表皮生长因子诱导骨髓间充质干细胞向视网膜神经细胞分化的体外研究

目的:研究表皮生长因子诱导骨髓间充质干细胞向视网膜神经细胞分化的可能性。方法:体外培养骨髓间充质干细胞,利用流式细胞仪分析其细胞表型。采用含EGF的培养液诱导骨髓间充质干细胞向视网膜神经细胞分化,并利用免疫荧光法进行鉴定。结果:从骨髓中分离培养的细胞具有成纤维细胞样形态,贴壁生长,表型相对均一,表面标志为CD90、CD44、CD147阳性;而CD34、CD38、CD45、CD14、HLA-DR阴性。体外诱导后可以得到神经干细胞标志物nestin、神经胶质细胞标志物GFAP和视网膜光感受器细胞标志物Rhodopsin呈阳性表达的细胞。结论:从骨髓中分离培养得到的间充质干细胞具有向视网膜神经细胞分化的潜能。     

生长因子TGF-β1和bFGF促兔骨髓间质干细胞向韧带样细胞分化的实验研究

摘要目的：采用生长因子TGF-β1和bFGF诱导体外培养的兔骨髓间质干细胞（MSCs）,转化为韧带样细胞,并研究此种韧带样细胞的生物特性。方法：自幼兔四肢骨抽取骨髓分离纯化MSCs并培养、增殖;采用特定浓度TGF-β1（10ng／ml）和bFGF（25ng／mL）对MSCs进行诱导分化,观察生长因子对MSCs生长、形态的影响,使用MTT法绘制细胞生长曲线,使用天狼腥红染色法定量对比MSCs分泌胶原蛋白量。单纯培养和单一因子诱导组作为对照。结果：TGF-β1和bFGF联合使用组,细胞形态优于空白组及单一因子组,细胞增殖率、胶原分泌量也均高于对照组。结论：联合使用生长因子TGF-β1和bFGF刺激兔MSCs,能够促使兔MSCs定向转化为韧带样细胞,对组织工程前交叉韧带的构建具有积极意义。     

Upregulation of FIk-1 by bFGF via the ERK pathway is essential for VEGF-mediated promotion of neural stem cell proliferation

Neural stem cells (NSCs) constitute the cellular basis for embryonic brain development and neurogenesis.The processis regulated by NSC niche including neighbor cells such as vascular and glial cells.Since both vascular and glial cellssecrete vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF),we assessed the effect ofVEGF and bFGF on NSC proliferation using nearly homogeneous NSCs that were differentiated from mouse embryonicstem cells.VEGF alone did not have any significant effect.When bFGF was added,however,VEGF stimulated NSCproliferation in a dose-dependent manner,and this stimulation was inhibited by ZM323881,a VEGF receptor (Flk-1)-specific inhibitor.Interestingly,ZM323881 also inhibited cell proliferation in the absence of exogenous VEGF,suggestingthat VEGF autocrine plays a role in the proliferation of NSCs.The stimulatory effect of VEGF on NSC proliferationdepends on bFGF,which is likely due to the fact that expression of Flk-1 was upregulated by bFGF via phosphoryla-tion of ERK1/2.Collectively,this study may provide insight into the mechanisms by which mieroenvironmental nichesignals regulate NSCs.     

Upregulation of Flk-1 by bFGF via the ERK pathway is essential for VEGF-mediated promotion of neural stem cell proliferation

Neural stem cells （NSCs） constitute the cellular basis for embryonic brain development and neurogenesis. The process is regulated by NSC niche including neighbor cells such as vascular and glial cells. Since both vascular and glial cells secrete vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF）, we assessed the effect of VEGF and bFGF on NSC proliferation using nearly homogeneous NSCs that were differentiated from mouse embryonic stem cells. VEGF alone did not have any significant effect. When bFGF was added, however, VEGF stimulated NSC proliferation in a dose-dependent manner, and this stimulation was inhibited by ZM323881, a VEGF receptor （Flk-1）- specific inhibitor. Interestingly, ZM323881 also inhibited cell proliferation in the absence of exogenous VEGF, suggesting that VEGF autocrine plays a role in the proliferation of NSCs. The stimulatory effect of VEGF on NSC proliferation depends on bFGF, which is likely due to the fact that expression of Flk-1 was upregulated by bFGF via phosphorylation of ERK1/2. Collectively, this study may provide insight into the mechanisms by which microenvironmental niche signals regulate NSCs.     

神经干细胞向少突胶质前体细胞的定向分化诱导

本研究采用神经胶质瘤细胞株(B104 neuroblatoma cells,B104 cells)培养上清(B104CM)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF),将冷冻复苏的大鼠胚胎脊髓神经干细胞(neural stem cells,NSCs)定向诱导为少突胶质前体细胞(oligodendrocyte precusor cells,OPCs)。形态学和免疫组化的结果显示,诱导后95％以上的细胞具有双极或多极突起的典型OPCs形态,并表达A285和血小板源生长因子受体-α(platelet derived growth factor receptor-α,PDGFR-α等0PCs标志,所有PDGFR-α阳性的OPCs均不表达β-Tublin Ⅲ,其中仅少量细胞表达胶质原纤维酸性蛋白(glia fibrillary acidic protein,GFAP)。在B104CM和bFGF共存的培养条件下,悬浮培养的OPCs可大量增殖形成少突胶质细胞球,该细胞球可通过传代继续扩增,且扩增的OPCs仍能维持其特有的形态和自我增殖的特性。撤去bFGF和B104CM后,OPCs能进一步分化为成熟的少突胶质细胞(oligodendrocytes,OLs)或Ⅱ型星形胶质细胞。实验表明,诱导NSCs产生的OPCs在形态、增殖以及分化格局等方面均与已报道的存在于胚胎脑区的O-2A前体细胞相类似。该培养系统可为实验性细胞移植的研究提供丰富的细胞来源。     

卵泡刺激素和表皮生长因子对小鼠精原细胞增殖的影响

利用生殖细胞-体细胞体外无血清共培养模型研究了卵泡刺激素(FSH)和表皮生长因子(EGF)对小鼠A型精原细胞增殖的影响。精原细胞在ITS培养液(添加胰岛素、转铁蛋白和亚硒酸钠的DMEM)中培养24h后进行c-kit免疫细胞化学鉴定和EGF及其受体(EGFR)免疫细胞化学检测,72h后测定其形成集落数的情况。结果表明:ITS培养液能维持生殖细胞的活性,增殖细胞核抗原(PCNA)的表达增高。A型精原细胞呈c-kit阳性,EGF和EGFR主要表达于精原细胞。单独的FSH(1～100ng/ml)或EGF(1～10ng/ml)显著促进精原细胞集落数的增加。此外,EGF(0.1ng/ml)联合FSH(10ng/ml)具有加性效应,但更高剂量的EGF(1～10ng/ml)则降低了FSH的刺激作用。结果说明FSH可联合适量的EGF促进精原细胞的增殖。     

FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles

The hypothesis was tested that bovine preantral follicles can be stimulated to grow in vitro by FSH and by the mitogens, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), but not by transforming growth factor-beta (TGFbeta), which generally inhibits EGF and bFGF action. Preantral follicles, 60 to 179 mum in diameter, were isolated from fetal ovaries by treatment with collagenase and DNase and cultured for 6 d in serum-free medium, with or without FSH and growth factors. Basic FGF (50 ng/ml), and to a lesser extent FSH (100 ng/ml) and EGF (50 ng/ml), stimulated thymidine incorporation by granulosa cells in bovine preantral follicles compared to control cultures (8-, 4- and 2.5-fold the labeling index of the controls; P < 0.05). Alone TGFbeta (10 ng/ml) had no effect on (3)H-thymidine incorporation, but it completely inhibited the bFGF- but not the FSH-stimulated increase in the labeling index and mean follicular diameter of preantral follicles (P < 0.05). By the end of the culture period oocytes in most treatments had degenerated, and the few surviving oocytes were in preantral follicles cultured with FSH or bFGF. Progesterone accumulation was greater (P < 0.05) in the presence of FSH (100 ng/ml) or EGF (50 ng/ml) than with bFGF, TGFbeta or control medium. Basic FGF strongly inhibited the effect of FSH on progesterone secretion (P < 0.05). Only FSH stimulated the conversion of exogenous testosterone to estradiol and both bFGF and TGFbeta markedly inhibited FSH-stimulated estradiol accumulation. These results indicate that proliferation of granulosa cells of bovine preantral follicles can be stimulated by bFGF, FSH and EGF, whereas TGFbeta inhibits growth, and that they are steroidogenically active in culture. Basic FGF and TGFbeta antagonize FSH-stimulated steroid production by granulosa cells of cultured bovine preantral follicles.     

低氧促进神经干细胞向多巴胺能神经元分化

神经干细胞（neural stem cells,NSCs）作为具有多向分化潜能的神经前体细胞,被广泛应用于细胞移植等研究,而低氧不但调节干细胞的体外增殖,在干细胞分化中也具有重要的作用。本文着重探讨了低氧对NSCs分化的调节作用。采用Wistar孕大鼠（E13．5d）,分离胚胎中脑NSCs,加入无血清DMEM／F12培养液（含20ng／mL EGF、20ng／mL bFGF、1％ N2和B27）,3～5d后传代,细胞培养至第三代进行诱导分化,分别在低氧（3％O2）和常氧（20％O2）条件下诱导分化3d,然后在常氧条件下分化成熟5～7d（DMEM／F12含1％FBS、N2和B27）后进行检测。Nestin、NeuN以及TH免疫组织化学鉴定NSCs;流式细胞术分析测定NSCs向TH阳性神经元方向的分化;高效液相色谱测定细胞培养上清液中多巴胺（dopamine,DA）含量。结果显示,分离培养的NSCs均为nestin阳性细胞;低氧可明显促进NSCs向神经元方向的分化;TH阳性神经元比例在常氧和低氧组分别为（10．25±1．03）％和（19．88±1．44）％。NSCs诱导分化7d后,低氧组细胞培养上清液中DA浓度明显增加,约为常氧组的2倍（P〈0．05,n=8）。上述结果表明,3％低氧可促进NSCs向神经元方向,特别是向DA能神经元方向分化。这为NSCs应用于临床治疗帕金森病提供了基础。     

Autocrine activation of EGF receptor promotes oscillation of glutamate-induced calcium increase in astrocytes cultured in rat cerebral cortex

We previously reported that astrocytes cultured for more than 2 days in a defined medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) showed calcium oscillation in response to glutamate, whereas the response pattern was transient in the absence of the exogenous growth factors. In the present study, we found that astrocytes showed glutamate-induced calcium oscillation, even in growth factor-free medium, if the cells had been cultured for more than 5 days. The calcium oscillation promoted by the prolonged culture period was suppressed by an inhibitor of EGF receptor tyrosine kinase, but not by a neutralizing antibody to bFGF, indicating that the accumulation of an autocrine factor that activates the EGF receptor leads to calcium oscillation. Astrocytes in our culture system expressed EGF, transforming growth factor alpha (TGFalpha), bFGF and acidic fibroblast growth factor (aFGF). Exogenous aFGF, which induced astrocyte immediate early gene expression to the same extent as EGF or bFGF, did not affect calcium oscillation. Exogenous EGF and bFGF promoted astrocyte hypertrophic morphology and proliferation, as well as calcium oscillation. In contrast, these properties did not accompany calcium oscillation induced by the prolonged culture period. These results suggest that astrocytes possess the ability to promote their own calcium oscillation, which is independent of hypertrophic changes to reactive astrocytes.     

Microenvironmental determinants of adult neural stem cell proliferation and lineage commitment in the healthy and injured central nervous system

The discovery of neural stem cells (NSC) which ensure continuous neurogenesis in the adult mammalian brain, has led to a conceptual revolution in basic neuroscience and to high hopes for clinical nervous tissue repair. However, several research issues remain to address before neural stem cells can be harnessed for regenerative therapies. The presence of NSC in a nervous structure is demonstrated in vitro by primary culture of dissociated adult nervous tissue in the presence of the specific mitogens EGF and bFGF. This leads to spherical masses of proliferating cells endowed with capacities for self-renewal and, after growth factor removal, differentiation into the three characteristic cell types of nervous tissue (neurons, astrocytes, oligodendrocytes). In vivo, neurogenesis per se, i.e. production of new neurons, occurs only in a small subset of NSC-endowed structures. The production of oligodendrocytes, i.e. myelinating glial cells, is similarly restricted. Such in vivo restrictions were formally demonstrated to arise from the tissular microenvironnement, which led to the emerging concept of "neurogenic niche". In this context, major challenges now consist in identifying the nature of tissue-specific extracellular signals that determine lineage commitment of NSC progeny, understanding why NSCs display weak in vivo reactivity to lesions compared to other stem cell types in adults, and identifying the factors behind the very high resistance to tumorigenesis displayed by NSCs. Altogether, the current data offer hope for the future use of adult NSCs in regenerative therapies, provided that tissue-specific signals are identified in view of counteracting the intrinsic repression of new cell genesis and/or stimulating endogenous NSC recruitment to lesion sites.     

Impact of peptide growth factors on the culture of small preantral follicles of domestic cats

Small preantral follicles (40 to 90 microm) of domestic cats were cultured in the presence or absence of epidermal growth factor (EGF), insulin-like growth factor I (IGF-I), and basic fibroblast growth factor (bFGF) for 5 d. The success of culture was estimated by in vitro incorporation of Brom-desoxyuridine (BrdU) into the oocytes and granulosa cells. Addition of EGF (4, 20, or 100 ng/ml) to the culture medium had no significant effect on the incidence of in vitro DNA synthesis. After supplementation with IGF-I and bFGF, BrdU-incorporation into the follicles and oocytes increased in correspondence to the concentration used, with 20 ng/ml IGF-I and 10 ng/ml bFGF giving the highest effect. In medium containing EGF, the IGF-I-induced increase in BrdU incorporation was suppressed, while the effect of bFGF was not decreased. Simultaneous addition of IGF-I and bFGF did not result in a further increase in DNA synthesis in the oocytes and granulosa cells. We conclude that bFGF mainly induces the proliferation of granulosa cells while IGF-I is involved in cellular activation of oocytes, which is modulated by EGF.