IL-1β诱导中脑神经干细胞向多巴胺能神经元分化的研究

在胚鼠中脑曲分离神经干细胞 ,研究 IL-1β对中脑神经干细胞诱导分化为多巴胺神经元的效能 ,运用体外分离、培养小鼠胚胎中的中脑神经经干细胞 ,加入 IL -1β诱导其分化 ,通过巢蛋白 ( Nestin)、神经元特异性烯醇化酶 ( NSE)、胶质纤维酸性蛋白( GFAP)、酪氨酸羟化酶 ( TH)免疫组化鉴定。结果显示 ,小鼠胚胎的腹侧中脑曲部位分离获得了大量未分化、呈球状悬浮生长的细胞 ,且能在有血清的培养基中分化为神经元和神经胶质细胞。经 IL-1β诱导后 ,多巴胺能神经元分化比例达 16%左右。     

胚胎大鼠神经干细胞体外分化的激光共聚焦显微镜观察

目的:采用激光扫描共聚焦显微镜观察体外培养胎鼠大脑皮质神经干细胞(neural stem cells,NSCs)分化情况。方法:利用无血清培养方法,分离培养胚胎大鼠大脑NSCs,进行体外扩增培养、传代;采用溴脱氧尿嘧啶核苷(bromodeoxyuridine,BrdU)掺入、双重免疫荧光细胞化学标记方法和激光扫描共聚焦显微镜,用神经细胞的特异性抗体(神经元β-微管蛋白、胶质纤维酸性蛋白),鉴定NSCs向神经元与星形胶质细胞分化的情况。结果:从胎鼠大脑皮质及皮质下分离的组织,经原代和传代培养均可形成细胞克隆,并表达神经上皮干细胞蛋白(Nestin)抗原。在血清诱导下,分化后的细胞表达神经元、星形胶质细胞2种神经细胞的特异性抗原,NSCs分化为星形胶质细胞神经元的比例分别为(43.70±8.55)%和(23.00±3.69)%。结论:从胎鼠大脑皮质分离出的细胞可获得呈集落样生长的神经干细胞团,并能表达NSCs的特异性抗原;激光扫描共聚焦显微镜可清晰地观察到培养细胞具有分化为神经元和星形胶质细胞的潜能。     

胎鼠脑神经干细胞的分离、培养和鉴定

目的 研究胎鼠脑皮质神经干细胞(NSCs)的分离、培养及鉴定方法。方法 从孕15d胎鼠的大脑皮层和海马区脑组织中获取NSCs,在含有B27、表皮生长因子(EGF)和碱性成纤维生长因子(bFGF)的DMEM/F12无血清培养液中培养;传代后用5%胎牛血清培养液诱导NSCs分化。结果 体外分离培养的NSCs在无血清培养液中形成大量的神经球。经3-5代传代的细胞生长稳定。经巢蛋白染色鉴定,大部分为阳性细胞,神经细胞球经胎牛血清培养液贴壁培养后可分化为神经元特异烯醇化酶、胶质纤维酸性蛋白和半乳糖脑苷脂表达阳性的细胞。结论 从孕15d胎鼠大脑皮质和海马组织分离出的NSCs具有自我更新能力和多向分化潜能,其在5%胎牛血清培养液中具有向神经元和神经胶质细胞分化的潜能。     

EGB对大鼠神经干细胞分化为星形胶质样细胞的影响

目的：观察银杏叶提取物（EGB）对大鼠海马神经干细胞（NSCs）增殖及分化为星形胶质样细胞的影响。方法：取新生24h内的Wistar大鼠海马组织的细胞进行体外培养，1周后将其接种在培养板中．观察不同浓度的EGB对细胞的生长作用．并检测胶质原纤维酸性蛋白（GFAP）的表达。结果：各实验组NSCs的生长明显好于对照组．免疫细胞化学显示：同一时间内，诱导NSCs分化为星形胶质样细胞的百分率增高；同一浓度的EGB，诱导神经干细胞分化为星形胶质样细胞的百分率随着时间的延长呈上升的趋势。结论：EGB能促进神经干细胞的存活、增殖及向星型胶质样细胞的方向分化。     

甲氨蝶呤对脂多糖诱导的大鼠脊髓神经胶质细胞pIκBα-NF-κBp65-炎性因子通路的影响

目的 观察甲氨蝶呤对脂多糖（lipopolysaccharide,LPS）诱导的大鼠脊髓神经胶质细胞pIκBα-NF-κBp65-炎性因子通路的影响。方法 脊髓组织块法培养神经胶质细胞。将分离的神经胶质细胞接种于多孔板培养48 h后,分为空白对照组、LPS组、LPS+pIκBα抑制剂组、LPS+甲氨喋呤组。随后应用免疫印迹法测定各组分的pIκBα与胞核及胞浆NF-κBp65水平变化,酶免疫法（ELISA）测定炎性因子TNF-α、IL^-1β、IL-6含量。结果 神经胶质细胞经LPS诱导后,pIκBα、胞核NF-κBp65和细胞上清液炎性因子TNF-α、IL^-1β、IL-6水平均显著增加（P<0.05或P<0.01）。甲氨喋呤可明显抑制经LPS诱导的神经胶质细胞pIκBα水平,显著降低胞核NF-κBp65水平和细胞上清液炎性因子TNF-α、IL^-1β、IL-6的含量（P<0.05）。结论 甲氨喋呤对LPS诱导的脊髓神经胶质细胞pIκBα-NF-κBp65-炎性因子通路有显著的抑制作用。     

神经鞘糖脂生物合成抑制剂D-PDMP对神经干细胞增殖、分化能力的影响

目的探讨神经鞘糖脂生物合成抑制剂DPDMP对神经干细胞增殖和分化能力的影响。方法用不同浓度的DPDMP处理体外培养的神经干细胞,通过神经球直径大小和数量、细胞计数、MTT分析和诱导分化实验,评价其对神经干细胞增殖和分化能力的影响。结果经DPDMP处理后培养的神经球减小,细胞计数和MTT分析都表明细胞增殖能力明显受到抑制,较高DPDMP浓度可导致神经干细胞死亡;神经干细胞的诱导分化能力也明显减弱,分化的细胞数较少,但仍可诱导成神经元和星形胶质细胞。结论神经鞘糖脂生物合成抑制剂DPDMP能抑制体外培养的神经干细胞增殖,甚至导致细胞死亡,并可减弱神经干细胞的诱导分化能力。     

心肌球培养基血清浓度优化研究

目的 通过比较含有不同胎牛血清浓度的心肌球培养基分离培养大鼠心肌干细胞（CSCs）的效果，探讨最优的心肌干细胞血清培养浓度。方法 选取15只健康新生Wistar大鼠（出生1天龄），无菌条件下取出心脏，经胰酶和Ⅱ型胶原酶反复消化后，种植于培养瓶中，应用含20%胎牛血清的完全培养基培养组织块源性心脏祖细胞（CPCs），将所得的培养外植物源性细胞（EDCs）随机分为A、B、C 3组，各组5瓶细胞（25 mL塑料培养瓶），分别应用含10%、11%、15%不同浓度胎牛血清的心肌球生长培养基培养，培养至第三代CSCs时行细胞表面抗原C-kit染色，鉴定C-kit阳性CSCs占所获得细胞的比率，比较所得的细胞数量，生长周期和生长曲线。结果 从新生大鼠心肌组织中成功分离培养出CSCs，C-kit阳性率87%。培养后所得细胞数量以细胞倍增水平（PDL）表示，各组细胞PDL呈匀速递增，PDL水平和其递增速率（生长曲线所示）比较：B组> C组> A组。应用含11%胎牛血清的心肌球生长培养基培养EDCs获得的CSCs，与其他两组相比，具有生长周期短（P<0.05），生长数量多的特点（P<0.05）。结论 含11%胎牛血清浓度是心肌球生长培养基培养原代大鼠心肌干细胞的最优血清培养浓度。     

丁苯酞对神经干细胞向神经细胞分化的促进作用

目的 研究丁苯酞通过PI3K-AKT信号通路促进神经干细胞向神经细胞分化的作用。方法 分离培养SD大鼠胚胎脑皮质层的神经干细胞,采用分化专用完全培养基培养后,通过光镜下观察和Nestin蛋白免疫荧光法进行鉴定。设置对照组和实验组,其中实验组添加丁苯酞辅助分化,分为丁苯酞低剂量组（1 μmol·L^-1）、中剂量组（5 μmol·L^-1）和高剂量组（10 μmol·L^-1）,作用24 h后,CCK-8法检测神经干细胞的细胞活力,碱性磷酸酶染色法检测神经干细胞的分化能力,免疫荧光法检测神经细胞分化程度,RT-qPCR法检测转录因子SOX2、PAX6和NeuN的mRNA表达水平,Western-blot及RT-qPCR检测PI3K-AKT信号通路的表达。结果 丁苯酞干预后神经干细胞活力提高,分化能力提高,碱性磷酸酶试验呈强阳性,转录因子SOX2、PAX6和NeuN的mRNA表达水平增高,PI3K-AKT信号通路蛋白和mRNA表达水平均增高,且呈现剂量依赖性。结论 丁苯酞可以促进神经干细胞向神经细胞分化,其作用机制可能与PI3K-AKT的激活有关。     

黄芪甲苷对体外培养神经干细胞分化作用的研究

目的观察黄芪甲苷对体外培养神经干细胞分化作用的影响。方法利用无血清培养技术在体外对新生Wistar大鼠海马区与神经干细胞进行培养,观察相同接种密度条件下,不同浓度黄芪甲苷（20mg／L,40ms／L,80mg／L）对神经干细胞分化作用的影响,采用免疫细胞化学法鉴定神经干细胞及其分化后细胞。结果从新生大鼠海马区分离出的神经干细胞经鉴定呈nestin阳性,且加入黄芪甲苷组所表达的神经干细胞和胶质纤维酸性蛋白阳性细胞较对照组明显增多（17．60±2．85vs15．65±2．29;24．10±3．13VS21．95±2．37;P〈0．05）。结论黄芪甲苷对体外培养大鼠海马区神经干细胞的分化有促进作用。     

SRC-1基因在小鼠神经干细胞分化过程中的变化

目的观察类固醇受体辅助活化因子-1(SRC-1)基因在小鼠神经干细胞(NSCs)体外培养分化过程中表达的变化。方法用机械分离和酶消化法从1~3 d小鼠大脑皮质获取NSCs原代细胞,体外培养获神经球,传代并消化,经血清诱导神经球细胞分化。形态学、细胞免疫荧光实验观察神经球形态、Nestin表达鉴定NSCs;抗体分别标记神经元、星形胶质细胞,检测细胞分化d 3、9的细胞类型。RT-qPCR和免疫印迹法检测细胞分化d 0、3、9 SRC-1基因mRNA和蛋白的表达。结果新生小鼠大脑皮质获取的细胞,体外培养能扩增形成神经球,Nestin阳性表达。NSCs分化d 3主要为神经元,d 9主要为星形胶质细胞。与NSCs分化d 0相比,SRC-1表达在分化d 3明显升高,在分化d 9明显降低。 结论 成功分离并获取新生小鼠皮质NSCs。在NSCs分化中,SRC-1表达量有明显变化,分布依次为神经元>NSCs>星形胶质细胞。     

Human neural progenitor and stem cells: US20080107633A1

Background: The application is in the field of neural stem cells (NSCs) and cellular therapy. Objective: It aims at establishing conditions for the isolation and propagation of neural progenitor and stem cells from human fetal tissue, with high rate of growth and high yields of differentiation into the neuronal, astroglial and oligodendroglial pathways. Methods: Neural progenitor and stem cells were isolated from fetal forebrain tissue and propagated as neurospheres, in defined medium in the presence of leukemia inhibitory factor (LIF). Three protocols were designed to differentiate human fetal neural progenitor and stem cells into their progenies. Results: The application claims the generation of human fetal neural progenitor and stem cells with a doubling rate between 5-10 days. It claims the differentiation of the neural progenitor and stem cells in vitro, into neurons, astrocytes and oligodendrocytes with high yields, e.g., 20 to 35% for neuronal cells. Conclusion: The establishment of human neural progenitor and stem cells with high rate of growth and high yields of differentiation provides a source of cells for therapy, particularly for the treatment of neurodegenerative diseases, like Alzheimer's and Parkinson's diseases.     

BFGF、EGF联合应用对新生大鼠海马神干细胞分化的影响

目的:探讨EGF和bFGF联合应用后对新生大鼠海马神经干细胞增殖分化的影响。方法:联合应用EGF和bFGF培养海马神经干细胞及诱导贴壁的细胞分化,并用免疫细胞化学技术研究神经干细胞的分化情况。结果:bFGF和EGF联合应用可以明显扩增海马神经干细胞,并诱导细胞向神经元和星形胶质细胞分化。结论:bFGF和EGF除了具有促神经干细胞有丝分裂作用外,还可以诱导神经干细胞向神经元和星形胶质细胞分化。     

Optimisation of culture conditions for differentiation of C17.2 neural stem cells to be used for in vitro toxicity tests

Here we present a multipotent neuronal progenitor cell line for toxicity testing as an alternative to primary cultures of mixed cell types from brain tissue. The v-myc immortalised C17.2 cell line, originally cloned from mouse cerebellar neural stem cells, were maintained as monolayer in cell culture dishes in DMEM supplemented with fetal calf serum, horse serum and antibiotics. Different media and exposure scenarios were used to induce differentiation. The optimal condition which generated mixed cultures of neurons and astrocytes included serum-free DMEM:F12 medium with N2 supplements, brain-derived neurotrophic factor and nerve growth factor. The medium was changed every 3rd or 4th day to fresh N2 medium with supplements. After 7 days, the culture contained two different morphological cell types, assumed to be neurons and glia cells. The presence of astrocytes and neurons in the culture was confirmed by RT-PCR and Western blot analyses, indicating increased mRNA and protein levels of the specific biomarkers glial fibrillary acidic protein (GFAP) and βIII-tubulin, respectively. Concomitantly, the expression of the neural progenitor cell marker nestin was down-regulated.     

红景天苷对神经干细胞作用的研究

目的:观察红景天苷药物血清对新生大鼠海马神经干细胞分化为神经元的影响。方法:从新生24h内的W istar大鼠脑中分离扩增获得大量神经干细胞后,加入低、中、高剂量的红景天苷药物血清,观察其对神经干细胞分化为神经元的影响,并通过免疫细胞化学染色检测神经干细胞分化为神经元的状况。结果:低、中、高剂量的红景天苷药物血清组单位视野内神经元特异烯醇化酶(N SE)阳性细胞个数与对照组相比有显著差异(P<0.01),并呈一定的剂量依赖性。结论:红景天苷药物血清在体外可促进神经干细胞向神经元方向分化,为红景天药物应用于相关神经系统疾病的临床治疗提供了试验依据。     

高-低密度法分离培养大胚龄人神经干细胞

目的 从大胚龄人胚脑中分离培养并鉴定神经干细胞。方法 采用高—低密度培养、无血清培养和单细胞克隆技术分离培养出神经干细胞，并应用免疫荧光染色鉴定。结果 从13周龄胚脑中成功分离出具有自我更新和多分化潜能的神经干细胞，它们具有连续克隆能力，可传代培养，表达神经巢蛋白抗原，分化后的细胞表达神经元细胞、胶质细胞和少校胶质细胞的特异性抗原。结论 高—低密度培养法能成功地分离和培养大胚龄入神经干细胞。     

Kir4.1 is coexpressed with stemness markers in activated astrocytes in the injured brain and a Kir4.1 inhibitor BaCl2 negatively regulates neurosphere formation in culture

Astrocytes are activated in response to brain damage. Here, we found that expression of Kir4.1, a major potassium channel in astrocytes, is increased in activated astrocytes in the injured brain together with upregulation of the neural stem cell markers, Sox2 and Nestin. Expression of Kir4.1 was also increased together with that of Nestin and Sox2 in neurospheres formed from dissociated P7 mouse brains. Using the Kir4.1 blocker BaCl₂ to determine whether Kir4.1 is involved in acquisition of stemness, we found that inhibition of Kir4.1 activity caused a concentration-dependent increase in sphere size and Sox2 levels, but had little effect on Nestin levels. Moreover, induction of differentiation of cultured neural stem cells by withdrawing epidermal growth factor and fibroblast growth factor from the culture medium caused a sharp initial increase in Kir4.1 expression followed by a decrease, whereas Sox2 and Nestin levels continuously decreased. Inhibition of Kir4.1 had no effect on expression levels of Sox2 or Nestin, or the astrocyte and neuron markers glial fibrillary acidic protein and β-tubulin III, respectively. Taken together, these results indicate that Kir4.1 may control gain of stemness but not differentiation of stem cells.     

脑缺血大鼠脑脊液对神经干细胞存活及分化的影响

目的：探讨脑缺血大鼠脑脊液对新生大鼠海马神经干细胞生存及分化的影响。方法：将脑缺血大鼠脑脊液加入神经干细胞的培养液中，观察神经干细胞存活及贴壁细胞团的分化．并应用免疫细胞化学技术对神经干细胞分化情况进行鉴定。结果：悬浮培养细胞实验组较对照组有明显差异；贴壁培养细胞分化的细胞数量较对照组明显增多。结论：脑缺血大鼠脑脊液可促进体外培养神经干细胞存活，并且促进神经干细胞向神经元和星形胶质细胞分化。     

Targeting nitric oxide in the subacute restorative treatment of ischemic stroke

Introduction: Stroke remains the leading cause of adult disability. Thus, it is imperative to develop restorative therapies for ischemic stroke designed specifically to treat the intact brain tissue to stimulate functional benefit. Therapies targeting amplification of brain repair processes with nitric oxide (NO) donors and phosphodiesterase type 5 (PDE5) inhibitors in preclinical studies are emerging and showing improvement of functional recovery after stroke.

Areas covered: This review will mainly cover the effect of NO donors, which produce NO, and PDE5 inhibitors, which elevate cyclic guanosine 3′,5′-monophosphate (cGMP), on neural restorative events in ischemic brain and highlight mechanisms underlying their restorative therapeutic activity.

Expert opinion: During stroke recovery, interwoven restorative events occur in ischemic brain, which include angiogenesis, neurogenesis, oligodendrogenesis, astrogliosis and neurite outgrowth. Emerging preclinical data indicate that restorative therapies targeting multiple parenchymal cells including neural stem cells, cerebral endothelial cells, astrocytes, oligodendrocytes, neurons would be more effective than agents with a single cell target. Preclinical data suggest that elevated cGMP levels induced by NO donors and PDE5 inhibitors act on cerebral endothelial cells, neural stem cells and oligodendrocyte progenitor cells to enhance stroke-induced angiogenesis, neurogenesis and oligodendrogenesis, respectively. These interacting remodeling events collectively improve neurological function after stroke.     

新生大鼠海马神经干细胞的分离培养及鉴定

目的：从新生大鼠海马分离培养并鉴定神经干细胞。方法：应用含有碱性成纤维细胞生长因子（bFGF)和表皮生长因子（EGF）的无血清条件培养基，采用无底物的悬浮培养法培养神经干细胞，并用免疫细胞化学技术鉴定传代后形成的细胞团。结果：EGF bFGF可能明显地促进神经干细胞分裂增殖，免疫细胞化学检测发现传代后形成的细胞团中的细胞均表达神经上皮干细胞蛋白（Nestin)。结论：我们成功地建立了新生大鼠海马神经干细胞培养模型。