成年小鼠嗅球神经干细胞的培养和鉴定

目的 建立完善的成年小鼠嗅球神经千细胞分离培养和鉴定方法,探索新的成年神经干细胞种子来源. 方法 用无血清方法 分离培养成年小鼠嗅球来源的神经干细胞;用克隆培养、5-溴2-脱氧尿嘧啶核昔(BrdU)整合的方法 检验培养细胞的干细胞特性;用免疫荧光细胞化学的方法 检测BrdU、神经干细胞标记物巢蛋白(nestin)和SOX2、分化的细胞标记物Tuj1、胶质纤维酸性蛋白(GFAP)、04的表达. 结果 从成年小鼠嗅球能够分离、培养出具有自我更新、增殖能力的神经球.构成神经球的细胞呈nestin和SOX2阳性,它们分化后产生TuJ1阳性的神经元、GFAP阳性的星形胶质细胞、04阳性的少突胶质细胞. 结论 成年小鼠嗅球存在神经干细胞,其能够在体外进行培养、增殖、分化.是神经干细胞的新的种子来源.     

血性脑脊液对胚胎神经干细胞增殖和分化的影响

目的 观察体外血性脑脊液培养对神经干细胞增殖和分化的影响,以期为临床治疗这类患者提供依据.方法 提取终止妊娠的16周人胚胎脑细胞,冻存于液氮中.组织复苏后,在DMEM/F12培养基(含EGF、bFGF、B27和N2)中培养14 d可获得形态完好的神经球(神经干细胞).从颅脑外伤患者和非外伤患者分别留取血性脑脊液和正常脑脊液.将制备的胚胎神经干细胞分为两组,分别用血性脑脊液和正常脑脊液培养.动态观察神经干细胞在两组脑脊液中生长、增殖和分化的情况.用免疫细胞化学技术对两组脑脊液中神经干细胞的分化进行标记和鉴定.结果 神经干细胞在两组脑脊液中均能存活、增殖和分化.但神经干细胞在血性脑脊液中分化速度较快,分化比例也较高.在血性脑脊液中,神经干细胞更倾向于向胶质细胞分化;而在正常脑脊液中,神经干细胞更倾向于向神经元分化.结论 血性脑脊液可能会影响神经干细胞的分化速度和分化方向.这一结果对采用神经干细胞治疗颅脑外伤和蛛网膜下腔出血等疾病有一定提示作用.     

快速老化小鼠海马神经元电压门控离子通道特点

目的：观察快速老化小鼠（Senescence-accelerated mouse,SAM)海马神经元的基本离子通道特点，并对抗快速老化亚系（SAM－resistance/1,SAMR1)与快速老化亚系（SAM－prone/8,SMAP8)的基本离子通道特点进行了比较，探讨了离子通道变化在衰老中的可能角度，方法：应用全细胞记录方式，观察并比较原代培养SAMR1和SAMP8海马神经元的电压门控离子通道及膜参数。结果：原代培养SAMR1和SAMP8海马神经元电压门控Na^2 通道电流（INa)和电压门控延迟整流K^ 通道电流（IK）的电学特点和幅度基本一致。SAMP8的电压门控Ca^2 通道电流（ICa)和瞬时外向K^ 通道电流（IA）的幅值则大于相同培养天数的SAMR1。经膜电容校正所得的ICa电流密度也表现出增大的变化规律。结论：SAMP8与SAMR1神经元间IA和ICa的差异可能与其神经系统变异而产生的学习记忆功能下降有关。     

昆明种小鼠胚胎神经干细胞的分离及培养**

目的：在神经干细胞的分离培养过程中，国内外的报道多数是用胰蛋白酶对组织细胞进行消化，但是消化时间比较难把握。采用胰酶消化与机械分离法相结合的方式对昆明种小鼠胚胎脑神经干细胞进行分离、培养和初步的免疫组织化学检测，为相关研究建立实验条件。 方法：实验于2006-10/2007-09在广西医科大学基础医学实验室完成。实验室为广西重点实验室、基础医学博士后工作站。 ①实验材料：孕14~16 d的昆明种小鼠由广西医科大学实验动物中心提供，实验过程中对动物处置符合动物伦理学标准。②实验方法：分离昆明种小鼠胎鼠的脑组织，经胰酶消化加机械吹打后，在加入碱性成纤维细胞生长因子和表皮细胞生长因子的B27无血清培养基中培养。③实验评估：用免疫细胞化学方法鉴定分离的神经干细胞。 结果：在无血清DMEM/F12培养液中，加入碱性成纤维细胞生长因子、表皮细胞生长因子及B27的条件下，培养24 h后可见细胞以悬浮方式生长，三五聚集成团块，48 h后形成由十数个至数十个细胞组成的，大小不等的细胞球，形态规则，细胞无突起，形成典型的神经球，可传代。免疫细胞化学法检测显示，细胞表达神经巢蛋白。 结论：①来自昆明种小鼠胎脑的神经干细胞能在体外培养、增殖并具有增殖传代能力。②无血清B27培养基添加表皮细胞生长因子、碱性成纤维细胞生长因子有利于神经干细胞的体外培养和促进其增殖。     

黄芪注射液对体外培养嗅鞘细胞增殖及其分泌神经营养因子的影响*☆

背景：单独黄芪注射或嗅鞘细胞移植均可促进多种神经元的存活及脊髓损伤后神经功能的恢复,若联合嗅鞘细胞移植和中药黄芪注射液治疗脊髓损伤,是否可达到更好临床效果？ 目的：观察黄芪注射液对嗅鞘细胞增殖及其分泌神经营养因子的影响。 方法：分离培养新生24 h内SD大鼠嗅鞘细胞并纯化,采用免疫组织化学方法鉴定。取培养至第8天的嗅鞘细胞,种植于多聚赖氨酸包被的96孔板,置于37 ℃、体积分数5%CO2培养箱饥饿24 h后,加入2 mg/L,20 mg/L,200 mg/L,2 g/L,20 g/L黄芪注射液作用24 h,采用MTT法和流式细胞仪检测黄芪注射液对嗅鞘细胞增殖的影响;采用ELISA法测定培养上清中胶质细胞源性神经营养因子的水平。以血清培养液组为阴性对照。 结果与结论：与阴性对照组比较,2,20 mg/L质量浓度黄芪注射液可明显促进嗅鞘细胞增殖(P < 0.05,P < 0.01);不同质量浓度黄芪注射液均增加嗅鞘细胞G1细胞比例,减少S、G2期细胞所占比例,但差异无显著性意义(P > 0.05),但各质量浓度黄芪注射液均显著减少嗅鞘细胞凋亡数量(P < 0.05); 2 mg/L质量浓度黄芪能对嗅鞘细胞分泌胶质细胞源性神经营养因子有显著的促进作用。结果说明黄芪注射液可协同嗅鞘细胞促进脊髓损伤的恢复。     

Prdx6对神经干细胞增殖和分化的影响研究

目的探讨过氧化物氧化还原酶6(Prdx6)对神经干细胞的增殖和分化的影响。方法 (1)采用悬浮培养法从新生SD大鼠脑分离培养神经干细胞,传至第三代后进行神经干细胞特异性标记蛋白巢蛋白(Nestin)的免疫荧光检测;(2)细胞实验分组:溶媒组、Prdx6 1 ng/m L、10 ng/m L、100 ng/m L、1μg/m L组;(3)采用台盼蓝细胞计数法计数各组细胞培养第1 d、第2 d、第3 d、第4 d和第5 d的细胞数,并绘制细胞生长曲线;(4)细胞增殖实验观测各组细胞形成神经球的直径,用Cell Counting kit-8(CCK-8)试剂盒检测各组细胞在450 nm处的吸光度值;(5)在细胞分化实验中,通过免疫荧光染色检测神经干细胞分化后神经元特异性烯醇化酶(neuron specific enolase,NSE)、胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)阳性细胞数。结果 (1)体外培养得到悬浮生长的神经干细胞,免疫荧光染色Nestin呈阳性;(2) Prdx6 100 ng/m L组和1μg/m L组神经干细胞形成神经球的直径、吸光度值,以及分化后的NSE、GFAP阳性细胞数与溶媒组比较,差异有统计学意义(均P 0. 05)。结论 Prdx6具有促进神经干细胞增殖和分化的作用。     

成年脑内的神经发生与抑郁症

哺乳动物成年后中枢神经系统内仍存在神经发生，成体脑内神经发生的调节因素及其与海马的功能联系是目前研究的热点。海马作为成体脑内神经发生最为活跃的区域之一，对各种应激刺激也最为敏感。同时，海马是参与情绪调控的主要脑区之一，是抑郁症机制研究的结构基础。影响神经发生的许多因素同时也和抑郁症的病因与预后有关。目前成体脑内神经发生和抑郁症的关系已成为抑郁症机制研究的新方向。     

硝普钠对小鼠神经干细胞增殖的抑制作用

目的：探讨外源性一氧化氮对小鼠神经干细胞增殖的抑制作用。方法：用含生长因子的培养基，原代培养神经干细胞。第5d时，取部分原代培养细胞于培养基中加入不同浓度的硝普钠进行培养。24h后用Griess还原法检测细胞上清液中一氧化氮的释放量。将这些细胞中的一半进行四甲基偶氮唑蓝比色法（MTT法）试验，另一半细胞换成无硝普钠的条件培养基继续培养24h，再行MTT试验。另取原代培养细胞经硝普钠作用24h后，行血清诱导分化试验。结果：24h后细胞上清液中一氧化氮释放量随硝普钠浓度的增加而增加；MTT试验反映经硝普钠作用后的神经干细胞活细胞数较对照组明显减少，其减少的程度与硝普钠浓度呈正相关；经硝普钠作用后的神经干细胞，在去除硝普钠后仍可保持旺盛的增殖能力并能被血清诱导分化为神经细胞样细胞。结论：外源性一氧化氮对培养状态下的小鼠神经干细胞增殖有抑制作用。     

TLR配体对大鼠脊髓神经干细胞增殖的影响

目的研究某些ToU样受体(TLR)的配体对脊髓神经干细胞(NPCs)增殖的影响。方法大鼠经单次腹腔注射TLR不同配体,用BrdU跟踪标记增殖的NPCs。结果硫代磷酸含CpG的寡核苷酸(PSCpG-ODN)未引起脊髓NPCs的明显增殖,而PolyI：C、LPS与R848可引起BrdU~ 细胞显著性增加。注射PolyI：C或R848的大鼠脊髓BrdU~ 细胞密度于注射后2～3 d达最高,随后快速降至正常水平。伴随NPCs增殖的还有中枢神经系统(CNS)先天免疫系统的激活,如神经小胶质细胞的活化。结论某些TLR的配体被TLR识别后可能参与脊髓内正常神经细胞的发生过程,提示机体的先天免疫反应可能与组织再生过程紧密相关,对开发促进脊髓损伤再生修复的药剂来说是一有价值的工具。     

实验性自身免疫性脑脊髓炎对大脑神经干细胞增殖的影响

目的探讨实验性自身免疫性脑脊髓炎(EAE)埘大脑神经干细胞(NSC)增殖能力的影响。方法将C57BL/6小鼠随机分为模型组、佐剂组和对照组,采用少突胶质细胞糖蛋白(MOG)抗原联合完全福氏佐剂(CFA)免疫模型组小鼠,佐剂组小鼠仅接受CFA免疫注射,对照组小鼠仅接受生理盐水注射。观察各组小鼠EAE症状评分,采用HE染色以及劳克坚牢蓝(LFB)髓鞘染色观察其脑和脊髓病理学改变,采用免疫荧光染色检测小鼠海马齿状回(DG)和侧脑室下区(SVZ)BrdU阳性细胞数量,以评价NSC增殖情况。结果模型组有14只(14/15)小鼠被成功诱导EAE,其症状达到高峰后短暂缓解,随后进入慢性持续期;而佐剂组和对照组小鼠均未出现任何EAE症状。模型组中枢神经系统存在大量炎性反应细胞浸润,在白质区可见多处LFB未着色的髓鞘脱失区;而佐剂组和对照组无明显病理学改变。佐剂组与对照组小鼠DG区和SVZ区BrdU阳性细胞数比较差异均无统计学意义。与佐剂组和对照组比较,模型组小鼠大脑SVZ区和DG颗粒下区BrdU阳性细胞均显著降低(均P0.05)。结论慢性持续型EAE可导致小鼠大脑NSC增殖能力下降。     

吡咯喹啉醌与神经干细胞的增殖

背景：目前国内外学者主要应用生长因子样物质来促进神经干细胞的分裂与增殖,所以作者推测吡咯喹啉醌小分子化合物也能促进神经干细胞的分裂增殖。 目的：体外观察氧化还原酶辅酶吡咯喹啉醌促进鼠成体神经干细胞增殖的作用。 方法：体外培养Wistar胎鼠成体神经干细胞成球体后,于培养液中加入1×10-8 mol/L吡咯喹啉醌,对照组加入同等剂量的Hanks溶液。应用流式细胞仪测细胞周期增殖率,应用免疫组化法和Western Blot测周期蛋白激酶(CDK2)和周期蛋白激酶抑制剂(P27)的表达。 结果与结论：与对照组相比,吡咯喹啉醌处理16 h和24 h后,S期和G2期细胞数的比例显著增高,而细胞的凋亡率降低;CDK2蛋白的表达显著增强,而P27的表达显著降低。结果显示低浓度的吡咯喹啉醌即能促进鼠成体神经干细胞的增殖。     

稳定增殖神经干细胞的实验研究

目的探讨稳定、可靠建立神经干细胞体外增殖的方法,并对增殖培养的细胞进行鉴定。方法获取胚胎大鼠的脑组织,通过加入神经生长因子和采用保留细胞联系技术操作,使脑组织中的神经干细胞在体外克隆增殖并稳定传代。以免疫荧光方法对增殖克隆的神经干细胞球进行鉴定。结果神经干细胞不断增殖形成神经干细胞球且神经干细胞能快速稳定传代增殖。培养的细胞为神经干细胞特异性巢蛋白(nestin)染色阳性细胞。结论在神经生长因子的作用下,利用保留细胞联系技术操作,神经干细胞可以在体外快速、稳定克隆增殖并传代。     

Effects of olfactory ensheathing cells on the proliferation and differentiation of neural stem cells

BACKGROUND:Olfactory ensheathing cells can promote oriented differentiation and proliferation of neural stem cells by cell-secreted neural factors. OBJECTIVE:To observe the effect of olfactory ensheathing cells on the differentiation and proliferation of neural stem cells. DESIGN,TIME AND SETTING:Cytology was performed at the Department of Neurology,Tongji Medical College,Huazhong University of Science and Technology,China,from September 2007 to October 2008. MATERIALS:Mouse anti-nestin polyclonal antibo...     

NOV对人神经干细胞增殖和分化的影响

目的 探讨NOV蛋白对人神经干细胞(HNSCs)增殖和分化的影响。方法 NOV基因重组质粒转染COS—7细胞，收集COS-7细胞和NOV基因修饰COS—7细胞的条件培养液(COS—CM和NOV—CM)，作用于培养的HNSCs，^3H—TdR掺入液闪检测HNSCs的增殖，免疫细胞化学和流式细胞仪检测HNSCs的分化。结果 (1)NOV—CM能明显促进HNSCs对^3H—TdR的摄入，说明NOV—CM能明显促进HNSCs的增殖，另外NOV—CM的促细胞增殖作用具有一定的量效关系；(2)免疫细胞化学和流式细胞仪结果显示，NOV—CM促进HNSCs向神经元方向分化。结论 NOV蛋白可能具有促进HNSCs增殖和分化的作用。     

Dnmt3a regulates both proliferation and differentiation of mouse neural stem cells

DNA methylation is known to regulate cell differentiation and neuronal function in vivo. Here we examined whether deficiency of a de novo DNA methyltransferase, Dnmt3a, affects in vitro differentiation of mouse embryonic stem cells (mESCs) to neuronal and glial cell lineages. Early‐passage neural stem cells (NSCs) derived from Dnmt3a‐deficient ESCs exhibited a moderate phenotype in precocious glial differentiation compared with wild‐type counterparts. However, successive passaging to passage 6 (P6), when wild‐type NSCs become gliogenic, revealed a robust phenotype of precocious astrocyte and oligodendrocyte differentiation in Dnmt3a^?/? NSCs, consistent with our previous findings in the more severely hypomethylated Dnmt1^?/? NSCs. Mass spectrometric analysis revealed that total levels of methylcytosine in Dnmt3a^?/? NSCs at P6 were globally hypomethylated. Moreover, the Dnmt3a^?/? NSC proliferation rate was significantly increased compared with control from P6 onward. Thus, our work revealed a novel role for Dnmt3a in regulating both the timing of neural cell differentiation and the cell proliferation in the paradigm of mESC‐derived‐NSCs. © 2012 Wiley Periodicals, Inc.     

Effect of Angelica sinensis on neural stem cell proliferation in neonatal rats following intrauterine hypoxia

BACKGROUND:Angelica sinensis is a widely used herb in Chinese traditional medicine.It has been shown to improve hypoxia in embryonic rats and reduce nestin expression in neural stem cells,resulting in proliferation of neural stem cells.OBJECTIVE:To study the protective effect of Angelica on neural stem cell proliferation in neonatal rats after intrauterine hypoxia.DESIGN,TIME AND SETTING:The randomized,controlled,experiment was performed at the Department of Histology and Embryology,Luzhou Medical College,China from July 2007 to January 2008.MATERIALS:Because gestational days 14-15 are a key stage in rat nervous system development,21 healthy,pregnant Sprague Dawley rats(14 days after conception)were used for this study.Nestin monoclonal primary antibody was obtained from Chemicon,USA.Angelica parenteral solution(250 g/L)was obtained from Pharmaceutical Preparation Section,Second Affiliated Hospital of Wuhan University,China.METHODS:Rats were randomly divided into a control group(n=5),a hypoxia group(n=8),and an Angelica group(n=8).Saline(8 mL/kg)was injected into the caudal vein of rats in the hypoxia group once a day for seven consecutive days.Intrauterine hypotonic hypoxia was induced using 13% O2 for two hours per day on three consecutive days.Rats in the Angelica group received injections of Angelica parenteral solution(250 g/L);all other protocols were the same as the hypoxia group.The control group procedures were identical to the hypoxia group,but under normal,non-hypoxic conditions.After birth,brain tissues were immediately obtained from neonatal rats and prepared for nestin immunohistochemistry.MAIN OUTCOME MEASURES:Nestin-positive cells in hippocampal CA3 area of neonatal rats in each group were quantified using image analysis to detect signal absorbance.RESULTS:The number of nestin-positive cells increased in the hippocampal CA3 area of neonatal rats in the hypoxia group.The number of nestin-positive cells was less in the Angelica group than in the hypoxia group.Integral absorbance of nestin-positive cells in the hippocampal CA3 area of neonatal rats was significantly higher in the hypoxia group,compared with the control group(P<0.05).The integral absorbance of nestin positive cells was lower in the Angelica group,compared with the hypoxia group(P<0.05).CONCLUSION:Intrauterine hypoxia,induced for 2 hours daily for three consecutive days,with an oxygen concentration of 13%,stimulated the proliferation of neural stem cells.Angelica injection has a protective effect on neural stem cells from neonatal rats following intrauterine hypoxia by decreasing proliferation of neural stem cells.     

Effect of Angelica sinensis on neural stem cell proliferation in neonatal rats following intrauterine hypoxia★

BACKGROUND： Angelica sinensis is a widely used herb in Chinese traditional medicine. It has been shown to improve hypoxia in embryonic rats and reduce nestin expression in neural stem cells, resulting in proliferation of neural stem cells.
OBJECTIVE： To study the protective effect of Angelica on neural stem cell proliferation in neonatal rats after intrauterine hypoxia.
DESIGN, TIME AND SETTING： The randomized, controlled, experiment was performed at the Department of Histology and Embryology, Luzhou Medical College, China from July 2007 to January 2008.
MATERIALS： Because gestational days 14-15 are a key stage in rat nervous system development, 21 healthy, pregnant Sprague Dawley rats （14 days after conception） were used for this study. Nestin monoclonal primary antibody was obtained from Chemicon, USA. Angelica parenteral solution （250 g/L） was obtained from Pharmaceutical Preparation Section, Second Affiliated Hospital of Wuhan University, China.
METHODS： Rats were randomly divided into a control group （n = 5）, a hypoxia group （n = 8）, and an Angelica group （n = 8）. Saline （8 mL/kg） was injected into the caudal vein of rats in the hypoxia group once a day for seven consecutive days. Intrauterine hypotonic hypoxia was induced using 13% O2 for two hours per day on three consecutive days. Rats in the Angelica group received injections of Angelica parenteral solution （250 g/L）; all other protocols were the same as the hypoxia group. The control group procedures were identical to the hypoxia group, but under normal, non-hypoxic conditions. After birth, brain tissues were immediately obtained from neonatal rats and prepared for nestin immunohistochemistry.
MAIN OUTCOME MEASURES： Nestin-positive cells in hippocampal CA3 area of neonatal rats in each group were quantified using image analysis to detect signal absorbance.
RESULTS： The number of nestin-positive cells increased in the hippocampal CA3 area of neonatal rats in the hypoxia group. The number of nestin-pos     

CNTF对神经干细胞体外增殖的影响

目的 探讨睫状神经营养因子(ciliary neurotrophic factor,CNTF)对神经干细胞体外增殖的影响.方法 原代培养新生SD大鼠的室下区神经干细胞并鉴定,经传代纯化后,用不同浓度CNTF (0.1、0.5、1、5、10、20、30 ng/ml)处理5～7 d,MTT实验检测神经干细胞增殖活性.不完全...