EGFP基因修饰大鼠胚胎中脑神经干细胞的建立

目的建立增强型绿色荧光蛋白（EGFP）基因修饰大鼠胚胎中脑神经干细胞。方法以质粒pEGFPNl转染培养第三代的大鼠胚胎中脑神经干细胞（mNSCs）,经G418筛选后,分别进行nestin免疫细胞化学鉴定和诱导分化后ρ-Ⅲ-／ubulin、GFAP、CNPase免疫细胞化学鉴定。结果EGFP在基因转染12h后开始表达,24h后明显增加,48h达到高峰,经G418筛选1月后有阳性克隆形成,EGFP基因修饰不影响大鼠胚胎mNSCs的增殖与分化。结论成功建立EGFP基因修饰大鼠胚胎mNSCs,为进一步开展帕金森病的细胞移植治疗研究奠定基础。     

NGF-β、GFP基因修饰大鼠胚胎中脑神经干细胞的建立

目的建立神经生长因子β（NGFβ）基因修饰大鼠胚胎中脑神经干细胞。方法以质粒pcDNA3-hNGFb、pEGFPN1共转染大鼠胚胎中脑神经干细胞，荧光显微镜观察绿色荧光蛋白（GFP）在细胞内的表达，免疫细胞化学、Westernblot鉴定NGF-β在细胞内的表达。体外诱导分化，免疫细胞化学鉴定其分化能力。结果GFP在基因转染12h后开始表达，免疫细胞化学、Westernblot结果表明NGF-β能在细胞中正确表达且NGF-β、GFP基因修饰不影响其增殖与分化。结论成功建立NGF-β、GFP基因修饰大鼠胚胎中脑神经干细胞。     

BDNF、EGFP基因修饰大鼠胚胎腹侧中脑神经干细胞的建立

目的建立脑源性神经营养因子（BDNF）、增强型绿色荧光蛋白（EGFP）基因修饰大鼠胚胎腹侧中脑神经干细胞（mNSCs）。方法以FuGENEHD转染试剂介导质粒pcDNA3-BDNF、pEGFPN1共转染第三代E14大鼠胚胎mNSCs。荧光显微镜观察EGFP表达情况；免疫细胞化学方法和Western blot鉴定BDNF的表达；体外诱导分化后免疫细胞化学鉴定其分化能力。结果基因转染12h后EGFP开始表达：免疫细胞化学、Western blot结果表明BDNF能在细胞中正确表达；体外诱导分化研究表明BDNF、EGFP基因修饰不影响大鼠胚胎mNSCs的增殖与分化。结论成功建立了BDNF、EGFP基因修饰大鼠胚胎mNSCs，可为进一步开展帕金森病的细胞移植治疗研究奠定基础。     

胚胎大鼠脑纹状体和中脑神经干细胞的培养

目的研究大鼠脑不同部位胚胎神经干细胞的增殖分化特性。方法采用无血清培养基分离和培养大鼠脑的纹状体和中脑的神经干细胞,通过巢蛋白(nestin)表达和5-溴脱氧尿嘧啶(5-bromo-2'-deoxyuridine BrdU)染色,鉴定细胞的增殖能力;通过新生神经元、星形胶质细胞和少突胶质细胞的特异性免疫细胞化学染色,鉴定培养细胞的多潜能性。通过酪氨酸羟化酶的染色(tyrosine hydroxylase,TH)鉴定多巴胺神经元。结果二者在体外培养都可增殖成球,并能分化成神经系统3个谱系的细胞。纹状体增殖传代3个月,中脑培养细胞增殖维持3周。中脑干细胞分化TH阳性细胞比例高于纹状体。结论培养的胎鼠脑细胞是神经干细胞。纹状体干细胞增殖能力高于中脑干细胞,中脑干细胞更倾向于分化成TH阳性细胞。     

重组大鼠质粒pEGFP-GDNF的构建及大鼠胚胎神经干细胞转染的研究

目的 探讨携带GDNF基因的神经干细胞表达载体的构建方法。方法 采用RT-PCR方法从大鼠胎脑组织总RNA中扩增出该基因的全序列cDNA,并克隆到增强型绿色荧光蛋白（EGFP）报告基因的真核表达载体pEGFP-C1中,经酶切鉴定及测序分析对重组质粒pEGFP-GDNF作进一步鉴定。采用阳离子脂质体将重组质粒pEGFP-GDNF转染至鼠胚胎神经干细胞。结果 大鼠GD-NF cDNA已正确地克隆到真核表达载体pEGFP-C1中,而构建成重组大鼠质粒pEGFP-GDNF,GDNF基因在细胞中可稳定表达。结论 神经干细胞可直接作为基因靶细胞,能被GDNF真核细胞表达载体pEGFP-GDNF有效的感染。     

大鼠胚胎腹侧中脑神经干细胞的分离培养与鉴定

目的探讨大鼠胚胎腹侧中脑神经干细胞分离、培养及鉴定的方法。方法解剖分离E14d大鼠胚胎腹侧中脑组织,经机械吹打制成单细胞悬液,接种于无血清培养基中培养,观察其增殖、分化并进行Nestin免疫细胞化学鉴定和诱导分化后β-Ⅲ-tubulin、GFAP、CNPase免疫细胞化学鉴定。结果原代培养7d后,可形成大量悬浮生长Nestin免疫阳性的神经球,经诱导分化后细胞呈GFAP、CNPase或β-Ⅲ-tubulin免疫阳性。结论建立大鼠胚胎腹侧中脑神经干细胞分离培养与鉴定的方法,为进一步开展帕金森病的细胞移植治疗研究奠定基础。     

SPIO、EGFP双标GDNF基因修饰中脑神经干细胞移植治疗帕金森病

目的探讨超顺磁氧化铁(SPIO)、增强型绿色荧光蛋白(EGFP)双标胶质细胞源性神经营养因子(GDNF)基因修饰中脑神经干细胞(mNSCs)移植对帕金森病(PD)大鼠的治疗作用。方法将PD大鼠随机分为对照组、GFP基因修饰mNSCs移植组和GDNF基因修饰mNSCs移植组,将相应细胞移植到PD大鼠纹状体。阿朴吗啡(APO)诱导PD大鼠旋转行为评估细胞移植的治疗作用。磁共振成像、免疫荧光组织化学研究移植细胞的存活、迁移和分化。结果GDNF基因修饰mNSCs移植能显著改善APO诱导PD大鼠的异常旋转行为;大多数移植细胞停留于移植原位,移植8w后,GDNF基因修饰mNSCs移植组有更多的细胞存活并分化为多巴胺神经元。结论MR I成像可对体内移植的SPIO标记细胞进行活体示踪。GDNF基因修饰胚胎mNSCs移植可显著改善PD大鼠的运动障碍,其分子机制有待进一步研究。     

小鼠胚胎干细胞诱导的神经前体细胞纹状体移植对PD大鼠的治疗作用

目的 观察来源于小鼠胚胎干细胞的神经前体细胞移植PD大鼠纹状体后的存活、分化以及细胞移植对PD大鼠的治疗作用。方法 采用无血清方法将小鼠胚胎干细胞定向诱导为神经前体细胞，免疫组化技术观察移植细胞的存活、分化。结果 胚胎体在N2选择性培养基选择生长5d后，85％以上的小鼠胚胎干细胞分化为nestin阳性的神经前体细胞。移植到PD大鼠纹状体后大部分神经前体细胞存活良好，移植细胞分别保持为未分化的nestin阳性的神经前体细胞和TH阳性的神经元。移植后3周，PD大鼠的旋转次数明显减少。结论 胚胎干细胞来源的神经前体细胞移植PD大鼠纹状体后能分化为TH阳性的神经细胞，对PD有治疗作用。     

NRTN与GDNF联合神经干细胞移植对AD大鼠CREB和TrkB表达的影响

神经干细胞因可分化为各类神经细胞,为临床很多疾病提供了一个崭新的研究视野[1].阿尔茨海默病患者脑内神经病理改变的主要原因之一是淀粉样β蛋白使神经干细胞诱导分化为胆碱能神经元的过程受到抑制而不能实现结构修复.外源性神经干细胞成为补充阿尔茨海默病患者中枢结构内缺损神经细胞的一个重要来源.Neurturin(NRTN)是近些年发现的一种对多种神经细胞的正常生存及分化具有维持和促进作用的神经营养类蛋白因子,尤其是能够诱导神经干细胞向胆碱能类记忆相关神经细胞的转化,CREB是NRTN下游作用的一个因子,其磷酸化与非磷酸化比例失衡与海马区记忆认知障碍直接相关[2].GDNF能够促使损伤的胆碱能神经元恢复,具有细胞来源的特异性.幼稚神经细胞能被GDNF维持存活及向某些类型神经细胞诱导分化但成熟的神经细胞GDNF仅表现促进存活而不能诱导其分化,TrkB是GDNF调节突触活性与细胞分化进而影响大鼠海马结构和神经行为的一个重要蛋白靶点[3].     

胚胎大鼠神经干细胞双向凝胶电泳方法的建立**◆

背景：神经干细胞是当今研究热点,双向电泳是蛋白质组学研究的技术基础,有必要建立稳定的神经干细胞蛋白质组双向凝胶电泳技术体系。目的：通过对神经干细胞双向凝胶电泳几个关键步骤的分析,建立稳定、重复性好的神经干细胞双向凝胶电泳的分离方法。方法：采用无血清体外细胞培养方法,培养胚胎大鼠神经干细胞并进行鉴定。提取样品蛋白并裂解,被动水化,采用不同IEF参数,使用PDQuest8.0软件对图谱进行分析和比较。观察不同等点聚焦参数下双向电泳图谱质量,蛋白质点的数目及重复性。结果与结论：不同IEF参数条件下的双向电泳图谱的质量有所不同,增加低电压的除盐步骤有助于去除电泳胶图上的横纹。在80 000 Vh聚焦总能量下,获得较理想的电泳图。通过选用适当的IEF参数,建立了适当的神经干细胞双向凝胶电泳方法。     

Developmental perspectives on human midbrain-derived neural stem cells

"Regenerative medicine" may hopefully provide some relief or therapy for diseases which we can currently not or only barely treat. This is also true for most diseases presenting with parkinsonism, including Parkinson's disease, in spite of the available effective symptomatic treatments. The replacement of dopaminergic neurons in patients with Parkinson's disease via implantation of embryonic midbrain tissue was taken from animal experiments to clinical applications showing limited efficacy. Today, it seems possible to generate functional dopaminergic neurons from a variety of stem cells including embryonic and neural stem cells. It is not clear which tissue source will be most appropriate to test in initial clinical trials. Such cells allow for prior extensive in-vitro and in-vivo testing as well as "good manufacturing production" to reduce the risks in subsequent clinical applications.     

Developmental perspectives on human midbrain-derived neural stem cells

Regeneration or restoration of lost or damaged neurons is very likely to profoundly alter the disability and needs of many patients. The replacement of dopaminergic (DA) neurons in patients with Parkinson's disease via implantation of embryonic midbrain tissue was taken from animal experiments to clinical applications. Ethical concerns related to the use of fetal tissue derived from abortions further argue for the search for alternative tissue sources. Today, it seems possible to generate functional DA neurons from a variety of stem cells, including embryonic and neural stem cells. Bone marrow stromal cells are another source for cell replacement. Neural stem cells derived from human fetal midbrain tissue maintain a considerable capacity to self-renew and to differentiate into DA neurons. Therefore, these cells may be a promising source to generate functional human DA neurons.     

In vitro culture and differentiation of rat embryonic midbrain-derived neural stem cells

BACKGROUND: Midbrain-derived neural stem cells (mNSCs) can differentiate into functional mature dopamincrgic neurons. The mNSCs are considered the ideal choice for cell therapy of Parkinson's disease. OBJECTIVE: To isolate rat embryonic mNSCs and to observe the differentiation characteristics of mNSCs induced by cell growth-promoting factors. DESIGN, TIME AND SETTING: An in vitro cell culture study based on the molecular biology of nerve cells was carried out at the Institute of Clinical Medicine, China-Japan Friendship Hospital (China) from March to November 2007. MATERIALS: Sprague Dawley rats at embryonic day 14 were used in this study. Nestin antibody, β-Ⅲ tubulin antibody, glial fibrillary acidic protein (GFAP) antibody and cyclic nucleotide 3'-phosphohydrolase (CNPase) antibody were provided by Abeam; DMEM/F12 medium and N2 supplement were provided by Invitrogen; epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF2) were provided by R＆D Systems. METHODS: The ventral mesencephalon was dissected from embryonic day 14 rat embryos. By trypsin digestion and mechanical separation, the brain tissue was triturated into a fine single-cell suspension. The cells were cultured in 5 mL serum-free medium containing DMEM/Fl2, 1% N2 supplement, 20 ng/mL EGF and FGF2. The mNSCs at the third generation were coated with 10 μg/mL polylysine and induced to differentiate in the DMEM/Fl2 supplemented with 1% fetal bovine serum and 1% N2. MAIN OUTCOME MEASURES: The neural spheres of the third passage were identified by nestin immunofluorescence; at the same time, the cells were induced to differentiate, and the types of differentiated cell were identified by immunofluorescence for βⅢ tubulin, GFAP and CNPase. RESULTS: Seven days after primary culture, a great many neurospheres could be obtained by successive pasage. Immunofluorescence assays showed that the neurospheres were nestin positive, and after differentiation, the cells expressed GFAP, CNPase and β -Ⅲ-tubulin. CONCLUSION: Embryonic day 14 rat mNSCs can differentiate into neuron-like cells and glial cells following induction by EGF, FGF2 and N2 additive.     

In vitro culture and differentiation of rat embryonic midbrain-derived neural stem cells*☆

BACKGROUND： Midbrain-derived neural stem cells （mNSCs） can differentiate into functional mature dopaminergic neurons. The mNSCs are considered the ideal choice for cell therapy of Parkinson＇s disease. OBJECTIVE： To isolate rat embryonic mNSCs and to observe the differentiation characteristics of mNSCs induced by cell growth-promoting factors. DESIGN, TIME AND SETTING： An in vitro cell culture study based on the molecular biology of nerve cells was carried out at the Institute of Clinical Medicine, China-Japan Friendship Hospital （China） from March to November 2007. MATERIALS： Sprague Dawley rats at embryonic day 14 were used in this study. Nestin antibody, β-Ⅲ tubulin antibody, glial fibrillary acidic protein （GFAP） antibody and cyclic nucleotide 3＇-phosphohydrolase （CNPase） antibody were provided by Abcam; DMEM/F12 medium and N2 supplement were provided by Invitrogen; epidermal growth factor （EGF） and fibroblast growth factor-2 （FGF2） were provided by R＆D Systems. METHODS： The ventral mesencephalon was dissected from embryonic day 14 rat embryos. By trypsin digestion and mechanical separation, the brain tissue was triturated into a fine single-cell suspension. The cells were cultured in 5 mL serum-free medium containing DMEM/FI 2, 1% N： supplement, 20 ng/mL EGF and FGF2. The mNSCs at the third generation were coated with 10ug/mL polylysine and induced to differentiate in the DMEM/F12 supplemented with 1% fetal bovine serum and 1% N2. MAIN OUTCOME MEASURES： The neural spheres of the third passage were identified by nestin immunofluorescence; at the same time, the cells were induced to differentiate, and the types of differentiated cell were identified by immunofluorescence for β Ⅲ tubulin, GFAP and CNPase. RESULTS： Seven days after primary culture, a great many neurospheres could be obtained by successive pasage. Immunofluorescence assays showed that the neurospheres were nestin positive, and after differentiation, the cells expressed GFAP, CNPase and β -Ⅲ-tu     

脑源性神经生长因子基因转染对脊髓神经干细胞分化的影响

目的 研究BDNF基因转染小鼠脊髓源性NSCs向神经元分化情况.方法 选取体外培养E14小鼠胚胎脊髓来源NSCs,构建整合有BDNF基因的逆转录病毒载体,感染体外培养的NSCs,诱导其向神经元分化.采用免疫细胞化学方法鉴定,确定NSCs的分化比例.结果 转染后诱导分化24 h后可见部分细胞贴壁分化,48 h后转染细胞大部分贴壁.BDNF转染NSCs分化为神经元比例较未转染NSCs明显增高,差异有统计学意义(P＜0.05).结论 逆转录病毒载体介导BDNF基因转染NSCs可促进细胞分化,且分化多为神经元方向.

Abstract：

Objective To study the differentiation potential of mouse spinal cord derived neural stem cells (NSCs) into neurons after being transfected with BDNF gene in vitro. Methods Spinal cord derived NSCs from the E14 fetus mouse were isolated and cultured in vitro; the retrovirus containing pLXSN-BDNF gene was established and transfected into thc above NSCs, and thea, spinal cord derived NSCs were induced to be differentiated into neuron-like cells. Immunohistochemistry was employed to detect and calculate the ratio of differentiation of NSCs into neurons. Results The NSCs cultured in vitro partly adhered to the wall and differentiated within 24 h of transfection with BDNF gene, and most of the NSCs adhered to the wall differentiated within 48 h of transfection. The level of neurons from spinal cord derived NSCs modified by BDNF gene was markedly increased as compared with that that from normal spinal cord derived NSCs (P＜0.05). Conclusion NSCs transfected by retroviral pLXSN-BDNF can promote the cell differentiation. BDNF gene can increase greatly the percentage of neurons in the course of inducing the differentiation of mouse NSCs.     

SPIO、GFP双标BDNF基因修饰中脑神经干细胞的研究

目的建立超顺磁氧化铁（SPIO）、绿色荧光蛋白（GFP）双标脑源性神经营养因子（BDNF）基因修饰中脑神经干细胞。方法以质粒pcDNA3-BDNF、pEGFPN1共转染第3代大鼠胚胎中脑神经干细胞,并用SPIO标记。荧光显微镜检测GFP的表达;免疫细胞化学、Westernblot鉴定BDNF的表达;普鲁士蓝染色、透射电镜鉴定SPIO标记。结果 GFP在基因转染12h后开始表达,24h明显增加,48h达顶峰。免疫细胞化学、Westernblot表明：细胞成功表达BDNF。普鲁士蓝染色显示：SPIO标记的中脑神经干细胞内有大量蓝染铁颗粒,细胞标记率达100%。透射电镜显示：SPIO颗粒位于吞饮小泡和细胞质内。结论成功建立SPIO、GFP双标BDNF基因修饰中脑神经干细胞,为进一步开展帕金森病的细胞移植治疗研究奠定基础。     

神经营养因子3基因修饰神经干细胞移植脊髓损伤的相关蛋白表达☆

背景：研究表明神经干细胞和神经营养因子3基因修饰的神经细胞联合移植能够在移植后存活并有效促进脊髓横断后脊髓的功能恢复,但神经营养因子3基因修饰的神经干细胞能否在脊髓受损部位发挥功能并促进脊髓损伤大鼠的功能恢复? 目的：观察神经营养因子3基因修饰胚胎脊髓神经干细胞移植后脊髓损伤大鼠的功能恢复情况及损伤局部的基因表达。 方法：将30只SD大鼠在T9水平进行脊髓半切后,随机分为3组,分别在受损脊髓内植入细胞培养液、神经干细胞及神经营养因子3基因修饰神经干细胞。另取10只仅行椎板切除设置为空白对照。移植后通过行为学测试评价脊髓功能的恢复,RT-PCR和Western blot检测脊髓损伤部位神经营养因子3和髓鞘碱性蛋白的表达。 结果与结论：移植神经营养因子3基因修饰神经干细胞组行为学测试结果最好,移植细胞培养液组行为学测试最差。与移植细胞培养液组相比,移植神经干细胞及神经营养因子3基因修饰神经干细胞组大鼠脊髓组织中神经营养因子3基因和髓鞘碱性蛋白基因的mRNA水平明显上调,在蛋白水平也有类似的结果,且神经营养因子3基因修饰神经干细胞组效果更明显。提示移植神经营养因子3基因修饰神经干细胞能促进脊髓受损部位出现更多向少突胶质细胞分化的细胞,并能更强的表达神经营养因子3。