绿色荧光蛋白转基因小鼠神经干细胞的培养与鉴定

目的:体外分离、培养及鉴定绿色荧光蛋白(green fluorescent protein,GFP)转基因小鼠神经干细胞(neu- ral stem cells,NSCs),为利用其示踪研究NSCs分化机制奠定基础。方法:从新生GFP小鼠海马组织分离NSCs,采用无血清培养、扩增及传代;免疫荧光和免疫细胞化学染色鉴定NSCs和神经细胞;荧光显微镜下观察GFP表达情况。结果:新生GFP小鼠大脑海马组织分离的NSCs具有自我增殖及分化成神经元、星形胶质细胞和少突胶质细胞的能力;连续传代后,神经球和分化后细胞的GFP表达不受影响。结论:成功分离并获得了GFP新生小鼠NSCs,该细胞具有自我更新能力和多向分化潜能,且稳定表达GFP。因此,源自GFP转基因小鼠的NSCs可以作为研究NSCs多向分化机制的有效示踪工具。     

Effects of engrafted neural stem cells in Alzheimer's disease rats

Cell therapy is thought to have a central role in restorative therapy, which aims to restore the function of the damaged nervous system. Neural stem cells (NSCs) can differentiate into neurons, astrocytes and oligodendrocytes. The purpose of this study was to evaluate the therapeutic effects of transplanting NSCs into rats which have the animal model of Alzheimer's disease (AD). NSCs from the hippocampus and NSCs-derived glial cells labeled with 5′-Bromo-2′-deoxyuridine (BrdU) were transplanted into two groups of transected rat basal forebrain. Nestin staining, glial fibrillary acidic protein (GFAP) staining and double-labeling immunofluorescence were used to detect the engrafted cells in the basal forebrain. Immunohistochemical detection of p75^NGFR showed that the number of cholinergic neurons of the NSCs-transplanted group was significant higher than that of the glia-transplanted group in medial septum (MS) and vertical diagonal branch (VDB) (P < 0.05). Learning and memory abilities were also measured by Y-maze test. The results indicate that transplanted NSCs can differentiate into cholinergic neurons, which may play an important role in the therapeutic effects of transplanted NSCs.     

Grafted neural stem cells migrate to substantia nigra and improve behavior in Parkinsonian rats

Neural stem cell (NSC) transplantation has the potential to treat neurodegenerative diseases such as Parkinson's disease (PD). In this study, we investigated the effect of transplanted NSCs in a PD animal model. NSCs isolated from the subventricular zone (SVZ) of E14 rats were cultured in vitro to produce neurospheres, which were subsequently infected with recombinant adeno-associated virus (rAAV₂) expressing enhanced green fluorescent protein (EGFP). The PD animal model was established by unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB) of Sprague–Dawley rats. Once the model was established, EGFP-expressing NSCs were transplanted into the substantia nigra pars compacta (SNc) or striatum of PD rats. We found that NSCs transplanted into either site significantly reduced apomorphine-induced circling behavior of PD rats. Pathological analysis revealed that the EGFP-expressing NSCs could be detected at both injection sites at 1, 2 and 4 months after transplantation. SNc transplanted cells dispersed within the SNc with a significant portion differentiated into tyrosine hydroxylase-positive neurons. Whereas cells transplanted into the striatum migrated ventrally and posteriorly towards the SNc. These results suggest that the 6-OHDA damaged brain area attracts grafted NSCs, which migrated from the striatum and survived for a long time in SNc, resulting in behavioral improvement of PD rats.     

Dopaminergic differentiation of grafted GFP transgenic neuroepithelial stem cells in the brain of a rat model of Parkinson's disease

Neuroepithelial stem cells (NEPs) possess multipotent potential for self-renewal and neuronal differentiation. Using green fluorescent protein (GFP) positive NEPs, we explored, firstly, the survival and differentiation of grafted NEPs in the host rat and, secondly, whether or not transplantation of NEPs is a feasible therapeutic option for treating Parkinson's disease. NEPs were harvested from the neural tube of enhanced GFP transgenic embryos. In culture, GFP(+) NEPs generated abundant neurospheres and differentiated into both neurons and glia. When stereotaxically transplanted into the 6-hydroxydopamine (6-OHDA)-lesioned striatum of rats, NEPs survived and tyrosine hydroxylase (TH)-positive cells were detected in the graft. Furthermore, these grafted GFP(+) NEPs significantly ameliorated Parkinsonian behavioral symptoms compared with controls which were treated only with normal saline. Our results suggest that transplanted NEPs accomplish dopaminergic differentiation may be used for treating Parkinson's disease.     

绿色荧光蛋白转基因小鼠不同源性间充质干细胞原代培养及生物学特性比较

 目的： 探讨绿色荧光蛋白转基因小鼠脐带和骨髓源性间充质干细胞 （MSCs）的体外分离培养方法、生物学特性、表面标志及多向分化潜能。方法：应用Ⅱ型胶原酶消化培养法分离脐带MSCs及密度梯度离心法分离骨髓MSCs进行体外培养。在倒置显微镜下观察2种细胞的生长特点,运用生长曲线和MTT法检测其原代细胞增殖能力,台盼蓝法测定细胞传代成活率,采用流式细胞术测定2种第3代（P3）细胞DNA周期及表面标志物的表达,并比较其向成脂细胞和成骨细胞的分化潜能。结果：酶消化法分离培养的脐带MSCs 1 d后,细胞贴壁呈成纤维形,2 d后呈漩涡状生长且增殖明显,3 d后达80%融合即可传代;应用密度梯度离心法分离骨髓MSCs,体外培养4 d后,细胞贴壁呈圆形、梭形和多角形生长,5 d后呈克隆样生长且增殖明显,7 d后达80%融合即可传代。原代培养的脐带MSCs生长曲线近似“S”形,骨髓MSCs 生长曲线较平缓;MTT法显示脐带MSCs在3～5 d增殖较明显,骨髓MSCs 7 d后细胞增殖较明显。2种P3细胞传代成活率均为96%以上,G0/G1期细胞均为85%以上,无明显差异（P>0.05）;2种P3细胞CD44、CD90和CD105阳性率均为(60.7±2.3)%以上高表达,CD45、CD19、CD14和CD79a均为(25.6±4.8)%低表达,两者无明显差异（P>0.05）;2种MSCs在体外均具有向成骨细胞和成脂细胞分化的潜能,脐带MSCs向成骨及成脂细胞分化率均为90%以上,与骨髓MSCs的分化潜能比较有显著差异（P<0.05）。结论：脐带MSCs较骨髓MSCs具有较强的增殖能力及分化潜能。绿色荧光蛋白转基因小鼠的脐带MSCs可作为较好干细胞示踪的细胞源。     

Increased survival and migration of engrafted mesenchymal bone marrow stem cells in 6-hydroxydopamine-lesioned rodents

Parkinson's disease is characterized by the loss of dopaminergic neurons in the substantia nigra. Attempted replacement of these neurons by stem cells has proved inconclusive. Bone marrow mesenchymal stem cells (MSC) are multipotent, differentiating into a variety of cells, including neuron-like cells. We used the 6-hydroxydopamine (6-OHDA) animal model of Parkinson's disease to assess migration and differentiation of transplanted MSC. We found in rodents that transplanted MSC survive better in the 6-OHDA-induced damaged hemisphere compared to the unlesioned side. Moreover, contralaterally engrafted MSC migrated through the corpus callosum to populate the striatum, thalamic nuclei and substantia nigra of the 6-OHDA-lesioned hemisphere. In conclusion, we demonstrate that 6-OHDA-induced damage increases the viability of transplanted MSC and attracts these cells from the opposite hemisphere.     

核受体相关因子1基因转染神经干细胞移植治疗帕金森病

目的 探讨转染核受体相关因子1(Nurr1)基因的神经干细胞(NSCs)移植至帕金森病(PD)大鼠纹状体后向多巴胺能神经元的分化及对PD大鼠行为的影响.方法 应用脑立体定位技术构建单侧PD大鼠模型.将PD大鼠随机分为3组,每组8只.假手术组注射生理盐水,NSCs组移植未转染的NSCs,Nurr1组先将重组质粒载体pEGFP-N1-Nurr1转染NSCs,用RT-PCR方法及免疫荧光染色检测Nurr1基因的表达效果,然后用转染Nurr1 基因的NSCs进行移植.细胞用DIL标记后移植至PD大鼠右侧纹状体中,术后2周起用阿朴吗啡诱发旋转试验观测移植后大鼠行为改善情况,12周后用免疫荧光技术检测移植细胞中酪氨酸羟化酶(TH)的表达.结果 重组质粒转染后Nurr1基因能在NSCs中过表达.移植后假手术组和NSCs组PD大鼠的旋转圈数均无下降趋势,两者差异无统计学意义(P＞0.05);NSCs组移植区可见DIL阳性细胞,但TH免疫阳性细胞较少,平均每视野为(3.21±0.40)个;Nurr1组移植后PD大鼠的旋转圈数从第6周开始下降,移植区DIL/TH双标神经元每视野达(9.28±1.09)个.结论 Nurr1基因过表达能诱导NSCs在PD大鼠纹状体内分化为TH阳性的多巴胺能神经元,并在一定程度上改善大鼠的行为功能.     

胚鼠神经干细胞培养与鉴定

目的　体外获取高浓度神经干细胞 (NSC)。方法　加入bFGF培养E12天大鼠胚脑的全部细胞 ,用荧光免疫组化法显示。结果　加bFGF的神经干细胞 (Neuralstemcell,NSC)大量分裂 ,形成大量的细胞分裂球 ,传到 4代时 ,90 %以上的细胞表达nestin。对照组分裂的细胞少 ,维持到第二代细胞逐渐死亡。结论　bFGF对胚胎E12天大鼠脑细胞的NSC有明显的促分裂作用 ,应用此种方法可获得高浓度的NSC     

Engraftable neural crest stem cells derived from cynomolgus monkey embryonic stem cells

Neural crest stem cells (NCSCs), a population of multipotent cells that migrate extensively and give rise to diverse derivatives, including peripheral and enteric neurons and glia, craniofacial cartilage and bone, melanocytes and smooth muscle, have great potential for regenerative medicine. Non-human primates provide optimal models for the development of stem cell therapies. Here, we describe the first derivation of NCSCs from cynomolgus monkey embryonic stem cells (CmESCs) at the neural rosette stage. CmESC-derived neurospheres replated on polyornithine/laminin-coated dishes migrated onto the substrate and showed characteristic expression of NCSC markers, including Sox10, AP2α, Slug, Nestin, p75, and HNK1. CmNCSCs were capable of propagating in an undifferentiated state in vitro as adherent or suspension cultures, and could be subsequently induced to differentiate towards peripheral nervous system lineages (peripheral sympathetic neurons, sensory neurons, and Schwann cells) and mesenchymal lineages (osteoblasts, adipocytes, chondrocytes, and smooth muscle cells). CmNCSCs transplanted into developing chick embryos or fetal brains of cynomolgus macaques survived, migrated, and differentiated into progeny consistent with a neural crest identity. Our studies demonstrate that CmNCSCs offer a new tool for investigating neural crest development and neural crest-associated human disease and suggest that this non-human primate model may facilitate tissue engineering and regenerative medicine efforts.     

鼠胚神经干细胞移植对帕金森模型大鼠的治疗作用

背景：干细胞移植是治疗帕金森的有潜力的方法之一。 目的：观察神经干细胞纹状体移植对帕金森模型大鼠旋转行为及脑内多巴胺含量的影响。 方法：采用6-羟基多巴胺定点注射毁损黑质纹状体的方法构建帕金森大鼠模型;向造模成功的大鼠纹状体内分别移植1×106(共计20 μL)的第3代胚鼠神经干细胞或等量生理盐水。 结果与结论：神经干细胞移植后,帕金森大鼠的旋转行为明显改善。干细胞移植后3周,免疫组化检测发现移植干细胞的帕金森大鼠脑黑质部位酪氨酸羟化酶阳性细胞数增多,纹状体内可见酪氨酸羟化酶阳性细胞;荧光显微镜下观察发现Hoechst 33324d标记神经干细胞在移植针道附近最为密集,并向远隔部位迁徙。干细胞移植后8周,高效液相色谱检测显示移植干细胞的帕金森大鼠纹状体内多巴胺含量明显增高(P < 0.01)。说明神经干细胞脑内移植能够减轻6-羟基多巴胺引起的大鼠中脑黑质多巴胺能神经元的损伤,改善大鼠的旋转行为。     

胚胎海马来源干细胞移植治疗缺血性脑梗死的研究

目的： 对脑梗死大鼠进行胚胎海马来源的干细胞移植治疗,观察其能否在梗死灶部位分化为成熟的神经元而发挥神经替代作用,并最终改善动物的肢体功能。方法: 从绿色荧光蛋白(GFP)转基因SD胚胎大鼠的海马提取细胞进行体外培养,免疫荧光染色观察这些细胞的特征。光化学法制作大脑皮层局灶缺血梗死模型, 即光血栓皮层损伤(PCI)。PCI后1 d将20只SD大鼠随机分为干细胞移植组和对照组,前者在梗死灶附近植入胚胎海马来源的干细胞,而后者不进行干细胞移植。在PCI后1、7、14、21 d,用旋转实验对动物的肢体功能进行测试和评分。在PCI后3周和12周,以抗神经元核抗体(NeuN)染色成熟的神经元,观察移植干细胞的存活及其分化为各种亚型神经细胞的情况。结果: 来自SD大鼠胚胎海马的细胞明显表现出神经干细胞的特征。移植的细胞可以在脑梗死动物模型中至少存活12周,并能分化为成熟神经元、星形胶质细胞等亚型的神经细胞。与移植后3周相比,12周时的NeuN+/GFP+ 密度和移植物体积均有所减少(P<0.05)。旋转实验结果表明,在PCI后7、14、21 d,移植组动物在平衡木上的停留时间均显著长于对照组(P<0.01)。结论: 来源于胚胎海马的细胞具有神经干细胞特征,其被移植入脑梗死灶周围后能存活12周以上,并可以分化为成熟的神经细胞,这可能与动物运动功能的改善有关。该结果提示由移植物分化的神经细胞可能对受损宿主细胞发挥了替代作用。     

Induced neural stem cells from Macaca fascicularis show potential of dopaminergic neuron specification and efficacy in a mouse Parkinson's disease model

Induced neural stem cells (iNSCs) can be reprogrammed from somatic cells and have shown potentials in treatment of various neurological diseases/disorders. Obtaining iNSCs of nonhuman primates serves as an important bridge for clinical translation using iNSCs. In the current study, cynomolgus (Macaca fascicularis) bone marrow mesenchymal stromal cells (MSCs) were reprogrammed into iNSCs by transduction of non-integrative Sendai virus encoding transgenes OCT4, SOX2, KLF4 and C-MYC. The obtained iNSCs showed characteristics of normal neural stem cells (NSCs) and could differentiate into neurons, astrocytes and oligodendrocytes. Furthermore, iNSCs could give rise to dopaminergic neural cells in vitro, which showed safety and efficacy after transplantation into the striatum of an immunodeficient mouse Parkinson’s disease (PD) model.     

人参皂苷Rg1对帕金森病模型小鼠室管膜下区神经干细胞的作用

目的：探讨人参皂苷Rg1促进帕金森病模型小鼠原住神经干细胞增殖分化的作用。方法：C57BL／6小鼠随机分成正常组、模型组和人参皂苷Rg1治疗组。采用MPTP腹腔注射建立帕金森病小鼠模型,利用免疫组化单标和双标技术观察人参皂苷Rg1对小鼠侧脑室室管膜下区神经干细胞增殖分化的影响。结果：人参皂苷Rg1治疗5d,侧脑室室管膜下区的5-溴脱氧尿苷（BrdU）、神经上皮干细胞蛋白（Nestin）阳性细胞较模型组明显增多,并可见大量从背外侧角沿胼胝体排列的BrdU、Nestin阳性细胞;人参皂苷Rg1治疗20d,BrdU／Nestin双标细胞的数量较模型组明显增多。结论：人参皂苷Rg1可促进帕金森病模型小鼠脑内神经干细胞的增殖和迁移。     

绿色荧光蛋白、菲立磁体外双标记神经干细胞及其活性的研究

目的探索用绿色荧光蛋白(green fluorescent protein,GFP)重组逆转录病毒和菲立磁(超顺磁性氧化铁,,Superparamagnetic Iron Oxide,SPIO)体外双标记神经干细胞的可行性及其对细胞活力的影响。方法用病毒介导的GFP基因转导大鼠胚胎神经干细胞。用SPIO和Lipofectamine体外磁化标记经GFP标记的胚胎神经干细胞。用荧光显微镜观察双标记神经干细胞,并行普鲁士蓝染色,了解转染成功率。MTT(四甲基偶氮唑蓝)法检测标记神经干细胞的细胞增殖活力。结果双标记神经干细胞分化的细胞荧光显微镜下观察见发出绿色荧光,普鲁士蓝染色呈阳性。而未标记神经干细胞荧光显微镜下观察未见发出绿色荧光,普鲁士蓝染色呈阴性。MTT法检测发现GFP和SPIO双标记神经干细胞的增殖活力与未标记神经干细胞相比无改变。结论绿色荧光蛋白重组逆转录病毒和菲立磁可以有效地标记体外分离培养的大鼠胚胎神经干细胞。双标记神经干细胞的增殖、分化活力与未标记神经干细胞相比无改变。     

神经干细胞分化调节机制与应用前景

Reynolds等（1992）将成鼠的纹状体在表皮生长因子（epidermal growth factor，EGF）存在下离体培养，发现可表达nestin的细胞，并证实该细胞具有增殖能力，可分化为神经元与胶质细胞。这一发现打破了以往认为的中枢神经系统（central neural system，CNS）神经元损伤不可再生的观点。神经干细胞（neural stem cells，NSCs）具有干细胞的一般特性：自我更新和多向分化，且具有潜在的迁移能力，这些良好的生物学特性，使其具有广泛的应用前景，对于NSCs的研究为脑功能的恢复带来了曙光。     

重组质粒pEGFP-Brn-4的构建及其在神经干细胞中的表达

目的构建神经发育转录因子Brn-4基因真核表达重组质粒,研究Brn-4在神经干细胞(NSC)中的表达情况。方法从新生鼠脑组织提取总RNA,利用RT-PCR的方法获得编码鼠Brn-4的基因片段,应用基因重组技术,将鼠Brn-4基因片段克隆到真核表达载体pEGFP-C2中,应用脂质体转染NSC,荧光显微镜观察该Brn-4基因在NSC中的表达。结果限制性内切酶酶切分析和PCR法鉴定表明为正确重组子,Brn-4基因在NSC中获得表达。结论新构建的真核表达重组质粒pEGFP-Brn-4通过鉴定,结构正确;Brn-4基因可在NSC中表达,可作为后续研究老年性痴呆动物模型转基因实验的基因来源。     

重组腺相关病毒对大鼠神经干细胞的体外转染及其对细胞生长的影响

目的 研究重组腺相关病毒（recombinant adeno-associated virus，rAAV）载体对原代培养的神经干细胞（neural stemcells，Nsc）的体外转染及其对细胞增殖、分化和迁移能力的影响。方法 取新生24h内的Wistar大鼠的海马进行神经干细胞原代培养，用不同滴度的rAAV为载体，以增强型绿色荧光蛋白（enhanced green fluorescent protein，EGFP）基因作为报告基因进行体外转染NSC，通过观察有绿色荧光的NSC的数量，测定rAAV对NSC的体外转染情况；用MTT法测定不同病毒滴度下rAAV对NSC增殖能力的影响，用免疫组化法鉴定NSC、神经元及神经胶质细胞，并在倒置荧光显微镜下直接测定细胞的迁移距离。结果 rAAV对NSC的体外转染效率随着病毒滴度的增加而提高，在转染后的第11天表达水平最高，用MOI为10^4、10^5、10^6的rAAV转染后第11天的转导率分别是9．81％、56．30％、64．67％；不同滴度rAAV转染NSC后不同时间点的MTT测定A值随着病毒滴度的增加而明显减小，差异均有统计学意义；但不同滴度rAAV转染NSC，其分化为神经元和神经胶质细胞的比率以及迁移的距离未见差异。结论 较高滴度（MOI为10^5和10^6）的rAAV可以在体外有效地转染神经干细胞，且不影响神经干细胞的分化和迁移能力，但对其增殖能力有明显抑制，并表现为病毒滴度依赖性。     

神经干细胞移植联合氨基甲酰促红细胞生成素对脑瘫大鼠的作用及机制

目的研究神经干细胞（NSCs）移植联合氨基甲酰促红细胞生成素（CEPO）对新生脑瘫大鼠脑损伤的保护作用。方法从孕14～16天大鼠获得神经干细胞,经过培养并传代后备用。利用7日龄wistar大鼠制作脑瘫模型,给予CEPO腹腔注射和神经干细胞移植,对神经干细胞移植后的分化、凋亡情况,免疫分子在移植前后的改变,大鼠神经系统功能检测等各项指标进行评估,并与单-神经干细胞移植、CEPO腹腔注射效果进行比较。结果缺氧缺血脑损伤后,IFN-γ和IL-1β的浓度显著提高（P〈0．05）,MHC的含量增加,大鼠学习能力下降（P〈0．05）。联合应用CEPO和神经干细胞移植可使移植细胞存活率提高并向神经元分化,降低血中IFN-γ和IL-1β水平,调节MHC抗原的表达,进而明显改善大鼠的神经运动功能。结论应用CEPO可促进移植细胞存活,调节免疫分子的分泌,改善细胞存活的环境,对移植细胞具有明显的保护作用。     

Functional dopaminergic neurons derived from human chorionic mesenchymal stem cells ameliorate striatal atrophy and improve behavioral deficits in Parkinsonian rat model

Human chorionic mesenchymal stem cells (HCMSCs) have been recognized as a desirable choice for cell therapy in neurological disorders such as Parkinson's disease (PD). Due to invaluable features of HCMSCs including their immunomodulatory and immunosuppressive properties, easily accessible and less differentiated compared to other types of MSCs, HCMSCs provide a great hope for regenerative medicine. Thus, the purpose of this study was to determine the in vitro and in vivo efficacy of HCMSCs-derived dopaminergic (DA) neuron-like cells with regard to PD. Initially, HCMSCs were isolated and underwent a 2-week DA differentiation, followed by in vitro assessments, using quantitative real-time polymerase chain reaction, immunocytochemistry, patch clamp recording, and high-performance liquid chromatography. In addition, the effects of implanted HCMSCs-derived DA neuron-like cells on the motor coordination along with stereological alterations in the striatum of rat models of PD were investigated. Our results showed that under neuronal induction, HCMSCs revealed neuron-like morphology, and expressed neuronal and DA-specific genes, together with DA release. Furthermore, transplantation of HCMSCs-derived DA neurons into the striatum of rat models of PD, augmented performance. Besides, it prevented reduction of striatal volume, dendritic length, and the total number of neurons, coupled with a diminished level of cleaved caspase-3. Altogether, these findings suggest that HCMSCs could be considered as an attractive strategy for cell-based therapies in PD.     

干预脑脂质结合蛋白表达对神经干细胞分化作用的影响

目的 探讨脑脂结合蛋白（BLBP）在大鼠海马神经干细胞（NSCs）向神经元分化中的作用。方法 构建BLBP过表达腺病毒和干扰慢病毒载体,并从大鼠胚胎海马组织中分离培养神经干细胞,采用Nestin免疫荧光鉴定NSCs;将体外培养的NSCs分为4组：腺病毒阴性对照组（Ad-NC组）,BLBP过表达腺病毒组（Ad-BLBP组）,慢病毒阴性对照组（LV-NC-RNAi组）和BLBP干扰慢病毒感染组(LV-BLBP-RNAi组)。Real-time PCR和Western blotting检测各组细胞中BLBP表达;病毒感染4 d后免疫荧光检测分化细胞中βⅢ微管蛋白（Tuj1）阳性神经元数量。结果 Ad-BLBP组较Ad-NC组NSCs分化为神经元的数量少,突起短而少;LV-BLBP-RNAi组较LV-NC-RNAi组NSCs分化为神经元的数量明显增多,突起多而长。结论 下调BLBP的表达能够促进体外培养的NSCs向神经元分化。