GFP转基因小鼠神经干细胞移植治疗大鼠帕金森病的实验研究

目的观察GFP(绿色荧光蛋白)转基因小鼠胚胎神经干细胞植入帕金森病大鼠纹状体后的存活、分化情况及治疗作用。方法建立PD模型大鼠及体外培养神经干细胞,然后将GFP转基因小鼠神经干细胞定向植入帕金森病大鼠毁损侧纹状体内,于移植后不同时间诱发旋转行为,并与对照组相比,观察症状的改善,并用酪氨酸羟化酶(TH)免疫组织化学染色方法检测移植GFP转基因小鼠神经干细胞的存活及分化状况。结果 GFP转基因小鼠神经干细胞脑内移植后,帕金森病大鼠的旋转行为明显改善。移植后2至4周时可检测到成片或散在的TH免疫阳性细胞。结论 GFP转基因小鼠神经干细胞移植至帕金森病大鼠纹状体后,可分化为多巴胺能神经元并能改善旋转症状。     

TH基因修饰的神经干细胞移植治疗帕金森病的实验研究

目的 探讨TH基因修饰的神经干细胞脑内移植对帕金森病(PD)的治疗作用。方法 构建pN：ATH逆转录病毒载体质粒，用PA317细胞包装，G418筛选阳性克隆，病毒上清感染神经干细胞，将表达TH的神经干细胞植入：PD大鼠纹状体内，测定：PD大鼠旋转行为改善，DA和DOPAC含量变化，以及TH在纹状体的表达。结果 TH基因修饰的神经干细胞移植8周时能显著降低PD大鼠旋转行为，增加纹状体DA和DOPAC含量，TH在纹状体内的表达增加，疗效好于单纯神经干细胞移植组。结论 TH基因修饰的神经干细胞移植对PD大鼠有明显的治疗作用，可望为PD治疗提供新的途径。     

胚胎干细胞来源的神经前体细胞移植β-淀粉样蛋白损伤大鼠海马后的靶向迁移与分化(英文)

目的观察小鼠胚胎干细胞来源的神经前体细胞移植β-淀粉样蛋白(amyloidβpeptide,Aβ)损伤大鼠海马后的靶向迁移及在体分化。方法采用无血清培养法将表达绿色荧光蛋白(enhanced green fluorescent protein,EGFP)的小鼠胚胎干细胞定向诱导为神经前体细胞,移植至Aβ_(1-40)、单侧损伤的大鼠海马;免疫荧光观察移植细胞的迁移距离、迁移方向与分化情况:对移植细胞的平均迁移距离与平均分化率作相关性分析。结果胚胎干细胞经改良的无血.清培养法生长可分化为nestin阳性神经前体细胞。移植后第16周,神经前体细胞平均迁移距离比第4周增长了约5倍。移植细胞中,胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)阳性细胞的平均分化率从(30.41±1.45)%增长到(49.25±1_23)%;神经丝蛋白200(neurofilament 200,NF200)细胞的平均分化率从(16.68±0.95)%增长到(27.94±1.21)%;靶向迁移至Aβ斑块同侧的GFAP阳性细胞的比例从60.2%增长到81.3%,靶向迁移至Aβ斑块的NF200阳性细胞的比例从61.3%增至84.1%。相关性分析结果显示平均迁移距离与神经元分化率之间存在显著线性相关(r=0.991),与胶质细胞分化率之间也存在显著线性相关(r=0.953)。结论胚胎干细胞来源的神经前体细胞移植Aβ损伤模型大鼠海马后能够靶向迁移至Aβ损伤区并分化为胶质细胞和神经元。     

Mes42细胞及细胞因子对中脑和纹状体神经前体细胞的分化作用

了解中脑神经前体细胞和纹状体神经前体细胞在Mes42细胞及细胞因子作用下的体外诱导分化情况。采用含细胞因子／和Mes42细胞条件培养基和细胞膜碎片的分化培养基培养中脑神经前体细胞和纹状体神经前体细胞，孵育7d后行免疫细胞化学染色了解其体外诱导分化情况。IL－1α（100pg/ml),IL-11(1ng/ml),GDNF(1μg/ml）和LIF(1ng/ml)可诱导中脑神经前体细胞分化成TH阳性神经元，增加分化的TH阳性细胞数目。Mes42细胞条件培养基和其细胞膜裂解碎片以及细胞因子IL－11，LIF，GDNF，IL－1α共同培养神经前体细胞，诱导分化的TH阳性细胞数目无明显增加，但TH阳性细胞的形态更趋成熟。而纹状体神经前体细胞在上述分化培养基内鲜见TH阳性细胞。中脑神经前体细胞可在体外被诱导分化成TH阳性神经元，可能成为帕金森病移植治疗的供体细胞。     

基因修饰的神经干细胞脑内移植后TH表达和神经递质的变化

目的：探讨经TH基因修饰的神经干细胞脑内移植后，PD模型动物脑内TH表达和神经递质的变化。方法：构建pN2ATH逆转录病毒载体质粒，用PA317细胞包装，G418筛选阳性克隆，病毒上清感染神经干细胞，将神经干细胞和表达TH的神经干细胞植入PD大鼠纹状体内，观察移植后TH的表达情况以及DA和DOPAC含量变化。结果：TH基因修饰的神经干细胞移植2月内，TH在纹状体内的表达增加，纹状体DA和DOPAC含量增高，好于单纯神经干细胞移植组和对照组，但比正常水平低，结论：TH基因修饰的神经干细胞，脑内移植能增加纹状体内TH的表达以及DA和DOPAC含量，为其脑内移植治疗PD提供了理论基础。     

胚胎干细胞来源的神经前体细胞移植β-淀粉样蛋白损伤大鼠海马后的靶向迁移与分化

目的观察小鼠胚胎干细胞来源的神经前体细胞移植β-淀粉样蛋（amyloidβpeptide,Aβ）损伤大鼠海马后的靶向迁移及在体分化。方法采用无血清培养法将表达绿色荧光蛋白（enhancedgreenfluorescentprotein,EGFP）的小鼠胚胎干细胞定向诱导为神经前体细胞,移植至Aβ1-40单侧损伤的大鼠海马;免疫荧光观察移植细胞的迁移距离、迁移方向与分化情况;对移植细胞的平均迁移距离与平均分化率作相关性分析。结果胚胎干细胞经改良的无血清培养法生长可分化为nestin阳性神经前体细胞。移植后第16周,神经前体细胞平均迁移距离比第4周增长了约5倍。移植细胞中,胶质纤维酸性蛋白（glialfibrillaryacidicprotein,GFAP）阳性细胞的平均分化率从（30．41±1．45）％增长到（49．25±1．23）％;神经丝蛋白200（neurofilament200,NF200）细胞的平均分化率从（16．68±0．95）％增长到（27．94±1．21）％;靶向迁移至Aβ斑块同侧的GFAP阳性细胞的比例从60．2％增长到81．3％,靶向迁移至Aβ斑块的NF200阳性细胞的比例从61．3％增至84．1％。相关性分析结果显示平均迁移距离与神经元分化率之间存在显著线性相关（r=0．991）,与胶质细胞分化率之间也存在显著线性相关（r=0．953）。结论胚胎干细胞来源的神经前体细胞移植Aβ损伤模型大鼠海马后能够靶向迁移至Aβ损伤区并分化为胶质细胞和神经元。     

胚胎干细胞分化的神经前体细胞移植治疗帕金森病的实验研究

目的探讨胚胎干细胞（embryonic stemcell,ESC）来源的神经前体细胞（Neural precursorcell）移植治疗帕金森病的可能性。方法将胚胎干细胞诱导分化到神经前体细胞阶段后移植到大鼠帕金森病模型纹状体中,并设生理盐水组做对照研究,观察两组移植后行为学改变及检查纹状体内DA、DOPAC的含量。结果移植组在2～4周后与对照组相比行为学上有明显改善（P〈0．01）,纹状体内DA、DOPAC的含量显著提高（P〈0．01）。结论胚胎干细胞经诱导分化成神经前体细胞可用于帕金森病的修复治疗,胚胎干细胞是良好的干细胞移植治疗用细胞来源。     

毒性损伤大鼠耳蜗核移植嗅球神经前体细胞的初步观察

目的体外培养大鼠嗅球神经前体细胞，并移植到谷氨酸诱导损伤的耳蜗核，观察其生存、分化过程。方法嗅球神经前体细胞取自孕15d胚胎大鼠，免疫荧光染色鉴定。耳蜗核定位注射谷氨酸制成损伤模型。伤后7d移植标记的神经前体细胞，不同时间测定听性脑干反应并取材观察移植细胞的存活及分化。结果巢蛋白阳性的嗅球神经前体细胞在体外可自我复制传代，并分化出神经元和神经胶质细胞。谷氨酸损伤和前体细胞移植均造成听性脑干诱发反应（ABR）阈值升高，并有部分的恢复。免疫荧光技术发现耳蜗核中Hoechst33342分别和神经元特异性烯醇化酶（NSE）、胶质纤维酸性蛋白（GFAP）、谷氨酸双重标记的移植细胞。结论嗅球神经前体细胞移植耳蜗核短期存活良好，所分化出的神经元可表达听觉神经递质。     

Mes42细胞及细胞因子对中脑和纹状体神经前体细胞的分化作用

了解中脑神经前体细胞和纹状体神经前体细胞在Mes42细胞及细胞因子作用下的体外诱导分化情况.采用含细胞因子或/和Mes42细胞条件培养基和细胞膜碎片的分化培养基培养中脑神经前体细胞和纹状体神经前体细胞,孵育7 d后行免疫细胞化学染色了解其体外诱导分化情况.IL-lα(100pg/ml),IL-11(1 ng/ml),GDNF(1μg/ml)和LIF(1 ng/ml)可诱导中脑神经前体细胞分化成TH阳性神经元,增加分化的TH阳性细胞数目.Mes42细胞条件培养基和其细胞膜裂解碎片以及细胞因子IL-11,LIF,GDNF,IL-1α共同培养神经前体细胞,诱导分化的TH阳性细胞数目无明显增加,但TH阳性细胞的形态更趋成熟.而纹状体神经前体细胞在上述分化培养基内鲜见TH阳性细胞.中脑神经前体细胞可在体外被诱导分化成TH阳性神经元,可能成为帕金森病移植治疗的供体细胞.     

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。     

Transfer of the von Hippel-Lindau gene to neuronal progenitor cells in treatment for Parkinson's disease

Neuronal progenitor cells (NPCs) may provide dopaminergic neurons for the treatment of Parkinson's disease (PD). However, transplantation of NPCs into the striatum by current methods has had limited success. It is possible to reverse the symptoms of PD in model rats but difficult to reverse them in humans because the number of dopaminergic neurons generated from NPCs is low. We transduced the von Hippel-Lindau (VHL) gene into NPCs isolated from embryonic rat brain. The NPCs with the transduced VHL gene efficiently differentiated into tyrosine hydroxylase-positive neurons in vitro. NPCs with the transduced VHL gene, which were labeled in advance with bromodeoxyuridine, were transplanted into the striatum of a rat model of PD. Numerous bromodeoxyuridine-tyrosine hydroxylase double-labeled cells were seen close to the transplant site, showing that the transplanted cells efficiently generated new dopaminergic neurons within the host striatum. Moreover, all of the animals with NPCs with VHL showed a remarkable decrease in apomorphine-induced rotations. These findings show that NPCs with the VHL gene can efficiently generate dopaminergic neurons and that a sufficient number of dopaminergic neurons can develop from them to reverse the symptoms of PD in humans. VHL gene transduction provides a new therapeutic approach for treatment of PD.     

Mesencephalic neural stem (progenitor) cells develop to dopaminergic neurons more strongly in dopamine-depleted striatum than in intact striatum

Epidermal growth factor (EGF)/fibroblast growth factor (FGF)-responsive stem (progenitor) cells from embryonic brain have self-renewing and multipotent properties and thus are good candidates for donor cells in neural transplantation. However, the survival and differentiation to mature neurons after grafting of stem cells into adult brain are rather poor. We hypothesize that the differentiation of stem cells to mature neurons, such as dopaminergic (DAergic) neurons, is dependent on environmental cues that control the ontogenic development. We compared the survival and differentiation between mesencephalic (MS) and cortical (CTx) stem (progenitor) cells, following grafting into bilateral striata of hemiparkinsonian model rats. MS and CTx stem cells were prepared from E12 rats and proliferated in serum-free medium with EGF or basic FGF for 2 weeks. One day after being primed to differentiate, the cell suspensions of both origins were grafted into the bilateral striata of adult rats that had unilateral 6-OHDA lesions in the substantia nigra. MS cells differentiated to tyrosine hydroxylase (TH)-positive neurons more strongly in DA-depleted striatum than in intact striatum, and methamphetamine-induced rotation was ameliorated in half of the grafted animals. Rosette-like cell aggregation and dysfunction of the blood-brain barrier (BBB) were less in and around the grafts in DA-depleted striatum, suggesting less proliferation and more differentiation of MS stem cells in DA-depleted striatum. Neither TH-positive neurons nor behavioral amelioration were detected following CTx stem (progenitor) cell transplantation in the striata. Data suggest that the DA-depleted striatum offers a suitable environment for MS stem (progenitor) cells to differentiate into mature DAergic neurons.     

Therapeutic effects of differentiated bone marrow stromal cell transplantation on rat models of Parkinson's disease

This study was to explore curative effect of bone marrow stromal cells (BMSCs) differentiated into nestin-positive cells transplantation on rat Parkinson's disease (PD) model. The PD rats were selected and allocated randomly into three groups. BMSCs with differentiation, BMSCs without differentiation and physiological saline were injected into right striatum of PD rat. The rotation test and immunofluorescence double staining were done. Frequency of rotation was significantly less in differentiated or non-differentiated BMSCs groups than that in normal saline group. Brdu/GFAP- and Brdu/NSE-positive cells appeared except BrdU/TH-positive cells. BMSCs differentiated had better effect than that of BMSCs without differentiated and physiological saline group.     

Optimal conditions for in vivo induction of dopaminergic neurons from embryonic stem cells through stromal cell-derived inducing activity

A method of inducing dopamine (DA) neurons from mouse embryonic stem (ES) cells by stromal cell-derived inducing activity (SDIA) was previously reported. When transplanted, SDIA-induced DA neurons integrate into the mouse striatum and remain positive for tyrosine hydroxylase (TH) expression. In the present study, to optimize the transplantation efficiency, we treated mouse ES cells with SDIA for various numbers of days (8-14 days). SDIA-treated ES cell colonies were isolated by papain treatment and then grafted into the 6-hydroxydopamine (6-OHDA)-lesioned mouse striatum. The ratio of the number of surviving TH-positive cells to the total number of grafted cells was highest when ES cells were treated with SDIA for 12 days before transplantation. This ratio revealed that grafting cell colonies was more efficient for obtaining TH-positive cells in vivo than grafting cell suspensions. When we grafted a cell suspension of 2 x 10(5), 2 x 10(4), or 2 x 10(3) cells into the 6-OHDA-lesioned mouse striatum, we observed only a few surviving TH-positive cells. In conclusion, inducing DA neurons from mouse ES cells by SDIA for 12 days and grafting cell colonies into mouse striatum was the most effective method for the survival of TH-positive neurons in vivo.     

Grafts of adult subependymal zone neuronal progenitor cells rescue hemiparkinsonian behavioral decline

Neuronal progenitor cells (NPCs) residing in the adult subependymal zone (SEZ) are a potential source of expandable cells for autologous transplantation to treat Parkinson's Disease and other types of brain injury. We have previously demonstrated the capacity of transplanted adult SEZ NPCs for heterotypic differentiation in the hippocampus. To further characterize the therapeutic potential of these cells, NPCs expanded from the adult rat SEZ were grafted to the striatum of normal and 6-OHDA lesioned adult rats. Grafted cells were assessed for neuronal differentiation, and lesioned animals were tested for amphetamine-induced rotational asymmetry. In addition, the effect of inducing differentiation in vitro prior to transplantation was assessed. Although grafted cells survived after 2 weeks in all animals, neither striatal deafferentation nor in vitro induction of differentiation resulted in significant neuronal differentiation of transplanted cells. Grafts, however, did produce a behavioral effect. While sham animals exhibited increased rotational behavior (+67%) from 2 to 4 weeks post-lesioning, grafted animals did not (-21%). Grafted cells continued to express nestin at the survival time point, and dopamine transporter (DAT) immunoreactivity was restored in the graft body. These results suggest that although neither the normal nor the deafferented striatum alone support the neuronal differentiation of transplanted adult SEZ NPCs, grafts maintaining a progenitor phenotype may produce a therapeutic benefit.     

Long‐term fate of grafted hippocampal neural progenitor cells following ischemic injury

The adult CNS has a very limited capacity to regenerate neurons after insult. To overcome this limitation, the transplantation of neural progenitor cells (NPCs) has developed into a key strategy for neuronal replacement. This study assesses the long‐term survival, migration, differentiation, and functional outcome of NPCs transplanted into the ischemic murine brain. Hippocampal neural progenitors were isolated from FVB‐Cg‐Tg(GFPU)5Nagy/J transgenic mice expressing green fluorescent protein (GFP). Syngeneic GFP‐positive NPCs were stereotactically transplanted into the hippocampus of FVB mice following a transient global cerebral ischemia model. Behavioral tests revealed that ischemia/reperfusion induced spatial learning disturbances in the experimental animals. The NPC transplantation promoted cognitive function recovery after ischemic injury. To study the long‐term fate of grafted GFP‐positive NPCs in a host brain, immunohistochemical approaches were applied. Confocal microscopy revealed that grafted cells survived in the recipient tissue for 90 days following transplantation and differentiated into mature neurons with extensive dendritic trees and apparent spines. Immunoelectron microscopy confirmed the formation of synapses between the transplanted GFP‐positive cells and host neurons that may be one of the factors underlying cognitive function recovery. Repair and functional recovery following brain damage represent a major challenge for current clinical and basic research. Our results provide insight into the therapeutic potential of transplanted hippocampal progenitor cells following ischemic brain injury. © 2014 Wiley Periodicals, Inc.     

Distinct efficacy of pre-differentiated versus intact fetal mesencephalon-derived human neural progenitor cells in alleviating rat model of Parkinson’s disease

Neural progenitor cells have shown the effectiveness in the treatment of Parkinson's disease, but the therapeutic efficacy remains variable. One of important factors that determine the efficacy is the necessity of pre-differentiation of progenitor cells into dopaminergic neurons before transplantation. This study therefore investigated the therapeutic efficacy of mesencephalon-derived human neural progenitor cells with or without the pre-differentiation in alleviating a rat model of Parkinson's disease. We found that a combination of 50 ng/ml fibroblast growth factor 8, 10 ng/ml glial cell line-derived neurotrophic factor and 10 microM forskolin facilitated the differentiation of human fetal mesencephalic progenitor cells into dopaminergic neurons in vitro. More importantly, after transplanted into the striatum of parkinsonian rats, only pre-differentiated grafts resulted in an elevated production of dopamine in the transplanted site and the amelioration of behavioral impairments of the parkinsonian rats. Unlike pre-differentiated progenitors, grafted intact progenitors rarely differentiated into dopaminergic neurons in vivo and emigrated actively away from the transplanted site. These data demonstrates the importance of pre-differentiation of human progenitor cells before transplantation in enhancing therapeutic potency for Parkinson's disease.     

Estrogen promotes differentiation and survival of dopaminergic neurons derived from human neural stem cells

To investigate the effect of estrogen on neuronal differentiation, especially on dopaminergic (DA) neurons, human neural stem cells (NSCs) were differentiated in the presence of 17beta-estradiol. NSCs gave rise to tyrosine hydroxylase (TH)-positive neurons in vitro, the proportion of which was increased by 17beta-estradiol. Increase in TH-positive neurons was abrogated by an estrogen receptor (ER) antagonist, ICI182780, suggesting ERs play a role in differentiation of DA neurons. The observation that ERs were expressed in both proliferating NSCs and postmitotic DA neurons suggested that increase in TH-positive neurons was due to induction and support of DA neurons. 17beta-Estradiol also increased the number of DA neurons derived from human NSCs in vivo when the cells were grafted into mouse brains. These results support a possible role for estrogen in the transplantation of NSCs for Parkinson's disease.