大鼠胚胎神经上皮细胞侧脑室移植后存活及分化的实验研究

目的：观察大鼠胚胎神经上皮细胞同种脑移植后的存活及生长分化状况。方法：孕12d大鼠剖腹取胚胎，剥离神经管，胰酶消化后获取神经管壁上的神经上皮细胞，然后植入其父体侧脑室中，分别于移植后10d,14d,21d,28d灌注取脑，肜HF染色及神经元特异烯醇化酶（NSE），胶质纤维酸性蛋白（GFAP）免疫组织化学方法检测胚胎神经上皮细胞移植后的存活及分化状况。结果：神经上皮细胞侧脑室移植后多贴附于脑室壁形成细胞团块，有的随脑脊液循环至第三脑室生长，随时间延长移植物增大，HE染色见脑室内有成团的移植细胞，免疫组织化学染色显示移植细胞团内既有NSE－免疫阳性细胞，也有GFAP－免疫阳性细胞，移植物周围多为GFAP－免疫阳性细胞。结论：胚胎神经上皮细胞侧脑室移植后能够贴附脑室壁存活，并能分化为神经元和神经胶质细胞。     

大鼠胚胎神经干细胞黑质内移植后的存活及分化

目的：观察胚胎神经于细胞脑内移植后的存活及生长分化状况。方法：从孕11d(E11d)大鼠胚胎神经管获取神经上皮细胞，经神经巢蛋白(nestin)染色鉴定干细胞；同时植入同种大鼠黑质内。于移植后7d、14d取脑，用神经元特异烯醇化酶(NSE)、酪氨酸羟化酶(TH)免疫组化方法检测移植细胞的存活及分化状况。结果：E11d神经管上皮细胞多数呈nestin染色阳性，黑质内移植后增殖形成细胞团并随时间延长而增大。免疫组化染色显示移植细胞团内有NSE及TH免疫阳性细胞。结论：胚胎神经上皮细胞多数为神经干细胞，黑质内移植后可以存活并分化为多巴胺能神经元。     

胚胎嗅鞘细胞和中脑腹侧细胞移植对帕金森病SD大鼠纹状体神经元存活和分化的影响

目的观察胚胎嗅球嗅鞘细胞(OECs)和胚胎中脑腹侧细胞(VMCs)联合移植对帕金森病(PD)SD大鼠纹状体神经元存活及分化的影响。方法 12只PD模型SD大鼠随机平均分成两组:单独VMCs组(在脑立体定位仪下将VMCs植入PD大鼠模型毁损侧纹状体内)和联合移植组(在脑立体定位仪下将OECs和VMCs植入PD大鼠模型毁损侧纹状体内)。OECs来源于5～7d绿荧光鼠的嗅球嗅鞘,VMCs来源于孕13～14d绿荧光胎鼠中脑腹侧的脑组织。于移植后4、14周灌注取脑,切片后做酪氨酸羟化酶(TH)免疫组织化学染色,检测胚胎中脑腹侧细胞的存活及分化状况。结果移植4周后单独VMCs组及联合移植组纹状体区都可检测到TH免疫阳性神经元,数量无统计学差异(P0.05)。移植14周后联合移植组纹状体区TH免疫阳性神经元数量比单独VMCs组明显增多(P0.05)。结论胚胎中脑腹侧细胞联合嗅鞘细胞移植入帕金森病大鼠纹状体促使纹状体神经元分化为多巴胺能神经元。     

胚胎神经上皮干细胞脑内移植治疗大鼠帕金森病

背景：研究表明胚胎干细胞移植可改善血管性痴呆大鼠的学习记忆功能,增强神经的可塑性,诱导自身定向迁移并分化为成熟神经元。 目的：观察胚胎神经上皮干细胞脑内移植治疗帕金森病大鼠及移植细胞的迁徙情况。 方法：将绿色荧光蛋白转基因鼠的胚胎神经上皮干细胞分别移植到帕金森病大鼠的黑质、纹状体和侧脑室内,移植后检测移植细胞的存活、迁徙与分化;利用高效液相色谱法检测实验动物脑内多巴胺神经递质的含量;对比实验动物旋转行为的改变,评估胚胎神经上皮干细胞移植对帕金森病大鼠的治疗作用。 结果与结论：移植细胞存活良好且分化出了酪氨酸羟化酶阳性细胞,向黑质纹状体环路迁徙趋势明显;脑内多巴胺含量增加,动物旋转行为改善明显。表明移植到帕金森病大鼠脑内的神经上皮干细胞多向黑质纹状体环路迁徙,且可增加脑内多巴胺神经递质的含量治疗帕金森病。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

大鼠胚胎中脑腹侧细胞和大脑皮质神经干细胞移植治疗帕金森病大鼠的比较

体外培养的大鼠胚胎中脑腹侧(VM)细胞和大脑皮质神经干细胞(NSCs)分别移植入帕金森病(PD)模型大鼠毁损侧纹状体。移植后2,4,8周采用脑微透析术结合高效液相色谱(HPLC)动态监测毁损侧纹状体内多巴胺水平,同期观察大鼠的旋转行为。移植后8周采用免疫组化法检测植入细胞的存活及分化情况。结果显示:移植后4,8周VM移植组多巴胺水平明显高于NSCs移植组或对照组,同期VM移植组较其余两组旋转行为有显著改善,而NSCs移植组与对照组多巴胺水平及旋转行为无显著差异。VM移植组较NSCs移植组植入的细胞易存活和分化。以上结果提示,大鼠胚胎VM细胞移植治疗PD模型的疗效明显优于胚胎大脑皮质NSCs。     

体外长期培养的人神经干细胞对大鼠帕金森病模型的药效初探

目的初步探讨体外长期连续培养的人神经干细胞(hNSCs)对大鼠帕金森病(PD)模型的药效。方法采用6-羟基多巴胺(6-OHDA)注射大鼠脑内黑质体部位,毁损黑质多巴胺能神经元建立PD大鼠模型。成模大鼠随机分为对照组、NSC混合、NSC高剂量、NSC低剂量四组,行脑内同侧纹状体部位移植h NSCs。行为学实验观察治疗效果,免疫荧光染色鉴定植入纹状体的hNSCs存活和分化情况。结果细胞移植后观测和检查:(1)NSC高剂量组在移植3个月后,大鼠的单侧旋转症状与移植前比较有显著的改善(P0.05);NSC低剂量组大鼠旋转症状也有改善,但与移植前比较无显著性差异(P0.05);NSC混合组大鼠旋转症状明显改善,与对照组比较有显著性差异(P0.05)。(2)6-OHDA能明显损伤大鼠脑内黑质部位,使其神经元数量减少;神经干细胞纹状体部位移植后可见移植的干细胞分化为神经元。结论 PD模型大鼠纹状体部位移植的hNSCs可分化为神经元,且对PD模型大鼠旋转症状有明显的改善。     

携带绿色荧光蛋白基因的神经上皮干细胞移植治疗帕金森病

背景：神经干细胞异体移植的细胞替代疗法是治疗帕金森病的热点,但先前的研究重点关注移植细胞的形态学信息和动物的行为改变,作用机制尚未明确。 目的：通过检测神经上皮干细胞脑内移植后帕金森病模型大鼠的旋转行为改变与其脑内多巴胺含量变化的关系,分析神经上皮干细胞移植治疗帕金森病的作用机制。 方法：建立帕金森病大鼠模型,造模后分为2组,实验组将携带绿色荧光蛋白基因的胚胎神经上皮干细胞克隆后移植到帕金森病模型大鼠的纹状体内,对照组注射等量的生理盐水。取材后检测移植细胞的存活与分化,对比分析大鼠行为改变与其脑内多巴胺含量变化的关系。 结果与结论：移植后实验组大鼠的旋转行为明显改善,移植细胞在宿主脑内存活良好,并分化出TH阳性细胞。实验组大鼠脑内多巴胺的含量明显高于对照组。提示携带绿色荧光蛋白基因的神经上皮干细胞脑内移植后帕金森病模型大鼠的旋转行为改善与其脑内多巴胺含量的变化成正比,分化出有功能的TH阳性细胞可能是神经干细胞移植治疗帕金森病的基本机制。 关键词：神经上皮干细胞;帕金森病;绿色荧光蛋白;转基因鼠;多巴胺 doi:10.3969/j.issn.1673-8225.2012.06.029     

神经干细胞移植对帕金森病样模型大鼠的治疗作用

目的 观察大鼠神经干细胞(NSC)植入帕金森病(PD)模型大鼠的脑纹状体后的存活、分化及功能状态.方法 体外分离培养NSC,单克隆培养,免疫细胞化学检测多向分化潜能和特异性标记nestin.建立PD大鼠模型,纹状体内植入DAPI标记的NSC,检测6-羟基多巴胺(6-HHDA)诱发的旋转行为,荧光检测标记细胞的分布情况,免疫细胞化学和高效液相色谱检测细胞分化和神经递质含量.结果 培养的NSC表达nestin,具有自我更新和多向分化能力;NSC植入PD模型大鼠纹状体后大鼠诱导旋转行为显著改善(P＜0.05);NSC在脑内迁移,并在纹状体内形成少量酪胺酸羟化酶(TH)阳性细胞、纤维;植入后纹状体内多巴胺(DA)及其代谢产物含量上升,2个月时分别增加3.6倍和2.8倍,4个月时增加3.4倍和2.4倍(P＜0.01).结论 神经干细胞在体外大量扩增后植入PD脑内能长期存活并分化、分泌神经递质,从而部分改善PD症状.     

胚胎干细胞移植治疗脑血管疾病

背景：胚胎干细胞移植是否能够成为脑血管疾病治疗有效的方法已成为目前研究的热点。 目的：探讨胚胎干细胞分化神经前体细胞移植治疗脑血管疾病的效果及可行性。 方法：分别建立帕金森病、缺血性脑损伤以及血管性痴呆大鼠模型,并进行胚胎干细胞体外培养,诱导分化为神经前体细胞,将胚胎干细胞分化神经前体细胞移植入相应脑血管疾病模型大鼠脑内,观察脑血管病变大鼠的旋转行为学变化、脑组织病理变化以及海马结构的变化和神经细胞数目的变化。 结果与结论：胚胎干细胞分化神经前体细胞移植入帕金森病大鼠脑内后,阿朴吗啡诱发的旋转次数逐渐减少,呈下降趋势,纹状体多巴胺的含量明显增高。胚胎干细胞分化神经前体细胞移植入缺血性脑损伤大鼠脑内后,能够长期存在于脑内,并迁移、分布于受损的海马,构成海马结构,进一步分化为神经元,并且受损海马内的神经细胞数量明显增加。说明移植的胶质细胞源性神经营养因子基因修饰的胚胎干细胞可改善血管性痴呆大鼠的学习记忆功能,增强神经的可塑性,诱导自身定向迁移并分化为成熟神经元。     

培养胚胎神经干细胞移植入纹状体后的形态学观察

本研究的目的是 :将体外培养的小鼠胚胎大脑皮层和脊髓的神经干细胞经单细胞悬液微移植后观察其在大鼠纹状体的存活、迁移和分化的状况。实验在无血清条件下将这些细胞扩增、培养再经活细胞荧光染料 Di I标记后 ,采用微移植的方法 ,通过脑立体定位仪上用微玻璃针将干细胞分别植入成年大鼠双侧纹状体的对称部位。大鼠存活 8周后 ,经灌注固定、恒冷箱切片 ,在荧光显微镜下观察标记的移植细胞的迁移状况 ;用星形胶质细胞特异性抗体观察移植区 GFAP的表达 ,以显示移植细胞分化状况。结果表明 :来源于胚胎小鼠大脑皮层和脊髓的体外培养神经干细胞经微移植后 ,均可在成年大鼠脑内纹状体区域存活 ,移植的神经干细胞可向周围的脑实质内迁移 ,迁移细胞沿特定的纹状体结构分布。神经干细胞主要分化成星形胶质细胞。本实验结果提示 ,移植的神经干细胞可在脑实质内存活、迁移和分化。     

止颤汤干预神经干细胞移植帕金森病大鼠脑内多巴胺及其代谢产物的变化

背景：如何促进脑内多巴胺含量的增加以及减少多巴胺的代谢,是治疗帕金森病的热点所在。 目的：从多巴胺代谢途径角度观察止颤汤对神经干细胞移植帕金森病大鼠的脑黑质中多巴胺及其代谢产物含量的变化。 方法：以大鼠脑立体定位和1-甲基-4-苯基-1,2,3,6-四氢吡啶建立帕金森病大鼠模型。应用高效液相色谱法测定帕金森病大鼠中脑多巴胺及其代谢产物的含量。 结果与结论：止颤汤可以提高神经干细胞移植后帕金森病大鼠中脑多巴胺及其代谢产物双羟苯乙酸的含量,但对代谢产物高香草酸无明显影响。通过促进帕金森病大鼠干细胞移植后神经干细胞的存活,使之定向分化为多巴胺能神经元并分泌多巴胺,同时抑制多巴胺分解达到治疗作用。     

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.     

Intracerebral transplantation of cultured neurons after reaggregation in a plasma clot

In order to be able to transplant neural cells which have been either manipulated in vitro or maintained in culture for the purpose of cell-type enrichment, we have developed a novel plasma clot method which permits reaggregation of previously dissociated cells such that they can be implanted as highly localized transplants rather than as dispersion-prone cell suspension grafts. To establish the method, enzymatically dissociated cells prepared from hippocampal primordia of late embryonic rats were immediately reaggregated into plasma clots and transplanted to the hippocampal formation of adult recipients. By using fluorescein-labelled bovine plasma to form the plasma clot grafts of reaggregated cells, the fate of the plasma clot protein matrix was followed at different post-operative survival times. Initially, 4-5 days post-operative, the plasma clot maintained the grafted cells in a loose sponge-like sack at the site of implantation. After 2-3 weeks, the transplanted cells were more compact and fused with the host neuropil, and the plasma clot matrix had largely been degraded. At 1 month or longer survival, there was no distinguishable boundary between transplant and host, and there was little or no evidence of any remaining plasma clot matrix or proteins. The plasma clot method was subsequently applied to the transplantation of cultures enriched in pyramidal cells. Enrichment for pyramidal cells was achieved by eliminating mitotic cells (dentate granule cells and glia) by brief (200 rad) irradiation of hippocampal primordia followed by dissociation and maintenance in monolayer culture for 4-6 days. Fibres from host dentate granule cells grew into the pyramidal cell-enriched transplants and established mossy fibre terminals on the donor cells. In transplants between embryonic and adult rats, donor cells were identified at long survival times by prelabelling donor cells in utero or in vitro with [3H]thymidine prior to transplantation. In transplants between embryonic and adult mice, donor tissue from A Thy-1.1 strain mice was transplanted to congenic A strain (Thy-1.2) mice such that the donor cells bearing the Thy-1.1 cell surface glycoprotein could be later identified by immunocytochemical staining with antibodies specific for the Thy-1.1 antigen. Reaggregation and transplantation of dissociated cells in a plasma clot thus provides a novel method whereby prior manipulation of neural tissue (separation of neurons and glia, enrichment for specific types of neurons, or glia etc.) can be used to great advantage in studying host-transplant connectivity and in assessing those factors which are critical in sustaining the survival of grafted neural tissue.     

bFGF和EGF对胚胎神经干细胞分化为神经元及神经胶质细胞的影响

目的：观察碱性成纤维生长因子（bFGF）和表皮生长因子（EGF）对体外培养胚胎神经管神经干细胞生长和分化的影响。方法：从孕12天大鼠胚胎神经管分离神经干细胞，进行原代培养，分为bFGF组、EGF组、bFGF＋EGF组及对照组：培养过程中观察干细胞的生长，培养2小时做nestin染色鉴定神经干细胞，培养第5天用免疫组化方法检测培养细胞神经元特异烯醇化酶（NSE）和胶质纤维酸性蛋白（GFAP）的表达，以观察神经干细胞分化为神经元及神经胶质细胞的状况。结果：取材细胞大部分为nestin免疫阳性细胞；各实验组均可促进培养细胞的生长和分 化。免疫组化中，EGF使神经干细胞增殖成团，增加GFAP的表达（P＜0．01）；bFGF能明显增加NSE及GFAP的表达（P＜0．01）；两种因子联合应用，神经元和神经胶质细胞均比对照组增多（P＜0．01）。结论：EGF和bFGF两类生长因子均能促进胚胎神经干细胞的生长，在分化方面，EGF倾向于诱导干细胞增并向着胶质细胞分化，bFGF则诱导干细胞分 成更多的神经元。     

Effects of engrafted neural stem cells derived from GFP transgenic mice in Parkinson's diseases rats

This study evaluated the therapeutic effect of neural stem cells (NSCs) transplanted into Parkinson's disease (PD) rats. NSCs were identified in vitro, then engrafted into the striatum of the PD rats. The rotational behavior was evaluated 1, 2, 4 and 6 weeks. A significant rotational behavior improvement was observed in PD rats subjected to cell transplantation. Transplanted NSCs not only express Nerve growth factor and Neurotrophin-3 in vitro, but also survive and partly differentiate into tyrosine hydroxylase (TH) positive cells in vivo. The results show that NSCs could be effective for PD treatment and the mechanisms might involve the neurotrophin expression and the neural differentiation.     

Results and outlooks of using cell technologies in the treatment of neurological diseases

An attempt was undertaken in the last decade of the 20th century to use a principally new approach to the treatment of neurological diseases--cell therapy. Main efforts were focused on developing a method related with replacement of neurons dying in neurodegenerative pathology, primarily, in Parkinson disease (PD). Outlined below are the key elements of the technology:--ensuring, in experiment, of a prolonged therapeutic effect in transplantation, to the affected part, first of embryonic neurons of the animal of the same species (allografting) and then of homologous embryonic neurons of man (heterografting);--obtaining, standardization and preparation (for transplantation) of embryonic nervous tissue of man; transplantation of embryonic nervous tissue of man to the brain of patient and evaluation, in situ, of the functional activity of its neurons; and evaluation of the therapeutic effect of grafting. Cell suspension of meseencephalon of 6-9 week human fetus containing around 10% of differentiating dopaminergic neurons was used for grafting in PD. Embryonic dopaminergic neurons, administered stereotactically into the striatum of patient, established synaptic links with neurons of the recipient, which was accompanied by the onset of synthesis and reverse uptake of dopamine (DA) as well as by the onset of spontaneous and stimulated release of DA. Neurografting ensured a temporary improvement of the condition in a part of PD patients but did not cure them. Moreover, such positive therapeutic effect was registered only in patients with the akineticorigid but not trembling variation of the disease. Hence, although there was a certain progress in clinical neurografting, the approach cannot be now recommended for introduction in neurology and neurosurgery. The limited therapeutic effect of the treatment method is primarily explained by a low rate of survival of transplanted dopaminergic neurons and, consequently, by the persisting DA deficit in patient's body. Therefore, the outlooks for perfecting the cell technology are related with increasing the survival rate of implanted dopaminergic neurons and with stimulating the innervation of target neurons in patient's striatum as well as with using the neural (glia) and non-neural (fibroblasts, myoblasts) cells with modified gene and stem cells. Finally, despite a certain progress of advancing the cell technology in neurology the approach still needs more research, which would enable further clinical trials.