脑出血大鼠脑内胚胎神经干细胞移植的运动神经功能改变和形态学研究

目的研究脑出血大鼠脑内胚胎神经干细胞移植对运动神经功能改善作用以及移植细胞分化后形态学改变。方法通过大鼠尾状核注射胶原酶IV制作脑出血模型大鼠,分离培养胚胎神经干细胞移植入脑出血大鼠脑内。对脑出血大鼠移植前后运动神经功能进行评价,并通过组织免疫荧光检测移植神经干细胞脑内分化后形态学改变情况。结果Hoechst标记的神经干细胞移植入脑出血大鼠后可见其在脑内存活,主要分布于血肿腔周边,免疫荧光检测显示移植细胞能进一步分化为神经元及星形胶质细胞;从移植术后第21天到第28天,神经干细胞移植组大鼠的神经功能改善显著好于培养液移植组和单纯脑出血组,有统计学意义(P<0.001)。结论胚胎神经干细胞脑出血大鼠脑内移植能促进偏瘫肢体功能恢复;胚胎神经干细胞脑出血大鼠脑内移植后能存活并进一步分化为神经元及星形胶质细胞。     

大鼠脑出血后神经干细胞移植的实验研究

目的探讨脑出血后大鼠胚胎神经干细胞移植治疗的可行性.方法从14d胚龄的大鼠胚胎脑组织中分离、培养神经干细胞,通过荧光免疫组化技术研究其特性.制作大鼠脑出血模型,分别于3d和7 d时将未分化的神经干细胞注入血同侧的尾状核内.分别记录模型制作后2 h和移植后2 h、4周的大鼠运动功能.移植4周后处死大鼠,观察移植后干细胞在体内生长情况.结果实验中分离、培养的神经干细胞体外能够被诱导分化成神经元、少突胶质细胞和星形胶质细胞.出血后7 d干细胞移植组的大鼠运动功能的改善显著好于3 d组和对照组.结论神经干细胞移植治疗能够显著改善脑出血动物的运动功能,是一种很有发展前途的方法,值得进行更深入的研究.     

大鼠脊髓神经干细胞的分离培养与鉴定

探讨胚胎大鼠神经干细胞（NSCs）的分离培养和鉴定方法。采用无血清培养液，从胚胎大鼠脊髓分离和培养NSCs，应用^3H-TdR掺入技术和免疫荧光技术进行细胞自我更新能力、巢蛋白表达和多向分化潜能的鉴定。结果表明，从E16胚胎大鼠脊髓分离培养的细胞具有NSCs特征，可在体外增殖存活，经1％胎牛血清诱导可分化为神经元、星形胶质细胞和少突胶质细胞。认为从胚胎大鼠脊髓成功分离培养的NSCs可在体外稳定地培养和传代，是研究细胞移植和基因治疗的理想细胞来源。     

骨髓间充质干细胞在脑缺血再灌注大鼠脑内存活并分化为神经元样细胞的实验研究

目的观察骨髓间充质干细胞(MSCs)经静脉移植在大脑中动脉缺血再灌注(MCAO)大鼠脑内存活并分化为神经元样细胞。方法常规方法分离、培养大鼠MSCs,根据Aspey方法制成MCAO模型,经尾静脉注射3×106溴脱氧尿嘧啶(BrdU)标记的大鼠MSCs,28d后处死大鼠,取脑组织行免疫荧光检测。结果共聚焦显微镜下观察到MSCs移植后脑梗死灶边缘聚大量BrdU阳性细胞,少量神经元特异性烯醇化酶(NSE)和BrdU双染细胞。结论经静脉移植的MSCs可移行至MCAO脑梗死灶周围并分化为神经元样细胞。     

大鼠心肌冷冻疤痕中胚胎心肌细胞移植物的观察

目的:观察胚胎心肌细胞移植物在大鼠冷冻心肌疤痕中存活的可行性.方法:以冷冻损伤造成16只大鼠左室游离壁心肌疤痕.于损伤后10天,将培养的胚胎心肌细胞或培养基注入心肌疤痕组织,5只动物冷冻后20天处死(疤痕对照组),细胞移植组7只,培养基对照组4只,移植后10天处死动物,取出心脏.结果:疤痕组和培养基对照组动物左室壁损伤局部组织变薄,细胞移植组室壁依然厚实.横纹肌α-肌动蛋白免疫组化染色证实,细胞移植组3只动物被证实有移植物存活.HE染色示移植物呈球形生长,移植细胞体积小,呈梭形,胞核数目多,核/质比例高,横纹不明显,排列不规则.移植物中及其周围未见淋巴细胞浸润.培养基对照组来见任何移植物生长征象.结论:胚胎心肌细胞移植在异体心肌疤痕中能够存活,该实验方法为修复心肌梗死疤痕,改善心脏功能提供了新的治疗策略.     

大鼠心肌冷冻疤痕中胚胎心肌细胞移植物的观察

目的：观察胚胎心肌细胞移植物在大鼠冷冻心肌疤痕中存活的可行性。方法：以冷冻损伤造成１６只大鼠左室游离壁心肌疤痕。于损伤后１０天，将培养的胚胎心肌细胞或培养基注入心股疤痕组织，５只动物冷冻后２０天处死（疤痕对照组），细胞移植组织７只，培养基对照组４只，移植后１０天死动物，取出心脏。结果：疤痕组和培养基对照组动物左室壁损伤局部组织变薄，细胞移植组室壁依然厚实。横纹肌α＾－肌动蛋白免疫组化染色证实，细胞     

人脐血间充质干细胞移植治疗新生鼠缺氧缺血性脑损伤的实验研究

建立大鼠缺氧缺血性脑损伤（HIBD）模型成功后，将大鼠分为假手术组、脑内移植组、颈静脉移植组、假移植组。将人脐血间充质干细胞（MSC）分别经脑及颈静脉注入脑内移植组、颈静脉移植组；假移植组体内仅注入生理盐水。用Y型迷宫观察新生鼠的学习记忆能力，免疫组化分析脑组织移植细胞的定位和分化情况。结果移植后2、4周，颈静脉移植组及脑内移植组正确反应率均高于假移植组（P均〈0．05），而两组之间无统计学差异。颈静脉移植组移植2、4周后脑组织胶质纤维酸性蛋白／5-溴-2-脱氧脲苷（GFAP／Brdu）双阳性细胞占Brdu阳性细胞的比率均高于脑内移植组（P均〈0．05）；脑组织免疫组化显示，MSC移植组中，进针注射部位存在大量移植细胞，并向周围迁移。提示MSC移植到HIBD大鼠脑内可分化为神经元和神经胶质细胞；MSC移植后不仅可以减轻新生大鼠缺氧缺血后近期的脑损伤，还能改善其远期预后；静脉移植较脑内移植效果好。     

骨髓基质细胞治疗局灶性脑缺血的研究进展

骨髓基质细胞（MSC）是指骨髓基质中具有自我复制和多向分化潜能的干细胞，在特定条件下不仅可分化为中胚层细胞，而且也可横向分化为外胚层起源的神经胶质细胞和神经元。缺血性脑损伤时，MSC可向缺血灶迁移并分化为神经细胞，从而减轻神经功能缺损。研究表明，MSC静脉移植促进脑缺血神经功能恢复并非由于移植后新分化的神经元与宿主神经环路发生整合，而是MSC分泌的各种生长因子介导的。MSC并不能取代损伤组织，而是增进其功能，提高残存组织的可塑性。另外，MSC还是外源基因转染和表达的良好载体。MSC具有很强的增殖能力，易于体外培养扩增，通过基因修饰MSC，可提高对缺血性脑损伤的修复作用。因此，MSC有望成为基因治疗的靶细胞，在基因工程方面有着广阔的应用前景。     

神经组织移植治疗脑皮质梗死的研究进展

神经组织移植治疗皮质梗死现正处于研究阶段。实验研究表明，胚胎脑组织植入皮质梗死区内，其神经元有分裂和分化能力，与宿主脑发生神经纤维联系。文章对神经组织移植治疗脑皮质梗死的方法、移植供体选择、宿主脑与移植物之间神经纤维连接、宿主与移植物的神经功能整合进行了讨论     

大鼠骨髓基质细胞体外培养转化为神经元细胞的实验研究

目的探讨体外原代培养骨髓基质细胞（BMSC）向神经元细胞转化情况.方法取成年Wistar大鼠BMSC,无血清和有血清培养基进行细胞克隆,获取骨髓间质干细胞（MSCs）,应用碱性成纤维细胞生长因子（bFGF）和表皮生长因子（EGF）进行细胞扩增及诱导分化.结果分离纯化后的BMSC经原代培养形成细胞克隆团,经传代后其数量明显增多,其分化细胞形态多样,包括神经元细胞、星形胶质细胞和少突胶质细胞,仍具有神经细胞所特有的性质.结论BMSC具有较强的自我更新能力和多分化潜能,在合适的环境条件下,可诱导分化出神经元细胞和神经胶质细胞,是较为理想的种子细胞.     

Transplanted fetal striatum in Huntington's disease: phenotypic development and lack of pathology

Neural and stem cell transplantation is emerging as a potential treatment for neurodegenerative diseases. Transplantation of specific committed neuroblasts (fetal neurons) to the adult brain provides such scientific exploration of these new potential therapies. Huntington's disease (HD) is a fatal, incurable autosomal dominant (CAG repeat expansion of huntingtin protein) neurodegenerative disorder with primary neuronal pathology within the caudate-putamen (striatum). In a clinical trial of human fetal striatal tissue transplantation, one patient died 18 months after transplantation from cardiovascular disease, and postmortem histological analysis demonstrated surviving transplanted cells with typical morphology of the developing striatum. Selective markers of both striatal projection and interneurons such as dopamine and c-AMP-related phosphoprotein, calretinin, acetylcholinesterase, choline acetyltransferase, tyrosine hydroxylase, calbindin, enkephalin, and substance P showed positive transplant regions clearly innervated by host tyrosine hydroxylase fibers. There was no histological evidence of immune rejection including microglia and macrophages. Notably, neuronal protein aggregates of mutated huntingtin, which is typical HD neuropathology, were not found within the transplanted fetal tissue. Thus, although there is a genetically predetermined process causing neuronal death within the HD striatum, implanted fetal neural cells lacking the mutant HD gene may be able to replace damaged host neurons and reconstitute damaged neuronal connections. This study demonstrates that grafts derived from human fetal striatal tissue can survive, develop, and are unaffected by the disease process, at least for 18 months, after transplantation into a patient with HD.     

Effects of transplants of fetal mediobasal hypothalamus on luteinizing hormone pulses impaired by hypothalamic deafferentation in adult ovariectomized rats.

Pulsatile luteinizing hormone (LH) secretion is impaired after posterior anterior-hypothalamic deafferentation (PAD), which separates the anterior part of the arcuate nucleus from the mediobasal hypothalamus (MBH). In the present study, we examined whether transplants of fetal brain tissue could prevent the effects of PAD. The brain tissue containing the MBH or the cerebral cortex taken from the fetal brain was transplanted into the third ventricle of ovariectomized rats. Four weeks after the brain transplantation, animals with or without the brain transplantation were subjected to PAD. One week after PAD, blood samples were collected every 6 min for 3 h through an indwelling atrial cannula. Rats bearing PAD without transplantation showed irregular pulsatile fluctuation of plasma LH, whereas LH pulses were maintained in rats bearing transplantation of the fetal MBH tissue. In rats which had been transplanted with the cerebral cortex, LH pulses were less apparent after PAD than in the MBH-transplanted or sham-deafferentated animals. No cell bodies of LH-releasing hormone (LHRH) neurons were found immunohistochemically in the MBH grafts. These results suggest that the graft containing the fetal MBH tissue maintains regular LH pulses after PAD and that the LHRH pulse generator may consist, at least in part, of a group of neurons in the MBH other than LHRH-producing neurons.     

The survival of brain transplants is enhanced by extracts from injured brain.

Fragments of rat embryo corpus striatum implanted into wound cavities in the cortex of neonatal rat hosts showed very poor survival when transplantation was performed immediately after the cavity was made. Consistent survival of the transplants was made possible by supplying the implant with extracts from the injured brain of neonatal rats. These extracts also supported in vitro survival of embryonic striatal neurons, suggesting that transplanted cells benefit from injury-induced neuronotrophic factors.     

Acetylcholine release from intrahippocampal septal grafts is under control of the host brain.

The activity of intrahippocampal transplants of cholinergic neurons was monitored by microdialysis in awake, freely moving rats. Fetal septal-diagonal band tissue was implanted into rats with a complete transection of the fimbria-fornix cholinergic pathway either as a cell suspension injected into the hippocampus or as a solid graft implanted in the lesion cavity. The grafts restored baseline acetylcholine release in the graft-reinnervated hippocampus to normal or supranormal levels. The graft-derived acetylcholine release was dependent on intact axonal impulse flow, and it was markedly increased during behavioral activation by sensory stimulation or by electrical stimulation of the lateral habenula. The results demonstrate that the septal grafts, despite their ectopic location, can become functionally integrated with the host brain and that the activity of the transplanted cholinergic neurons can be modulated from the host brain during ongoing behavior. Anatomical observations, using immunohistochemistry and retrograde tracing, indicate that direct or indirect brainstem afferents to the graft could mediate this functional integration. Host afferent control of the graft may thus play a role in the recovery of lesion-induced functional deficits seen with these types of transplants.     

Intracerebral xenografts of human mesencephalic tissue into athymic rats: immunochemical and in vivo electrochemical studies.

Intracerebral allografts of fetal neurons have been studied in both rodents and nonhuman primates. Such research has been directed towards problems in developmental neurobiology and in animal models of neurological diseases. Whether intracerebrally transplanted human fetal neurons are capable of forming synapses and releasing neurotransmitters are key questions in any application of this approach to human brain development and dysfunction. We studied these questions by examining the immunocytochemical and in vivo electrochemical properties of xenografts of human mesencephalic dopaminergic neurons placed into athymic "nude" rats. The transplanted neurons survive, continue to express human-specific Thy-1 immunoreactivity, and extend neuronal processes into the host brain where morphologically identifiable synapses form. Potassium-evoked release of monoamines occurs in the vicinity of the graft but is absent in more remote areas of the host neuropil. These results indicate that human fetal tissue fragments can provide a source of viable neuroblasts for transplantation. Further, synapses form between pre- and postsynaptic elements expressing different species-specific cell surface markers; thus, these markers do not play a determining role in synaptogenesis.     

Transplants of Schwann cell cultures promote axonal regeneration in the adult mammalian brain.

Transplantation of embryonic brain tissue or mature peripheral nerves into the adult mammalian central nervous system promotes axonal regrowth from axotomized central nervous system neurons; however, the cellular origin and molecular nature of the factors promoting axonal growth in vivo are unknown. To further characterize cellular environments that facilitate regeneration of central nervous system axons, we developed a methodology whereby cultured cell preparations can be transplanted into the brain of mature mammals. For this procedure, lesions are produced in the septal-hippocampal system of adult rats, and selected regions from collagen-supported Schwann cell/neuron cultures (consisting of Schwann cells, extracellular matrix, and degenerating neuronal processes and myelin but devoid of neuronal perikarya and fibroblasts) are positioned within the intracephalic cavity so that they bridge the lesion gap (approximately 3 mm) separating the septum and hippocampus. At various time up to 3 weeks after transplantation, specimens were prepared for acetylcholinesterase histochemistry and the immunocytochemical localization of laminin (an extracellular matrix protein) and C-4 (a Schwann cell membrane antigen). All specimens (from uninjured controls and from animals with either acellular collagen or mature Schwann cell/extracellular matrix transplants) contained laminin immunoreactivity associated with the meninges, choroid plexus, ependyma, and cerebral blood vessels. All animals with transplants showed prominent laminin staining on astrocytic processes along the intracephalic cavity, but only the Schwann cell/extracellular matrix transplants exhibited dense laminin and C-4 immunoreactivity within the cellular portion of the transplants. Regeneration of acetylcholinesterase-positive septal fibers occurred only in animals containing Schwann cell/extracellular matrix transplants. By 6 days after transplantation, acetylcholinesterase-positive fibers were observed both on laminin-positive cellular tissue strands connecting the septum and the Schwann cell/extracellular matrix transplants and on the initial portions of the transplants. By day 14, acetylcholinesterase-positive fibers traversed the entire lesion cavity in intimate association with the laminin- and C-4-positive cellular layer of the transplants and reinnervated the host hippocampus. However, cholinergic fibers were not associated with all laminin-containing processes along the lesion cavity nor did they grow along acellular collagen transplants. These results indicate the presence of factors in transplants of cultured Schwann cells and their associated extracellular matrix that promote rapid regeneration of central nervous system cholinergic axons in vivo.     

Revascularization of skin transplanted into the brain: Source of the graft endothelium

Transplantation of tissues into the brain is becoming a feasible therapeutic approach to some neurological diseases. The fate of the graft vasculature is not well understood. The purpose of the present study was to determine the source of the endothelium in the revascularized skin grafts transplanted into the brain. We hypothesized that if the skin endothelium were replaced by brain endothelium then we should observe in the grafts the following: (a) degenerating endothelium soon after grafting, (b) regenerating endothelium subsequently, (c) a time course for reestablishment of circulation that is consistent with the time required for vessel growth and invasion, and (d) doubling of the vascular basement membrane a few weeks later. We found only a few degenerating endothelial cells soon after transplantation and no evidence of regenerating endothelium or of double layers of basement membrane even after prolonged survival. The vessels within mature grafts had morphological characteristics typical of normal skin vessels. We concluded that when tissue fragments are transplanted to the brain, native graft vessels survive and anastomose with host vessels.     

体外增殖培养的骨髓间充质干细胞移植后在脑梗死大鼠脑内的存活

目的观察应用维生素C（Vc）和碱性成纤维因子（bFGF）对骨髓间充质干细胞（BMSCs）体外增殖培养,移植到脑梗死大鼠体内后,在脑内的存活情况。方法①采用密度梯度离心及贴壁法分离大鼠BMSCs。按培养液中加入的不同因子分为4组：对照组、Vc组（50μg/m1）、bFGF组（1μg/L）以及Vc＋bFGF组（50μg/ml Vc和1μg/L bFGF）,在体外对BMSCs进行培养并观察各组细胞的增殖情况。采用MT丁比色法分别于培养第1、2、3、5和7天,测定细胞在450nm波长处的吸光度（A）值;采用流式细胞仪检测培养96h后的细胞周期。②采用线栓法制备20只大鼠右侧大脑中动脉闭塞2h模型,并随机分为2组：联合Vc＋bFGF培养的BMSCs移植组（10只）和对照BMSCs移植组（10只）。将相应组别的BMSCs细胞移植人脑梗死24h后的大鼠体内。分别于移植后第1、2和3周,制备大鼠脑组织切片并行BrdU免疫组化染色。结果①形态学观察显示,Vc＋bFGF组的BMSCs增殖最快,对照组则相对缓慢。②各组细胞的A值于培养第2天开始增加,Vc组和bFGF组在第3天达到高峰（F=728．52和F=197．18,P〈0．05）,Vc＋bFGF组在第5天达到高峰（F=1771．32,P〈0．05）,第7天均有所下降。第2天起,bFGF组、Vc组与bFGF＋Vc组的A值均高于对照组;第3天起,Vc＋bFGF组高于Vc组与bFGF组,差异均有统计学意义（P〈0．05）。③Vc组、bFGF组与Vc＋bFGF组细胞处于增殖期（s＋G2＋M期）的百分比均较对照组高,差异有统计学意义（P〈0．05）。④BMSCs移植后第2周,联合Vc＋bFGF培养的BMSCs移植组大鼠脑组织切片中BrdU阳性细胞计数较对照组高;第3周两组阳性细胞计数均有下降,但前者仍较后者高。差异均有统计学意义（P〈0．01）;Brdu阳性细胞主要分布在梗死灶周围。结论Vc与bFGF均可促进大鼠BMSCs的增殖,二者联用较单用其一的效果更好;脑梗死大鼠移植增殖培养后的BMSCs,其在脑组织中的成活能力明显增强。