神经细胞微囊偏侧帕金森病猴脑内移植的组织学观察

通过脑内移植脉络膜细胞(CP)海藻酸盐微囊观察偏侧帕金森病样猴脑神经组织学改变,以探讨微囊化CP对灵长类动物纹状体神经元的保护作用.用海藻酸盐多聚鸟氨酸微囊包裹猪脉络膜细胞,移植至偏侧帕金森病样猴黑质-纹状体通道,移植后6个月进行神经病理学检查,观察脑组织学变化.脉络膜细胞微囊脑内移植能改善偏侧帕金森病猴行为,能促进多巴胺能纤维增生和活动增强.     

用微囊化转基因细胞治疗PD猴的初步研究

用海藻酸钠／多聚赖氨酸（ＡＬＧ／ＰＬＬ）方法制备了包裹转 酪氨酸羟化酶基因的大鼠在肌细胞微囊。在在体外,微囊内的细胞能长时间存活及生长,并能产生ＴＨ蛋白。将这种微囊移植于正常猴的纹状体,１个月后仍能在微囊内观察到活细胞,而且移植处凶胶质细胞明显增生,在此基础上,将这种微囊移植于患帕金森病（ＰＤ）的模型猴的纹状体,初步观察到的其病理性旋转行为有明显改善。     

SGA微囊制备及其与大鼠胰岛生物相容性的体外观测

目的:制备硫酸酯化葡甘聚糖-海藻酸钡(SGA)微囊,包裹大鼠胰岛,对比观测SGA微囊的体外生物相容性.方法:溶液共混法制备硫酸酯化葡甘聚糖-海藻酸盐混合凝胶液,改良Omer法制作SGA微囊.X射线衍射分析SGA二元共混膜结构;SGA微囊化SD大鼠胰岛,体外培养14d后,胰岛素刺激释放实验对比SGA微囊和APA、ABa(海藻酸钡)微囊对大鼠胰岛的影响.结果:衍射图谱显示硫酸酯化葡甘聚糖-海藻酸钡共混膜在原属于海藻酸钡的2θ 11°、20.3°结晶峰变小,在20 29.8.出现新结晶峰,说明SGA二元共混膜相互作用强烈,融合较好;胰岛素刺激释放实验显示,体外培养后SGA微囊对大鼠胰岛的影响与APA、ABa微囊无差别(P＞0.05).结论:SGA微囊膜结构稳定,融合度较好,对大鼠胰岛体外生物相容性较高,可以为下一步体内移植所应用.     

胶质细胞源神经营养因子

胶质细胞源神经营养因子陈哲宇何成王成海（第二军医大学神经生物教研室,上海２００４３３）关键词胶质细胞源神经营养因子多巴胺能神经元运动神经元神经营养因子是指能够促进神经细胞存活、生长和分化的一类蛋白质。胶质细胞源神经营养因子（ＧＤＮＦ）,因其最初从大鼠...     

有望控制帕金森氏病的新的特异性神经营养因子

Synergen Inc.公司(Boulder,CO)分离出一种新的、特异性刺激中脑多巴胺生产细胞的神经营养因子。公司打算用这种因子来治疗帕金森氏病。帕金森氏病的病因是由于这类多巴胺吸附和分泌细胞的降解。这种因子称为神经胶质细胞系衍生的神经营养因子(GDNF),是从大鼠中提取后用一种基因探针     

体液蛋白质组学的研究和应用

蛋白质组学是目前生命科学的研究热点之一.体液中的蛋白质是来源于与其密切接触组织或者细胞的分泌或渗漏,体液蛋白质组的变化能反映这些组织的生理或者病理改变,因此以寻找疾病相关生物标记为主要目标的比较蛋白质组学是蛋白质组学研究的核心内容之一.对近年来各种体液蛋白质组学的研究状况和应用及存在挑战作一综述.     

微囊化新生大鼠卵巢组织体外及异体移植

为探讨海藻酸钠-聚左赖氨酸-海藻酸钠(APA)微囊化新生大鼠卵巢组织用于治疗实验性卵巢功能丧失大鼠的可行性,应用高压静电法,用海藻酸钠-聚左赖氨酸-海藻酸钠(APA)生物膜包裹新生大鼠卵巢组织,体外培养微囊,用免疫化学分析法检测雌二醇(E2)、孕酮(P)分泌情况,透射电镜观察卵巢组织形态,并将微囊移植到去势大鼠(切除双侧卵巢的雌性大鼠)腹腔中,检测大鼠血清中雌、孕激素变化情况,同时用阴道涂片观察大鼠动情周期恢复情况,并在不同时间回收观察微囊。结果显示在相同条件下制得的微囊粒径均匀、表面光滑;体外培养条件下持续分泌E2、P;卵巢组织中颗粒细胞发育成为粒性黄体细胞;大鼠腹腔移植微囊后无异常,E2、P水平上升,动情周期未恢复;回收的微囊大部分形态完整。提示用高压静电法制备的APA微囊化新生大鼠卵巢组织能持续稳定释放E2、P,明显改善大鼠卵巢功能,在大鼠体内有良好的生物相容性。     

微囊化胰岛B细胞系体外生长和分泌功能的研究

目的：研究海藻酸钠－多聚赖氨酸－海澡酸钠（APA）微囊化胰岛B细胞系BTC6－F7的生长和分泌规律,探索其作为生物人工胰岛的可能性。方法：以微囊静电液滴发生器制作APA微囊化BTC6－F7细胞,体外培养并定期测定微囊化细胞的生长和胰岛素分泌。结果：在实验观察的90d内,BTC6－F7细胞可在微囊内以细胞团的形式生长、存活。囊内细胞总数随培养时间的延长而增加,但细胞活率呈下降趋势,胰岛素分泌与囊内活细胞数的变化规律一致,最初呈上升趋势,然后较长时间维持在相对恒定的水平。结论：本研究所制备的APA微囊化胰岛B细胞可在较长时间内保持生长、存活和分泌功能,为进一步发展生物型人工胰岛奠定了基础,并可用于糖尿病的发病机理和治疗研究。     

大鼠骨髓基质细胞向Schwann细胞分化后蛋白表达变化的研究

近年来很多研究报道大鼠骨髓基质细胞（bone marrow stromal cells,MSCs）可以向神经细胞或神经胶质细胞分化,其中多数研究观察了细胞形态以及几种神经相关蛋白的免疫反应,也有报道用基因组学方法分析MSCs诱导过程中基因谱的表达变化.尚未见报道用蛋白质组学方法分析MSCs在诱导过程中蛋白谱的表达变化.本研究利用蛋白质组学技术分析MSCs向Schwann细胞样细胞诱导分化过程中蛋白谱的表达变化.从成年SD大鼠的股骨和胫骨中分离培养MSCs,应用复合诱导因子体外连续诱导MSCs向Schwann细胞样细胞分化.采用2-DE技术分离未诱导和诱导分化后MSCs总蛋白,应用基质辅助激光解吸电离飞行时间质谱得到相应的肽质量指纹图谱,搜索数据库分析差异蛋白质点.所得到的MSCs蛋白谱有792个蛋白点,通过PDQuest软件分析诱导前后MSCs的蛋白谱,初步分析发现有74个蛋白质的表达发生了明显的变化,其中43个蛋白表达上调,31个蛋白表达下调.本研究通过质谱分析并进行网上数据库搜索匹配,初步分析发现这些蛋白主要包括骨架和结构蛋白、生长因子类的蛋白、代谢相关蛋白及酶类、伴侣蛋白、受体类蛋白、细胞周期蛋白、钙结合蛋白以及其他蛋白等.研究表明,MSCs体外条件诱导分化过程中有较多蛋白表达发生变化;其中与神经细胞和神经胶质细胞相关蛋白：BDNF,CNTF,GFAP等在MSCs体外条件诱导分化后中表达明显上调;本研究从蛋白质水平为MSCs体外向Schwann细胞样细胞条件诱导提供了新的研究资料。     

创伤后应激障碍机制的研究进展

创伤后应激障碍(Post-traumatic stress disorder;PTSD)是一种由严重强烈的伤害事件造成的精神障碍,随着近年来社会应激事件的增多和自然灾害的发生,创伤应激障碍的发病率逐渐增高。同时为了研究对应的治疗方法,人们对创伤应激障碍的机制进行了更深入的探索,也有了新的进展。本文着重从激素、神经营养因子、免疫系统等方面来总结创伤后应激障碍发生的生物学机制。激素方面,PTSD主要与交感肾上腺髓质系统(Sympatho-adrenomedullarysystem,SAS)和下丘脑-垂体-肾上腺轴(Hypothalamicpituitary-adrenal axis,HPA)的功能异常有关;神经营养因子方面,其产生与分泌的异常增加或减少可能是PTSD产生的重要机制;免疫系统方面,PTSD可能与免疫系统相关的蛋白质、细胞的数量和功能变化有关。整合神经生物学与分子生物学、表观遗传学、蛋白质组学及分子影像学的成果将对PTSD的研究产生推动作用。     

Transplantation of adult neural progenitor cells transfected with vascular endothelial growth factor rescues grafted cells in the rat brain

Growth factors are currently evaluated as therapeutics in stroke and neurodegeneration. Besides direct neurotrophic effects, they promote proliferation, survival, and differentiation of both transplanted and endogenous neural precursor cells (NPCs). In the current study, we investigated whether NPCs expressing Vascular Endothelial Growth Factor VEGF-A165 are a useful vehicle for growth factor delivery after transplantation into the caudate putamen of the rat brain. We found an increased survival of adenovirally transfected NPCs after 11 days, but not after 24 hours or 4 days. Additional brain immunohistochemistry revealed increased expression of the endothelial cell marker PECAM-1 (CD31) after 24 hours, 4 day, and 11 days after transplantation. In conclusion, we show that the graft itself is a useful vehicle for growth factor delivery, promoting the survival of NPCs. Moreover, transplantation of VEGF-expressing NPCs supports angiogenesis in the brain, which may contribute to potential brain repair.     

Human Microglia Transplanted in Rat Focal Ischemia Brain Induce Neuroprotection and Behavioral Improvement

Background and Purpose

Microglia are resident immunocompenent and phagocytic cells of central nervous system (CNS), which produce various cytokines and growth factors in response to injury and thereby regulate disease pathology. The purpose of this study is to investigate the effects of microglial transplantation on focal cerebral ischemia model in rat.

Methods

Transient middle cerebral artery occlusion (MCAO) in rats was induced by the intraluminal filament technique. HMO6 cells, human microglial cell line, were transplanted intravenously at 48 hours after MCAO. Functional tests were performed and the infarct volume was measured at 7 and 14 days after MCAO. Migration and cell survival of transplanted microglial cells and host glial reaction in the brain were studied by immunohistochemistry. Gene expression of neurotrophic factors, cytokines and chemokines in transplanted cells and host rat glial cells was determined by laser capture microdissection (LCM) and quantitative real time-PCR.

Results

HMO6 human microglial cells transplantion group demonstrated significant functional recovery compared with control group. At 7 and 14 days after MCAO, infarct volume was significantly reduced in the HMO group. In the HMO6 group, number of apoptotic cells was time-dependently reduced in the infarct core and penumbra. In addition, number of host rat microglia/macrophages and reactive astrocytes was significantly decreased at 7 and 14 days after MCAO in the penumbra. Gene expression of various neurotrophic factors (GDNF, BDNF, VEGF and BMP7) and anti-inflammatory cytokines (IL4 and IL5) was up-regulated in transplanted HMO6 cells of brain tissue compared with those in culture. The expression of GDNF and VEGF in astrocytes in penumbra was significantly up-regulated in the HMO6 group.

Conclusions

Our results indicate that transplantation of HMO6 human microglial cells reduces ischemic deficits and apoptotic events in stroke animals. The results were mediated by modulation of gliosis and neuroinflammation, and neuroprotection provided by neurotrophic factors of endogenous and transplanted cells-origin.     

Transplantation of human umbilical cord blood cells mediated beneficial effects on apoptosis, angiogenesis and neuronal survival after hypoxic-ischemic brain injury in rats

Transplantation of human umbilical cord blood (hucb) cells in a model of hypoxic-ischemic brain injury led to the amelioration of lesion-impaired neurological and motor functions. However, the mechanisms by which transplanted cells mediate functional recovery after brain injury are largely unknown. In this study, the effects of hucb cell transplantation were investigated in this experimental paradigm at the cellular and molecular level. As the pathological cascade in hypoxic-ischemic brain injury includes inflammation, reduced blood flow, and neuronal cell death, we analyzed the effects of peripherally administered hucb cells on these detrimental processes, investigating the expression of characteristic marker proteins. Application of hucb cells after perinatal hypoxic-ischemic brain injury correlated with an increased expression of the proteins Tie-2 and occludin, which are associated with angiogenesis. Lesion-induced apoptosis, determined by expression of cleaved caspase-3, decreased, whereas the number of vital neurons, identified by counting of NeuN-positive cells, increased. In addition, we observed an increase in the expression of neurotrophic and pro-angiogenic growth factors, namely BDNF and VEGF, in the lesioned brain upon hucb cell transplantation. The release of neurotrophic factors mediated by transplanted hucb cells might cause a lower number of neurons to undergo apoptosis and result in a higher number of living neurons. In parallel, the increase of VEGF might cause growth of blood vessels. Thus, hucb transplantation might contribute to functional recovery after brain injury mediated by systemic or local effects.     

Differentiation and migration of long term expanded human neural progenitors in a partial lesion model of Parkinson's disease

Human neural progenitor cells (HNPCs) can be expanded in large numbers for significant periods of time to provide a reliable source of neural cells for transplantation in neurodegenerative disorders such as Parkinson's disease (PD). In the present study, HNPCs isolated from embryonic cortex were expanded as neurospheres in cell culture for 10 months. Just prior to transplantation, a proportion of the HNPCs were treated in a "predifferentiation" protocol in combination with the neurotropic factor NT4, in order to yield significant numbers of neurons. For transplantation, either undifferentiated HNPCs, or predifferentiated HNPCs were transplanted into the substantia nigra of a rat model of Parkinson's disease. At 12 weeks, there was good survival with proliferation of transplanted HNPCs occurring after transplantation but ceasing before the animals were sacrificed. Transplants of predifferentiated cells contained a higher proportion of neurons. The presence of a lesion in the striatum had a significant influence on the migration of transplanted cells from the substantia nigra into the striatum. There was no significant behavioural recovery or effect of transplanted HNPCs on the loss of dopaminergic cells from the host brain. In conclusion, HNPCs may provide a source of cells for use in the treatment of Parkinson's disease.     

Release of growth factors by neuronal precursor cells as a treatment for diseases with tau pathology

The intraneuronal accumulation of the microtubule associated protein tau in a hyperphosphorylated state and the extracellular deposit of ?amyloid protein constitute the defining neuropathological signature of Alzheimer's disease, the most common type of dementia in ageing Homo sapiens.There is accumulating evidence suggesting that transplantation of embryonic and adult derived neuronal precursor cells (NPCs) has a major role for cell based repair strategies in models of acute and chronic injury. In order to determine whether NPCs could rescue tau related neuronal cell death NPCs were transplanted into the transgenic mouse cortex of transgenic mice expressing human P301S tau protein at 2 month of age and the effect followed 2 and 3 months after transplantation. The results demonstrated that following transplantation mouse NPCs differentiated into astrocytes and exerted a neuroprotective effect. In particular, the expression of ciliary neurotrophic factor, nerve growth factor and glial cell derived neurotrophic factor was increased near the transplanted cells. A nonsignificant increase of brain derived neurotrophic factor expression was instead found in the area of the cortex where neuronal death was rescued.     

Neuroprotective Effects of Oligodendrocyte Progenitor Cell Transplantation in Premature Rat Brain following Hypoxic-Ischemic Injury

Periventricular leukomalacia (PVL) is a common ischemic brain injury in premature infants for which there is no effective treatment. The objective of this study was to determine whether transplanted mouse oligodendrocyte progenitor cells (OPCs) have neuroprotective effects in a rat model of PVL. Hypoxia-ischemia (HI) was induced in 3-day-old rat pups by left carotid artery ligation, followed by exposure to 6% oxygen for 2.5 h. Animals were assigned to OPC transplantation or sham control groups and injected with OPCs or PBS, respectively, and sacrificed up to 6 weeks later for immunohistochemical analysis to investigate the survival and differentiation of transplanted OPCs. Apoptosis was evaluated by double immunolabeling of brain sections for caspase-3 and neuronal nuclei (NeuN), while proliferation was assessed using a combination of anti-Nestin and -bromodeoxyuridine antibodies. The expression of brain-derived neurotrophic factor (BDNF) and Bcl-2 was examined 7 days after OPC transplantation. The Morris water maze was used to test spatial learning and memory. The results showed that transplanted OPCs survived and formed a myelin sheath, and stimulated BDNF and Bcl-2 expression and the proliferation of neural stem cells (NSC), while inhibiting HI-induced neuronal apoptosis relative to control animals. Moreover, deficits in spatial learning and memory resulting from HI were improved by OPC transplantation. These results demonstrate an important neuroprotective role for OPCs that can potentially be exploited in cell-based therapeutic approaches to minimize HI-induced brain injury.     

Neural Stem-Like Cells Derived from Human Amnion Tissue are Effective in Treating Traumatic Brain Injury in Rat

Although human amnion derived mesenchymal stem cells (AMSC) are a promising source of stem cells, their therapeutic potential for traumatic brain injury (TBI) has not been widely investigated. In this study, we evaluated the therapeutic potential of AMSC using a rat TBI model. AMSC were isolated from human amniotic membrane and characterized by flow cytometry. After induction, AMSC differentiated in vitro into neural stem-like cells (AM-NSC) that expressed higher levels of the neural stem cell markers, nestin, sox2 and musashi, in comparison to undifferentiated AMSC. Interestingly, the neurotrophic factors, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT-3), glial cell derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) were markedly upregulated after neural stem cell induction. Following transplantation in a rat TBI model, significant improvements in neurological function, brain tissue morphology, and higher levels of BDNF, NGF, NT-3, GDNF and CNTF, were observed in the AM-NSC group compared with the AMSC and Matrigel groups. However, few grafted cells survived with minimal differentiation into neural-like cells. Together, our results suggest that transplantation of AM-NSC promotes functional rehabilitation of rats with TBI, with enhanced expression of neurotrophic factors a likely mechanistic pathway.     

Comparison of intracerebral transplantation effects of different stem cells on rodent stroke models

In the present study, induced pluripotent stem cells (iPSCs), induced neural stem cells (iNSCs), mesenchymal stem cells (MSCs) and an immortalized cell line (RMNE6), representing different characteristics of stem cells, were transplanted into normal and/or injured brain areas of rodent stroke models, and their effects were compared to select suitable stem cells for cell replacement stroke therapy. The rat and mice ischaemic models were constructed using the middle cerebral artery occlusion technique. Both electrocoagulation of the artery and the intraluminal filament technique were used. The behaviour changes and fates of grafted stem cells were determined mainly by behaviour testing and immunocytochemistry. Following iPSC transplantation into the corpora striata of normal mice, a tumour developed in the brain. The iNSCs survived well and migrated towards the injured area without differentiation. Although there was no tumourigenesis in the brain of normal or ischaemic mice after the iNSCs were transplanted in the cortices, the behaviour in ischaemic mice was not improved. Upon transplanting MSC and RMNE6 cells into ischaemic rat brains, results similar to iNSCs in mice were seen. However, transplantation of RMNE6 caused a brain tumour. Thus, tumourigenesis and indeterminate improvement of behaviour are challenging problems encountered in stem cell therapy for stroke, and the intrinsic characteristics of stem cells should be remodelled before transplantation. Copyright © 2015 John Wiley & Sons, Ltd.     

Behavior and Differentiation of the Neural Stem Cells in vivo

We studied the behavior and differentiation of human and rat neural stem cells after transplantation in the adult rat brain without immunosuppression. The rat stem cells were isolated from the presumptive neocortex of 15-day-old embryos. The human cells were isolated from the ventricular brain zone of 9-week-old embryos and cultivated for two weeks before transplantation. The results of histomorphological studies suggest that the microenvironment factors did not suppress the growth or development of transplanted stem cells. Both rat and human embryonic multipotent neural cells showed similar behavior and differentiation into neurons and glial cells. After transplantation, they continued to mitotically divide and migrated from the graft area to the surrounding tissue of a recipient brain. The presumptive glial cells migrated preferentially along the capillaries and fibrous structures of the recipient brain. Similar behavior of the rat and human neural stem cells in the microenvironment of the recipient adult rat brain and the absence of immune reaction suggest that the transplantation into the rat brain may serve as a model for studying the developmental biology of the human stem cells.