脑内移植骨髓间质干细胞治疗帕金森病模型大鼠的实验研究

目的 探讨骨髓间质干细胞(mesenchymal stem cells,MSCs)移植治疗帕金森病(parkinson's disease,PD)大鼠的可行性及可能的机制.方法 移植Brdu标记的MSCs到模型大鼠的纹状体内.术后4个月中,定期对大鼠进行旋转行为学实验测试.并分别在术后2周和4个月时进行脑内针道处及移植区免疫组化检测TH和Brdu的表达.结果 Brdu标记的MSCs移植到模型大鼠的纹状体内,术后2周时移植针道处及针道周围可见Brdu阳性外源MSCs,并有外源性细胞表达TH,术后4个月时移植针道内仍可以看到MSCs存活.MSCs移植的PD模型大鼠症状较PBS注射组行为学明显改善.结论 移植MSCs到大鼠PD模型的纹状体内能成活,且分化细胞能表达TH蛋白,大鼠PD模型行为学症状明显改善.     

骨髓间质干细胞移植对大鼠颅脑损伤后氧化应激的影响

目的观察骨髓间质干细胞移植对大鼠外伤性脑损伤的治疗效应和对大鼠颅脑损伤后氧化应激的影响。方法体外分离培养MSCs;建立大鼠TBI模型;经颈内动脉移植MSCs,观察大鼠神经功能的改善状况和两组大鼠脑组织内氧化应激指标的差异。结果MSCs在体外可长期培养扩增,生物学特性稳定;经颈动脉移植4周后,移植治疗的大鼠其神经功能状况改善较好。与对照组相比较,实验组大鼠脑组织内SOD的活性较高,羟自由基的含量较低。结论移植MSCs对TBI所造成的大鼠神经功能缺损有一定的治疗效应。而移植MSCs后能上调脑组织的抗氧化能力,这可能是产生治疗效应的机制之一。     

超顺磁氧化铁标记骨髓基质干细胞移植治疗帕金森病鼠的实验研究

目的:研究应用超顺磁氧化铁(SPIO)标记骨髓基质干细胞(MSCs)移植治疗帕金森病(PD)大鼠后的在体MRI观察。方法:分离、获取大鼠骨髓基质细胞,脂质体转染法将SPIO标记MSCs;制作PD模型,SPIO标记的MSCs移植到PD大鼠右侧纹状体区,应用MRI在体观察脑内移植的骨髓基质细胞的存活和迁徙情况。结果:体外SPIO标记的骨髓基质细胞普鲁蓝染色阳性;脑内移植SPIO标记MSCs的PD大鼠磁共振T2和T2GRE扫描检查显示在移植区呈低信号改变。随时间的延长,移植区信号向周围扩大。脑纹状体区的铁染色也可见SPIO标记MSCs从移植部位向四周迁移。结论:SPIO可用于体外标记MSCs,通过MRI技术可以对标记细胞脑内移植后进行初步的活体示踪,有利于MSCs移植治疗PD后的疗效观察。     

胚胎中脑黑质脑内移植治疗帕金森病6例随访观察

本报告采用胚胎中脑黑质细胞脑内移植治疗6例晚期帕金森病，包括1例脑立件定向丘脑毁损后震颤复发，经移植治疗后获得良好效果。本组病例韦氏计分平均从21分降至11分。其中2例经术后3、9，14个月CT、MRI随访，脑内移植物体积增大．症状和体征明显改善。     

自体骨髓间充质干细胞不同移植途径对脑冷冻伤大鼠神经功能改善的影响

目的探索自体骨髓间充质干细胞(BMMSCs)不同移植途径对脑冷冻伤大鼠神经功能改善的影响。方法将Wistar大鼠自体骨髓间充质干细胞在体外扩增并经Brdu标记后,通过颈内动脉和脑室系统注射移植入冷冻伤脑水肿动物体内,以免疫组化和神经功能评分评价BMMSCs自体移植的疗效。结果经颈内动脉和脑室系统注射的Brdu标记的大鼠BMMSCs细胞可向脑损伤区域迁移。经颈动脉注射BMMSCs治疗组和经脑室注射BMMSCs治疗组Brdu阳性细胞的数量和神经功能恢复的程度均高于常规治疗组,且有明显统计学差异(P0.05)。两移植组BMMSCsBrdu阳性细胞的数量和神经功能恢复的程度相比无明显差异(P0.05)。结论本实验研究提示,BMMSCs通过循环系统和脑室系统移植后可向脑损伤区迁移而发挥治疗作用,但二者无统计学差异。     

脑移植在老年痴呆研究中的现状与展望

脑移植不仅可在老年动物及痴呆模型动物的脑内存活、发育、与宿主脑细胞建立相互联系,而且可发挥功能效应,影响宿主动物的学习、记忆等脑活动。通过基因工程获得的遗传修饰细胞和16三体胚胎海马组织的脑内移植为这一领域的研究开辟了新途径。此外,脑移植作为一种治疗手段,可能为临床上治疗老年痴呆病人提供了新的、有效的方法。     

颈上交感神经节脑内移植治疗帕金森病的实验研究

目的研究治疗帕金森病的理想脑内移植物.方法建立SD大鼠帕金森病动物模型,选取SD模型大鼠30只分成3组(移植组12只,对照组12只,空白组6只)用同种成年大鼠颈上交感神经节(SCG)制成细胞悬液,用微量注射器、立体定向仪将细胞悬液植入移植组模型鼠毁损侧尾核头部.每2周观察动物旋转行为变化.6个月后处死动物,制成脑切片,行TH免疫组化及HE染色,切片光镜观察.结果SCG移植后6个月,移植组大鼠旋转行为明显减少.组织学观察发现移植区有TH样阳性细胞存活,且存活数与治疗效果成正比.结论同种异体成年大鼠的SCG脑内移植,移植物能存活并对帕金森病模型鼠有短期治疗作用.     

骨髓基质细胞成年大鼠脑内移植

目的 研究骨髓基质细胞脑内移植后的分布和移行，为细胞移植治疗疾病奠定基础。方法 常规培养大鼠骨髓基质细胞，应用免疫组织学方法对细胞进行鉴定，Hoechst33258标记细胞，立体定向移植到大鼠的纹状体，经过一段时间后处死大鼠，脑组织切片，直接在荧光显微镜下检查存活的细胞。结果 细胞移植到大鼠脑内能够长时间存活，移植细胞与宿主细胞有很好的相容性，宿主脑组织的结构无破坏，移植细胞能够移行一段距离，说明脑内存在的信号诱导细胞向一定的方向迁徙。结论 骨髓基质细胞脑内移植后，能够与宿主脑组织整合在一起，无细胞过度增生和胶质瘢痕形成，这种细胞可能成为中枢神经系统自体移植的细胞来源。     

帕金森氏病的脑内移植治疗进展

自1982年脑内移植治疗帕金森氏病(PD)在临床取得疗效以来,这一领域的研究迅猛发展。目前这项课题已发展成为神经科学的前沿和热点之一。各国学者先后将肾上腺髓质、胚胎黑质、交感神经节及培养的细胞系细胞植入脑内,以期恢复黑质纹状体多巴胺系统功能。特别是近年来开展的包被细胞及遗传修饰细胞移植,为PD治疗开辟了新途径。本文综述了该领域当前的现状及最新进展,提出了存在的问题和前景展望。     

神经干细胞立体定向脑内移植治疗大鼠重型颅脑损伤

目的探讨神经干细胞立体定向脑内移植对颅脑损伤大鼠的治疗效果。方法采用Feeney等人的方法制成大鼠脑损伤模型,伤后24h将体外培养的胚胎神经干细胞经立体定向移植到脑损伤灶内。伤前24h、伤后24h及伤后1、2周行动物神经学缺损评分。结果移植后1和2周,接受神经干细胞移植的大鼠神经学缺损评分明显低于对照组（P〈0.05）;且其脑组织切片中的神经元数量较对照组明显增多（P〈0.01）。结论经立体定向移植到脑内损伤灶的神经干细胞可存活、增殖、分化、并可明显改善重型颅脑损伤后大鼠的神经学功能。     

骨髓间充质干细胞自体移植治疗大鼠脑冷冻伤的实验研究

目的探索骨髓间充质干细胞（MSCs）对脑损伤的治疗作用。方法将Wistar大鼠自体骨髓MSCs在体外扩增并经Brdu标记后，通过颈内动脉注射将其植入冷冻伤脑水肿动物体内，从组织化学和神经功能评分两个方面观察骨髓MSCs自体移植的治疗作用。实验动物分为4组；A组给予常规治疗；B组给予干细胞自体移植；C组在进行干细胞自体移植的同时给予神经节苷脂和丹参注射液治疗；D组在干细胞自体移植前给予罂粟碱开放血脑屏障。结果经颈内动脉注射的Brdu标记的大鼠自体骨髓MSCs细胞可向脑损伤区域迁移。B、C、D组Brdu阳性细胞的数量和神经功能恢复的程度均高于A组（P〈0．05）。与B组相比，C组未增加Brdu阳性细胞的数量，但是神经功能恢复的程度明显升高（P〈0．05）。D组Brdu阳性细胞的数量、神经功能恢复的程度均高于B组（P〈0．05），但神经恢复的程度与C组相比无明显差异（P〉0．05）。结论骨髓间充质干细胞可以通过循环系统向脑损伤区迁移而发挥其治疗作用。     

经枕大池神经干细胞移植治疗大鼠创伤性脑损伤的研究

目的 将神经干细胞经枕大池移植到创伤性脑损伤模型大鼠蛛网膜下腔中并观察其存活、迁移和分化,从而为神经干细胞的体内存活、迁移和分化机理研究和临床应用提供实验依据.方法 体外培养BrdU标记的胚胎神经干细胞并应用免疫荧光细胞化学染色对BrdU、神经干细胞标记物nestin的表达进行鉴定:采用Feeney自由落体撞击法制做大鼠脑损伤模型,伤后24 h将BrdU标记的胚胎神经十细胞经立体定向注射移植到蛛网膜下腔;制作大鼠脑绢织石蜡切片,应用免疫组织化学染色检测BrdU、微管相关蛋白2(MAP2)、胶质纤维酸性蛋白(GFAP)表达;伤前24h、伤后24 h及1、2周行动物运动神经功能评分.结果 免疫荧光检测显示神经球的表面细胞表达nestin及BrdU:免疫组织化学染色检测到脑内损伤灶存在BrdU阳性神经干细胞、MAP2阳性神经元和GFAP阳性胶质细胞;接受神经十细胞移植的大鼠神经运动功能评分的恢复较对照组有明显提高,差异有统计学意义(P<0.05).结论 经枕大池移植到脑损伤大鼠蛛网膜下腔中的神经干细胞能存活且具有远距离迁移能力,并明显有助于脑损伤大鼠神经运动功能的恢复.     

Mesenchymal stem cells expressing neural antigens instruct a neurogenic cell fate on neural stem cells

The neurogenic response to injury in the postnatal brain is limited and insufficient for restoration of function. Recent evidence suggests that transplantation of mesenchymal stem cells (MSCs) into the injured brain is associated with improved functional recovery, mediated in part through amplification in the endogenous neurogenic response to injury. In the current study we investigate the interactions between bone marrow-derived MSCs and embryonic neural stem cells (NSCs) plus their differentiated progeny using an in vitro co-culture system. Two populations of MSCs were used, MSCs induced to express neural antigens (nestin+, Tuj-1+, GFAP+) and neural antigen negative MSCs. Following co-culture of induced MSCs with differentiating NSC/progenitor cells a significant increase in Tuj-1+ neurons was detected compared to co-cultures of non-induced MSCs in which an increase in astrocyte (GFAP+) differentiation was observed. The effect was mediated by soluble interactions between the two cell populations and was independent of any effect on cell death and proliferation. Induced and non-induced MSCs also promoted the survival of Tuj-1+ cell progeny in long-term cultures and both promoted axonal growth, an effect also seen in differentiating neuroblastoma cells. Therefore, MSCs provide instructive signals that are able to direct the differentiation of NSCs and promote axonal development in neuronal progeny. The data indicates that the nature of MSC derived signals is dependent not only on their microenvironment but on the developmental status of the MSCs. Pre-manipulation of MSCs prior to transplantation in vivo may be an effective means of enhancing the endogenous neurogenic response to injury.     

Protective effects of bone marrow stromal cell transplantation in injured rodent brain: synthesis of neurotrophic factors

Several groups have suggested that transplantation of marrow stromal cells (MSCs) promotes functional recovery in animal models of brain trauma. Recent studies indicate that tissue replacement by this method may not be the main source of therapeutic benefit, as transplanted MSCs have only limited ability to replace injured central nervous system (CNS) tissue. To gain insight into the mechanisms responsible for such effects, we systematically investigated the therapeutic potential of MSCs for treatment of brain injury. Using in vitro studies, we detected the synthesis of various growth factors, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and neurotrophin-3 (NT-3). Enzyme-linked immunosorbent assay (ELISA) demonstrated that MSCs cultured in Dulbecco's modified Eagle medium (DMEM) produced substantial amounts of NGF for at least 7 weeks, whereas the levels of BDNF, GDNF and NT-3 remained unchanged. In studies in mice, after intraventricular injection of MSCs, NGF levels were increased significantly in cerebrospinal fluid by ELISA, confirming our cell culture results. Further studies showed that treatment of traumatic brain injury with MSCs could attenuate the loss of cholinergic neuronal immunostaining in the medial septum of mice. These studies demonstrate for the first time that by increasing the brain concentration of NGF, intraventricularly transplanted MSCs might play an important role in the treatment of traumatic brain injury.     

Consequences of noggin expression by neural stem, glial, and neuronal precursor cells engrafted into the injured spinal cord

Bone morphogenetic proteins (BMPs) are a large class of secreted factors, which serve as modulators of development in multiple organ systems, including the CNS. Studies investigating the potential of stem cell transplantation for restoration of function and cellular replacement following traumatic spinal cord injury (SCI) have demonstrated that the injured adult spinal cord is not conducive to neurogenesis or oligodendrogenesis of engrafted CNS precursors. In light of recent findings that BMP expression is modulated by SCI, we hypothesized that they may play a role in lineage restriction of multipotent grafts. To test this hypothesis, neural stem or precursor cells were engineered to express noggin, an endogenous antagonist of BMP action, prior to transplantation or in vitro challenge with recombinant BMPs. Adult rats were subjected to both contusion and focal ischemic SCI. One week following injury, the animals were transplanted with either EGFP- or noggin-expressing neural stem or precursor cells. Results demonstrate that noggin expression does not antagonize terminal astroglial differentiation in the engrafted stem cells. Furthermore, neutralizing endogenous BMP in the injured spinal cord significantly increased both the lesion volume and the number of infiltrating macrophages in injured spinal cords receiving noggin-expressing stem cell grafts compared with EGFP controls. These data strongly suggest that endogenous factors in the injured spinal microenvironment other than the BMPs restrict the differentiation of engrafted pluripotent neural stem cells as well as suggest other roles for BMPs in tissue protection in the injured CNS.     

骨髓间充质干细胞向脑胶质瘤趋向性的初步研究

背景骨髓间充质干细胞(MSCs)是存在于骨髓中的非造血类干细胞,具有自我更新及多向分化潜能.在大鼠脑外伤模型中的迁移能力已得到证实,而其对于脑胶质瘤的趋向性的研究尚处于初期.本实验通过将标记的MSCs移植到大鼠脑胶质瘤模型体内,观察它的迁移情况.方法采用全骨髓细胞培养法,利用MSCs的贴壁生长及可在体外长期培养的特性,获取纯化的MSCs.采用流式细胞术鉴定其表面抗原及细胞周期.在处于对数生长期的MSCs培养皿中加入BrdUrd(终浓度10μg/mL),培养24～48 h后进行移植.采用立体定向技术建立大鼠脑胶质瘤模型,3 d后移植标记好的MSCs在大脑半球组,BrdUrd标记的MSCs被注入大鼠脑胶质瘤模型肿瘤对侧大脑;在颈内动脉组,BrdUrd标记的MSCs被注入大鼠脑胶质瘤模型肿瘤同侧的颈内动脉.分别以BrdUrd标记的3T3细胞作为对照组.2周后,处死大鼠取脑组织制作病理切片,进行抗BrdUrd免疫组化及免疫荧光染色,观察MSCs的迁移情况.结果全骨髓培养法获得了纯化的MSCs.移植到脑组织及颈内动脉的BrdUrd标记的MSCs表现出了明显的向脑胶质瘤迁移的特性.在大脑半球组,MSCs主要集中在肿瘤与正常脑组织的交界部位,在瘤内只有少量分布.在颈内动脉组,MSCs主要分布于肿瘤内部,在肿瘤与正常脑组织的交界位置有少量分布.结论MSCs具有明显的向脑胶质瘤迁移的特性,同时亦可通过血脑屏障,有可能成为胶质瘤基因治疗的理想载体.     

脐带血间充质干细胞移植对大鼠脑创伤的影响

目的 探讨脐带血间充质于细胞(CB-MSC)移植对脑创伤大鼠的治疗作用及其在体内分化为神经元样细胞的可行性.方法 健康Wistar大鼠采用随机数字表法分为3组:(1)损伤组,开颅钻孔打击脑组织不移植细胞;(2)移植对照组,开颅创伤脑组织后在创伤区注射生理盐水1.25μ;(3)CB-MSC移植组,开颅创伤脑组织后在创伤区注射含CB-MSC混悬液.每组各18只.CB-MSC从脐带血中分离、培养得到,采用BrdU标记.分别于移植后3 d及10 d进行大鼠行为学评分,2周和4周行Y迷宫试验.移植后2周和5周对植人脑内的CB-MSC进行免疫组织化学检测,镜下观察胶质纤维酸性蛋白(GFAP)和神经元特异性烯醇化酶(NSE)阳性细胞.结果移植后10d 3组大鼠行为学评分差异有统计学意义(p<0.05),移植后2周和4周大鼠学习、记忆评分差异亦有有统计学意义(P<0.05).移植后2周和5周在CB-MSC移植组细胞移植区均发现BrdU-GFAP和BrdU-NSE阳性细胞,其他2组均未发现.结论 CB-MSC移植可促进大鼠脑创伤恢复,提高学习和记忆能力,CB-MSC在体内可以向神经元样细胞分化.     

Protective Effects of BDNF Overexpression Bone Marrow Stromal Cell Transplantation in Rat Models of Traumatic Brain Injury

Bone marrow stromal cells (MSCs) were used as cell therapy for various diseases in recent years. Some reports showed that transplanted MSCs promote functional recovery in animal models of brain trauma. But other studies indicate that tissue replacement by this method may not be the main source of therapeutic benefit. Neurotrophic factors such as brain-derived neurotrophic factor (BDNF) therapeutic potential may contribute to the recovery of function after trauma. Our previous study showed that BDNF–MSCs could promote the survival of neurons in neuronal injured models in vitro. The present study was undertaken to explore the therapeutic effects of MSCs transfected with BDNF in vivo. After intraventricular injection of MSCs–BDNF, BDNF levels were increased significantly in cerebrospinal fluid by ELISA. Further studies showed that treatment of traumatic brain injury with MSCs–BDNF could attenuate neuronal injury as measurement of biological behavior assessment. These studies demonstrate that by increasing the brain concentration of BDNF, intraventricularly transplanted MSCs–BDNF might play an important role in the treatment of traumatic brain injury and might be an optional therapeutic strategy.     

Human bone marrow stromal cell cultures conditioned by traumatic brain tissue extracts: growth factor production

Treatment of traumatic brain injury (TBI) with bone marrow stromal cells (MSCs) improves functional outcome in the rat. However, the specific mechanisms by which introduced MSCs provide benefit remain to be elucidated. Currently, the ability of therapeutically transplanted MSCs to replace injured parenchymal CNS tissue appears limited at best. Tissue replacement, however, is not the only possible compensatory avenue in cell transplantation therapy. Various growth factors have been shown to mediate the repair and replacement of damaged tissue, so trophic support provided by transplanted MSCs may play a role in the treatment of damaged tissue. We therefore investigated the temporal profile of various growth factors, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF), within cultures of human MSCs (hMSCs) conditioned with cerebral tissue extract from TBI. hMSCs were cultured with TBI extracts of rat brain in vitro and quantitative sandwich enzyme-linked immunosorbent assays (ELISAs) were performed. TBI-conditioned hMSCs cultures demonstrated a time-dependent increase of BDNF, NGF, VEGF, and HGF, indicating a responsive production of these growth factors by the hMSCs. The ELISA data suggest that transplanted hMSCs may provide therapeutic benefit via a responsive secretion of an array of growth factors that can foster neuroprotection and angiogenesis.     

Intravenous transplantation of bone marrow mesenchymal stem cells promotes neural regeneration after traumatic brain injury

To investigate the supplement of lost nerve cells in rats with traumatic brain injury by intravenous administration of allogenic bone marrow mesenchymal stem cells, this study established a Wistar rat model of traumatic brain injury by weight drop impact acceleration method and administered 3 × 106 rat bone marrow mesenchymal stem cells via the lateral tail vein. At 14 days after cell transplantation, bone marrow mesenchymal stem cells differentiated into neurons and astrocytes in injured rat cerebral cortex and rat neurological function was improved significantly. These findings suggest that intravenously administered bone marrow mesenchymal stem cells can promote nerve cell regeneration in injured cerebral cortex, which supplement the lost nerve cells.