摘要
脊髓损伤(Spinal cord injury，SCI)是一种严重伤病，其致残率极高。SCI的传统治疗方法有脊柱骨折脱位的复位固定、解除压迫、对症、预防并发症及功能康复锻炼治疗。近年来，随着神经病理生理及神经发育学研究的不断深入，神经组织不能再生的传统观念受到了挑战，神经组织细胞移植逐渐应用于SCI的治疗并取得了肯定的效果。神经干细胞(Neural stem cells，NSCs)特别是骨髓基质细胞(Bone marrow stromal cells，BMSCs)源性NSCs分化调控和移植修复SCI成为研究重点。
BMSCs是骨髓细胞中除去造血干细胞之外的细胞部分，目前已证实BMSCs在特定的培养条件下可分化成多种细胞，如间充质细胞、成纤维细胞、成骨细胞、软骨细胞、脂肪细胞、肌肉细胞、内皮细胞和神经元及神经胶质细胞等，具有克隆样增殖特性。其可向神经细胞方向分化的潜能，为NSCs的来源增加了一个新的途径，同时因为其获取方便、来源充足、可进行自体移植、避免了使用胚胎干细胞所带来的伦理争议和免疫排斥反应等优势，因此成为SCI后细胞移植替代治疗最理想的种子细胞来源之一。
目前国内外众多实验结果表明：NSCs移植可以促进SCI后组织修复及功能改善，但目前尚未有移植细胞与宿主细胞问形成突触联系的报道，即尚未发现移植细胞可以重建神经环路、恢复中断的神经纤维。究其原因，主要是目前的细胞标记方法仅能标记细胞胞体，但不能有效标记移植细胞发出的轴突、树突等结构，尤其是在电镜观察超微结构时，不能区分轴突或者树突的来源。目前Ferumoxides是作为移植细胞的标记物受到广泛的关注，其和多聚赖氨酸(poly 1 lysine，PLL)形成的FE-PLL复合物可以有效标记移植细胞，且由于其颗粒细小，有可能会随着胞浆在轴突、树突中的运输过程而扩散到轴突、树突的末端，在电镜下可以区分轴突及树突是来源于移植细胞还是来源于宿主细胞。而TrkB受体作为细胞膜上的受体，在正常的神经组织细胞中有少量分布，并且分布于轴突、树突的末端甚至突触部位，因此，如果给予外源性TrkB一个宿主细胞不存在的标记物，并且此标记物可以在电镜下清楚显示，也可以据此在电镜下辨别轴突及树突的来源。
本研究以BMSCs源性NSCs为移植细胞，建立脊髓全横断损伤大鼠模型，通过脂质体法转染pEGFP-C_2-TrkB至NSCs，其中以EGFP作为外源性TrkB的报告基因，并在细胞移植前再以FE-PLL进行了标记，以方便影像学活体追踪及动物实验的电镜下细胞辨认。将携带pEGFP-C2-TrkB质粒并用FE-PLL标记的NSCs移植到损伤SCI处，获取病理组织，通过胶体金标记的EGFP免疫组化及含铁颗粒在细胞内分布来观察移植的NSCs存活、分化及与宿主细胞间是否存在突触或其它方式的联系，并通过生物素化的葡聚糖胺(Biotinylated dextran amine，BDA)顺行追踪皮质脊髓束以观察神经纤维能否再通，即神经环路能否重建，从而观察NSCs移植促进SCI组织修复的超微结构证据。
第一部分大鼠骨髓基质细胞体外诱导分化成神经元样细胞的实验研究
目的：
观察9L／fisher944同源基因型大鼠BMSCs在体外培养及诱导分化获得神经元样细胞的可行性。
材料及方法：
(一)、无菌条件下获取大鼠骨髓，梯度密度离心BMSCs。
(二)、在体外应用NSCs培养基(专利号：ZL02134314.4)和细胞因子进行BMSCs向NSCs、神经元样细胞的诱导分化。细胞培养中分别加入维甲酸(retinoic acid，RA)等细胞因子。
(三)、观察培养细胞：CK2型倒置相差光学显微镜(OLYMPAS，JAPAN)追踪观察细胞生长情况并拍照；采用SABC法进行免疫细胞化学检测(一抗分别为鼠抗-Nestin、鼠抗-NSE，二抗试剂盒为生物素化羊抗鼠IgG，链霉亲和素-生物素-过氧化酶复合物，DAB显色)。
结果：
(一)、贴壁细胞培养48h后有分裂增殖，然后逐渐形成岛屿状细胞克隆团，给予适当的分化条件10～20d后，这些细胞能分化成具有细长突起、多种形态的细胞。
(二)、经免疫细胞化学鉴定，早期的培养细胞在没有给予诱导分化时可阳性表达Nestin，表明具有干细胞特性；培养细胞经诱导分化后有神经元特异性烯醇化酶(neuron-specific enolase，NSE)阳性表达的细胞出现。
结论：
大鼠BMSCs是具有较强自我更新能力和多向分化潜能的细胞，在一定条件下，可分化为NSCs并分化为神经元样细胞，表达神经组织细胞特异性抗原。RA在BMSCs向神经元样细胞分化的过程中起着积极作用。BMSCs可作为NSCs的来源细胞。
第二部分：大鼠骨髓基质细胞源性神经干细胞的TrkB基因转染
目的：通过基因转染，获得能长期稳定表达TrkB基因的NSCs，同时评价脂质体法的转染效率及转基因后蛋白的表达情况。
方法：采用LIPOEECTAMINE．2000脂质体介导法，将真核表达载体pEGFP-C_2-TrkB导入NSCs。转染24h后，荧光显微镜下观察标记蛋白—EGFP的表达情况，计算转染效率。应用含G418的选择培养基对细胞进行筛选，获得转染阳性细胞。通过半定量RT-PCR法测定转染阳性NSCs中TrkB mRNA的表达水平。通过TrkB免疫细胞化学荧光染色观察目的基因在蛋白水平的表达情况。取同期培养未转染及经相同方法转染pEGFP-C_2的NSCs作为对照组。
结果：经LIPOEECTAMINE．2000脂质体法转染后24h小时后，荧光显微镜下可观察到绿色荧光蛋白的表达，表达率约18.12±0.63％，随着培养时间的延长，表达绿色荧光蛋白的细胞数量逐渐增多，于一周左右达到高峰，表达率约40.19±0.70％，继续观察一个月未发现荧光表达减弱。转染一周后，更换含有浓度为500ng／L G418的选择培养基进行筛选，常规培养，5d后未转染组的细胞全部死亡，转染pEGFP-C2-TrkB及pEGFP-C_2组中可见较多细胞从培养瓶壁上脱落、死亡。5d后开始以浓度为300ng／L G418的选择培养基继续筛选20d以上。半定量法RT-PCR法测定细胞中TrkB mRNA相对表达水平，结果显示转染pEGFP-C_2-TrkB组明显高于转染pEGFP-C_2组及未转染组(P＜0.001)。TzkB免疫细胞化学荧光染色后共聚焦显微镜检测发现，转染pEGFP-C_2-TrkB质粒后细胞上的TrkB表达主要集中在细胞膜上。
结论：本实验通过脂质体介导的方法将pEGFP-C_2-TrkB转染入NSCs中，获得稳定表达外源TrkB基因的抗G418细胞克隆，并从蛋白及mRNA水平证实了转染后的NSCs可大量表达外源TrkB，表达主要集中在细胞膜上。LIPOEECTAMINE．2000脂质体法转染操作简便、转染效率可以满足实验要求；EGFP可以作为大鼠TrkB有效的基因表达标记。
第三部分：菲立磁标记大鼠神经干细胞的细胞生物学研究
目的：利用神经影像学在体追踪移植入宿主体内的NSCs无疑能够帮助我们正确评价移植细胞对功能恢复的调节情况。Ferumoxides是一种超顺磁性氧化铁，是磁共振的对比造影剂。本研究旨在初步探索利用Ferumoxides标记大鼠NSCs及对其活力、增殖的影响，并观察标记成功后含铁颗粒在细胞里的分布情况，为临床和基础研究利用磁共振成像追踪标记细胞及根据含铁颗粒在细胞内的分布以区分移植细胞和宿主细胞的轴突及树突奠定基础。
方法：按第一部分方法获得NSCs，将终浓度25μg／ml的Ferumoxides和终浓度0.75μg／ml PLL混合振荡30min形成Ferumoxi des和PLL的复合物(FE-PLL)，应用FE-PLL标记NSCs，采用普鲁士兰染色和投射电镜对细胞内铁分布和标记效率进行检测，采用四唑盐(MTT)法鉴定FE-PLL标记NSCs活力。应用流式细胞仪对细胞凋亡情况进行分析，分析该标记方法对NSCs活力、增殖的影响。实验分组：A．纯NSCs组；B．PLL(0.75μg／ml)组；C．FE(25μg／ml)组；D．FE(25μg／ml)+PLL(0.75μg／ml)组。
结果：倒置相差显微镜观察：FE-PLL标记组NSCs呈黄色，而PLL孵育组、FE组及正常未标记组细胞未见黄染，之间区别明显。经FE—PLL标记的NSCs普鲁士兰染色阳性率均在90％以上。倒置显微镜下观察，普鲁士蓝染色阳性标记的NSCs胞质内存在细小的蓝色铁颗粒，细胞呈蓝色，主要集中在胞体，而PLL孵育组、FE组及正常未标记组细胞未见细胞蓝染。透射电镜结果显示，FE-PLL标记阳性的NSCs胞质内含有许多包裹铁颗粒的囊泡，直径在0.8～1.5μm，囊泡内有浓聚细小的铁颗粒，主要集中在胞体。MTT结果显示，FE-PLL标记细胞与PLL孵育组、FE组及正常未标记组细胞相比，吸光度值差异无显著性，说明FE-PLL复合物在此浓度下对细胞活力无显著影响，表明应用FE-PLL作为NSCs的标记物是安全的。流式细胞仪检测结果显示，FE-PLL标记细胞与单纯PLL孵育组、单纯FE组及正常未标记组细胞相比，细胞凋亡率的差异无显著性，说明FE-PLL复合物在实验浓度下对细胞的凋亡无显著影响。
结论：FE-PLL标记的NSCs经普鲁士蓝染色及电镜检测均显示其胞质内含铁颗粒的存在，含铁颗粒主要分布于胞体上。MTT及流式细胞仪结果结果显示，Ferumoxides终浓度25μg／ml、PLL终浓度为0.75μg／ml时，FE-PLL复合物对细胞增殖及凋亡没有明显影响；含铁颗粒分布在胞浆内，可能会随着胞浆运输而分布至细胞的轴突或者树突中，依此可能区分轴突及树突的来源，但也需体内实验的进一步证实；用FE-PLL体外标记NSCs是一种可行的实验方案，可考虑用于移植细胞的活体追踪。
第四部分：TrkB基因修饰NSCs移植促进SCI大鼠组织修复超微结构观察
目的：建立9L／fisher944同源基因型大鼠胸8节段脊髓横断损伤模型，将体外FE-PLL标记的携带pEGFP-C_2-TrkB基因的NSCs经单细胞悬液微移植入体内，以体外FE-PLL标记的携带pEGFP-C_2的NSCs以及单纯FE-PLL标记的NSCs移植为对照组，观察移植细胞在脊髓损伤处的存活、迁移、分化情况及与宿主组织细胞的整合情况，尤其是移植细胞与宿主细胞间联系的存在情况，为细胞移植治疗SCI寻找超微结构的证据。
方法：采用全横断法制作胸8节段SCI动物模型，并根据处理方式的不同将实验动物分为五组：A组：正常对照组；B组：单纯SCI组；C组：SCI+单纯NSCs移植组；D组：SCI+携带pEGFP—C2的NSCs移植组；E组：SCI+携带pEGFP-C2-TrkB的NSCs移植组。在细胞移植后4周应用磁共振成像对移植细胞进行活体示踪(C、D、E组)，A、B组作为对照组。同时对移植细胞生存、分化状态检测：D、E组中，荧光显微镜下挑选有EGFP表达的组织切片，行抗-NSE及抗-GFAP免疫组化荧光染色(Cy3)；细胞存活数，移植细胞分化的NSE、GFAP阳性细胞计数、GFAP阳性细胞计数，其中GFAP阳性细胞计数比较中A、B、C组作为对照。通过普鲁士蓝染色及透射电镜对移植细胞内铁进行鉴定和观察。应用BDA法顺行追踪皮质脊髓束观察中断的神经纤维再通的情况。实验重点在于移植细胞与宿主细胞间整合情况的超微结构观察，即是否存在突触等联系，应用方法用两种：第一种，应用透射电镜观察含铁颗粒是否能分布至轴突、树突，能否以此来观察移植细胞与宿主细胞间整合情况的超微结构：第二种方法GFP免疫组化(二抗为胶体金标记)来观察外源性TrkB的标记物一EGFP的蛋白表达情况，根据胞膜、轴突、树突上是否存在胶体金颗粒来区分移植细胞及其轴突、树突，以此来观察移植细胞与宿主细胞间整合情况的超微结构。
结果：FE-PLL细胞移植入体内后，可以在MRI上清楚显像，在T1W1序列上主要表现为损伤区域轻度升高的信号，而单纯的损伤区为低信号改变。MRI显像区域经组织切片普鲁士蓝染色及透射电镜观察证实了细胞内铁的存在。移植的细胞在体内可以存活并分化为表达NSE、GFAP的细胞，其中以表达GFAP的细胞数量为多，TrkB的转染可以增加移植细胞的存活及向NSE阳性细胞的分化、减少向GFAP阳性细胞的分化，可以在一定程度上减轻脊髓损伤后胶质瘢痕的程度，间接发挥了BDNF的生物学效应，有利于神经组织的再生及修复。BDA法证实细胞移植可以促进神经纤维的再通及中断的神经环路的重建，而TrkB的转染可以增加这种作用。超微结构的观察发现应用FE-PLL标记细胞的方法可以在电镜下有效的辨别出移植细胞胞体，但由于含铁颗粒不能随胞浆运输而转运至轴突及树突内，因此，不能区分轴突及树突的来源，只能确切的观察移植细胞与宿主细胞胞体问的联系。而通过给予外源性TrkB受体一个宿主自身没有的标记蛋白-EGFP，通过免疫组化定位EGFP蛋白，由于TrkB受体可以分布至轴突、树突甚至突触前膜上，因此应用此方法可以在电镜下确切的区分出移植细胞形成的轴突及树突，据此可以明确的了解移植细胞与宿主细胞间的整合情况。透射电镜下发现部分细胞膜有散在的高密度胶体金颗粒，分布基本均匀一致，部分细胞突起上存在类似的高密度金属颗粒，胶体金标记的细胞突起与铁标记的细胞胞体间有较多的接触，胶体金标记的细胞突起与没有铁标记的细胞胞体间形成的接触少，胶体金标记的细胞突起与无胶体金标记的细胞突起间也存在着接触，但均未形成典型的突触样结构。
小结：
1．FE-PLL标记的NSCs移植入体内后可以通过MRI活体观察、追踪，但在电镜下不能区分轴突及树突的来源；
2．给予外源性TrkB特殊的标记蛋白—EGFP后，予胶体金标记的免疫组化蛋白定位，可以在电镜下区分轴突及树突是否来源于移植细胞，并可以依此观察移植细胞与宿主细胞间的整合情况；
3．NSCs移植入大鼠SCI模型后可以在移植部位能在存活，可分化为表达NSE及GFAP的细胞，但分化的细胞以表达GFAP者为多；通过转染TrkB，可以间接的发挥BDNF的生物学作用，促使移植的NSCs分化为表达NSE细胞的比例增高，减少向表达GFAP细胞分化的比例，在一定程度上减少胶质瘢痕的程度；
4．移植入体内的NSCs与宿主细胞间可以形成接触、联系，但未发现典型的突触样结构，BDA追踪皮质脊髓束证实，中断的皮质脊髓束在细胞移植后可以不同程度的得以再通，神经环路得以重新构建，转染TrkB到移植细胞上可以强化神经纤维的再通；
5．NSCs移植治疗SCI有一定的疗效，但仍有许多问题需要解决。
Spinal cord injury (SCI) is a kind of extremely severe damage that having high disability rate. The traditional treatment to SCI usually included spine fracture-dislocation stabilization, relieve spinal cord compression, symptom therapy and rehabilitative treatment. On recent, following development of study of neuropathophysiology and auxology, conventional idea that nervous tissue do not regenerative had challenged, the transplantation of nervous tissue and cells as a new therapy method is gradually being applied to the treatment of SCI, and some satisfactory results were found. Nerual stem cells (NSCs), especially the cells derived from bone marrow stromal cells(BMSCs) become study emphasis.
The BMSCs are a part of bone marrow remove hemopoietic stem cells. It was confirmated recently that the BMSCs in under definite culture condition can differentiate into a variety of cell, for instance mesenchymal cell, fibroblast, osteoblast, chondrocyte, adipocyte muscle Cells, endothelial cell and neuron, glial cell and so on, passing clone proliferation. Detected of BMSCs pluripotency and succeed of toward nerve cell directional differentiation, increase new pathway for NSCs source. Meanwhile, it possess convenience harvested, adequacy resource, autografting, avoidance ethics dispute and immunological rejection that applying embryonic stem cell (ESCs), also provide a new idea for solve "self-NSCs" transplantation project cell quantity short of problem.
Multitude study exterior and interior were indicated that transplant multi-origin NSCs into SCI animal model could promoted tissue repair and function improvement. However, it was not found that the synapse contact between grafted cells and host cells, to say in other words, nerve circuit loop interrupted not found to get reablement and rebuild. We could observe neuraxon or dendric under electron microscope when we observe ultramic-structure, but we did not distinguished it derived from grafted cells or host cells. Why? It was the chief reason that we could not labeled the neuraxon or dendric notwithstanding we could labeled transplanted cells applied many sign methods.
Ferumoxides(FE), take as labeled marker of grafting cells, were obtained extensive attention. It could effective labeled transplanting cells when it combined with poly 1 lysine(PLL) formed FE-PLL compound. Because its bead small, average diameter is 80nm, diameter of ferric oxide lied in cores is 20nm, it can possible diffusing to extremity of neuraxon and bendritic of labeled cells following endochylema transportation. Tyrosine kinase receptor B(TrkB), as specificity receptor of brain-derived neurotrophic factor(BDNF) lied in cellular membrane, have a small quantity disposition in common nerve cells and can distribute extremity of neuraxon and bendritic even synapse following cellular membrane extension. If labeled grafting cells applying FE-PLL or exogenous TrkB carried marker do not lied in host cells, we could distinguish the source of neuraxon and bendritic under electron microscope.
Based on these, we take NSCs derived from BMSCs as transplantation cells, established rat SCI model, and transfect pEGFP-C2-TrkB into the grafting cells, supplied symbol protein-EGFP to labeling exogenous TrkB receptor, labeled cells with FE-PLL before transplanted. Harvested patho-tissue at 4 weeks after cells transplanted, observing cells survival, differentiation, and observing neuraxon and bendritic of grafted cells and synapse contact between host cells and transplanted cells by Ferumoxides or EGFP immunohistochemistry labeled by colloidal gold under electron microscope, and observing nerve circuit loop interrupted did or not get reablement and rebuild applying biotinylated dextran amine(BDA) tracing corticospinal tract and to explore ultrastructural evidence of tissue repair of SCI after NSCs transplanted.
Part one: the induction and differentiation of BMSCs into NSCs in rat
Objective: To study the feasibility of induction and differentiation of BMSCs into NSCs in 9L/fisher homologous genes rat in vitro.
Methods: Under the sterile condition, BMSCs, which isolated from bone marrow stromal of 9L/fisher homologous genes rat and were separated by gradient density centrifugation, were cultured, induced and differentiated into neural stem cells and neuron-like cells with cytokines consisted of retinoic acid(RA, 0.5μg/ml) and special culture medium confected by our lab(patent number: ZL02134314.4), and then evaluated by immunocytochemistry (SABC, first antibodys were Rat anti-Nestin, Rat anti neuron-specific enolase (NSE), Rat antineuron specific nuclear protein (NeuN). Second antibodys were biotinylated Goat anti Rat IgG and streptavidin peroxidase.).
Results: We can see division and proliferation in adherent cells in culture, which can form cell clones like islands. When continuous cultured, and give appropriate differentiated condition, these cells can differentiate into poly-form cells with some long and thin apophysis. Evaluate in immunocytochemical: in earlier period, the cultured cells without induce and differentiate condition can express Nestin positive. It is indicated the cells had some character of stem cell; when these cells differentiated, there were some cells express NSE and NeuN positive.
Conclusions: BMSCs of rat could proliferate and differentiate into the cells expressing Nestin and NSE and NeuN antigen. Considering its source convenience, the BMSCs could be considered as one of the ideal seed cells of the NSCs.
Part two: Transfection of exogenous TrkB into rat neural stem cells
Objective: To obtain the rat NSCs expressing exogenous TrkB(tyrosine kinase receptor B) gene stably and in long term and to evaluate the transfection efficiency with liposomes-mediated transfection method in vitro.
Methods: The eukaryotic expression vector pEGFP-C_2-TrkB (plasmid-enhanced green fluorescent protein-C_2-TrkB) was transfect into NSCs with LIPOEECTAMINE. 2000 liposomes-mediated transfect method. The transfect efficiency was calculated via expression of EGFP, which was detected with fluorescent microscope at 24h after transfect. After G418 screening, the TrkB mRNA expression of positive NSCs were analyzed with semi-quantitative RT-PCR. The NSCs were cultured and either transfect with pEGFP-C_2-TrkB or pEGFP-C_2 by LIPOEECTAMINE. 2000 or uninfected in vitro respectively, analyzed the protein level expression information after plasmid pEGFP-C_2-TrkB transfected by means of TrkB immunocytochemistry Cy3 fluorescent staining.
Results: EGFP were successfully expressed 24 hours after nucleofection, the rate of positive EGFP expression was 18.124±0.63%. The positive EGFP expression was enhanced gradually alone with the prolonged culture time, and showed the strongest one week after marked, with about40.194±0.70%. The expression intensity of EGFP did not attenuate even one month after marked. Cell culture till one week, 500ng/L G418 was added into medium. Five days later, all of the untransfect NSCs came to death and some of NSCs contained exogenous pEGFP-C_2-TrkB or pEGFP-C_2 were fall off from the culture bottle and dead. The concentration of G418 was changed to 300ng/L at 5th day and its effect was maintained over 20 days. The expression of TrkB in pEOFP-C2-TrkB was higher significantly than those in pEGFP-C_2 NSCs and untransfeted cells with half-quantitation RT-PCR determine TrkB mRNA. The expression of TrkB in protein level in NSCs transfect plasmid pEGFP-C_2-TrkB was higer significantly than in NSCs transfect pEOFP-C2 and untransfeted cells under confocal microscopy after TrkB immunocytochemistry Cy3 fluorescent staining and protein expression most concentrates in cellular membrane.
Conclusions: The study obtain anti-G418 cell clone that stabilize express ectogenous TrkB by means of transfection pEOFP-C_2-TrkB into NSCs with LIPOEECTAMINE. 2000 liposomes-mediated transfect method, and confirmed NSCs transfect could multiplicity express TrkB in mRNA and protein level, and TrkB expression most concentrate in cellular membrane in protein level. LIPOEECTAMINE. 2000 is a convenient and efficient gene transfect method for the introduction of the genes into rat NSCs, the pEGFP-C_2-TrkB gene transferred could stabilize expression, EGFP plays an symbol role in the expression of the pEGFP-C~2-TrkB gene.
Part three: Cytobiology study on intracellular labeling of neural stem cells derived from bone marrow of rat with Ferumoxides
Objective: The ability to track NSCs transplanted into host by neuroimaging in vivo will undoubtedly aid our understanding of how these cells mediate functional recovery after cells transplantation. Ferumoxides is a class of dextran-coated superoparamagnetic iron oxides (SPIO) nanoparticle used as MRI contrast agent for hepatic imaging. The purpose of this study is to explore effect of Ferumoxides on NSCs biological abilities to survive and proliferate and to observing distribution information of iron partical laid in NSCs successfully labeled by Ferumoxides, and provide a basis of clinical and lab study application for MRI tracing labeled cells and to observeing and distinguishing neuraxon and cytodendrite were derived from transplanted cells or host cells.
Methods: The culture, induction and differentiation of NSCs derived from the rat BMSCs in vitro were completed. Admixture and vibrate 25μg/ml final concentration Ferumoxides and 0.75μg/ml final concentration Poly-1-lysine (PLL) 30 minutes so as to form Ferumoxides and PLL compound(FE-PLL), and applying FE-PLL compound labeled NSCs. The efficiency of FE-PLL labeled NSCs were evaluated by Prussian blue staining and electron microscopy. Cellular viability of labeled NSCs was investigated by methyl thiazolyl tetrazolium (MTT) test. The cellular apoptosis of labeled NSCs were studied by flow cytometry. The study eventually divided into four groups: A group, single NSCs labeled group; B group, 0.75μg/ml final concentration poly-1-lysine labeled group; C group, 25μg/ml final concentration Ferumoxides labeled group; D group, 0.75μg/ml final concentration poly-1-lysine and 25μg/ml final concentration Ferumoxides (FE-PLL) labeled group.
Results: The observe results under inverted phase contrast microscope after FE-PLL labeled NSCs were demonstrated that the NSCs labeled by FE-PLL compound were yellow, contradictory, the NSCs labeled by single FE and by single PLL and did not labeled were not yellow, it was obviously difference among four groups cells. The observe results under inverted microscope were demonstrated that NSCs could be effectively labeled and labeling efficiency were above 90%. Prussian blue staining showed numerous blue stained fine particles in the cytoplasm of NSCs labeled FE-PLL, the labeled cells were show blue, but the NSCs labeled by single FE and by single PLL and did not labeled were not show blue. Transmission electron microscopy of FE-PLL labeled NSCs revealed the presence of numerous vesicles which are spreaded in intracytoplasm and filled with the electron-dense magnetic iron particles, these vesicles diameter were from 0.8 to 1.5μm, these vesicles principal concentrate in cell body. The results of MTT are demonstrated that labeled cell viability was not affected by FE-PLL when the concentration of ferumoxides is 25μg/ml and PLL is 0.75μg/ml, it was not significant distinguish among the FE-PLL groups and the groups labeled by single FE and by single PLL and did not labeled. Results of flow cytometry were suggested that the cells apoptosis would not be affected when the concentration of Ferumoxides is 25μg/ml and PLL is 0.75μg/ml, it was not significant distinguish among the FE-PLL groups and the groups labeled by single FE and by single PLL and did not labeled.
Conclusion: The results of Prussian blue and electron microscope were show that numerous blue stained fine particles in chief lied in cell body in vitro. Results of MTT and flow cytometry were suggested that the cells viability ability and apoptosis would not be affected when the concentration of Ferumoxides is 25μg/ml and PLL is 0.75μg/ml. The iron particles main spread into intracytoplasm, we could possible observe and distinguish the neuraxon and cytodendrite were derived from the transplanted cells after cells labeled by FE-PLL transplanted into host, however, it need further observation in vivo. The above results suggested that FE-PLL labeled NSCs is a feasible, efficient and safe method. It could be make use of tract transplanted cells in vivo. We are possible to distinguish the neuraxon and dendritic derived from transplanted cells or host cells by means of FE-PLL labeling cells.
Part four: Ultramicrostructure observation of the spinal cord injury rat after transplanted neural stem cells modified with protein-tyrosine kinase receptor B gene
Objective: The 9L/fisher homologous genes rat model of spinal cord injury was set via transsection under microscopy. Transfected pEGFP-C2-TrkB plasmid to rat NSCs derived from BMSCs, applying FE-PLL labeled NSCs before it transplanted. Investigate the cells survival, migration, differentiation and incorporation, especially synapse present information after transplanted NSCs transacted by above-mentioned into host tissue with single cell suspension by autologous micro-transplantation method. Taken cells carried pEGFP-C2-TrkB or pEGFP-C2 or no gene transplanted as control group. Search ultramicrostructure of cell contact and connection between transplanted and host cells so as to provided a ultramicrostructure witness for incorporation between transplanted and host cells, establish a foundation for cells transplantation treatment to central nervous system disease.
Methods: The 9L/fisher homologous genes rat model of spinal cord injury was set via transsection under microscopy. The rats were divided into 5 groups as follows: Group A: normal control; Group B: simple spinal cord injury; Group C: spinal cord injury and simple NSCs transplantation; Group D: spinal cord injury and NSCs carried pEGFP-C2 transplantation; Group E: spinal cord injury and NSCs carried pEGFP-C2-TrkB transplantation. 4 weeks after cells transplantation, tracing transplanted cells in vivo applying magnetic resonance imaging (Group C、D、E), took A and B groups as control. Meanwhile, transplanted cells survival and differentiation in D and E groups: Choose GFP positive cells to carry out anti-NSE and anti-GFAP immunocytochemistry fluorescent staining(second antibodys were cy3), and record express NSE or GFAP positive cells, compared NSE or GFAP positive express rate between D and E groups, and compared NSE or GFAP positive cells in D and E groups, and compared GFAP positive cells among A、B、C、D、E groups to get the message about glial scar forming. Detect and observe iron particles in transplanted by prussion blue staining and transmission electron microscope. Tracing corticospinal tract cured off by means of BDA method so as to observe neurofibra whether could recanalization and rebuild or not. The emphasis of this exprement on searching ultramicrostructure of cell contact and connection between transplanted and host cells, especially synapse structure, so as to provided a ultramicrostructure witness for incorporation between transplanted and host cells applying two methods. The first, applying transmission electron microscope to observe the cells containing iron particles and whether this particles could dispersed into neuraxon and cytodendrite. According the method, we will observe ultramicrostructure of cell contact and connection between transplmated and host cells. The second, provided a specificness tumor marker, EGFP, to exogenous TrkB that in chief express in cellular membrane, applied GFP immunocytochemistry staining(second antibodys labeled by gold colloid), we can observe ultramicrostructure of cell contact and connection, esprislly synapse contact, between transplanted and host cells though observe gold colloid dispersion information in cellular membrane and neuraxon and cytodendrite.
Results: We could found the transplanted cells labeled by can clear visualization under with MRI detection of animal model: FE-PLL labeled transplants clearly demarcated the injection site on SET1- and SET2-weighted MR images 4 weeks postgrafting. The high density of labeled cells in injury spinal cord in a strong signal and the dark appearance in do not injection site in all scans. Surrounding the cells injection site, in the T1-weighted scans, a hyperintense border was apparent around the dark transplant region. MRI in T2- weighted scans sequence showed remarkable low signal change in the cells injection site. Among T2-weighted scans, the most significant change occurred in GRE T2-weighted scans sequence. The tissue and cells visualization under MRI were contain iron particles confirmed by prussion blue staining and transmission electron microscope. The cells transplanted could survival and differentiated into cells expressing NSE or GFAP, quantity of GFAP positive cells were more than the NSE positive cells. It is profit to promote the cells survival and differentiated into NSE positive cells and decrease differentiated into GFAP positive cells though transfect TrkB into transplanted cells, and lighten glial scar after SCI in some extent, and educe indirectly biological effect of BDNF so as to promote injury tissue regeneration and reparation. Results of BDA tracing corticospinal tract were indicated neurofibra breakdown could recanalization and nerve ring rebuild. It could strengthen this action though transfect TrkB. Results of ultramicrostructure observe were indicated we could effective distinguish transplanted cells and host cells by means of labeled cells with FE-PLL and observe the contact between transplanted cells body and host cells body, however, we did not distinguish whether neuraxon and cytodendrite were derived from transplanted cells or host cells because the iron particle did not spread into neuraxon and cytodendrite follow as endochylema transportation. Meanwhile, we could effective distinguish transplanted cells and host cells and their neuraxon and cytodendrite though immunocytochemistry staining(second antibody labeled by gold colloid) evaluated exogenous TrkB specificness tumor marker, EGFP, gold colloid particles can distributed into neuraxon and cytodendrite and presynaptic membrane. We found the high density gold particle uniformity distributed in cellular membrane and cells ecphyma and presynaptic membrane. There were contact between transplanted cells body or ecphyma and host cells body or ecphyma, the contact between transplanted cells ecphyma and body more than it between transplanted cells ecphyma and host cells body, however, these contact were not posses typical synaptic structure.
Conclusion:
1、The FE-PLL compound could effective labeled NSCs, and we could observe and tract transplanted NSCs labeled by FE-PLL with MRI,but,we did not distinguish whether neuraxon and cytodendrite were derived from transplanted cells or host cells so that did not effective observe contact between transplanted cells and host cells;
2、Provided a specificness tumor marker, EGFP, to exogenous TrkB, we could effective distinguish transplanted cells and host cells and their neuraxon and cytodendrite though immunocytochemistry staining(second antibody labeled by gold colloid);
3、The transplanted NSCs could survival and differentiated into cells expressing NSE or GFAP in host vivo, quantity of GFAP positive cells were more than the NSE positive cells, could promote the cells survival and differentiated into NSE positive cells, decrease differentiated into GFAP positive cells, lighten glial scar after SCI in some extent and educe indirectly biological effect of BDNF so as to promote injury tissue regeneration and reparation;
4、The transplanted NSCs could formed contact with host cells, these contact were not posses typical synaptic structure. BDA tracing corticospinal tract were indicated neurofibra breakdown could recanalization and nerve ring rebuild, could strengthen this action though transfect TrkB to transplanted NSCs.
5、there were a lot of question need to solve notwithstanding it had invariably therapeutic effect applying NSCs transplanted treat SCI.
引文
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