大鼠脊髓匀浆上清诱导骨髓间充质干细胞分化为神经元样细胞的实验研究

目的:观察大鼠脊髓匀浆上清诱导骨髓间充质干细胞(mesenchymal stem cells,MSCs)形成的神经元样细胞形态特征.方法:通过贴壁法培养分离大鼠骨髓MSCs,体外扩增纯化后加入正常大鼠脊髓匀浆上清诱导72h,倒置显微镜下观察诱导前后细胞的形态结构.激光共聚焦显微镜观测钙离子细胞形态和荧光强度变化,免疫细胞化学方法鉴定诱导后细胞的表型特征.结果:倒置显微镜下可见MSCs呈纺锤形和多角形,核居中,有1-2个核仁,诱导后细胞呈神经元样,细胞伸出较长的轴突样和树突样突起.免疫细胞化学法显示NSE(神经元特异性烯醇化酶)、NF(神经丝蛋白)阳性,GFAP(神经胶质细胞酸性蛋白)阴性.共聚焦显微镜扫描脊髓匀浆上清诱导前细胞形态呈细长的梭形,细胞核不明显,胞体染色强,突起染色弱,荧光像素值低;诱导后,细胞呈现神经元样形态,胞体大,有多个突起,胞体及各突起染色强,荧光像素值高.结论:大鼠脊髓匀浆上清液可在体外诱导骨髓间充质干细胞分化为神经元样细胞.     

Notch信号在盐酸法舒地尔诱导大鼠骨髓间充质干细胞向神经元分化过程中的作用

目的:探讨Notch信号通路在盐酸法舒地尔诱导大鼠骨髓间充质干细胞(MSCs)向神经元分化中的作用。方法:实验分为未转染组、转染组(转染Rn-Notch1-siRNA)、阳性对照组(转染Rn-MAPK-1 Control siRNA)及阴性对照组(转染Negative Control siRNA)等4组。采用盐酸法舒地尔诱导大鼠MSCs分化为神经元。倒置荧光显微镜下观察MSCs转染后荧光表达情况;RT-PCR检测Notch1、Hes1和MAPK1 mRNA的表达变化;免疫细胞化学法检测Notch1、神经元烯醇化酶(NSE)、神经微丝蛋白亚单位(NF-M)和胶质纤维酸性蛋白(GFAP)的表达变化;MTT方法检测细胞存活率。结果:①siRNA转染72h,MSCs荧光表达最强,转染率可达91.3%±4.2%;同时,转染组MSCs的Notch1和Hes1 mRNA转录下降(P0.05);MTT提示转染组细胞存活率也显著减少(P0.05)。②盐酸法舒地尔可以诱导MSCs向神经元分化,其中以转染组诱导效果最佳,NSE、NF-M的表达率显著的高于其它各组(P0.05)。结论:盐酸法舒地尔在诱导大鼠MSCs向神经元分化过程中,可能存在Notch信号通路与RhoA/Rho激酶通路信号的协同作用,共同促进MSCs向神经元分化。     

骨髓间充质干细胞源神经细胞移植治疗帕金森病大鼠模型

目的探讨骨髓间充质干细胞（mesenchymal stemcells,MSCs）源神经细胞脑内移植对帕金森病（Parkinson s disease,PD）大鼠的治疗作用。方法贴壁培养法分离、培养大鼠骨髓MSCs,脑匀浆上清诱导第3代MSCs向神经细胞分化,采用免疫细胞化学法鉴定诱导分化后细胞的性质,激光共聚焦显微镜检测诱导前后细胞Ca2＋浓度变化,6只PD大鼠行纹状体内MSCs源神经细胞移植作为细胞移植组,6只PD大鼠作为对照组。细胞移植术后4周检测PD大鼠的行为变化,观察移植细胞在脑内的分布情况。结果倒置显微镜下可见MSCs呈纺锤形和多角形,有1～2个核仁,MSCs经脑匀浆上清诱导后其胞体折光性增强,发出数个细长突起,互相交织成网,有的似轴突。诱导后细胞表达神经元特异性标志物神经元特异性烯醇化酶（NSE）和神经丝蛋白（NF）,胞质Ca2＋荧光强度显著增强,可推测诱导后的细胞为MSCs源神经细胞,将BrdU标记的MSCs源神经细胞移植到PD大鼠纹状体治疗4周后,可见细胞散在分布于注射侧脑组织,有少量细胞可迁移到对侧脑组织,PD大鼠的旋转行为得到显著改善。结论MSCs源神经细胞移植治疗帕金森病大鼠可使其旋转行为得到改善。     

腺病毒介导siRNA抑制全反式维甲酸诱导的骨髓间充质干细胞RARβ表达

构建携带针对大鼠维甲酸受体β(Retinoic acid receptorβ,RARβ)基因的siRNA重组腺病毒,并感染全反式维甲酸(All-trans retinoic acid,ATRA)处理的骨髓间充质干细胞(Mesenchymal stem cells,MSCs),检测其对RARβ的表达及MSCs成神经分化的影响。设计针对大鼠RARβ的4对siRNA的DNA序列,体外退火形成双链,定向克隆至含有U6/H1双启动子的腺病毒穿梭质粒pSES-HUS,随后与腺病毒骨架质粒pAd-Easy1在BJ5183细菌中同源重组,并在HEK293细胞中包装获得重组腺病毒Ad-siRARβ。腺病毒感染大鼠MSCs后经ATRA处理24 h,Real-time、Western blotting及免疫荧光检测RARβ的表达情况。改良神经诱导培养基(Modified neuronal induction medium,MNM)诱导MSCs神经分化,Real-time PCR及免疫荧光检测神经相关蛋白表达。PCR、酶切及测序鉴定均证实siRNA正确克隆至腺病毒质粒中,腺病毒感染大鼠MSCs后可观察到60%以上的细胞有红色荧光蛋白(Red fluorescent protein,RFP)表达。经ATRA处理24 h,Real-time、Westernblotting及免疫荧光检测发现RARβ表达定位于细胞核,ATRA作用后MSCs中RARβ表达增高16.5±2.34倍(P<0.05),有3组siRNA能有效抑制ATRA诱导的RARβ表达增强,抑制率分别为(66.26±9.12)%、(48.70±5.78)%、(64.09±0.53)%(P<0.05),且以pool组效果最强,抑制率为(78.09±4.24)%(P<0.01)。ATRA联合MNM诱导MSCs成神经样细胞,表达相关神经特异蛋白Nestin、NSE、MAP-2、Tau,免疫荧光结果显示神经标志蛋白Nestin、NSE、Tju1表达阳性细胞率为(50-88)%,而腺病毒介导的siRARβ能有效抑制MSCs的神经标志物表达水平及阳性细胞率(P<0.05)。成功构建了携带针对大鼠RARβ基因的siRNA重组腺病毒,能有效感染MSCs并显著抑制ATRA诱导的RARβ表达增强和MSCs的神经分化。     

神经妥乐平体外诱导大鼠骨髓基质细胞分化为神经元样细胞

目的探索神经妥乐平(NT)体外诱导大鼠骨髓基质细胞(bone marrow stromal cells,rMSCs)分化为神经元样细胞的可行性,以期为临床应用MSCs治疗神经系统疾病奠定基础。方法取一月龄SD大鼠骨髓,分离出MSCs进行培养、扩增、纯化。用NT诱导MSCs分化为神经元样细胞。用神经元特异性烯醇化酶(NSE)、神经胶质纤维酸性蛋白(GFAP)免疫细胞化学染色鉴定阳性细胞。结果MSCs经诱导后胞体变圆,伸出细长突起,呈神经元样形态。免疫组化鉴定显示(31.50±7.32)%的细胞表达NSE阳性,(45.30±9.38)%的细胞表达GFAP阳性。结论MSCs在体外可被NT诱导分化为神经元样细胞。     

脱细胞神经移植物对骨髓间充质干细胞的诱导分化

目的探讨脱细胞神经移植物诱导大鼠骨髓间充质干细胞分化为施旺细胞样细胞的可行性。方法将分离纯化的SD大鼠骨髓间充质干细胞进行体外培养扩增,行表型鉴定后,取第5代细胞,诱导组采用脱细胞神经移植物匀浆进行诱导,非诱导组加入等量无血清培养基,倒置相差显微镜观察诱导后细胞形态变化,免疫细胞化学染色检测诱导后细胞S-100,神经胶质纤维酸性蛋白(glial fibrillary acidic protein GFAP)的表达情况。结果BMSCs表型鉴定为CD44+、CD54+、CD34-,免疫细胞化学染色GFAP、S-100的阳性表达率分别为为(42±4)%和(64±5)%。结果 脱细胞神经移植物可诱导骨髓间充质干细胞分化为施旺细胞样细胞。     

川芎嗪体外诱导小鼠骨髓间充质干细胞分化为神经元样细胞的研究

为了探讨川芎嗪体外诱导小鼠骨髓间质干细胞（BMSCs）分化为神经元样细胞的作用,以小鼠骨髓间充质干细胞为研究对象,实验分为空白对照组、β-巯基乙醇（BME）阳性对照组和川芎嗪诱导组。采用荧光免疫化学和Western blot方法,分别检测神经干细胞巢蛋白（nestin）和经元特异性烯醇化酶（NSE）的表达;RT-PCR检测诱导不同时间对神经细胞相关基因Nestin、NSE、β-微管蛋白III（β-Tubulin III）和核受体相关因子-1（Nurr1）mRNA表达的影响。结果显示川芎嗪诱导间充质干细胞24 h后,细胞形态发生显著改变,细胞突起形成且数目不等,形成神经元样细胞。细胞死亡率低于β-巯基乙醇诱导组。免疫荧光化学法和western blot结果显示：川芎嗪诱导后的细胞nes-tin和NSE蛋白表达呈阳性,且表达丰度显著高于β-巯基乙醇诱导组。川芎嗪作用不同时间的BMSCs表达神经细胞相关基因Nestin、β-Tubulin III、NSE和Nurrl。结果表明川芎嗪能定向诱导小鼠骨髓间充质干细胞分化为神经元样细胞,是较理想的诱导剂。     

骨髓间充质干细胞向神经细胞分化中Tau蛋白的表达

目的：观察骨髓间充质干细胞（MSCs）分化为神经细胞过程中,神经元微管相关蛋白Tau及其磷酸化位点pSer202的表达和含量的差异,探讨Tau蛋白在此过程中的作用。方法：使用EGF和bFGF联合诱导第4、第8和第12代的MSCs向神经细胞分化;14d后,免疫细胞化学法检测Tau蛋白和pSer202的表达;ELISA法分析各代细胞Tau蛋白含量。结果：第4、第8和第12代未诱导组Tau蛋白阳性细胞均〈6％;诱导14d后,各代MSCs在形态上均分化为类似神经元样细胞,Tau蛋白阳性细胞率较未诱导组显著升高（P〈0．05）,但各代之间无显著性,而pSer202在各代MSCs未诱导组和诱导组中均未见表达。ELISA法检测发现Tau蛋白含量在诱导过程中呈上升趋势,14d时各代细胞分化后的Tau蛋白升高程度无显著性差异。结论：MSCs向神经细胞分化过程中Tau蛋白表达量增加且可能尚未发生磷酸化,将有助于神经细胞的正常分化和突触形成。     

人骨髓间充质干细胞在成年大鼠脑内的迁移及分化

骨髓间充质干细胞 (mesenchymalstemcells,MSCs)是目前备受关注的一类具有多向分化潜能的组织干细胞 ,体外可以分化为骨、软骨、脂肪等多种细胞。因此 ,MSCs是细胞治疗和基因治疗的种子细胞之一。为了探索MSCs的迁移和分化趋势 ,为帕金森病 (Parkinsondisease,PD)的干细胞治疗提供理论和实验依据 ,本实验将体外扩增并转染增强型绿色荧光蛋白 (enhancedgreenfluorescentprotein ,EGFP)的人骨髓MSCs注入PD大鼠脑内纹状体 ,观察了人骨髓MSCs在大鼠脑内的存活、迁移、分化以及注射MSCs前后大鼠的行为变化。结果表明 ,人骨髓MSCs在大鼠脑内可存活较长时间 ( 10周以上 ) ;随着时间的延长 ,MSCs迁移范围扩大 ,分布于纹状体、胼胝体、皮质以及脑内血管壁 ;免疫组化法检测证实MSCs在大鼠脑内表达人神经丝蛋白 (neurofilament,NF)、神经元特异性烯醇化酶 (neuron specificeno lase,NSE)以及胶质原纤维酸性蛋白 ( glialfibrillaryacidprotein ,GFAP) ;PD大鼠的异常行为有所缓解 ,转圈数由 8 86±2 0 9r/min下降到 4 87± 2 0 6r/min ,统计学分析P <0 0 5为差异显著。以上观察结果表明 ,骨髓MSCs有望成为治疗PD的种子细胞     

茂丹通脉片含药血清体外诱导 S 分M化C为 内皮细胞的作用

目的：观察芪丹通脉片含药血清体外诱导大鼠骨髓间充质干细胞（MSCs）向内皮细胞分化的作用。方法：灌胃法制备芪丹通脉片含药血清和对照血清。采用密度梯度离心法分离和培养大鼠MSCs,取第三代MSCs,采用10wg／LVEGF预诱导24h后,分别加入15％芪丹通脉片含药血清与对照血清体外时MSCs诱导分化,至第7天,利用相差显微镜观察细胞形态改变,透射电镜观察细胞超微结构。免疫荧光方法检测内皮细胞特异性表面标志CD31、Ⅷ因子的表达。结果：至第7天,合15％芪丹通脉片合药血清组诱导后的MSCs形态发生明显改变,呈“卵石样”改变,透射电镜下细胞胞浆内可见Weible-Palade小体,共聚焦显微镜下可见CD31、Ⅷ因子阳性细胞。对照血清组MSCs形态仍呈长梭型,电镜下胞浆内无Weible-Palade小体,共聚焦显微镜下无CD31、Ⅷ因子阳性细胞。结论：益气活血复方芪丹通脉片含药血清具有体外诱导大鼠MSCs向内皮细胞定向分化的作用。     

肿瘤干细胞及肝癌肿瘤干细胞

肿瘤干细胞理论认为只有存在于肿瘤中的少量干细胞性质的细胞群体对肿瘤发生和发展起着决定作用,肿瘤是由干细胞突变积累而形成的无限增殖的异常组织,这一理论的提出使人们对肿瘤发生机制的认识上升到了一个新的高度,也引起了研究者的广泛关注;肝癌是我国常见的恶性肿瘤之一,我国肝癌死亡率居世界之首,目前对肝癌的研究是我国恶性肿瘤防治的重点工作,现对当前肿瘤干细胞与肝癌肿瘤干细胞相关方面的最新研究进展作一概述。     

Fusion of Tumour Cells with Host Cells

THE A9 cell is an 8-azaguanine-resistant derivative of the L cell line¹. It lacks the enzyme inosinic acid pyrophosphorylase and is thus unable to grow in media such as HAT² in which endogenous synthesis of nucleic acid is blocked by aminopterin. The A9 line has little ability to grow progressively in vivo. Inocula of 5 × 10⁴ to 2 × 10⁶ cells produced progressive tumours in only 12% of X-irradiated newborn syngeneic C3H mice³. One of these tumours was explanted as a cell suspension into Eagle's minimal essential medium containing 15% foetal calf serum and then subcultivated in this medium with 5% foetal calf serum. At each passage, cells were inoculated into X-irradiated newborn syngeneic C3H or semi-allogeneic C3H×X F₁ mice (X designates a number of different allogeneic parents). Between 80 and 90% of the inoculated animals developed progressive tumours. The cell line was therefore designated A9HT (high take incidence). The karyotype of the A9HT line was found to be similar to that of the A9 line, but with a slightly reduced total chromosome number. The modal chromosome number of A9HT was about 53, compared with about 57 for A9 (see ref. 4). A9 and A9HT both had between 20 and 30 bi-armed chromosomes and a number of marker chromosomes in common. A detailed comparison of the karyotypes of the two lines examined by the quinacrine fluorescence technique has been made⁵. The A9HT line, like its A9 parent, lacks inosinic acid pyrophos-phorylase and is unable to grow in HAT medium.     

体细胞重编程逆转为干细胞的研究进展

目前细胞和发育生物学上的研究成果为生物医学研究提供了广泛的前景.将完全分化的细胞重编程,不经过胚胎逆转为多能干细胞状态,这点燃了再生医学应用的新希望,这一成果从法律、道德、伦理等不同方面被人们所接受.通过体细胞克隆胚胎获得干细胞所面临的破坏胚胎的伦理限制,促使研究者去寻求将分化细胞重编程逆转为干细胞的新方法.主要论述了体细胞重编程的原理、过程及不经过胚胎逆转为多能干细胞的方法.     

Place Cells,Grid Cells,and Memory

The hippocampal system is critical for storage and retrieval of declarative memories, including memories for locations and events that take place at those locations. Spatial memories place high demands on capacity. Memories must be distinct to be recalled without interference and encoding must be fast. Recent studies have indicated that hippocampal networks allow for fast storage of large quantities of uncorrelated spatial information. The aim of the this article is to review and discuss some of this work, taking as a starting point the discovery of multiple functionally specialized cell types of the hippocampal–entorhinal circuit, such as place, grid, and border cells. We will show that grid cells provide the hippocampus with a metric, as well as a putative mechanism for decorrelation of representations, that the formation of environment-specific place maps depends on mechanisms for long-term plasticity in the hippocampus, and that long-term spatiotemporal memory storage may depend on offline consolidation processes related to sharp-wave ripple activity in the hippocampus. The multitude of representations generated through interactions between a variety of functionally specialized cell types in the entorhinal–hippocampal circuit may be at the heart of the mechanism for declarative memory formation.The scientific study of human memory started with Herman Ebbinghaus, who initiated the quantitative investigation of associative memory processes as they take place (Ebbinghaus 1885). Ebbinghaus described the conditions that influence memory formation and he determined several basic principles of encoding and recall, such as the law of frequency and the effect of time on forgetting. With Ebbinghaus, higher mental functions were brought to the laboratory. In parallel with the human learning tradition that Ebbinghaus started, a new generation of experimental psychologists described the laws of associative learning in animals. With behaviorists like Pavlov, Watson, Hull, Skinner, and Tolman, a rigorous program for identifying the laws of animal learning was initiated. By the middle of the 20th century, a language for associative learning processes had been developed, and many of the fundamental relationships between environment and behavior had been described. What was completely missing, though, was an understanding of the neural activity underlying the formation of the memory. The behaviorists had deliberately shied away from physiological explanations because of the intangible nature of neural activity at that time.Then the climate began to change. Karl Lashley had shown that lesions in the cerebral cortex had predictable effects on behavior in animals (Lashley 1929, 1950), and Donald Hebb introduced concepts and ideas to account for complex brain functions at the neural circuit level, many of which have retained a place in modern neuroscience (Hebb 1949). Both Lashley and Hebb searched for the engram, but they found no specific locus for it. A significant turning point was reached when Scoville and Milner (1957) reported severe loss of memory in an epileptic patient, patient H.M., after bilateral surgical removal of the hippocampal formation and the surrounding medial temporal lobe areas. “After operation this young man could no longer recognize the hospital staff nor find his way to the bathroom, and he seemed to recall nothing of the day-to-day events of his hospital life.” This tragic misfortune inspired decades of research on the function of the hippocampus in memory. H.M.’s memory impairment could be reproduced in memory tasks in animals and studies of H.M., as well as laboratory animals, pointed to a critical role for the hippocampus in declarative memory—memory, which, in humans, can be consciously recalled and declared, such as memories of experiences and facts (Milner et al. 1968; Mishkin 1978; Cohen and Squire 1980; Squire 1992; Corkin 2002). What was missing from these early studies, however, was a way to address the neuronal mechanisms that led information to be stored as memory.The aim of this article is to show how studies of hippocampal neuronal activity during the past few decades have brought us to a point at which a mechanistic basis of memory formation is beginning to surface. An early landmark in this series of investigations was the discovery of place cells, cells that fire selectively at one or few locations in the environment. At first, these cells seemed to be part of the animal’s instantaneous representation of location, independent of memory, but gradually, over the course of several decades, it has become clear that place cells express current as well as past and future locations. In many ways, place cells can be used as readouts of the memories that are stored in the hippocampus. More recent work has also shown that place cells are part of a wider network of spatially modulated neurons, including grid, border, and head direction cells, each with distinct roles in the representation of space and spatial memory. In this article, we shall discuss potential mechanisms by which these cell types, particularly place and grid cells, in conjunction with synaptic plasticity, may form the basis of a mammalian system for fast high-capacity declarative memory.     

Dendritic Cells: Migratory Cells that are Attractive

Dendritic cells (DC) are professional antigen presenting cells, playing an important role in the initiation of T- and T cell dependent immune responses. DC are highly mobile cells and the sequential migration of DC in and out of tissues is accompanied by phenotypical as well as functional changes instrumental to their function as sentinels of the immune system. Herein, we will review recent progress in understanding the origin of DC, their migratory behaviour and their capacity to attract and interact with lymphocytes, with emphasis on the chemokine system.     

IDS Transfer from Overexpressing Cells to IDS-Deficient Cells

Iduronate sulfatase (IDS) is responsible for mucopolysaccharidosis type II, a rare recessive X-linked lysosomal storage disease. The aim of this work was to test the ability of overexpressing cells to transfer IDS to deficient cells. In the first part of our work, IDS processing steps were compared in fibroblasts, COS cells, and lymphoblastoid cell lines and shown to be identical: the two precursor forms (76 and 90 kDa) were processed by a series of intermediate forms to the 55- and 45-kDa mature polypeptides. Then IDS transfer to IDS-deficient cells was tested either by incubation with cell-free medium of overexpressing cells or by coculture. Endocytosis and coculture experiments between transfected Lβ and deleted fibroblasts showed that IDS transfer occurred preferentially by cell-to-cell contact as IDS precursors are poorly secreted by transfected Lβ. The 76- and 62-kDa IDS polypeptides transferred to deleted fibroblasts were correctly processed to the mature 55- and 45-kDa forms. Lβ were not able to internalize the 90-kDa phosphorylated precursor forms excreted in large amounts in the medium of overexpressing fibroblasts. Enzyme transfer occurred only by cell-to-cell contact, but the precursor forms transferred in Lβ after cell-to-cell contact were not processed. This absence of maturation was probably due to a mistargeting of IDS precursors in these cells.     

Oocyte-like Cells Induced from Mouse Spermatogonial Stem Cells

ABSTRACT: BACKGROUND: During normal development primordial germ cells (PGCs) derived from the epiblast are the precursors of spermatogonia and oogonia. In culture, PGCs can be induced to dedifferentiate to pluripotent embryonic germ (EG) cells in the presence of various growth factors. Several recent studies have now demonstrated that spermatogonial stem cells (SSCs) can also revert back to pluripotency as embryonic stem (ES)-like cells under certain culture conditions. However, the potential dedifferentiation of SSCs into PGCs or the potential generation of oocytes from SSCs has not been demonstrated before. RESULTS: We report that mouse male SSCs can be converted into oocyte-like cells in culture. These SSCs-derived oocytes (SSC-Oocs) were similar in size to normal mouse mature oocytes. They expressed oocyte-specific markers and give rise to embryos through parthenogenesis. Interestingly, the Y- and X-linked testis-specific genes in these SSC-Oocs were significantly down-regulated or turned off, while oocyte-specific X-linked genes were activated. The gene expression profile appeared to switch to that of the oocyte across the X chromosome. Furthermore, these oocyte-like cells lost paternal imprinting but acquired maternal imprinting. CONCLUSIONS: Our data demonstrate that SSCs might maintain the potential to be reprogrammed into oocytes with corresponding epigenetic reversals. This study provides not only further evidence for the remarkable plasticity of SSCs but also a potential system for dissecting molecular and epigenetic regulations in germ cell fate determination and imprinting establishment during gametogenesis.     

Dendritic Cells and Regulatory T Cells in Atherosclerosis

Although macrophages and other immune system cells, especially T cells, have been shown to play disease-promoting roles in atherosclerosis, less is known about the role of antigen presenting cells. Functional, immune stimulating dendritic cells (DCs) have recently been detected in aortic intima, the site of origin of atherosclerosis. We had compared DCs with macrophages in mice with experimental atherosclerosis, to clearly define cell types by developmental and functional criteria. This review summarizes recent advances in studies of DCs in humans and in mouse models of atherosclerosis, as well as providing a simple strategy to measure regulatory T (Treg) cells in the mouse aorta.