成年大鼠嗅鞘细胞与嗅觉神经成纤维细胞的原代培养及鉴定

目的 建立一种原代提取嗅鞘细胞与嗅觉神经成纤维细胞混合培养的方法.方法 自2.5月龄SD大鼠嗅球最外两层分离嗅鞘细胞和嗅觉神经成纤维细胞进行混合培养,并不进行纯化,分别于7 d、10 d、14 d行免疫细胞化学鉴定,并计算各个时间点嗅鞘细胞的纯度.结果 体外培养的嗅鞘细胞主要呈两极或多极状,而嗅觉神经成纤维细胞则成扁平的像成纤维细胞的形态,免疫细胞化学结果显示嗅鞘细胞呈p75 NGFR阳性,嗅觉神经成纤维细胞呈fibronectin阳性,两种细胞都呈vimentin阳性,在7 d、10 d、14 d各个时间点嗅鞘细胞分别占混合培养的34.1%、25.6%、8.6%.结论 从成年大鼠嗅球最外两层分离的培养中主要包含嗅鞘细胞和嗅觉神经成纤维细胞,嗅鞘细胞在混合培养中所占的比例随培养时间的延长而逐渐降低.     

神经上皮干细胞的分离培养及其体外分化特性的观察

目的探讨大鼠胚胎神经管神经上皮干细胞的分离培养条件,并观察其在体外的分化特性.方法采用显微解剖、机械吹打、无血清悬浮培养方法分离培养神经上皮干细胞,采用巢蛋白(nestin)免疫细胞化学染色技术检测神经上皮干细胞,用NSE和GFAP免疫组化染色检测并计数神经细胞和神经胶质细胞.结果大鼠胚胎神经管神经上皮干细胞在无血清培养基中可形成大量呈nestin抗原阳性细胞构成的神经球,经传代有血清培养后分化为NSE阳性和GFAP阳性细胞,其中NSE阳性细胞占细胞总数的47.7%,GFAP阳性细胞占细胞总数的39.8%.结论胎鼠神经管神经上皮干细胞在无血清培养中可增殖和传代,在有血清培养中可分化为神经细胞和神经胶质细胞,两者之比为47.7∶39.8.     

甘丙肽对嗅成鞘细胞三种神经营养因子mRNA表达的影响

实验从新生大鼠嗅球中分离出嗅成鞘细胞,进行体外培养。运用半定量RT—PCR方法检测甘丙肽对体外培养的嗅成鞘细胞中三种神经营养因子（LIF、CNTF和GDNF）mRNA表达的影响。实验以甘油醛-3-磷酸脱氢酶（G3PDH）作为内参照。结果显示：体外培养的嗅成鞘细胞表达此三种神经营养因子的mRNA;当甘丙肽作用于细胞3d后,嗅成鞘细胞中LIF和GDNF表达量明显降低,而CNTF的表达量则没有发生明显变化。     

人胚胎干细胞分化为神经干细胞诱导方法的对比研究

目的探讨人胚胎干细胞分化为神经干细胞过程中,经拟胚体（embryonic body,EB）法和直接分化法的不同效率。方法人胚胎干细胞常规培养消化后,分为两组：A组,经EB法分化;B组,添加noggin和ITSFn直接分化法。倒置相差显微镜观察细胞形态变化,RT-PCR检测细胞各阶段标志物,免疫荧光及流式细胞仪观察两组细胞Nestin阳性细胞率。神经干细胞继续分化,免疫荧光、RT-PCR法检测MAP2、GFAP表达。结果RT-PCR检测到OCT4、nestin表达。B组nestin阳性细胞率明显高于A组,差异有统计学意义（P〈0.01）,且诱导周期短于A组。神经干细胞继续分化,得到不同数量的神经元和胶质细胞,MAP2、GFAP分别阳性。结论在体外采用定向分化诱导,人胚胎干细胞不经EB,可直接定向分化为神经干细胞,且诱导效率比EB法高。因此直接分化法是一种经济实用的诱导方法。     

HCMV感染抑制人海马神经干细胞分化

研究HCMV感染对体外培养的人海马源性神经干细胞(Neural stem cells,NSCs)分化的影响。体外分离、培养人海马NSCs,应用免疫荧光方法检测其NSCs标记物-巢蛋白(Nestin)的表达。10%胎牛血清诱导NSCs贴壁分化,同时用MOI为5的HCMV AD169株感染NSCs,7d后使用激光共聚焦显微镜免疫荧光方法检测Nestin、神经胶质纤维酸性蛋白(GFAP)和HCMV即刻早期蛋白(IE)的表达,计算阳性细胞比率。本实验所培养的细胞(4～6代)95±8%表达Nestin;分化诱导7d后,感染组86±12%细胞表达IE,未感染组和感染组Nestin阳性率分别为50±19%和93±10%(t=6.03,P<0.01),GFAP阳性细胞率分别为81±11%和55±17%(t=3.77,P<0.01)。以上结果表明分化过程中的NSCs是HCMV的容许细胞;HCMV感染可以抑制NSCs的分化。     

一氧化氮促进体外培养大鼠脑室下区神经干细胞的分化

目的：探讨一氧化氮（NO）对新生大鼠体外培养的神经干细胞（NSCs）分化的作用。方法：采用常规方法分离新生大鼠脑室下区（SVZ）组织,进行NSCs体外培养。用DETA／NO作为NO供体,用L-NAME作为一氧化氮合酶（NOS）抑制剂。免疫荧光法检测NSCs标志物-巢蛋白（nestin）、神经元标志物-8Ⅲ型微管蛋白（Tuj-1）和星型胶质细胞标志物-胶质原纤维酸性蛋白（GFAP）的表达,还检测了神经元型NOS的表达。用Greiss还原法检测培养液中总NO的浓度。结果：培养的神经球均为nestin阳性、BIdu阳性和nNOS阳性。NSCs和40μmol／L、50μmol／L、60μmol／LDEFA／N0共培养5d,实验组培养液中N0浓度较对照组显著增高（P〈0．01）,相应实验组分化的神经元数和星型胶质细胞数较对照组明显增加（P〈0．01和P〈0．05）。NSCs和100μmol／L、150μmol／L、200μmol／LL-NAME共培养5d,实验组培养液中NO浓度较对照组降低（P〈0．05）,相应实验组分化的神经元数和星型胶质细胞数也较对照组减少（P〈0．05）。结论：NO能直接促进大鼠SVZ体外培养的NSCs分化。     

甘丙肽及其受体在嗅成鞘细胞中的表达及对细胞增殖的影响

本实验从新生大鼠嗅球中分离出嗅成鞘细胞,进行体外培养。运用RT-PCR方法检测甘丙肽及其受体在体外培养的嗅成鞘细胞中的表达;运用MTT法检测甘丙肽及其受体激动剂、拮抗剂对嗅成鞘细胞增殖的影响。结果显示:嗅成鞘细胞表达甘丙肽(GAL)及其受体GalR2,而不表达其他两种受体GalR1和GalR3;甘西肽及两种受体激动剂GAL1-11和GAL2-11能够明显地抑制体外培养的嗅成鞘细胞的增殖,这一效应可被非特异性甘丙肽受体拮抗剂M35所阻断。     

壮通饮对体外培养新生大鼠海马神经干细胞增殖分化的影响

探讨壮通饮对体外培养大鼠大脑海马区内源性神经干细胞(neural stem cells,NSCS)分化作用的影响。利用无血清DMEM/F12(含20 ng/mL bFGF和EGF及2%B27)体外培养技术从新生Wistar大鼠大脑海马区中培养NSCS,在NSCS分化过程中添加不同剂量(20、40和80 mg/L)壮通饮进行干预,以免疫荧光及细胞化学方法对NSCS及其分化后的细胞进行鉴定。从大脑海马区分离培养的NSCS能够表达巢蛋白(Nestin),并具有分化为神经元、星形胶质细胞及少突胶质细胞的能力。在同一接种密度条件下,不同剂量壮通饮干预组的Nestin阳性细胞表达数分别为:20 mg/L组:21.5±2.9;40 mg/L组:18.6±2.4;80 mg/L组:16.7±2.2,与对照组(24.8±3.1)比较明显减少(P0.05)。而壮通饮干预组表达神经元核心抗原(NeuN)、胶原纤维酸性蛋白(GFAP)、髓鞘碱性蛋白(MBP)的阳性细胞数较对照组明显增加(P0.05)。通过本实验证实壮通饮能诱导大脑海马区NSCS分化,具有一定的促进神经再生的作用。     

成年转基因小鼠嗅鞘细胞的培养、纯化及生物学特性

已有多项研究表明,嗅鞘细胞具有修复中枢及外周神经损伤的潜能。我们选用了表达增强型绿色荧光蛋白（enhanced green fluorescent protein,eGFP）的成年小鼠,分离其双侧嗅球嗅神经纤维层及嗅小球层细胞,体外原代培养并予以纯化。同时结合共聚焦、相差显微镜,细胞增殖分析及免疫组织化学鉴定等技术,对其生物学活性进行研究。结果表明：（1）原代培养转基因成年小鼠嗅球嗅鞘细胞（Olfactory ensheathing cells,OECs）15d后,主要存在两种不同形态和免疫组织化学特征的细胞。一种是带有长突起的双极或多极OECs,表达P75^NTR（P75 low affinity neurotrophic receptor）、S100和胶质原纤维酸性蛋白（glial fibrillary acidic protein,GFAP）。另一种则是对Thy 1．1抗体免疫反应阳性,呈扁平或内皮样形态的成纤维细胞。（2）根据不同类型细胞在未覆层的培养器皿上贴壁速度的差异,我们建立了一种简单易行、不需任何抗体或昂贵仪器的细胞纯化方法,获得了大量高纯度的OECs。（3）在连续纯化培养22d后,OECs仍能保持较高的增殖活性。本实验支持和丰富了OECs发育的相关理论,为进一步体内移植修复CNS损伤提供了理想的材料。     

嗅鞘细胞治疗脊髓损伤的研究进展

本文通过广泛查阅近几年嗅鞘细胞治疗脊髓损伤的国内外相关文献,发现嗅鞘细胞可以分泌众多的神经营养因子并表达相应受体,且能够调节星型胶质细胞的反应性,降低其神经胶质酸性蛋白和硫酸软骨素糖蛋白的表达水平,且能与其更好的融合在一起.在临床应用方面,嗅鞘细胞供应来源、应用时机及纯度正引起关注及研究.嗅鞘细胞对于治疗脊髓损伤有巨大的应用前景,对嗅鞘细胞进行基因改造、联合应用其他有促进作用的治疗方法将是未来的研究方向.     

Production of hematopoietic cells from ES cells

Murine embryonic stem (ES) cells are cell lines established from blastocyst which can contribute to all adult tissues, including the germ-cell lineage, after reincorporation into the normal embryo. ES cell pluripotentiality is preserved in culture in the presence of LIF. LIF withdrawal induces ES cell differentiation to nervous, myocardial, endothelial and hematopoietic tissues. The model of murine ES cell hematopoietic differentiation is of major interest because ES cells are non transformed cell lines and the consequences of genomic manipulations of these cells are directly measurable on a hierarchy of synchronized in vitro ES cell-derived hematopoietic cell populations. These include the putative hemangioblast (which represents the emergence of both hematopoietic and endothelial tissues during development), myeloid progenitors and mature stages of myeloid lineages. Human ES cell lines have been recently derived from human blastocyst in the USA. Their manipulation in vitro should be authorized in France in a near future with the possibility of developing a model of human hematopoietic differentiation. This allows to envisage in the future the use of ES cells as a source of human hematopoietic cells.     

Homing of annexin-labeled stem cells to apoptotic cells

Ischemic diseases are characterized by the presence of pro-apoptotic stimuli, which initiate a cascade of processes that lead to cell injury and death. Several molecules and events represent detectable indicators of the different stages of apoptosis. Among these indicators is phosphatidylserine (PS) translocation from the inner to the outer leaflet of the plasma membrane, which can be detected by annexinV (ANXA5) conjugation. This is a widely used in vivo and in vitro assay marking the early stages of apoptosis. We report here on an original method that employs PS-ANXA5 conjugation to target stem cells to apoptotic cells. Mesenchymal stem cells (MSCs) from GFP-positive transgenic rats were biotinylated on membrane surfaces with sulfosuccinimidyl-6-(biotinamido) hexanoate (sulfo-NHS-LC-biot) and then bound to avidin. The avidin-biotinylated MSCs were labeled with biotin conjugated ANXA5. Bovine aortic endothelial cells (BAE-1 cells) were exposed to UVC to induce caspasedependent apoptosis. Finally, we tested the ability of ANXA5-labeled MSCs to bind BAE-1 apoptotic cells: suspended ANXA5-labeled MSCs were seeded for 1 hour on a monolayer of UV-treated or control BAE-1 cells. After washing, the number of MSCs bound to BAE-1 cells was evaluated by confocal microscopy. Statistical analysis demonstrated a significant increase in the number of MSCs tagged to apoptotic BAE-1 cells. Therefore, stem cell ANXA5 tagging via biotin-avidin bridges could be a straightforward method of improving homing to apoptotic tissues. A. Gerasimou, R. Ramella and A. Brero contributed equally to this paper.     

Transdifferentiation of mouse BM cells into hepatocyte-like cells

BACKGROUND: During the past few years multiple studies have revealed that adult stem cells, including BM origin stem cells, can be transdifferentiated into various cell types, including hepatocyte-like cells, under proper treatments or in a suitable microenvironment. However, little is known about the mechanism of the transdifferentiation, and the treatments employed seem to be very complicated and require simplification. It is important to determine the suitable conditions in which BM cells would be efficiently differentiated into hepatocytes. METHODS: Mouse BM cells were isolated from femurs and tibias and cultured in IMDM supplemented with 10% FBS. Hepatic differentiation was induced in a differentiation medium containing 20 ng/mL HGF, 10 ng/mL FGF-4, 10 ng/mL Oncostatin M (OSM) and different concentrations of liver-injured mouse sera. The differentiated hepatic cells were characterized by the expression of liver-associated mRNA and proteins and morphologic and functional features. RESULTS: BM cell-derived polygonal cell colonies appeared after several days of culture, and these hepatocyte-like cells expressed AFP, HNF-3beta, CK19, CK18, ALB, TAT and G-6-Pase at mRNA and protein levels, and the cells also had some hepatic cellular functions, such as glycogen storage and urea production. Interestingly, suitable concentrations of sera from liver-injured mice added to this system showed strong stimulation on the in vitro transdifferentiation of mouse BM cells into hepatocytes. DISCUSSION: In the present study we have established an effective hepatic differentiation system by a combination of HGF, FGF-4, OSM and liver-injured mouse sera in vitro. Accordingly, it will be a useful resource not only for understanding the mechanisms of transdifferentiation but also for efficient amplification of hepatocyte progenitor cells of BM origin.     

Progenitor cells of the testosterone-producing Leydig cells revealed

The cells responsible for production of the male sex hormone testosterone, the Leydig cells of the testis, are post-mitotic cells with neuroendocrine characteristics. Their origin during ontogeny and regeneration processes is still a matter of debate. Here, we show that cells of testicular blood vessels, namely vascular smooth muscle cells and pericytes, are the progenitors of Leydig cells. Resembling stem cells of the nervous system, the Leydig cell progenitors are characterized by the expression of nestin. Using an in vivo model to induce and monitor the synchronized generation of a completely new Leydig cell population in adult rats, we demonstrate specific proliferation of vascular progenitors and their subsequent transdifferentiation into steroidogenic Leydig cells which, in addition, rapidly acquire neuronal and glial properties. These findings, shown to be representative also for ontogenetic Leydig cell formation and for the human testis, provide further evidence that cellular components of blood vessels can act as progenitor cells for organogenesis and repair.     

Phosphatidylserine-dependent adhesion of T cells to endothelial cells

Phosphatidylserine (PS) was exposed at the surface of human umbilical vein endothelial cells (HUVECs) and cultured cell lines by agonists that increase cytosolic Ca(2+), and factors governing the adhesion of T cells to the treated cells were investigated. Thrombin, ionophore A23187 and the Ca(2+)-ATPase inhibitor 2, 5-di-tert-butyl-1,4-benzohydroquinone each induced a PS-dependent adhesion of Jurkat T cells. A23187, which was the most effective agonist in releasing PS-bearing microvesicles, was the least effective in inducing the PS-dependent adhesion of Jurkat cells. Treatment of ECV304 and EA.hy926 cells with EGTA, followed by a return to normal medium, resulted in an influx of Ca(2+) and an increase in adhering Jurkat cells. Oxidised low-density lipoprotein induced a procoagulant response in cultured ECV304 cells and increased the number of adhering Jurkat cells, but adhesion was not inhibited by pretreating ECV304 cells with annexin V. PS was not significantly exposed on untreated Jurkat cells, as determined by flow cytometry with annexin V-FITC. However, after adhesion to thrombin-treated ECV304 cells for 10 min followed by detachment in 1 mM EDTA, there was a marked exposure of PS on the Jurkat cells. Binding of annexin V-FITC to the detached cells was inhibited by pretreating them with unlabelled annexin V. Contact with thrombin-treated ECV304 cells thus induced the exposure of PS on Jurkat cells and, as Jurkat cells were unable to adhere to thrombin-treated ECV304 cells in the presence of EGTA, the adhesion of the two cell types may involve a Ca(2+) bridge between PS on both cell surfaces. The number of T cells from normal, human peripheral blood that adhered to ECV304 cells was not increased by treating the latter with thrombin. However, findings made with several T cell lines were generally, but not completely, consistent with the possibility that adhesion to surface PS on endothelial cells may be a feature of T cells that express both CD4(+) and CD8(+) antigens. Possible implications for PS-dependent adhesion of T cells to endothelial cells in metastasis, and early in atherogenesis, are discussed.     

Separation of mitogen-induced suppressor cells of human antibody-producing cells

Human antibody-forming cells were demonstrated by a plaque in agar technique following in vitro stimulation with either pokeweed mitogen or Cowan I strain of protein A-positive Staphylococcus aureus bacteria. We evaluated the effects on this antibody formation caused by the addition of cells which had been stimulated with PH A or Con A. Both Con A and PHA cells harvested after 3 days showed strong inhibition of pokeweed-induced plaque formation. The majority of the suppression could be accounted for by a blast fraction separated on 1g sedimentation gradients from the Con A or PHA cultures. Small cells from such cultures showed inhibition of PFC when added at high ratios (1:2), but this suppressive activity diluted out much more rapidly than that of the blast cells. No helper activity was noted with either small cells or blasts. Our studies indicate a T-cell blast as the suppressive fraction in Con A- or PHA-stimulated human lymphoid cells. While this T-cell suppression applies to T-dependent responses such as antibody stimulation with pokeweed mitogen, it does not have a substantial effect on Cowan I-induced plaque-forming responses. The finding that Cowan I-induced plaques could not be inhibited by Con A or PHA blasts indicates the T independence of this response.     

Stabilization of apoptotic cells: generation of zombie cells

Apoptosis is characterized by degradation of cell components but plasma membrane remains intact. Apoptotic microtubule network (AMN) is organized during apoptosis forming a cortical structure beneath plasma membrane that maintains plasma membrane integrity. Apoptotic cells are also characterized by high reactive oxygen species (ROS) production that can be potentially harmful for the cell. The aim of this study was to develop a method that allows stabilizing apoptotic cells for diagnostic and therapeutic applications. By using a cocktail composed of taxol (a microtubule stabilizer), Zn²⁺ (a caspase inhibitor) and coenzyme Q₁₀ (a lipid antioxidant), we were able to stabilize H460 apoptotic cells in cell cultures for at least 72 h, preventing secondary necrosis. Stabilized apoptotic cells maintain many apoptotic cell characteristics such as the presence of apoptotic microtubules, plasma membrane integrity, low intracellular calcium levels and mitochondrial polarization. Apoptotic cell stabilization may open new avenues in apoptosis detection and therapy.Apoptosis, also known as programmed cell death, is central to homoeostasis and normal development and physiology in multicellular organisms, including humans.¹ The dysregulation of apoptosis can lead to the destruction of normal tissues in a variety of disorders, including autoimmune and neurodegenerative diseases (increased apoptosis) or cancer (reduced apoptosis). In addition, effective therapy of tumors requires the iatrogenic induction of apoptosis by radiation, chemotherapy or both. In particular, many antineoplasic drugs such as campothecin, a topoisomerase I inhibitor, kill tumor cells by inducing apoptosis.Apoptosis is thought to be physiologically advantageous because apoptotic cells are removed by phagocytosis before they lose their permeability barrier, thus preventing induction of an inflammatory response to the dying cells and potential harmful secondary effects. However, when massive cell death overwhelms macrophage clearance, as for example in early postchemotherapy or viral infection,² apoptotic cells may progress to secondary necrosis characterized by cell membrane degradation with spillage of intracellular contents to the extracellular milieu.³ Similarly, cells undergoing apoptosis in vitro cannot usually be cleared by phagocytes and undergo a late process of secondary necrosis.⁴In the execution phase of apoptosis, effector caspases cleave vital cellular proteins, leading to the morphological changes that characterize apoptosis. These changes include destruction of the nucleus and other organelles, DNA fragmentation, chromatin condensation, cell shrinkage, cell detachment and membrane blebbing.⁵ In apoptosis, all the degradative processes are isolated from the extracellular space by the plasma membrane that remains impermeable. However, the mechanisms involved in plasma membrane and associated protein protection from the action of caspases are not completely understood. In contrast, necrosis is accompanied by disruption of plasma membrane integrity with the subsequent release of all intracellular compounds to the intercellular space, thus inducing inflammation and more toxic effects to adjacent cells.^{6, 7}To allow the dramatic morphological changes that accompany the execution phase, an apoptotic cell undergoes a series of profound cytoskeletal breakdowns/rearrangements. Previous evidence suggests that the actomyosin cytoskeleton plays an essential role in apoptotic cell remodeling during the early events of the execution phase, whereas all other cytoskeleton elements (microtubules and intermediate filaments) are dismantled.⁸ However, during the course of the execution phase and after actininomyosin ring contraction, the actomyosin filaments are also depolymerized by a caspase-dependent mechanism. In this situation, the apoptotic cell forms a network of apoptotic microtubules that becomes the main cytoskeleton element of the apoptotic cell. The presence of microtubules in apoptotic cells has previously been reported.^{9, 10} Moreover, more recent results indicate that microtubules during apoptosis assist in the dispersal of nuclear and cellular fragments,^{11, 12} and may help to preserve the integrity of plasma membrane of the dying cell.¹³Reactive oxygen species (ROS) are also important mediators of apoptosis. ROS have been shown to play a major role in apoptosis signaling.^{14, 15, 16} Electron leak in the presence of oxygen during the process of oxidative phosphorylation make mitochondria the major endogenous source of ROS in the cell. Although mitochondria have been identified as a key player, the mechanism connecting ROS and apoptosis remains unclear.¹⁷ It has been debated whether increased ROS during apoptosis is a cause or a consequence of impaired mitochondrial function, and whether ROS are a death signal to the mitochondria or are produced as effector molecules by the mitochondria in response to apoptosis signal.^{18, 19} Hyperproduction of ROS in execution stages of apoptosis is thought to be caused by the disruption of the mitochondrial respiratory chain after release of cytochrome c into the cytosol.²⁰The main objective of this work was to develop a method for the stabilization of apoptotic cells for proper apoptosis detection or safer potential therapeutic applications. Our results show that apoptotic cells can be stabilized by a cocktail of a microtubule stabilizer (taxol), a caspase inhibitor such (Zn²⁺) and an antioxidant (coenzyme Q₁₀ (CoQ)).     

Recognition of virus infected cells by NK cells

NK cells show cytotoxicity against virus-infected cells and tumor cells and play an important role in host defense. Although mecheanism of target cell recognition by NK cells have been unclear for a long time, it has recently been elucidated that certain NK cell receptors specifically recognize virus products. Furthermore, expression pattern of NK cell receptors, which consist of activating and inhibitory receptors, determines susceptibility to virus-infection. Here, we review recent progress of mechanism of recognition of virus-infected by NK cells.