大鼠睾丸支持细胞对体外共微囊化肝细胞的双重保护作用

目的 观察大鼠睾丸支持细胞(Sertoli cell)对混合共微囊化肝细胞的营养支持和免疫豁免(immune privilege)的双重保护作用.方法 气流法制备含不同比例肝细胞与Sertoli细胞混合的微囊进行体外培养,测定培养上清中白蛋白、尿素的含量,结合形态学判断肝细胞的功能和活性,并选择细胞最佳混合比例.CCK-8法测定Sertoli细胞对肝细胞引起的脾细胞增殖的影响.结果 与纯肝细胞微囊组相比,一定比例的Sertoli细胞可促进共微囊化肝细胞的白蛋白和尿素合成(P＜0.01),肝细胞存活时间也相应延长.其中,肝细胞和Sertoli细胞以20:1混合时即可达最佳效果.在脾细胞增殖反应中,与对照组相比,微囊化肝细胞仍可引起明显的脾细胞增殖(P＜0.01),而Sertoli细胞可显著抑制这一反应.结论 Sertoli细胞与肝细胞混合共微囊化能明显改善囊内肝细胞的形态、功能与寿命,并使后者得到一定的免疫豁免保护作用.     

胚胎干细胞体外诱导分化为成血管血液干细胞的研究进展

成血管血液干细胞的概念早在20世纪就已经有人提出来,如今它的存在已经在小鼠和人的胚胎干细胞分化形成的类胚体中得到证实.在胚胎正常发育过程中,原始成血管血液干细胞具有分化形成血管内皮细胞和造血细胞2个谱系的双向分化潜能,是它们共同的前体细胞;而体外诱导分化胚胎干细胞形成成血管血液干细胞的模型为研究血管内皮细胞及造血细胞发育、分化过程和调控以及之间的关系提供了有效的途径.就近年这方面的一些研究成果作一综述. Abstract： The concept of hemangioblast was proposed a century ago. The existence of hemangioblasts has been demonstrated recently in vitro by differentiation of mouse and human embryonic stem (ES) cell into em-bryoid bodies(EBs). In the developing embryo, a common progenitor, termed "hemangioblast", generates both hematopoietic and endothelial cell lineages. The in vitro differentiation of embryonic stem cells to hemangioblast is a powerful approach for studying the commitment of the hematopoietic and endothelial lineages. This review will summarize recent development in the studies on hemangioblast.     

诱导胚胎干细胞向软骨细胞分化研究进展

胚胎干细胞因其具有体外无限增殖能力和体内外分化发育的全能性,有望为细胞治疗或组织工程化组织构建提供可靠的种子细胞来源.如何诱导胚胎干细胞向目的细胞分化是目前面临的一大难题.首先回顾软骨细胞的体内发生、发育过程,继而对目前已知的能在体外培养环境中影响胚胎干细胞向软骨细胞分化的各类因素进行分析综述,并探讨进一步的研究方向.     

小鼠睾丸支持细胞对大脑皮质细胞的体外营养作用

目的通过观察睾丸支持细胞(SCs)对大脑皮质细胞体外的营养作用,探讨移植的SCs在体内对神经组织营养作用的方式。方法将原代分离的小鼠大脑皮质细胞单独培养(对照组)或与小鼠SCs共培养(实验组),比较两组中神经元、神经干细胞(NSC)及神经胶质细胞的生长情况。结果实验组的神经元数及其突起数、神经元特异性烯醇化酶光密度(P<0.01)和神经胶质细胞数均较对照组高,两组各时期的神经元胞体面积也不相同(P<0.05),实验组于第3天出现NSC的集落形成。结论SCs在体外对大脑皮质的神经元、NSC和神经胶质细胞均有较强的营养作用,在中枢神经系统疾病的细胞移植治疗中将具有很大的应用价值。     

人胚胎干细胞建系培养及体外诱导分化的研究进展

人胚胎干细胞具有发育全能性,在特定条件下能分化成多种类型的细胞.人胚胎干细胞的研究对人胚胎发育机制、人基因功能研究和治疗性克隆有着重大的意义.本文从人胚胎干细胞建系、培养及体外诱导分化等方面作一综述.     

Phenotypical changes of embryonic chick adrenal medullary cells in vitro induced by nerve growth factor and ciliary neuronotrophic factor

This study investigates the survival properties and changes in the morphological phenotype of adrenal medullary (chromaffin and neuronal) cells cultured from embryonic chicks at different developmental ages (embryonic days E8 to E16) in response to nerve growth factor (NGF) and ciliary neuronotrophic factor (CNTF). The 4-day survival of medullary cells from all embryonic ages except E8 was about 80% of the seeded cells and was only slightly enhanced by the addition of saturating doses of CNTF (10 ng/ml). With no factors, after 4 days 10-30% of the surviving medullary cells extended neurites. NGF (100 ng/ml) and, even more, CNTF (10 ng/ml) and their combination substantially increased the proportions of neurite-bearing cells (up to 70%). The effect of the factors were maximal at E10 and E12 and declined at older developmental ages. Neurite growth was virtually unaffected by NGF and CNTF at E8. These results show that in vitro survival and neurite growth of chick adrenal medullary cells in response to trophic factors is developmentally regulated.     

比较体外不同条件诱导胚胎干细胞分化为心肌细胞的实验研究

目的:比较体外不同分化条件下胚胎干细胞(ESC)分化为心肌细胞的分化比率。方法:用布法罗大鼠肝细胞(BRL)条件培养基抑制ESC分化及保持其增殖,以维甲酸(RA)、二甲基亚砜(DMSO)、转化生长因子β₁(TGF-β₁)及激活素-A(activin-A)为分化诱导剂,组合成6种分化培养基,采用悬滴悬浮、贴壁三步法诱导ESC分化为心肌细胞,比较各组分化比率。结果:6种分化条件均能使ESC分化为心肌细胞,其中以TGF-β₁(2 μg/L)、activin-A(20 μg/L)及20%胎牛血清(FCS)组成的分化培养基可以使高达92.80%±2.22%的ESC分化为心肌细胞,分化比率显著高于其它各组(P＜0.01)。结论:以BRL条件培养基培养,TGF-β₁、activin-A作分化诱导剂可作为一种理想的ESC体外分化体系。     

体外诱导小鼠胚胎干细胞分化为心肌细胞的初步研究

目的：5-氮胞苷作为分化诱导剂，初步探讨其单独或联合全反式维甲酸应用时对小鼠胚胎干细胞（mESC）分化为心肌细胞的影响，旨在建立一种体外诱导mESC分化为心肌细胞的实验方法。方法： 采用 MTT法确定5-氮胞苷的非细胞毒性参考剂量。设计不同条件培养基（5-氮胞苷单独或配伍全反式维甲酸应用）对mESC 进行诱导分化，并通过免疫组化技术及RT-PCR方法等对分化细胞进行鉴定。结果： 5-氮胞苷的非细胞毒性参考剂量为8 μmol/L，能够诱导mESC分化为心肌合胞体（与阴性对照组比较，P<0.01），诱导分化率可达50%。配伍全反式维甲酸持续诱导的结果等同于单独应用全反式维甲酸的作用效果（P>0.05）：即对ESC向心肌细胞的诱导分化没有促进作用。结论：5-氮胞苷能够诱导mESC分化为心肌合胞体，从而得以建立一种体外诱导mESC分化为心肌细胞的方法。     

Probing glucocorticoid-dependent osteogenesis in rat and chick cells in vitro by specific blockade of osteoblastic differentiation with progesterone and RU38486

Glucocorticoids and sex-steroids can modulate osteogenesis in vivo and in vitro. Although the effects of glucocorticoids on bone cells in vitro have been described in detail, the role of sex-steroids is not as well defined. We examined whether sex-steroids influence bone metabolism indirectly by regulating glucocorticoid effects on bone. Interactions of the sex-steroid progesterone or its analog RU38486 with the glucocorticoid dexamethasone (dex) were studied in functional assays of osteogenesis. Three osteoblastic models were evaluated:(1) the rat bone marrow stromal cell (RBMC) nodule system; (2) the chick periosteal osteogenesis (CPO) model; and (3) ROS 17/2.8 cells. RU38486, progesterone, and unlabelled dex competitively inhibited ³H-dex uptake by ROS 17/2.8 cells as well as its (³H-dex) binding to cytosol preps. Both RU38486 and progesterone inhibited dex-induced increases in alkaline phosphatase in CPO cultures, in RBMC cultures, and in ROS 17/2.8 cells. Dex-induced decreases in cell proliferation in ROS 17/2.8 cells were reversed by RU38486 but dex-induced increases in proliferation in the CPO model were not affected. In CPO cultures, dex-induced increases in collagen synthesis were inhibited completely by RU38486 and progesterone, Dex-dependent nodule formation in the RBMC was blocked by RU38486. Both RU38486 and dex mediated reduction of calcium uptake in the CPO model but did not affect mineralized tissue area. The data indicate that RU38486 and progesterone competitively inhibit dex-mediated stimulation of osteogenesis in vitro; this inhibition is exerted on early but not late stage differentiation events of osteoprogenitor cells. © 1995 Wiley-Liss, Inc.     

IL-18在体外培养系统中诱导肿瘤快速杀伤效应及肿瘤特异性CTL

目的应用rhlL-18在体外培养系统(Coculture system in vitro，CCs)中诱导快速肿瘤杀伤效应及诱导肿瘤特异性细胞毒性T淋巴细胞(Cytotoxic T Lymphocyte，CTL)。方法 采用StemSep^TM免疫磁性细胞分离法分离人外周血NK细胞、T细胞及树突细胞(DCs)，流式细胞仪分析细胞表型，125I-UdR标记的细胞毒实验检测杀伤活性，ELISA方法检测IFN-7的产生量。结果 在CCs中，rhIL-18诱导出快速肿瘤杀伤效应，这种杀伤效应无抗原特异性、不受MHC限制，DCs和T细胞的存在与否对其无明显影响。在同一培养系统中，肿瘤抗原存在的条件下，96h后，rhIL-18能够诱导并促进CTL介导的肿瘤特异性杀伤效应。结论 rhIL-18能够在体外培养系统中相继诱导肿瘤快速杀伤效应及肿瘤特异性CTL。     

胚胎干细胞体外分化为心肌细胞诱导因素的探讨

目的:用胚胎干细胞(ESC)体外诱导分化为心肌细胞,从分子水平上研究早期心脏发育相关基因及其功能。方法:(1)胚胎干细胞的培养。(2)胚胎干细胞的分化培养。(3)被分化的心肌细胞鉴定:RNA的提取;心脏特异性引物的合成和RT-PCR反应;探针标记、纯化和比放射活性测定;RNA斑点杂交。结果:用最适条件培养液对ESC定向诱导分化,可使80%以上的ESC分化为心肌细胞。心肌细胞以同步的方式进行收缩。反转录PCR和斑点杂交的结果显示:心肌在早期发育就开始表达其特异性基因。结论:胚胎干细胞体外能诱导分化为心肌细胞。胎牛血清、二甲基亚砜和维甲酸的浓度及它们之间的组成不同,对ES细胞定向诱导为心肌细胞均有影响。最佳诱导条件是用2 nmol/L维甲酸、0.6%二甲基亚砜和20%胎牛血清组成的条件培养液。     

大鼠骨髓间充质干细胞与Sertoli细胞体外共培育的免疫负调作用

目的 探讨骨髓间充质干细胞（BMSCs）与Sertoli细胞（SCs）体外共培育时对免疫应答的调节作用,为两者联合应用于移植修复提供线索.方法 用密度梯度离心法分离SD大鼠脾脏单个核细胞（简称L）,羧基荧光素二醋酸盐琥珀酰亚胺酯（CFSE）标记细胞后,将其加入按照不同的比例混合的BMSCs与SCs共培育体系,设立L细胞为对照组,L＋刀豆蛋白A（ConA）、BMSCs＋L＋ConA、SCs＋L＋ConA、BMSCs＋SCs＋L＋ConA四大组细胞为实验组.培养4 d,通过流式细胞术分析T淋巴细胞的增殖情况.结果 单个核细胞培养体系中加入ConA可以显著刺激T淋巴细胞增殖.BMSCs、SCs均可抑制淋巴细胞增殖,抑制作用随加入BMSCs和SCs的比例增加而增强,BMSCs和SCs混合培养后对淋巴细胞增殖抑制作用进一步增强,且在某些浓度时呈现协同性.结论 BMSCs与SCs体外共培育时可增强免疫抑制作用.     

体外定向诱导小鼠胚胎干细胞发育为造血干/祖细胞方法的初步研究

目的:探讨体外定向诱导胚胎干细胞(ESC)发育为造血干细胞(HSC)的方法。方法:将小鼠E14胚胎干细胞在含干细胞生长因子(SCF)和血管内皮生长因子(VEGF)的甲基纤维素培养基中首先诱导发育为胚胎体(EB),再将EB置于均含SCF、VEGF、IL-3、IL-6及促红细胞生成素(EPO)的3种不同培养体系中定向分化为HSC,并观察HSC表面标志性抗原、造血集落形成及瑞氏-姬姆萨染色的结果。结果:经两阶段诱导ESC分化为HSC,发现在甲基纤维素半固体培养体系中HSC发育缓慢,分化14d后CD34+/Sca-1+细胞数最高为(31.5±4.7)%;而在骨髓基质细胞饲养层上HSC发育较快,细胞数量较多,分化第10dCD34+/Sca-1+细胞数即达到峰值,为(47.8±6.3)%;骨髓基质细胞饲养层+胎肝基质细胞上清培养体系中HSC发育同样迅速,所产生的CD34+/Sca-1+细胞数量在3个体系中最高,为(53.6±7.2)%。经瑞氏-姬姆萨染色证实上述细胞为早期造血细胞,均有形成各系造血细胞集落的能力。结论:使用骨髓基质细胞饲养层+胎肝基质细胞上清培养体系及SCF、VEGF、IL-3、IL-6及EPO等细胞因子,通过两阶段诱导分化,可从小鼠ESC获得较高比例的HSC。     

星形胶质细胞条件培养液促进胚胎干细胞分化为神经元样细胞的实验研究

目的:探讨星形胶质细胞条件培养液在体外能否促进胚胎干细胞（ESCs）向神经元方向分化。 方法:在Bain等建立的4-/4+方案基础上，分成两组：单用ATRA组和ATRA联合胎脑星形胶质细胞条件培养液组，分3阶段诱导ESCs定向生成神经元样细胞。形态学观察及畸胎瘤形成试验对ESCs的全能性进行鉴定；免疫组化染色法检测nestin、GFAP、NSE和NF-200的表达对诱导过程进行动态监测。 结果:（1）体外培养的小鼠ESC-D3细胞在胚胎成纤维饲养层细胞上连续传代培养，仍保持向3胚层分化的能力。（2）在诱导分化阶段，联合诱导组分化细胞NF-200和NSE的表达阳性率高于单用ATRA组。（3） 联合诱导组最终诱导ESCs生成纯度高达73.5%神经元样细胞。 结论:采用胚胎脑星形胶质细胞条件培养液可较高产率及纯度地促进ESCs生成神经元样细胞，值得推广应用。     

Changes in the arrangement of actin filaments in myoid cells and sertoli cells of rat testes during postnatal development and after experimental cryptorchidism

Background: Abundant actin filaments are present in myoid cells and Sertoli cells in the testis. In the adult rat, the filaments form a lattice arrangement within the myoid cell, and show a hexagonal pattern in the basal junctional regions of Sertoli cells. Methods: Isolated seminiferous tubules and frozen sections were prepared from juvenile to adult Wistar rat testes, stained with FITC-conjugated phalloidin, and observed by confocal microscopy. Unilateral cryptorchidism was induced in adult rats, and seven days later, their testes were also examined. Results: In the myoid cell, parallel actin filaments running circularly around the seminiferous tubules were observed at 15 and 20 days of age. Then, at 30 days, actin filaments arranged longitudinally along the tubular long axis appeared in addition to the circular bundles. A lattice arrangement of actin-filament bundles in myoid cells became obvious at 40 days, when elongated spermatids are found in the tubule. Actin filaments in the basal junctional regions of Sertoli cells did not acquire the hexagonal pattern seen in the adult testis until 30 days of age. In the cryptorchid testes, the arrangement of actin filaments in the both cells showed a remarkable change compared to the control testis; the filaments became thinner and disrupted. Conclusions: A lattice arrangement of the actin filaments in the myoid cell appear at around 30 days, before the completion of spermatogenesis. A hexagonal pattern of the filaments in the junctional regions of Sertoli cells has already developed at this age. Cryptorchidism affects the actin filaments of the both cells. © 1995 Wiley-Liss, Inc.     

Expression of HNK1 epitope by the cardiomyocytes of the early embryonic chick: In situ and in vitro studies

Monoclonal antibody HNK1 reacts with a carbohydrate epitope in cell surface glycoproteins and glycolipids. During development, in various species the HNK1 epitopes are expressed in migrating neural crest cells and in the developing conduction cardiomyocytes. The conduction system is generally thought to be developed from cardiomyocytes, but some investigators have hypothesized that it is derived from the neural crest because conduction myocytes express neural antigens, including HNK1. Using immunohistochemistry, we examined the spatiotemporal expression of HNK1 in early chick cardiogenesis (stages 4 to 18) and whether cultured precardiac mesoderm does or does not express HNK1 as well as sarcomeric myosin (MF20). HNK1 was first expressed in the premyocardium at stage 8. At stage 10, HNK1‐positive cardiomyocytes were scattered along the straight heart tube. By stage 18, HNK1‐positive cardiomyocytes had become restricted to the atrium and sinus venosus. Atrioventricular cushion mesenchyme also expressed an HNK1 epitope. Immunostaining of HNK1 and MF20 in cultured precardiac mesoderm showed that there are at least three types of cells: 1) cardiomyocytes without HNK1 expression, 2) cells possessing both HNK1‐ and MF20‐immunoreactivity, and 3) mesenchymal cells with HNK1. Immunogold electron microscopy showed that cardiomyocytes containing sparsely distributed myofibrils associated with the Z‐band react with anti‐HNK1 antibody. Our observations showed a direct evidence for the first time that the precardiac mesoderm generates HNK1‐positive cardiomyocytes with morphological features similar to those of conduction cardiomyocytes. Anat Rec 263:326–333, 2001. © 2001 Wiley‐Liss, Inc.     

Bio-engineering insulin-secreting cells from embryonic stem cells: A review of progress

According to the Edmonton protocol, human islet transplantation can result in insulin independency for periods longer than 3 years. However, this therapy for type 1 diabetes is limited by the scarcity of cadaveric donors. Owing to the ability of embryonic stem cells to expandin vitro and differentiate into a variety of cell types, research has focused on ways to manipulate these cells to overcome this problem. It has been demonstrated that mouse embryonic stem cells can differentiate into insulin-containing cells, restoring normoglycaemia in diabetic mice. To this end, mouse embryonic stem cells were transfected with a DNA construct that provides resistance to neomycin under the control of the regulatory regions of the human insulin gene. However, this protocol has a very low efficiency, needing improvements for this technology to be transferred to human embryonic stem cells. Optimum protocols will be instrumental in the production of an unlimited source of cells that synthesise, store and release insulin in a physiological manner. The review focuses on the alternative source of tissue offered by embryonic stem cells for regenerative medicine in diabetes and some key points that should be considered in order for a definitive protocol forin vitro differentiation to be established.     

体外培养系统中IL-18诱导的快速肿瘤杀伤效应影响因素的研究

目的：应用rhIL-18在体外培养系统(Coculture system in vitro，CCs)中诱导快速肿瘤杀伤效应，并对这种快速杀伤效应的影响因素进行了探讨。方法：采用Stem Sep^TM免疫磁性细胞分离法分离人外周血NK细胞、T细胞及树突状细胞(DCs)，流式细胞仪分析细胞表型，^125I-UdR标记的细胞毒实验检测杀伤活性。结果：rhIL-18在CCs中能够诱导快速肿瘤杀伤效应，这种杀伤效应与rhIL-18含量呈剂量依赖关系，DCs和T细胞的存在与否对这种杀伤效应无明显影响，同时这种快速杀伤效应无抗原特异性、不受MHC的限制。结论：在体外培养系统中只要有rhIL-18和NK细胞的存在，即能产生对肿瘤细胞的快速杀伤效应，即这种快速杀伤效应细胞为NK细胞。