SD大鼠视网膜神经细胞培养上清液对胚胎干细胞体外分化的诱导作用

目的 探讨视网膜神经细胞培养的上清液对胚胎干细胞（embryonic stem cells, ES）体外分化的诱导作用。 方法 收集SD大鼠视网膜神经细胞培养上清液，抽滤后按2∶3比例与DMEM培养液混合，用该混合液进行ES 细胞的诱导分化，每天倒置相差显微镜观察ES细胞的生长及分化情况,对诱导分化后的细胞进行神经丝蛋白(nellcofilament protein，NFP)免疫组织化学检查。 结果 加入了视网膜神经细胞培养上清液的ES细胞生长出类似神经细胞突起样结构，NFP免疫组织化学染色阳性。 结论 SD大鼠视网膜神经细胞培养的上清液具有诱导ES细胞向神经细胞分化的作用。 (中华眼底病杂志, 2002, 18: 134-136)     

蛋白激酶Cα在小鼠胚胎干细胞定向分化中对发育基因表达的调控作用

目的探讨蛋白激酶Cα(PKCα)对小鼠胚胎干细胞(ESCs)定向诱导分化中发育基因(pax-6、slit-2、netrin-1)表达的影响.方法 ES-BALB/c细胞株用无白血病抑制因子(mLIF)的ESCs培养液培养4 d,形成胚胎体(EBs),再分别种植于预置有盖玻片的6孔板和直径100 mm的培养皿,经5×10-7 mol/L视黄酸 (RA)诱导后,用神经细胞特异性抗原NSE和NF-200来鉴定分化细胞的类型,在诱导后1、3、5、7及14 d用逆转录聚合酶链反应(RT-PCR)方法测定细胞的PKCα和发育基因的mRNA表达情况 ,并观察了PKC激活剂佛波酯(PMA)和PKC抑制剂D-鞘氨醇对它们表达的影响.结果免疫组化发现在诱导后第3 d大多数细胞NSE和NF-200染色阳性,RT-PCR 证实诱导后1 d PKCα、pax-6和netrin-1的mRNA表达急剧下降,3～7 d逐渐升高,至14 d恢复至正常水平,而slit-2在诱导前后均无明显表达.PMA可使PKCα、 pax-6、netrin-1 的mRNA水平在诱导后细胞中的表达明显增强,而D-鞘氨醇的作用相反, 使它们的表达显著降低,而对slit-2无明显影响.结论发育基因pax-6和netrin-1在ESCs定向分化为神经样细胞中有重要的调控作用,可能是通过PKCα信号通路起作用.(中华眼科杂志,2005,41123-127)     

蛋白激酶Cα在小鼠胚胎干细胞诱导分化为神经样细胞中的表达变化

目的研究小鼠胚胎干细胞(embryonic stem cells,ESC)定向诱导分化为神经样细胞过程中蛋白激酶Cα(protein kinase Cα,PKCα)的表达变化,探讨可能涉及ESC定向分化的信号机制.方法ES-D3细胞株用不含小鼠白血病抑制因子(mouseleukemiainhibitoryfactor,mLIF)的ESC培养液培养4 d,形成胚胎体(embryoid bodies,Ebs),再分别种植于预置有盖玻片的6孔板和直径100mm的培养皿,经5×10-7 mol/L维甲酸(retinoic acid,RA)诱导后,用神经特异性烯醇化酶(NSE)和NF-200来鉴定分化细胞的类型,在诱导后第1、3、5、7和14天用Western印迹和逆转录聚合酶链反应(RT-PCR)方法检测PKCα亚型的表达变化.结果诱导前免疫组化发现PKCα在ES-D3细胞有广泛的棕黄色的阳性表达,以细胞浆和细胞膜最明显;Western印迹发现PKCα出现一条蛋白印迹条带,分子量约为84kD;诱导后第3天,NSE和NF-200开始表达,第7天达到高峰;诱导后Western印迹和RT-PCR方法都显示PKCα表达量急剧下降,以后逐渐升高,至第14天基本恢复至正常水平.结论在RA诱导ESC定向分化为神经样细胞过程中,PKCα有独特的时空表达特点,它可能对ESC的分化有非常重要的调控作用.     

角膜基质诱导胚胎干细胞定向分化的初步实验研究

目的:探索角膜基质接触诱导胚胎干细胞(embryonic stem cells,ES.细胞)定向分化的可能性。方法:分别在去上皮的新西兰白兔表层角膜缘基质上、晶状体上皮细胞饲养层上培养ES-D_3细胞,裸鼠皮下移植使其形成复层细胞,一段时间后,行扫描电镜和光镜观察。结果:I.ES细胞在新鲜表层角膜缘基质上增殖缓慢,2～3周形成较大细胞集落,光镜下细胞形态单一,体积较正常ES细胞大,电镜下细胞核已演变为细长形,移植到裸鼠皮下2周形成上皮样细胞复层,电镜下可见微绒毛,而角膜基质非上皮面的ES细胞仍保持其小细胞状态不变。2.晶状体上皮细胞与ES细胞共培养只能延缓其分化时间,不能诱导其定向分化。结论:表层角膜缘基质具有诱导ES细胞定向分化的潜能,体外培养条件下可能不发生晶状体诱导的第三级胚胎诱导。眼科学报1999;15:195-198。     

Differentiation of Embryonic Stem Cells into Neurons and Retina—like Structure in Nude Mice

Purpose: To investigate the intraocular growth and biological characteristics of mice embryonic stem cells in nude mice.Methods: Murine embryonic stem cells (D3 cell line) were cultured and maintained in an undifferentiated state in vitro, then transplanted into the anterior chamber of nude mice. Mophological and immunohistochemical examinations were implemented. Results: Two to three days after transplantation, yellow-white floating granules, sheets and masses were seen inside the anterior chamber and vitreous cavity, and enlarged gradually. 14 - 20 days later, the mice were executed. Morphological examination showed that there were undifferentiated cells and some round or polygonal differentiated cells in anterior chamber and vitreous cavity. The morphology of these differentiated cells were similar to that of the retina. The cells were highly positive in NSE staining. Conclusion: The tranplanted embryonic stem cells could grow in the eyes of nude mice with tendency to differentiate into neurons and r     

骨髓间充质干细胞移植治疗视网膜病变

目前,对视网膜病变的治疗仍缺乏有效的药物.为解决视网膜损伤的修复问题,有研究者用胚胎干细胞、胚胎视网膜祖细胞、成体哺乳动物眼干细胞等进行了实验,但这些方法都存在着伦理学和移植学上的不足.骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSC)是骨髓中一类具有多向分化潜能的细胞群,在体外可诱导分化为神经元样细胞,进行BMSC移植,为视网膜病变的治疗提供了一条新的途径.现就BMSC生物学特征、体外的分离和纯化、向神经元样细胞的诱导分化及在视网膜病变治疗中的研究进展作一综述.     

视黄酸联合视网膜细胞共培养对胚胎干细胞体外分化的诱导作用

目的 探讨胚胎干细胞(ESC)在视黄酸联合视网膜细胞共培养诱导条件下的分化特征。方法 将ESC自液氮中复苏、培养，传1代后进行拟胚体培养。将部分3．5d拟胚体离心重悬后加入含有视黄酸的24孔板中进行诱导，另一部分加入已经培养有视网膜混合细胞的培养瓶中，培养液中同时也加入视黄酸。视网膜混合细胞中仅加入视黄酸未加拟胚体作为对照。倒置相差显微镜下观察细胞在整个诱导过程中形态学的改变并使用免疫细胞化学检测诱导细胞中巢蛋白、胶质纤维酸性蛋白、广谱细胞角蛋白、微管相关蛋白-2、视紫质蛋白表达情况。结果 (1)形态学改变仅由视黄酸诱导，诱导细胞呈多种形态；在共培养条件下，绝大多数拟胚体分化出来的细胞形态非常单一，呈透明圆形。部分原贴壁的视网膜细胞出现了明显的网状结构。(2)免疫细胞化学显示两种诱导条件均可见大部分诱导细胞MAP-2阳性，并可见Nestin阳性细胞。共培养诱导尚可见GFAP阳性、Cytokeratin阳性和Rhodopsin阳性的细胞。结论 视黄酸可以诱导大部分细胞成为神经细胞，在视黄酸和与视网膜细胞共培养诱导条件下，可以获得更为纯化的形态一致的神经样细胞，部分诱导细胞表达视网膜细胞的特性。     

Pluripotent Embryonic Stem Cells Developed into Medulloepithelioma in Nude Mice Eyes

Purpose:The pluripotent embryonic stem cells can differentiate into various kinds of normal tissues.There is no previous preport on the differentiation of embryonic stem cell in the intraocular environment .In this paper,the authors tried to investigate the intraocular growth character of mice embryonic stem cells in nude mice.Methods:Murine embryonic stem cells were cultured and maintained in anundifferentiated state in vitro.They were transplanted into the right eyes of 20 nude mice by microinjection under operating microscope.Animal eye observation,light microscope and immunohistochemical examinations were implemented.Results:Two to three days after transplantation,Small pieces of gray-white material could be viewed in the vitreous cavity.Between the 15th and 20th day,the gray-white mage grew into the anterior chamber in 4 nude mice eyes.Then,the mass at the anterior chamber extended extraocularly.On the 30th day,a remarkable proptosis was observed in two of the four nude mice.In 6 to 45 days,the mice were exeuted for morphological examination which showed the following typical structures:(1) Undifferentiated cells with prominent nucleolius.(2) Flexner-Wintersteiner-like rosettes.(3) Medulloepithe-liome-like structure:the cells were arranged in sheets,cords,tubes,and cysts.(4)Large,spindle-or astrocyte-like cells.(5) Cartilage-like structure,Immunohistochemically,most of the cells were highly positive in NSE staining and a few cells were moderately positive in GFAP staining.Conclusions:Both animal eye findings and morphologic examinations certificated that the transplanted embryonic stem cells could grow in the eyes of nude mice and dofferentiate into intraocular medulloepithelima.     

Revisiting nestin expression in retinal progenitor cells in vitro and after transplantation in vivo

The purpose of this study is to characterize the co-expression of nestin--a neuroectodermal stem cell and a reactive glial marker-with various mature retinal cell markers in retinal progenitor cells (RPCs) expanded in vitro, followed either by in vitro induction or subretinal transplantation. Rat RPCs derived from embryonic day (E) 17 rat retina were expanded in serum free defined culture, and induced to differentiate by all-trans retinoic acid (RA). Following induction, cells were stained for nestin in combination with retinal neuronal and glial markers. Cultured cells were collected for quantitative RT-PCR gene expression analysis prior to and after induction. In a second series, passage 2 RPCs were transplanted into the subretinal space of S334ter-3 retinal degeneration rats at postnatal day 28. After 1-4 weeks, sections through the transplant were double immunostained for nestin and various retinal specific neuronal markers. The cultured RPCs treated with RA exhibited nestin co-expression with various retinal specific markers, including protein kinase C alpha (PKC), neurofilament 200 (NF200), cellular retinaldehyde binding protein (CRALBP), and rhodopsin. Following RA induction, quantitative RT-PCR analysis demonstrated downregulation of nestin, PAX-6, thy1.1, and PKCalpha, and upregulation of rhodopsin, glial fibrillary acidic protein (GFAP), and CrX. No nestin coexpression was observed with any of the retinal specific neuronal markers in RPC transplants in vivo except for some nestin-immunoreactivity overlapping with GFAP positive cells in the host retina. The role of nestin as a unique neural stem/progenitor cell marker should be reconsidered. Nestin expression during RPC maturation appears to be different in vitro versus in vivo.     

Induction of the differentiation of lentoids from primate embryonic stem cells

PURPOSE: To produce lens cells from primate embryonic stem (ES) cells in a reproducible, controlled manner. METHODS: Cynomologus monkey ES cells were induced to differentiate by stromal cell-derived inducing activity (SDIA). The lentoids produced by this treatment were processed for immunohistochemical and immunoblotting analysis. The effect of varying the concentration of fibroblast growth factor (FGF)-2 and the density of the ES colonies plated during the differentiation process were also examined. RESULTS: After a 2- to 3-week induction period, lentoids were produced by a subpopulation of ES colonies. Western blot analysis and immunohistochemistry revealed that these lentoids expressed alphaA-crystallin and Pax6. The number of lentoids resulting from treatment increased with increasing FGF-2 concentration and plated colony density. CONCLUSIONS: The differentiation of primate ES cells into lentoids can be achieved by treatment with SIDA. ES cells can be used to facilitate a greater understanding of the mechanisms functioning in differentiation in vivo and in vitro.     

视黄酸联合视网膜细胞培养上清液对胚胎干细胞体外分化的诱导作用

目的：探讨经过初步诱导的拟胚体在视黄酸和视网膜细胞培养上清液作用下的分化特征。方法：将胚胎干细胞(embryonic stem cells,ESC)自液氮中复苏、培养，传1代后进行拟胚体(embry-onic bodies,EB)培养。3天半的EB离心重悬后不作消化，使用视黄酸、视黄酸加鼠视网膜胶质细胞和神经细胞培养上清液、视黄酸加人视网膜色素上皮培养上清液、视黄酸加人胎儿视网膜胶质细胞培养上清液(分别记为A、B、C、D组)等进行诱导。观察诱导过程中形态学改变，培养3周时使用免疫细胞化学检测Nestin、GFAP、cytokeratin、MAP-2、rhodopsin等在诱导后细胞中的表达情况。结果：(1)形态学改变：4种条件下的早期改变基本相同，均可见多种形态的细胞；3周后：拟胚体结构基本已散开，A组：见多种形态的细胞，细胞边界欠清，饱满度下降；B组：细胞以透明度较高的圆形细胞为主；C组：拟胚体及拟胚体周围的细胞中出现了含有明显色素的细胞；D组诱导：细胞胞体较大，形态结构较为单一；(2)免疫细胞化学：4种条件下均表现为大部分细胞MAP—2阳性，未见Nestin阳性细胞；此外，A组中，未见GFAP、Cytokeratin、Rhodopsin阳性细胞；B组中，可见GFAP、Cytokeratin、Rhodopsin阳性细胞；C组仅见Cytokeratin阳性细胞；D组仅见GFAP阳性细胞。结论：在KSC的次级诱导中，视黄酸可以诱导大部分细胞成为神经细胞，多种视网膜细胞的上清液在诱导中具有一定的作用，ESC能被诱导形成与上清液来源细胞相关的细胞。眼科学报2003；19：122-125     

我国眼内干细胞研究现状与展望

干细胞为机体组织中的一个极小的未分化细胞亚群，具有增生、自我维持、自我更新、能产生大量具有功能的子代细胞。对胚胎干细胞、人成体视网膜干细胞及眼内肿瘤干细\胞研究发现，人胚胎及成体视网膜均存在有干细胞，眼内肿瘤如视网膜母细胞瘤、脉络膜黑色素瘤内也有肿瘤干细胞的存在；将不同干细胞移植到视网膜下或玻璃体腔，均能分化成神经元或视网膜结构。干细胞有望成为疾病发病机制及治疗研究突破的最新靶点。(中华眼底病杂志, 2007, 23: 83-86)     

胚胎干细胞在裸鼠眼内向神经元和视网膜样结构分化

目的 研究小鼠胚胎干细胞在裸鼠眼内的生长特性。 方法 将未分化的胚胎干细胞移植到Balb/c裸小鼠眼内。14～20 d处死裸鼠,观察其形态学和免疫组织化学变化。 结果 胚胎干细胞移植到裸鼠眼内,在眼前房和玻璃体腔内能见到黄白色物,逐渐增大。光镜检查：在前房和玻璃体腔内均能见到未分化细胞和已分化的细胞,部分细胞的形态和排列方式与视网膜组织中的一些结构相似,其免疫组织化学抗神经元特异性烯醇化酶染色大部分已分化细胞呈强阳性反应。 结论 胚胎干细胞在裸小鼠眼内的特定环境中,向神经元和视网膜样结构分化。（中华眼底病杂志,2000,16：213-284）     

An in vitro mouse model for retinal ganglion cell replacement therapy using eye-like structures differentiated from ES cells

The aim of this study was to investigate the developmental potential of embryonic stem (ES) cell-derived eye-like structures as a replacement cell therapy model for retinas with N-methyl-d-aspartate (NMDA)-induced damage. For this purpose mouse ES cells were induced to differentiate into eye-like structures in vitro for 10 days and co-cultured with adult mouse retina treated with or without NMDA treatment. NMDA induces excitotoxic neuronal cell death in the inner neural retina, specifically within the ganglion cell layer. After 10 days of co-culture, the specimens were fixed, embedded in paraffin wax and analyzed by immunohistochemistry. Transplanted eye-like structures differentiate into Tuj1-positive neurons when co-cultured with an adult mouse retina and the cells then migrate into the ganglion cell layer. When co-cultured with an NMDA-treated retina, most of the cells migrating into the ganglion cell layer express the ganglion cell-specific markers Hu and Brn3b. Murine ES cell-derived eye-like structures contain cell populations that can differentiate into ganglion-like cells in the host ganglion cell layer in vitro. Moreover, their contribution to the ganglion cell layer was more prominent when ganglion cell specific damage was induced by NMDA administration. These findings suggest that cells prepared from the eye-like structures generated from ES cells may be useful for cell replacement therapy and may also serve as a model system for such therapies.     

大鼠尾芽胚视杯干细胞的分布与特征

目的探索视杯干细胞在大鼠尾芽胚(第12.5天胚龄)的分布与特征。方法采用免疫组织化学技术，检测视杯干细胞在大鼠尾芽胚(第12.5天胚龄)视杯组织中的分布；分离视杯细胞，体外无血清培养，应用免疫细胞化学技术分析其增生能力以及血清诱导分化前后CHX10和多种成熟视网膜细胞特异性标记蛋白的表达，以了解这一发育时期视杯组织的分化特点。结果大鼠尾芽胚(第12.5天胚龄)的视杯干细胞主要分布在视杯的内外层和边缘层，不表达成熟视网膜细胞特异性标记蛋白。从尾芽胚视杯中分离出的细胞具有单细胞克隆能力，CHX10表达阳性，血清诱导后表达多种成熟视网膜细胞特异性标记蛋白：Thy1.1、神经胶质纤维酸性蛋白(GFAP)、蛋白激酶C(PKC)α和rhodopsin。结论大鼠尾芽胚(第12.5天胚龄)视杯主要由未分化的细胞组成，视杯干细胞的分布集中在视杯内层和边缘层。体外培养的视杯干细胞增生能力强，经诱导分化后表达多种成熟视网膜细胞特异性标记蛋白。(中华眼底病杂志,2005,21:159-162)     

无动物源性成分培养体系快速诱导人胚胎干细胞向视网膜色素上皮细胞的分化

背景 随着视网膜色素上皮(RPE)细胞视网膜下腔移植治疗年龄相关性黄斑变性(AMD)研究的开展,需要优化无动物源性成分(xeno-free)培养体系快速定向诱导人胚胎干细胞(hESCs)向RPE细胞分化以满足日渐增长的科研及临床需要. 目的 建立xeno-free培养体系,优化快速诱导hESCs向RPE细胞分化的方法. 方法 将hESCs克隆团接种至Vitronectin XFTM,在培养液中加入50 ng/ml noggin、10 ng/ml DKK-1以及10 ng/ml胰岛素样生长因子-1(IGF-1)培养2d,第2～4天将培养液中noggin质量浓度减至10 ng/ml,并加入5 ng/ml碱性成纤维细胞生长因子(bFGF),第4～6天移除培养液中的noggin和bFGF,第8～14天培养液中加入1 μmol/L CHIR99021.倒置显微镜下观察ESCs在分化为RPE细胞过程中的形态变化,免疫荧光染色检测细胞内特异性抗原的表达以对分化细胞进行鉴定,实时荧光定量PCR (qRT-PCR)法检测细胞分化过程中RPE细胞特异性蛋白mRNA的相对表达变化量.结果 分化培养第14天,部分细胞呈多角形并呈现铺路石样排列,且细胞内可见色素颗粒;培养第35天,诱导分化的细胞内表达RPE细胞特异性抗原Mitf及RPE65;培养至第60天,细胞内富含黑色素颗粒且呈规则六边形.与诱导分化前比较,诱导分化第7天和第14天hES-RPE细胞中Mitf mRNA的表达量分别增加了(3.43±2.77)倍和(8.91±2.83)倍,而RPE65 mRNA的表达量分别增加了(14.60±3.94)倍和(87.16±9.32)倍,分化第7天和第14天细胞中的Mitf mRNA和RPE65mRNA的相对表达量均明显高于分化前,差异均有统计学意义(均P＜0.05). 结论 hESCs可在含尼克酰胺、DKK-1、noggin、IGF-1和CHIR99021的xeno-free优化培养体系中快速分化为RPE细胞.     

干细胞诱导分化为视网膜色素上皮细胞的途径探讨

近年来,干细胞在眼科领域的研究及应用受到高度关注,胚胎干细胞(ES)、成体干细胞能够被定向诱导分化成视网膜色素上皮细胞,由此可获得转分化的大量的视网膜色素上皮细胞源,通过体内干细胞及视网膜色素上皮细胞移植有望应用于各种视网膜退行性疾病的细胞替代治疗。本文就各种干细胞诱导分化为视网膜色素上皮细胞的途径及应用进行探讨。     

Expression of embryonic markers in pterygium derived mesenchymal cells

BackgroundDestruction of the limbal epithelium barrier is the most important mechanism of pterygium formation (conjunctiva proliferation, encroaching onto the cornea). It is thought to arise from activated and proliferating limbal epithelial stem cells. The objective of this study is to evaluate the presence of undifferentiated mesenchymal cells (stem cells) in cultured cells extracted from human pterygium.Material and methodsCells from 6 human pterygium were isolated by explantation and placed in cultures with amniomax medium. Once the monolayer was reached the cells were seeded onto 24 well microplates. The cells were studied in the second sub-culture. The immunohistochemical expression of different embryonic stem cell markers, OCT3/4 and CD9, was analysed. The differentiated phenotypes were characterised with the monoclonal antibodies anti-CD31, α-actin and vimentin.ResultsAll the cell populations obtained from pterygium showed vimentin expression. Less than 1% of the cells were positive for CD31 and α-actin markers. The majority of the cell population was positive for OCT3/4 and CD9.ConclusionsThe cell population obtained from pterygium expressed mesenchymal cell phenotype and embryonic markers, such us OCT3/4 and CD9. This undifferentiated population could be involved in the large recurrence rate of this type of tissue after surgery.