Mesenchymal stem cells in the human corneal limbal stroma

Purpose. Peripheral and limbal corneal stromal cells (PLCSCs), which contain keratocytes, have a complex phenotype. Knowledge of keratocyte cell properties, function, and origin is limited. Evidence available thus far has suggested both mesenchymal stromal and hematopoietic characteristics. Multipotent mesenchymal stromal cells (MSCs) are found in an increasing number of tissues and are the subject of considerable interest and investigation in the disciplines of tissue engineering, immunology, gene therapy, and oncology. Methods. Isolated PLCSCs were characterized by markers aldehyde dehydrogenase and keratocan, cultured, and analyzed against a set of criteria for the identification of MSCs developed by the International Society of Cellular Therapy (ISCT). PLCSCs were directly compared to fetal liver MSCs (flMSCs). Additional cell surface markers were also used to quantify differentiation, which was also performed on both cell types. Results. PLCSCs were found to be plastic adherent, displayed the correct profile and proportions of CSMs, and demonstrated trilineage potential in accordance with the ISCT guidelines. Furthermore, PLCSCs displayed a high degree of similarity to flMSCs and this likeness extended into the non-ISCT MSC cell surface markers and trilineage differentiation, which were often but not always comparable. Conclusions. Herein we report a novel observation that PLCSCs conform to all the ISCT criteria and are therefore MSCs. Furthermore, this study has identified the limbal stroma as yet another MSC niche and presents a new perspective on the role of the PLCSC.     

体外构建含细胞组织工程角膜基质的方法

目的:研究体外构建组织工程角膜基质的方法.方法:用去垢剂联合酶的方法脱细胞处理猪角膜基质,在去细胞猪角膜基质材料上接种体外培养的兔角膜基质细胞,并将兔角膜基质细胞进行PKH26荧光标记,在体外构建组织工程角膜基质.用倒置荧光显微镜;HE染色光学显微镜;扫描电镜观察.结果:利用异种去细胞角膜基质组织为支架材料而构建的组织工程角膜基质具有近似正常角膜基质的三维立体结构.结论:以去细胞猪角膜基质材料为支架,利用组织工程技术可成功的在体外构建出含细胞的组织工程角膜基质组织.     

角膜上皮干细胞的体外生长特征

目的　了解角膜上皮干细胞的生长特性。方法　用体外细胞培养的方法 ,从生长曲线、细胞倍增时间、细胞 DNA合成的活跃程度等几个方面 ,观察人角膜缘部、周边部及中央部角膜上皮细胞的生长特征。结果　角膜缘部细胞群体倍增时间最短 (4 9.79h±1.2 6 h) ,周边部次之 (6 4.89h± 2 .18h) ,中央部最长 (78.86 h± 1.38h) ,3组两两比较 P<0 .0 1;角膜缘部角膜上皮细胞 DNA合成最活跃 (CPM值 92 7.75± 47.94) ,周边部次之(CPM值 711.75± 2 9.47) ,中央部最弱 (CPM值 5 14.0 0± 72 .82 ) ,3组两两比较 P <0 .0 1。结论　结果进一部证实了角膜上皮干细胞存在于角膜缘的观点 ;也反映了角膜缘在角膜上皮创伤愈合 ,角膜上皮完整与稳定性维持过程中起着重要的作用     

A preliminary study of mesenchymal stem cell-like cells derived from murine corneal stroma

Background  

Mesenchymal stem cells can be isolated from various tissues besides bone marrow and can differentiate into cells of three germ layers. Recent studies indicate that some cells in corneal stroma express stem cell markers and can also differentiate into chondrocytes and neurocytes. This study was carried out to investigate whether mesenchymal stem cells reside in the murine corneal stroma.     

复方中药金芪滴眼剂对兔角膜上皮及基质细胞增殖的影响

目的：观察复方中药金芪滴眼剂对兔角膜上皮细胞及兔角膜基质层细胞增殖的影响。方法：建立体外培养兔角膜上皮细胞，兔角膜基质层细胞系，用MTT法检测不同浓度金芪滴眼剂对细胞增殖活性的影响。结果：金芪滴眼剂对兔角膜上皮细胞有显著的促进增殖作用。且随浓度的增加。增殖率加大，对兔角膜基质层细胞无促增殖作用。高浓度时有抑制作用。结论：复方中药金芪滴眼剂对兔角膜上皮细胞有较好的保护作用。     

The organization of collagen in the corneal stroma

The cornea has evolved to fulfil the dual functions of enclosing and protecting the inner contents of the eye, and focussing light onto the retina with minimum scatter and optical degradation. It does this by means of the arrangement of the constituent collagen fibrils, an arrangement that is unique in connective tissues. This article reviews our current knowledge about the detailed organization of collagen in the corneal stroma, and presents new data suggesting that a significant proportion of collagen fibrils running across the cornea, change direction near the limbus and fuse with the circumferential limbal collagen.     

组织工程角膜治疗兔无菌性角膜溃疡

目的:探讨利用组织工程技术制备角膜基质进行板层角膜移植治疗无菌性角膜溃疡的可行性和疗效。方法:用去垢剂联合胰酶、DNA-RNA酶去除猪角膜基质细胞后制备成组织工程角膜基质;将16只兔的角膜基质内植入壳聚糖膜使之形成角膜溃疡,随机从16只兔中选8只行组织工程角膜基质板层移植;另外8只作为对照组,行新鲜的同种异体板层角膜移植。术后对角膜进行裂隙灯、HE染色光学显微镜检查。结果:组织工程角膜基质移植后无免疫排斥发生,术后8～10wk角膜溃疡完全修复,角膜恢复透明性,与对照组疗效相同。结论:组织工程角膜基质具有良好的生物相容性和治疗作用。     

人角膜基质细胞诱导人骨髓间充质干细胞分化为角膜上皮细胞的初步研究

目的 探讨人骨髓间充质干细胞（MSC）体外分化为角膜上皮细胞的可行性.方法 实验研究.分别取第3代人MSC和自行培养的第3代人角膜基质细胞共同培养1周,实验组在Transwell共培养体系中培养,对照组不放置Transwell小室培养.1周后,观察实验组和对照组中人MSC光镜特征、间接免疫细胞化学染色和电镜结构,对被诱导分化的细胞进行综合鉴别.结果 第3代人MSC和人角膜基质细胞在体外培养条件下均能够较快贴壁生长.两种细胞共同培养1周后,可见部分细胞形态上呈上皮细胞特征,单克隆抗体AE1染色呈阳性,电镜下可见微绒毛、桥粒和张力丝等典型上皮细胞结构特征.结论 体外培养的人MSC在人角膜基质细胞的诱导下可能会分化为人角膜上皮样细胞.     

人角膜缘干细胞在体外不同载体上培养的实验研究

目的 寻找人角膜缘干细胞体外培养的载体。方法 人角膜缘干细胞原代培养单细胞层传代到羊膜、卵壳膜、聚乳酸膜、藻酸盐凝胶4种载体和6种培养板中,倒置显微镜观察、记录细胞生长情况。对6孔板传代细胞行生物特性检测,对载体上培养细胞行组织学检测。结果 6孔板中传代细胞对AE－5,4G10．3表达阳性。羊膜、卵壳膜和聚乳酸膜上有细胞生长,7天左右形成单层;藻酸盐凝胶载体上未见细胞生长。结论 传代细胞具有角膜缘干细胞生物特性;羊膜、卵壳膜和聚乳酸膜适合角膜缘干细胞传代培养。     

小鼠胚胎干细胞条件培养液培养的人角膜内皮细胞在脱细胞猪角膜基质上单层细胞片的构建

背景 目前角膜供体来源短缺,且体外培养的人角膜内皮细胞(HCECs)难以再生和扩增,为临床上角膜移植的开展带来了很大困难,组织工程角膜的构建仍是研究的主要课题.前期研究已证实,小鼠胚胎干细胞条件培养基(ESC-CM)能够促进体外HCECs的增生,脱细胞猪角膜基质(APCS)是较好的支架材料,但ESC-CM培养的HCECs是否能在APCS上融合成单层细胞鲜见研究. 目的 研究ESC-CM培养的HCECs在APCS上是否能够形成单层细胞. 方法 用小鼠ESC-CM培养液培养小鼠ES-E14细胞,收集培养上清液,离心后按体积比1∶3与人角膜内皮细胞培养液(CEM)混合,制备体积分数25％ ESC-CM液.取穿透角膜移植术后剩余的供体人角巩膜缘组织,采用组织块培养法用ESC-CM对HCECs进行培养和传代,采用逆转录PCR法检测HCECs中Ⅷ型胶原蛋白(ColⅧ)与神经元特异性烯醇化酶(NSE)的表达以鉴定细胞.取猪角膜,利用磷脂酶A2和碳酸氢盐溶液脱细胞法制备APCS,将第2代HCECs混合液按照800/mm2的密度种植于灭菌的APCS上进行培养,于倒置相差显微镜下观察细胞形态,采用苏木精-伊红染色法观察培养细胞的组织结构;采用免疫荧光法检测构建的HCECs单细胞片中闭锁小带蛋白-1(ZO-1)和Na+-K+-ATP酶的表达. 结果 体外培养的HCECs呈典型的六角形,内皮特异性标志物ColⅧ和NSE mRNA表达阳性.脱细胞APCS呈白色透明状,苏木精-伊红染色显示APCS中无角膜细胞,但角膜胶原纤维排列规则.第2代HCECs复水后可在APCS上生长并贴附,培养后7d融合成单层细胞片,细胞片上HCECs的细胞密度为(2 694-± 143)/mm2.构建的HCECs片中内皮细胞泵功能相关蛋白ZO-1和Na+-K+-ATP酶均呈阳性表达,呈红色荧光. 结论 25％ESC-CM可促进HCECs的生长并维持细胞的正常形态,APCS可为HCECs片的构建提供支架和较好的生存微环境.在APCS形成的HCECs单细胞片可表达HCECs泵功能,是角膜移植的良好供体.     

体外诱导人骨髓间充质干细胞向角膜上皮细胞分化的初步研究

目的 探讨在体外损伤微环境下,人骨髓间充质干细胞(MSCs)分化为角膜上皮细胞的可行性. 方法体外分离培养人MSCs和人角膜缘干细胞(LSCs),检测细胞CD34、CD44、细胞角蛋白3(CK3)、细胞角蛋白19(CK19)的表达情况.制作人LSCs热损伤模型,加入增强型绿色荧光蛋白基因(EGFP)标记的人MSCs共同培养,2周后检测细胞CK19的表达情况.结果 人MSCs CD44表达阳性、CD34表达阴性,流式细胞术显示CD44阳性率为92.59%,CD34阳性率为0.01%;人LSCs CK19表达阳性,CK3表达阴性.共同培养后部分人MSCs CK19表达阳性,并有多核细胞现象.结论 在损伤微环境下,人MSCs可以转化为类角膜上皮细胞的细胞,在此过程中存在细胞融合现象.     

人脂肪组织来源的干细胞体外诱导为角膜上皮样细胞的分化潜能

目的 初步探讨人脂肪组织来源的干细胞(human adipose-derived stem cells,hADSC)体外诱导为角膜上皮样细胞的分化潜能.方法 分离、纯化、体外扩增hADSC,流式细胞术鉴定所获得细胞的CD29+、CD34-、CD49d+、CD105+、CD106-的表达情况.成脂、成骨诱导鉴定其多向分化潜能.在DMEM/F12体系、KM体系及上述二者等体积混合的KM/DMEM/F12体系内添加不同梯度浓度的生长因子对hADSC进行体外诱导:0μg·L-1、10μg·L-1、20μg·L-1、30μg·L-1、40μg·L-1、50μg·L-1的表皮生长因子(epidermal growth factor,EGF),及50μg·L-1 EGF+10μg·L-1 碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)和50μg·L-1 EGF+20 μg·L-1 bFGF.连续诱导21 d,观察hADSC形态学的改变及第21天时角膜特异性蛋白AE5(CK3/CK12)的免疫化学表达情况,比较不同培养体系及不同浓度生长因子对hADSC诱导作用的差异.结果 流式细胞仪测定传3-5代的细胞CD34-、CD106-、CD29+、CD49d+、CD105+.成脂、成骨体外诱导14 d后.油红O、碱性磷酸酶染色阳性率分别为74.6%和29.3%.KM体系中加入终浓度为0～50μg·L-1 EGF诱导21d后,hADSC AE5阳性细胞率均占90%以上.其中,40μg·L-1EGF诱导下的AE5阳性细胞率为98.7%,50μg·L-1 EGF可达到100%.而加入bFGF的hADSC 则为AE5弱阳性.而另2个体系对各浓度ECF、bFGF诱导的hADSC的作用均为阴性.结论 人吸脂术废弃液中可获得大量高纯度脂肪组织来源的干细胞,经体外条件诱导具备向角膜上皮样细胞分化潜能.添加EGF的角质细胞培养液有利于其分化,并具有浓度依赖性,bFGF则对分化有抑制性作用.     

增龄对角膜基质载体培养的角膜缘干细胞生物学特性的影响

目的 观察增龄对新西兰兔角膜缘干细胞在同种异体角膜基质上生长、增殖的影响.方法 用酶消化法将不同年龄新西兰兔角膜缘组织制成细胞悬液培养,以相同的细胞密度种植于不同年龄的新西兰兔的角膜基质上,观察角膜缘干细胞在角膜幕质上的生长情况.原位杂交检测ABCG2、ANp63的表达,IPP5.1软件分析图像,流式细胞术测细胞周期,运用SPSS13.0软件对析因设计资料进行方差分析.结果 角膜缘干细胞供体年龄各水平差异有统汁学意义,相同时间内角膜基质培养的青年兔角膜缘干细胞在角膜基质单位面积卜的牛长总而积均较中年和老年高,增殖期细胞所占比例大;而角膜基质供体年龄各水平差异与角膜缘干细胞和角膜基质的交互作用均无统计学意义.结论 在同种异体角膜基质载体上培养的角膜缘干细胞的增殖能力随着年龄的增加而降低.     

Isolation of putative corneal epithelial stem cells from cultured limbal tissue

PURPOSE: To investigate methods of isolating putative corneal epithelial stem cells from cultured limbal tissue. METHODS: Three extraction techniques were compared to identify an efficient method of obtaining a large number of viable corneal epithelial stem cells from the limbus. Limbal tissues were extracted by incubation at 37 degrees C or 4 degrees C for 1 or 16 hours, respectively, with 1.2 U/ml dispase/trypsin or by treatment with 0.05% trypsin and 0.01% ethyldiaminetetraacetic acid (EDTA) at 37 degrees C in single procedure. Collected cells were cultured on NIH/3T3-seeded plates, and colony forming efficiency (CFE) was evaluated. Fluorescence activated cell sorting (FACS) was performed with a Coulter EPICS 753 after incubation with Hoechst 33342 and propidium iodide (PI). Hoechst negative cells were obtained using gates exhibiting low Hoechst blue with a 424/44 nm BP filter. Gated cells of each fraction were re-cultured to assess the capability of colony formation. RESULTS: The mean numbers of viable cells obtained from treatment with dispase and trypsin was 3 x 10(4) cell/ml and 8.06 x 10(5) cell/ml at 37 degrees C and 4 degrees C incubations; the number increased to 1.21 x 10(6) cell/ml with a trypsin/EDTA treatment (p < 0.05). CFE was 9.67 +/- 2.13% and 6.63 +/- 2.35% in rabbit and human cells, respectively. Likewise, the Hoechst negative fraction was 3.61 +/- 0.42% and 5.21 +/- 4.91% in rabbit and human cells, respectively. The sorted Hoechst negative cells were cultured through four passages, forming small round colonies. In rabbit cells, the CFEs of Hoechst negative and positive fractions after FACS, were 12.67 +/- 2.24% and 1.17 +/- 6.13%, respectively (p < 0.05). CONCLUSIONS: Putative corneal epithelial stem cells were efficiently isolated from limbal tissue using a trypsin/EDTA extraction and FACS. This technique may be very useful in tissue engineered stem cell therapy.     

人牙周膜干细胞、脐带间充质干细胞对体外培养角膜缘干细胞的影响

目的　比较人牙周膜干细胞（ｈｕｍａｎｐｅｒｉｏｄｏｎｔａｌｌｉｇａｍｅｎｔｓｔｅｍｃｅｌｌｓ,ＨＰＤＬＳＣｓ）、人脐带间充质干细胞（ｈｕｍａｎｕｍｂｉｌｉｃａｌｃｏｒｄｍｅｓｅｎｃｈｙｍａｌｓｔｅｍｃｅｌｌｓ,ＨＵＣＭＳＣｓ）作为饲养层培养角膜缘干细胞（ｌｉｍｂａｌｓｔｅｍｃｅｌｌｓ,ＬＳＣｓ）的优越性,从而筛选出扩增ＬＳＣｓ的最佳饲养层。方法　体外分离培养ＨＰＤＬＳＣｓ、ＨＵＣＭＳＣｓ,诱导细胞成脂、成骨分化,并检测细胞表面标志物的表达;将兔ＬＳＣｓ与ＨＰＤＬＳＣｓ、ＨＵＣＭＳＣｓ、ＮＩＨ-３Ｔ３共培养和无饲养层单独培养,比较各组克隆形成率,免疫荧光比较各组ＬＳＣｓ克隆团ＡＢＣＢ５、ＩＰＯ１３、ＣＫ３／１２的表达情况。结果　体外培养ＨＰＤＬＳＣｓ、ＨＵＣＭＳＣｓ均可向脂肪、成骨细胞分化,两种细胞均高表达间充质来源的表面标志ＣＤ９０,不表达ＣＤ４５;三组饲养层与兔ＬＳＣｓ共培养均可形成小克隆团, ＨＰＤＬＳＣｓ组、ＨＵＣＭＳＣｓ组、ＮＩＨ-３Ｔ３组、无饲养层组克隆形成率分别为（４．９０±０．９６）％、（４．１０±０．５６）％、（４．６７±０．７６）％、（０．８３±０．３５）％,四组间总体比较差异有统计学意义（Ｆ＝２２．０４７,Ｐ＝０．００）;ＨＰＤＬＳＣｓ组、ＨＵＣＭＳＣｓ组与ＮＩＨ-３Ｔ３组的克隆形成率差异均无统计学意义（均为Ｐ＞０．０５）;ＨＰＤＬＳＣｓ组、ＨＵＣＭＳＣｓ组、ＮＩＨ-３Ｔ３组与无饲养层组间差异均有统计学意义（均为Ｐ＜０．０１）。免疫荧光化学检测三种饲养层ＬＳＣｓ标志物ＡＢＣＢ５、ＩＰＯ１３呈高表达,ＣＫ３／１２呈低表达。结论　ＨＰＤＬＳＣｓ、ＨＵＣＭＳＣｓ均可作为替代饲养层用以培养ＬＳＣｓ。     

壳聚糖构建的组织工程角膜基质的实验研究

目的 利用角膜基质细胞与壳聚糖支架共培养构建组织工程角膜基质.方法 将壳聚糖、胶原蛋白、硫酸软骨素、透明质酸钠等按照一定的比例混合,加入原花青素交联剂,构建壳聚糖支架,检测获得的壳聚糖支架的特点及生物相容性.在羊膜上培养兔角膜基质细胞.兔角膜基质细胞与原花青素交联的壳聚糖支架共培养构建初步组织工程角膜基质,行兔角膜板层移植.结果 兔角膜基质细胞可在支架表面上生长,获得初步的组织工程角膜基质.兔角膜板层移植2周后,壳聚糖支架全部脱落.结论 原花青素交联的壳聚糖支架具有良好的生物相容性,在羊膜上培养的兔角膜基质细胞可以保持正常的生物学特性,兔角膜基质细胞和壳聚糖支架可以构建初步的组织工程角膜基质,但不能用于移植.     

Longterm survival of transplanted human corneal epithelial cells and corneal stem cells

PURPOSE: To investigate the survival of donor-derived epithelial cells in conventional penetrating keratoplasty (PKP) and in homologous penetrating central limbal keratoplasty (HPCLK). METHODS AND PATIENTS: Epithelial cells from 26 eyes of 26 patients were analysed. All cases were sex-mismatched (i.e. the transplant and patient were of different genders). At suture removal more than 1 year post surgery, epithelial cells were obtained by gently wiping the removed sutures on glass slides. The cell samples were analysed using fluorescent in situ hybridization (FISH) of the sex chromosomes. This technique makes it possible to allocate the origin of each cell nucleus to either the donor or the recipient. RESULTS: All 19 conventional PKPs were clear and seven had donor-derived epithelial cells at suture removal. Five of the seven HPCLK grafts were clear at the time of investigation (365-1355 days post surgery), and donor-derived epithelial cells were found in two grafts. CONCLUSION: Harvesting cells from removed sutures in combination with FISH enables the clinical study of cell survival in corneal transplants without jeopardizing functioning grafts. From the limited sample investigated, the following tentative conclusions can be made. Donor-derived epithelial cells can remain in conventional PKP for over 1 year. In combined stem cell and corneal grafts (HPCLK), donor-derived epithelial cells may also be retrieved at 1 year or beyond following surgery but the correlation between their presence and a remaining clear graft is uncertain.     

小鼠胚胎干细胞条件培养基体外促进人角膜内皮细胞增殖的研究

目的　观察小鼠胚胎干细胞条件培养基（ｍｏｕｓｅｅｍｂｒｙｏｎｉｃｓｔｅｍｃｅｌｌｓｃｏｎｄｉｔｉｏｎｅｄｍｅｄｉｕｍ,ＥＳＣ-ＣＭ）是否可以在体外促进人角膜内皮细胞（ｈｕｍａｎｃｏｒｎｅａｌｅｎｄｏｔｈｅｌｉａｌｃｅｌｌｓ,ＨＣＥＣｓ）的增殖。方法　利用角膜内皮后弹力层组织块方法进行原代培养Ｐ０ＨＣＥＣｓ。实验组使用含有２５％ＥＳＣ-ＣＭ的培养液进行培养,对照组使用普通角膜内皮细胞培养液（ｃｏｒｎｅａｌｅｎｄｏｔｈｅｌｉｕｍｍｅｄｉ-ｕｍ,ＣＥＭ）进行培养。倒置相差显微镜、反转录聚合酶链反应（ｒｅｖｅｒｓｅ-ｔｒａｎｓｃｒｉｐｔｉｏｎｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ,ＲＴ-ＰＣＲ）鉴定ＨＣＥＣｓ;倒置相差显微镜观察细胞的形态及萌出时间;ＷｅｓｔｅｒｎＢｌｏｔ、免疫组织化学法观察ＨＣＥＣｓ的泵相关功能蛋白（ｚｏｎａｏｃｃｌｕ-ｄｅｎｓｐｒｏｔｅｉｎ-１,ＺＯ-１）及Ｎａ＋-Ｋ＋-ＡＴＰ酶的表达。Ｇｉｅｍｓａ染色细胞克隆实验、免疫组织化学及流式细胞学检测Ｋｉ６７阳性率的方法比较ＨＣＥＣｓ的增殖能力;流式细胞学方法检查细胞周期及细胞凋亡情况。ＷｅｓｔｅｒｎＢｌｏｔ和免疫组织化学方法检测细胞周期负性调节蛋白Ｐ２１的水平,初步探讨其可能的作用机制。结果　原代培养时,２５％ＥＳＣ-ＣＭ组培养的ＨＣＥＣｓＰ２细胞爬出,细胞形态呈典型多角形结构。ＣＥＭ在Ｐ２时细胞形态变大,失去了多角形结构。２５％ＥＳＣ-ＣＭ 组和ＣＥＭ组均表达ＺＯ-１、Ｎａ＋-Ｋ＋-ＡＴＰ酶。２５％ＥＳＣ-ＣＭ组的Ｋｉ６７阳性率、克隆形成数量、进入到细胞周期Ｓ期和Ｇ２期的比例均高于ＣＥＭ组（均为Ｐ＜０．０５）。２５％ＥＳＣ-ＣＭ组的细胞凋亡数量和Ｐ２１阳性率均低于ＣＥＭ组（均为Ｐ＜０．０５）。结论　２５％ＥＳＣ-ＣＭ组可显著促进ＨＣＥＣｓ增殖;其作用可能是通过抑制Ｐ２１蛋白的表达和抑制细胞凋亡实现的,为ＨＣＥＣｓ体外大量扩增提供了一种新方法。