【作者】 李柳；

【导师】 侯光辉；

【作者基本信息】 暨南大学 ， 眼科学， 2012， 硕士

【摘要】 1.目的（1）提高体外分离hUC-MCs的成功率（2）探讨hUC-MCs作为组织工程角膜种子细胞的可能性（3）改良冻存VECs的方法（4）探讨VECs作为组织工程角膜种子细胞的可能性2.方法（1）用多片脐带组织片悬浮法联合Ⅳ型胶原酶消化法、单纯多片脐带组织片悬浮法及脐带组织片悬浮法这三种方法体外分离hUC-MCs，并比较其成功率，鉴定细胞表面标记并诱导其分化（2）以猪角膜基质后板层为载体培养hUC-MCs构建组织工程角膜后板层并行动物移植，观察角膜透明情况，并行hUC-MCs表面特异内皮标志物NSE和ZO-1的检测（3）采用改良及传统方法冻存VECs，4周后复苏分别比较其存活率、形态、生长曲线，流式细胞术检测其凋亡率（4）以猪角膜基质后板层为载体培养VECs构建组织工程角膜后板层，扫描电镜观察其表面结构3.结果（1）多片悬浮脐带片法联合Ⅳ型胶原酶消化法及单纯多片悬浮脐带片法成功率高于悬浮脐带片法（2）种植hUC-MCs移植组角膜较非种植组透明，hUC-MCs表面特异内皮标志物NSE和ZO-1表达阳性（3）改良的冻存法VECs复苏后形态上较传统组好，存活率、生长曲线及凋亡率与传统组无明显差异(4）扫描电镜可见hUC-MCs在猪角膜基质后板层表面生长良好，连接成片4.结论（1）本研究探讨了hUC-MCs体外分离的几种方法，提供了一种较为高效的培养方法（2）hUC-MCs可向CECs方向转化，为组织工程后板层的构建提供了新的种子细胞来源（3）改良的VECs冻存法能大幅度缩短冻存所需时间达到与传统方法类似的效果(4）VECs或能为组织工程后板层的构建提供了新的种子细胞来源更多还原

【Abstract】 Objective (1) To improve the success rate of hUC-MCs separation in vitro(2) To explore the possibility of hUC-MCs as engineering corneal tissueseed cells(3)To improve the VECs cryopreserving method(4) To explore the possibility of VECs as engineering corneal tissue seedcellsMethods (1) Separate the human umbilical cord-mesenchymal stem cells in vitrowith three methods: Multi-chip of umbilical cord tissue suspended combine with Ⅳcollagenase digestion method,Simple multi-chip of umbilical cord tissue suspendedmethod, umbilical cord tissue suspended method. After isolation,the cells’ shape andsurface markers is observed and detectde through the use of flow cytometry,andinduced to be adipocaytes and osteoblasts.(2)Cultivate the hUC-MSCs on porcine corneal stroma to build tissueengineering corneal lamellar， observe the corneal stroma whether it istransparent,detect it’s surface marker for NSE and ZO-1which is specific for cornealendothelial cells(3)Cryopreserve the VECs with two kinds of methods,compare theiractivity after4weeks through their survival rates,growth curve and apoptosis rate byflow cytometry(4)Cultivate the VECs on porcine corneal stroma to build tissueengineering corneal lamellar,scaned it’s surface structure by electron microscopyResults (1)The success rate of the Multi-chip suspended umbilical cord slicescombined with type IV collagenase digestion method and Simple multi-chip ofumbilical cord tissue suspended method were higher than umbilical cord tissuesuspended method(2)The corneas with hUC-MSCs are transparenter than withoutit,surface-specific endothelial markers NSE and ZO-1expression were positive on hUC-MSCs(3)The reformed method is better than traditional method in morphology,andthere was no significant difference in thei survival rate,growth curve and the apoptosis ratebetween these two kinds of methods(4) Scanning electron microscopy showed the surface of the corneas withVECs grew wellConclusion (1) In this research,we study sever kinds of methods to isolatehUC-MSCs in vitro,and create a more effcient method(2)The hUC-MSCs can converse into CECs(3)The reformed cryopreserved method is convenient and feasible(4)The VECs may be new seed cells to establish engineering corneal更多还原