【摘要】 目的：组织工程人角膜内皮（tissueengineered human corneal endothelia,TEHCE）的体外重建及其形态结构。方法：用有限稀释法从HCE细胞系筛选出单克隆细胞（mcHCE细胞），用常规染色体标本制作和核型分类学方法进行核型分析。用羊膜的胰酶倒置消化和细胞外基质蛋白包被方法制备去上皮层修饰羊膜（mdAM）。以核型正常的对数期mcHCE细胞为种子细胞，以平铺于24孔培养板孔底的mdAM为载体支架，用200mL/L胎牛血清DMEM/F12培养液在37℃，50mL/L CO2培养箱中进行TEHCE的体外重建。用茜素红染色、冰冻切片HE染色、倒置显微镜和扫描电镜观察种子细胞的形态、细胞连接的形成情况、细胞单层的完整性及其与mdAM结合的紧密程度。用透射电镜方法鉴定种子细胞的超微结构以及细胞连接的形成情况。用免疫荧光技术检测种子细胞对不同细胞连接蛋白的表达模式。结果：从非转染HCE细胞系中筛选出了7个核型正常（2n=46）的单克隆细胞株。在启动重建30h后，mcHCE种子细胞在mdAM上形成了完整的细胞单层，细胞密度高达3413/mm2。HCE细胞呈多角形细胞形态，形成了完整的细胞单层，且在细胞细胞以及细胞mdAM间形成了多种细胞连接，种子细胞在超微结构上与在体HCE细胞类似，胞质中含有许多线粒体，并具有紧密连接蛋白1、钙黏蛋白、间隙连接蛋白43和整联蛋白αv/β5的阳性表达。结论：体外重建的TEHCE在结构和功能上与在体HCE相似，有望作为HCE的替代物用于临床角膜内皮移植。
【关键词】 人角膜内皮细胞；去上皮层修饰羊膜；组织工程；体外重建；形态；结构
In vitro reconstruction of tissueengineered human corneal endothelium and characterization of its morphology and structureTingJun Fan, Jun Zhao, Jing Wang, RiShan Cong, XiuXia Yang, WeiYun Shi, YiQiang WangFoundation items: National High Technology Research and Development Program ("863" Program) of China (No.2006AA02A132); Opening Program from The State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology (No.2006K05) Key Laboratory for Corneal Tissue Engineering, Ocean University of China, Qingdao 266003, Shandong Province, China; Shandong Eye Institute, Shandong Academy of Medical Sciences, Qingdao 266071, Shandong Province, ChinaAbstract AIM: To reconstruct tissueengineered human corneal endothelia (TEHCE) in vitro and characterize them in morphology and structure. METHODS: Monoclonal HCE cells (mcHCE cells) were cloned from untransfected HCE cell line by limited dilution, and their karyotypes were analyzed by routine methods of chromosomal preparing and karyosystematics. Modified denuded amniotic membranes (mdAMs) were prepared from amniotic membrane by inverted trypsin denudation and coated with extracellular matrix proteins. TEHCEs were in vitro reconstructed by using mcHCE cells at logarithmic phase as seed cells and mdAMs tiled on well bottoms of a 24well culture plate as scaffold carriers, which were cultured in 200mL/L fetal bovine serum (FBS)containing DMEM/F12 medium at 37℃ in a 50mL/L CO2 incubator. The morphology of seed cells, formation of cell junctions, integrality of endothelial monolayer and its integrated status to mdAM were investigated by Alizarin red staining, freezesection's hematoxylineosin (HE) staining, inverted microscopy and scanning electron microscopy. The ultrastructure of seed cells on mdAM and formation of cell junctions were examined by transmission electron microscopy. The expression patterns of different cell junction proteins of TEHCE seeder cells were detected by immunofluorescent techniques.RESULTS: Seven mcHCE cell strains with normal karyotype (2n=46) were screened out from the untransfected HCE cell line. About 30 hours after reconstruction initiation, mcHCE seeder cells formed an integrated monolayer on mdAM with a cell density as high as 3413/mm2. Most of seed cells were in polygonal morphology, integral endothelial monolayer was reconstructed with various cellcell and cellmdAM junctions. And the ultrastructure of seed cells was similar to that of HCE cells in vivo, with a lot of mitochondria scattered in cytoplasm. Besides, the seed cells maintained positive expression of cell junction proteins such as zonula occludens protein 1, Ncadherin, connecxin43 and integrin αv/β5. CONCLSUION: The TEHCEs, with similar morphology and structure to those of HCE in vivo, were successfully reconstructed, and can be used promisingly as HCE equivalents for clinical corneal endothelium transplantation.
KEYWORDS: human corneal endothelial cell; modified denuded amniotic membrane; tissueengineered human corneal endothelium; in vitro reconstruction; morphology; structure
0 引言
人角膜内皮（human corneal endothelial，HCE）层在维持角膜正常厚度和透明度中具有不可替代的作用，是人眼发挥正常视功能的必要条件。角膜内皮盲（primary corneal endotheliopathy）是HCE细胞密度低于维持角膜内皮层完整性及其生理功能的临界密度后所引起的不可逆病变，绝大多数（约999‰）患者因得不到可用于移植的捐献角膜而无法复明[1]。角膜组织工程的兴起为组织工程人角膜内皮（tissueengineered human corneal endothelia,TEHCE）的体外重建和患者通过临床角膜内皮移植重见光明带来了希望。许多学者利用癌基因转染的永生化HCE细胞、原代培养HCE细胞或仅传4～5代的HCE细胞在体外成功重建出了人角膜内皮的组织类似物，为组织工程角膜内皮的体外重建开辟了道路，但由于所用永生化转染的种子细胞具有潜在致瘤性而无法用于临床角膜内皮移植[2，3]，所重建的HCE类似物移植后仅使新西兰兔角膜维持透明1wk左右，离角膜内皮盲的临床移植应用还差得很远[49]。去上皮层修饰羊膜（mdAM）为来自人胎盘羊膜的修饰物，目前已广泛用于眼表损伤、角膜基质炎、角膜溃疡、大泡性角膜病变及新生血管性青光眼的移植治疗[10]。我们以非转染HCE细胞系的单克隆细胞株为种子细胞、以mdAM为载体支架进行了TEHCE的体外重建，并对其形态结构进行了鉴定，旨在为形态结构正常TEHCE的体外重建及其作为角膜内皮替代物进行临床角膜内皮移植奠定基础。
1 材料和方法
1.1 材料
无眼疾健康新西兰兔，体质量2.0～2.5kg，不分雄雌，由山东省眼科所实验动物中心提供与饲养；非转染HCE细胞系，由本实验室自行建立，现已传至第282代；新鲜羊膜（AM）由山东省眼科所友情惠赠。用胰酶消化法收获对数期第24代HCE细胞，经Casy细胞计数仪计数后用200mL/L胎牛血清（FBS）DMEM/F12（1∶1）培养液将细胞密度调整为8×103个/L，接种于预铺有饲养层细胞（feeder cell）的96孔板内（0.1mL/孔），置37℃，50mL/L CO2培养箱中进行克隆化培养。每周换培养液1次，每3d观察细胞克隆的形成情况，2～3wk后挑出细胞单克隆进行扩增培养，对每个细胞单克隆进行核型分析，筛选出具有正常核型（2n=46）的单克隆细胞株，扩增后作为种子细胞备用。另新鲜羊膜经生理盐水冲洗后用硫酸妥布霉素液浸泡消毒，经DHanks漂洗后让羊膜上皮面朝下用胰蛋白酶 EDTA于37℃倒置消化，获得去除上皮细胞的dAM，经DHanks液漂洗后用眼科剪将羊膜剪成直径1.56cm的圆片，使其上皮面朝上平铺于24孔培养板中，滴加dAM专用包被液处理过夜，吸出包被液晾干后，作为mdAM载体支架使用。
1.2 方法
单克隆细胞株种子细胞用200mL/L FBSDMEM/F12（1∶1）培养液将细胞密度调整为4.2×109/L，按1mL/孔将种子细胞悬液接种于预铺有dAM的24孔培养板中，置37oC，50mL/L CO2培养箱中进行体外重建培养，每2d换液1次，每日观察种子细胞的形态、增殖和细胞单层形成情况，待种子细胞长成紧密单层后用于形态结构鉴定。形成细胞单层后的TEHCE，置Nikon Eclipse TS 100倒置显微镜下观察，随机选取5个视野，利用网格目微尺统计单位面积的细胞数，计算出TEHCE单层的细胞密度。形成细胞单层后的TEHCE，用生理盐水轻轻漂洗后，滴加10g/L茜素红染液染色15min，吸除染液后用生理盐水轻轻冲洗，在Nikon Eclipse TS 100倒置显微镜下观察并照相。从培养孔中取出TEHCE单层，经冰冻胶低温固定冰冻切片厚5～6μm，甲醇固定后空气干燥30min，水洗后用苏木素染色30s，经10mL/L盐酸乙醇分化和水洗后伊红染色10s，脱水封片，光镜下观察并照相。从培养孔中取出TEHCE单层，经40g/L戊二醛蔗糖添加二甲胂缓冲液固定后，进行CO2临界点干燥和喷金处理，使用JSM 2840扫描电镜进行观察和照相。从培养孔中取出TEHCE单层，经40g/L戊二醛蔗糖添加二甲胂缓冲液固定后，按常规方法进行包埋和超薄切片，利用H700透射电镜进行观察和照相。形成细胞单层后的TEHCE，经PBS轻轻漂洗后，用40g/L多聚甲醛于4℃固定10min，吸出固定液后用PBS洗涤2次，用40g/L小牛血清于37℃湿盒内封闭处理30min，分别加入山羊抗人ZO1（zonula occludens protein 1，紧密连接蛋白1）（1∶50）、小鼠抗人N钙黏蛋白（Ncadherin）（1∶50）、小鼠抗人间隙连接蛋白43（connecxin43）（1∶250）和小鼠抗人整联蛋白αv/β5（integrin αv/β5）（1:50）mAb（第1抗体）于4℃温盒中过夜孵育，用PBS液冲洗5次后再加入FITC荧光素标记的兔抗羊IgG（1∶10）或羊抗鼠（1∶100）IgG第2抗体，于37℃湿盒内下避光孵育2h，经PBS冲洗5次后置Nikon Eclipse Ti倒置荧光显微镜观察并照相。用PBS代替第一抗体作为阴性对照。
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