目的:组织工程人角膜内皮(tissue-engineered human corneal endothelia,TE-HCE)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...目的:组织工程人角膜内皮(tissue-engineered human corneal endothelia,TE-HCE)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/LCO2培养箱中进行TE-HCE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TE-HCE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。展开更多
To evaluate the therapeutic efficiency of tissue-engineered human corneal endothelia (TE-HCEs) on rabbit primary corneal endotheliopathy (PCEP),TE-HCEs reconstructed with monoclonal human corneal endothelial cells (mc...To evaluate the therapeutic efficiency of tissue-engineered human corneal endothelia (TE-HCEs) on rabbit primary corneal endotheliopathy (PCEP),TE-HCEs reconstructed with monoclonal human corneal endothelial cells (mcHCECs) and modified denuded amniotic membranes (mdAMs) were transplanted into PCEP models of New Zealand white rabbits using penetrating keratoplasty.The TE-HCEs were examined using diverse techniques including slit-lamp biomicroscopy observation and pachymeter and tonometer measurements in vivo,and fluorescent microscopy,alizarin red staining,paraffin sectioning,scanning and transmission electron microscopy observations in vitro.The corneas of transplanted eyes maintained transparency for as long as 200 d without obvious edema or immune rejection.The corneal thickness of transplanted eyes decreased gradually after transplanting,reaching almost the thickness of normal eyes after 156 d,while the TE-HCE non-transplanted eyes were turbid and showed obvious corneal edema.The polygonal corneal endothelial cells in the transplanted area originated from the TE-HCE transplant.An intact monolayer corneal endothelium had been reconstructed with the morphology,cell density and structure similar to those of normal rabbit corneal endothelium.In conclusion,the transplanted TE-HCE can reconstruct the integrality of corneal endothelium and restore corneal transparency and thickness in PCEP rabbits.The TE-HCE functions normally as an endothelial barrier and pump and promises to be an equivalent of HCE for clinical therapy of human PCEP.展开更多
文摘目的:组织工程人角膜内皮(tissue-engineered human corneal endothelia,TE-HCE)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/LCO2培养箱中进行TE-HCE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TE-HCE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。
基金Project (Nos.2001AA625050 and 2006AA02A132) supported by the National High-Tech R&D Program (863) of China
文摘To evaluate the therapeutic efficiency of tissue-engineered human corneal endothelia (TE-HCEs) on rabbit primary corneal endotheliopathy (PCEP),TE-HCEs reconstructed with monoclonal human corneal endothelial cells (mcHCECs) and modified denuded amniotic membranes (mdAMs) were transplanted into PCEP models of New Zealand white rabbits using penetrating keratoplasty.The TE-HCEs were examined using diverse techniques including slit-lamp biomicroscopy observation and pachymeter and tonometer measurements in vivo,and fluorescent microscopy,alizarin red staining,paraffin sectioning,scanning and transmission electron microscopy observations in vitro.The corneas of transplanted eyes maintained transparency for as long as 200 d without obvious edema or immune rejection.The corneal thickness of transplanted eyes decreased gradually after transplanting,reaching almost the thickness of normal eyes after 156 d,while the TE-HCE non-transplanted eyes were turbid and showed obvious corneal edema.The polygonal corneal endothelial cells in the transplanted area originated from the TE-HCE transplant.An intact monolayer corneal endothelium had been reconstructed with the morphology,cell density and structure similar to those of normal rabbit corneal endothelium.In conclusion,the transplanted TE-HCE can reconstruct the integrality of corneal endothelium and restore corneal transparency and thickness in PCEP rabbits.The TE-HCE functions normally as an endothelial barrier and pump and promises to be an equivalent of HCE for clinical therapy of human PCEP.
