组织工程人角膜上皮的体外重建、鉴定及其在新西兰兔角膜上皮移植中的作用研究
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摘要
人角膜上皮（Human Corneal Epithelium，HCEP）位于角膜最前端，由多层细胞构成，其组织学结构的完整性对于维持角膜的生理功能至关重要。人角膜上皮细胞（Human Corneal Epithelial Cell，HCEPC）是角膜抵御外来致病因子侵害的第一道重要防线，不仅能维持角膜的透明性，还能吸收氧气和营养为角膜供氧/养。HCEP具有自我更新能力，其完整性和更新能力取决于位于角膜缘基底部的角膜缘干细胞（LimbalStem Cell，LSC）的不断增殖与分化。眼球表面的烧伤、灼伤、严重机械创伤、病原体感染、角膜缘部位冷冻治疗、接触镜的长期佩戴以及眼瘢痕性类天疱疮等各种原因均可导致LSC损伤或功能障碍。角膜缘干细胞缺陷症（Limbal Stem Cell Deficiency，LSCD）表现为LSC增殖能力丧失，导致持续性角膜上皮缺损、角膜缘屏障功能下降，致使结膜上皮侵入角膜、新生血管形成，进而影响角膜的光学特性，导致视力受损或致盲。在以前的眼表重建治疗中，一般采用角膜上皮成形术以及自体或异体角膜缘移植的方法，但因供体角膜来源严重不足以及术后排斥反应发生率较高，从而限制了其在临床中的广泛应用。近年来，角膜组织工程的兴起为组织工程人角膜上皮（Tissue-engineered Human Corneal Epithelium，TE-HCEP）的体外重建以及患者通过临床角膜上皮移植而重见光明带来了希望。TE-HCEP体外重建的关键要素包括结构功能正常的HCEPC种子细胞的大量获得和生物相容性理想的载体支架的制备。在种子细胞方面，由于自体LSC来源与数量的限制，而连续性HCEPC细胞系因能提供出大量细胞故被认为是TE-HCEP规模化体外重建的种子细胞的有效来源。在业已建立的HCEPC细胞系中，癌基因转染的永生化HCEPC细胞系因具有潜在的致瘤性无法作为种子细胞用于TE-HCEP的体外重建尤其是临床移植，而非转染HCEPC细胞系因能为理论研究和TE-HCEP提供出足量的正常细胞，故被认为是TE-HCEP种子细胞的理想来源。近年来，国内学者成功建立了非转染的连续性人角膜缘上皮细胞系，但至今仍未见其用于TE-HCEP体外重建的研究报道。在载体支架方面，羊膜（Amniotic Membrane，AM）作为一种天然高分子生物材料，为半透明且抗原性低的薄片组织，其基质中含有大量的生长因子和蛋白酶抑制因子以及抗炎症、抗成纤维化、抗菌、抗血管生成等的相关蛋白，与HCEPC具有理想的生物相容性并能促进HCEPC的细胞分化，已被广泛应用于眼表重建，因此被认为是TE-HCEP的理想载体支架之一。本文在本实验室自主建立的非转染HCEPC细胞系的基础上，对此细胞系的属性和致瘤性进行进一步鉴定，首次以该细胞为种子细胞建、以去上皮层羊膜（denuded AM，dAM）为载体支架，开展TE-HCEP的体外重建研究，并利用LSCD新西兰兔模型对TE-HCEP在角膜上皮移植中的作用进行鉴定，旨在建立TE-HCEP体外重建的技术工艺条件，获得可长期维持兔角膜透明的TE-HCEP，为TE-HCEP替代捐献角膜用于角膜上皮异常疾病的临床治疗创造条件。
为了进一步鉴定非转染HCEPC细胞系的属性、功能蛋白的表达及潜在致瘤性，本文对第80代HCEPC进行了复苏、扩增培养和鉴定。属性检测结果显示，复苏的第80代HCEPC透明度高，形态饱满，呈圆形或卵圆形，镶嵌排列呈铺路状，细胞群体倍增时间为40.75h，生长状态良好，分裂旺盛，特征性染色体数目仍为2n=46。功能蛋白的表达检测结果显示，该细胞能够表达HCEP特异性标志蛋白——角蛋白（keratin）3和12，以及紧密连接蛋白ZO-1、E-钙粘蛋白、间隙连接蛋白-43、整联蛋白β1，表明该细胞仍具有角膜上皮的表型，且具有形成完整HCEP结构的潜能。致瘤性检验结果显示，该细胞无致瘤性。因此，该细胞为TE-HCEP体外重建提供了种子细胞。
为了获得TE-HCEP体外重建的理想载体支架，本文以新鲜AM为材料，采用胰酶倒置消化法对其进行去上皮层处理，再使用Ⅳ型胶原进行包被，并通过接种HCEPC研究dAM的生物相容性。通过光镜、电镜以及石蜡切片HE染色等方法对处理后的AM以及HCEPC在经包被的dAM上的生长情况进行了观察和鉴定，结果显示利用胰酶于37℃倒置消化20min，再经Ⅳ型胶原包被后获得的dAM表面平整，无上皮细胞残留，且有基底膜结构；dAM对HCEPC具有理想的生物相容性，且光学性能良好。因此，dAM能够作为理想的载体支架进行TE-HCEP的体外重建研究。
为了建立TE-HCEP体外规模化重建的技术工艺条件，本文以生长状态良好的HCEPC为种子细胞，以dAM为载体支架，采用气-液界面培养法体外重建TE-HCEP。利用石蜡切片HE染色、免疫组织化学检测及电镜观察等方法对重建的TE-HCEP形态结构、潜在功能等方面进行了鉴定。结果显示TE-HCEP经气-液界面法培养5d后即可形成6~7层的复层结构，结构与正常HCEP结构最为相近，且光学性能良好，表层的HCEPC平整呈卵圆形，且形成了丰富的微绒毛结构。TE-HCEP仍具有HCEP属性以及形成细胞间、细胞与dAM间连接的潜能，超微结构与正常的角膜上皮层结构相近。因此该方法能够获得理想的TE-HCEP，有望在将来进行规模化生产。
为了研究体外重建的TE-HCEP在新西兰兔模型角膜上皮移植中的作用，本文利用DiI标记的第80代HCEPC在经包被的dAM上进行体外重建。根据改良的三种方法分别制作了LSCD新西兰兔模型，并利用TE-HCEP分别对其进行角膜上皮移植。利用角膜测厚、裂隙灯显微镜观察、印迹细胞学检查等方法跟踪记录了创伤以及移植后兔模型在体角膜的厚度、透明度、上皮完整性和新生血管情况；利用石蜡切片HE染色、免疫组织化学、电镜观察等方法进行TE-HCEP角膜上皮移植效果的离体检测。结果显示利用碱烧伤、机械创伤和联合法均能获得LSCD的新西兰兔模型；利用TE-HCEP对碱烧伤的LSCD模型进行角膜上皮移植，术后80d角膜仍为白瓷色，有新生血管；利用TE-HCEP对机械创伤获得的LSCD模型进行角膜上皮移植，术后第12d角膜开始透明，厚度明显下降，新生血管减少，第25d角膜厚度恢复到正常眼水平，且能够长时间保持较透明状态。术后120d的离体检测结果显示角膜上皮细胞来源于体外重建的TE-HCEP，约4~5层，角蛋白3表达呈阳性，上皮细胞之间存在紧密连接结构，与对照组存在显著性差异；利用TE-HCEP对联合法获得的LSCD模型进行角膜上皮移植，术后第12d角膜厚度明显下降，第49d角膜新生血管明显减少，第70d角膜厚度恢复到正常眼水平，角膜长时间保持较透明状态。术后147d的离体检测结果显示角膜上皮细胞来源于TE-HCEP，约3~4层，角蛋白3表达呈阳性，上皮细胞之间存在紧密连接结构并有基底膜结构，与对照组存在显著性差异。新西兰兔角膜上皮移植实验的结果表明，所移植TE-HCEP形成了形态结构较正常的角膜上皮层，新生血管明显减少，并能够使角膜长时间保持较透明状态。
综上所述，本文首次以非转染、无致瘤性的第80代HCEPC为种子细胞，以dAM为载体支架，体外重建出了形态结构和潜在功能与正常HCEP相近的TE-HCEP，其移植后能够长时间保持LSCD兔角膜较透明状态。因此，该TE-HCEP有望作为角膜上皮的替代物从根本上解决角膜供体材料严重不足的现状，为角膜上皮异常疾病的患者带来重见光明的希望，这不仅具有重要的理论意义，而且能产生巨大的社会效益和经济效益。
Human corneal epithelium (HCEP) is a multi-cellular membrane located at the anteriorend of cornea. As the first defence barrier against foreign invasion, HCEP cells (HCEPCs)are crucial for the maintenance of corneal transparency and oxygen and nutrients absorbing.HCEPCs are in dynamic equilibrium state that is maintained by a population of unipotentlimbal stem cells located at the limbus. Chemical or thermal burns, multiple surgicalprocedures, severe microbial infection, cryotherapy in the limbal region, contact lenses orocular cicatricial pemphigoid may lead to limbal stem cell deficiency (LSCD), which resultsin conjunctivalization and vascularization of the cornea thus impairing visual clarity.Although the treatment for ocular surface reconstruction including epithelial keratoplasty,limbal autograft and allograft transplantation has made some progress, the application ofthese treatments in clinic was limited due to severe shortage of donor cornea and hightransplant rejection percentage. However, corneal tissue engineering has opened a new pathfor in vitro reconstruction of tissue-engineered human corneal epithelium (TE-HCEP) whichwill cure LSCD by clinical corneal transplantation recently. HCEP cell lines as a powerfulresearch tool can provide a readily available source of HCEPCs for long-term studies onTE-HCEP. Unfortunately, there is no report on the application of HCEP cell lines as seedercells to treat LSCD because most of cultured HCEP cell lines have only been established bytransfection. These immortalized cell lines cannot be used for clinical purpose due to theirabnormal phenotypes, latent risk of tumorigenicity and decreased potency to reconstructmultilayered epithelia. Nor did spontaneously derived HCEP cell line which was establishedthrough serial culture of limbal cells from a normal human limbus be applied to the studieson TE-HCEP. Amniotic membrane facilitates epithelialisation, inhibits fibrosis, possessesanti-inflammatory, anti-angiogenic, anti-microbial and anti-viral properties, and it also has ahigh hydraulic conductivity and shows low or no immunogenicity. These advantages makeamniotic membrane an extremely useful biomaterial for ophthalmologcial surgery. Therefore,TE-HCEP was in vitro reconstructed with passage80HCEPCs presenting normal karyotype and based on the molecular identification from a continuous untransfected HCEP cell lineestablished by our laboratory as seeder cells and epithelium-denuded amniotic membrane(dAM) as scaffold carrier, and to examine their functions by corneal epitheliumtransplantation in LSCD rabbit models in this thesis.
Growth properties, chromosome morphological observation, RT-PCR, immuno-cytochemistry analysis and tumorigenesis assay were used to identify the properties,functions and latent risk of tumorigenicity of passage80HCEPCs. The results showed thatthe cells, with high transparency and cobblestone appearance in shape, proliferated activelyand constantly with a population doubling time of40.75hours. Predominant chromosomenumber of the cells is46. The HCEPCs express keratin3/12positively, which is a specificmarker of HCEPCs. This provestheir HCEP origin. Results of immunocytochemistry showedthat HCEPCs expressed zonula occludens1, E-cadherin, connexin-43, and integrin β1positively, which suggested that the HCEPCs still had normal phenotypes and the potentialto form normal HCEP. Besides, the cells had no tumorigenicity. Therefore, the passage80HCEPCs can be used as seeder cells for in vitro reconstruction of TE-HCEP.
A reverse trypsin digestion and scraping by scraper were used to get dAM. Then thedAM was coated collagen IV coating on its epithelial side. By microscopy, paraffin sectionHE, scanning electron microscopy (SEM) and transmission electron microscopy (TEM), theresults showed that the surface of processed dAM was smooth and acellular for20min aftertrypsin digestion at37°C. Besides, the dAM with high transparency had goodbiocompatibility to HCEPCs, which can be used as scaffold carriers for in vitroreconstruction of TE-HCEP.
TE-HCEP was reconstructed in vitro by using passage80HCEPCs as seeder cells anddAM as scaffold carries in air-liquid interface culture system. Light microscope observation,paraffin section HE, immunohistochemical staining, SEM and TEM were used to identifythe TE-HCEP morphology and potential functions. The results showed that HCEPCs, withhigh transparency and cobblestone shape, formed a6~7layers of an HCEP-like structure ondAM in air-liquid interface culture for5days. Under scanning electron microscopy andtransmission electron microscopy, the HCEPCs were rich in microvilli on apical surface, andconstructed numerous intercellular cell junctions including desmosomes and cell-dAMhemidesmosomes. Results of immunohistochemistry showed that the seeder cells ofTE-HCEP still had HCEP properties, and could potentially form tight junction, anchoringjunction and communicating junction. All these indicated that the reconstructed TE-HCEPhad almost the same morphology and histological structure as that of innate HCEP, providing a promise for scale production of TE-HCEP.
New Zealand white rabbit LSCD models were made for corneal epitheliumtransplantation to examine the functions of in vitro reconstructed TE-HCEP. There werethree improved menthod used for producing LSCD models. All of alkaline burning,mechanical trauma and composite method succeeded establishing LSCD models based oncorneal opacity, epithelial fluorescence staining, neovascularization and impression cytology.TE-HCEPs, reconstructed with CM-DiI labeled passage80HCEPCs as seeder cells anddAM as scaffold carries in air-liquid interface culture, were transplanted into LSCD rabbitmodels. The results showed that in alkaline burning LSCD group, the cornea had beenentirely opaque and covered with blood vessels, because the corneal limbus, cornealepithelium, even stroma and endothelium had been totally damaged, which are not suitablefor making LSCD pathological model because of unpredictable subsequent lesions; inmechanical removed LSCD group, the corneal stroma began to become transparent and newblood vessels formation reduced on day12after transplantation. The corneal thicknessreturned to normal level at day25. The HCEPCs with DiI labels, formed4~5layers structureand constructed tight cell junctions. Keratin3was expressed at day120, but the surfaceretained a little vascular ingrowth; in composite method LSCD group, the corneal stromabegan to become transparent and the new blood vessels reduced on day49aftertransplantation. The corneal thickness returned to normal level at day70. The HCEPCs withDiI labels, formed3~4layers structure and constructed tight cell junctions. Keratin3wasexpressed at day147, but the surface retained a little vascular ingrowth. Therefore, the invitro reconstructed TE-HCEP can make cornea of the transplanted eyes of LSCD modelstransparent for a long time.
In conclusion, TE-HCEP was reconstructed in vitro by using passage80HCEPCs asseeder cells and dAM as scaffold carries in this thesis. The TE-HCEP with normal structureand function can make cornea of the transplanted eyes of LSCD models transparent for along time, which can be used promisingly as LESC equivalents for clinical cornealepithelium transplantation. Therefore, successful in vitro reconstruction of TE-HCEP andtheir future clinical applications have not only great theoretical significance, but also veryimportant social and economic benefits.
为了进一步鉴定非转染HCEPC细胞系的属性、功能蛋白的表达及潜在致瘤性，本文对第80代HCEPC进行了复苏、扩增培养和鉴定。属性检测结果显示，复苏的第80代HCEPC透明度高，形态饱满，呈圆形或卵圆形，镶嵌排列呈铺路状，细胞群体倍增时间为40.75h，生长状态良好，分裂旺盛，特征性染色体数目仍为2n=46。功能蛋白的表达检测结果显示，该细胞能够表达HCEP特异性标志蛋白——角蛋白（keratin）3和12，以及紧密连接蛋白ZO-1、E-钙粘蛋白、间隙连接蛋白-43、整联蛋白β1，表明该细胞仍具有角膜上皮的表型，且具有形成完整HCEP结构的潜能。致瘤性检验结果显示，该细胞无致瘤性。因此，该细胞为TE-HCEP体外重建提供了种子细胞。
为了获得TE-HCEP体外重建的理想载体支架，本文以新鲜AM为材料，采用胰酶倒置消化法对其进行去上皮层处理，再使用Ⅳ型胶原进行包被，并通过接种HCEPC研究dAM的生物相容性。通过光镜、电镜以及石蜡切片HE染色等方法对处理后的AM以及HCEPC在经包被的dAM上的生长情况进行了观察和鉴定，结果显示利用胰酶于37℃倒置消化20min，再经Ⅳ型胶原包被后获得的dAM表面平整，无上皮细胞残留，且有基底膜结构；dAM对HCEPC具有理想的生物相容性，且光学性能良好。因此，dAM能够作为理想的载体支架进行TE-HCEP的体外重建研究。
为了建立TE-HCEP体外规模化重建的技术工艺条件，本文以生长状态良好的HCEPC为种子细胞，以dAM为载体支架，采用气-液界面培养法体外重建TE-HCEP。利用石蜡切片HE染色、免疫组织化学检测及电镜观察等方法对重建的TE-HCEP形态结构、潜在功能等方面进行了鉴定。结果显示TE-HCEP经气-液界面法培养5d后即可形成6~7层的复层结构，结构与正常HCEP结构最为相近，且光学性能良好，表层的HCEPC平整呈卵圆形，且形成了丰富的微绒毛结构。TE-HCEP仍具有HCEP属性以及形成细胞间、细胞与dAM间连接的潜能，超微结构与正常的角膜上皮层结构相近。因此该方法能够获得理想的TE-HCEP，有望在将来进行规模化生产。
为了研究体外重建的TE-HCEP在新西兰兔模型角膜上皮移植中的作用，本文利用DiI标记的第80代HCEPC在经包被的dAM上进行体外重建。根据改良的三种方法分别制作了LSCD新西兰兔模型，并利用TE-HCEP分别对其进行角膜上皮移植。利用角膜测厚、裂隙灯显微镜观察、印迹细胞学检查等方法跟踪记录了创伤以及移植后兔模型在体角膜的厚度、透明度、上皮完整性和新生血管情况；利用石蜡切片HE染色、免疫组织化学、电镜观察等方法进行TE-HCEP角膜上皮移植效果的离体检测。结果显示利用碱烧伤、机械创伤和联合法均能获得LSCD的新西兰兔模型；利用TE-HCEP对碱烧伤的LSCD模型进行角膜上皮移植，术后80d角膜仍为白瓷色，有新生血管；利用TE-HCEP对机械创伤获得的LSCD模型进行角膜上皮移植，术后第12d角膜开始透明，厚度明显下降，新生血管减少，第25d角膜厚度恢复到正常眼水平，且能够长时间保持较透明状态。术后120d的离体检测结果显示角膜上皮细胞来源于体外重建的TE-HCEP，约4~5层，角蛋白3表达呈阳性，上皮细胞之间存在紧密连接结构，与对照组存在显著性差异；利用TE-HCEP对联合法获得的LSCD模型进行角膜上皮移植，术后第12d角膜厚度明显下降，第49d角膜新生血管明显减少，第70d角膜厚度恢复到正常眼水平，角膜长时间保持较透明状态。术后147d的离体检测结果显示角膜上皮细胞来源于TE-HCEP，约3~4层，角蛋白3表达呈阳性，上皮细胞之间存在紧密连接结构并有基底膜结构，与对照组存在显著性差异。新西兰兔角膜上皮移植实验的结果表明，所移植TE-HCEP形成了形态结构较正常的角膜上皮层，新生血管明显减少，并能够使角膜长时间保持较透明状态。
综上所述，本文首次以非转染、无致瘤性的第80代HCEPC为种子细胞，以dAM为载体支架，体外重建出了形态结构和潜在功能与正常HCEP相近的TE-HCEP，其移植后能够长时间保持LSCD兔角膜较透明状态。因此，该TE-HCEP有望作为角膜上皮的替代物从根本上解决角膜供体材料严重不足的现状，为角膜上皮异常疾病的患者带来重见光明的希望，这不仅具有重要的理论意义，而且能产生巨大的社会效益和经济效益。
Human corneal epithelium (HCEP) is a multi-cellular membrane located at the anteriorend of cornea. As the first defence barrier against foreign invasion, HCEP cells (HCEPCs)are crucial for the maintenance of corneal transparency and oxygen and nutrients absorbing.HCEPCs are in dynamic equilibrium state that is maintained by a population of unipotentlimbal stem cells located at the limbus. Chemical or thermal burns, multiple surgicalprocedures, severe microbial infection, cryotherapy in the limbal region, contact lenses orocular cicatricial pemphigoid may lead to limbal stem cell deficiency (LSCD), which resultsin conjunctivalization and vascularization of the cornea thus impairing visual clarity.Although the treatment for ocular surface reconstruction including epithelial keratoplasty,limbal autograft and allograft transplantation has made some progress, the application ofthese treatments in clinic was limited due to severe shortage of donor cornea and hightransplant rejection percentage. However, corneal tissue engineering has opened a new pathfor in vitro reconstruction of tissue-engineered human corneal epithelium (TE-HCEP) whichwill cure LSCD by clinical corneal transplantation recently. HCEP cell lines as a powerfulresearch tool can provide a readily available source of HCEPCs for long-term studies onTE-HCEP. Unfortunately, there is no report on the application of HCEP cell lines as seedercells to treat LSCD because most of cultured HCEP cell lines have only been established bytransfection. These immortalized cell lines cannot be used for clinical purpose due to theirabnormal phenotypes, latent risk of tumorigenicity and decreased potency to reconstructmultilayered epithelia. Nor did spontaneously derived HCEP cell line which was establishedthrough serial culture of limbal cells from a normal human limbus be applied to the studieson TE-HCEP. Amniotic membrane facilitates epithelialisation, inhibits fibrosis, possessesanti-inflammatory, anti-angiogenic, anti-microbial and anti-viral properties, and it also has ahigh hydraulic conductivity and shows low or no immunogenicity. These advantages makeamniotic membrane an extremely useful biomaterial for ophthalmologcial surgery. Therefore,TE-HCEP was in vitro reconstructed with passage80HCEPCs presenting normal karyotype and based on the molecular identification from a continuous untransfected HCEP cell lineestablished by our laboratory as seeder cells and epithelium-denuded amniotic membrane(dAM) as scaffold carrier, and to examine their functions by corneal epitheliumtransplantation in LSCD rabbit models in this thesis.
Growth properties, chromosome morphological observation, RT-PCR, immuno-cytochemistry analysis and tumorigenesis assay were used to identify the properties,functions and latent risk of tumorigenicity of passage80HCEPCs. The results showed thatthe cells, with high transparency and cobblestone appearance in shape, proliferated activelyand constantly with a population doubling time of40.75hours. Predominant chromosomenumber of the cells is46. The HCEPCs express keratin3/12positively, which is a specificmarker of HCEPCs. This provestheir HCEP origin. Results of immunocytochemistry showedthat HCEPCs expressed zonula occludens1, E-cadherin, connexin-43, and integrin β1positively, which suggested that the HCEPCs still had normal phenotypes and the potentialto form normal HCEP. Besides, the cells had no tumorigenicity. Therefore, the passage80HCEPCs can be used as seeder cells for in vitro reconstruction of TE-HCEP.
A reverse trypsin digestion and scraping by scraper were used to get dAM. Then thedAM was coated collagen IV coating on its epithelial side. By microscopy, paraffin sectionHE, scanning electron microscopy (SEM) and transmission electron microscopy (TEM), theresults showed that the surface of processed dAM was smooth and acellular for20min aftertrypsin digestion at37°C. Besides, the dAM with high transparency had goodbiocompatibility to HCEPCs, which can be used as scaffold carriers for in vitroreconstruction of TE-HCEP.
TE-HCEP was reconstructed in vitro by using passage80HCEPCs as seeder cells anddAM as scaffold carries in air-liquid interface culture system. Light microscope observation,paraffin section HE, immunohistochemical staining, SEM and TEM were used to identifythe TE-HCEP morphology and potential functions. The results showed that HCEPCs, withhigh transparency and cobblestone shape, formed a6~7layers of an HCEP-like structure ondAM in air-liquid interface culture for5days. Under scanning electron microscopy andtransmission electron microscopy, the HCEPCs were rich in microvilli on apical surface, andconstructed numerous intercellular cell junctions including desmosomes and cell-dAMhemidesmosomes. Results of immunohistochemistry showed that the seeder cells ofTE-HCEP still had HCEP properties, and could potentially form tight junction, anchoringjunction and communicating junction. All these indicated that the reconstructed TE-HCEPhad almost the same morphology and histological structure as that of innate HCEP, providing a promise for scale production of TE-HCEP.
New Zealand white rabbit LSCD models were made for corneal epitheliumtransplantation to examine the functions of in vitro reconstructed TE-HCEP. There werethree improved menthod used for producing LSCD models. All of alkaline burning,mechanical trauma and composite method succeeded establishing LSCD models based oncorneal opacity, epithelial fluorescence staining, neovascularization and impression cytology.TE-HCEPs, reconstructed with CM-DiI labeled passage80HCEPCs as seeder cells anddAM as scaffold carries in air-liquid interface culture, were transplanted into LSCD rabbitmodels. The results showed that in alkaline burning LSCD group, the cornea had beenentirely opaque and covered with blood vessels, because the corneal limbus, cornealepithelium, even stroma and endothelium had been totally damaged, which are not suitablefor making LSCD pathological model because of unpredictable subsequent lesions; inmechanical removed LSCD group, the corneal stroma began to become transparent and newblood vessels formation reduced on day12after transplantation. The corneal thicknessreturned to normal level at day25. The HCEPCs with DiI labels, formed4~5layers structureand constructed tight cell junctions. Keratin3was expressed at day120, but the surfaceretained a little vascular ingrowth; in composite method LSCD group, the corneal stromabegan to become transparent and the new blood vessels reduced on day49aftertransplantation. The corneal thickness returned to normal level at day70. The HCEPCs withDiI labels, formed3~4layers structure and constructed tight cell junctions. Keratin3wasexpressed at day147, but the surface retained a little vascular ingrowth. Therefore, the invitro reconstructed TE-HCEP can make cornea of the transplanted eyes of LSCD modelstransparent for a long time.
In conclusion, TE-HCEP was reconstructed in vitro by using passage80HCEPCs asseeder cells and dAM as scaffold carries in this thesis. The TE-HCEP with normal structureand function can make cornea of the transplanted eyes of LSCD models transparent for along time, which can be used promisingly as LESC equivalents for clinical cornealepithelium transplantation. Therefore, successful in vitro reconstruction of TE-HCEP andtheir future clinical applications have not only great theoretical significance, but also veryimportant social and economic benefits.
引文
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