| 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. |
PDF Full Text Request