Survival Of Donor Cells After Ex-vivo Expanded Allogeneic Limbal Epithelial Cells On Amniotic Membrane Transplantation

Part1the Establishment, Evaluation and Pathological Changes of the Rabbit Alkali Burn Related Limbal Stem Cell DeficiencyObjective:To establish the rabbit model of alkali burn related limbal stem cell deficiency (LSCD) which can simulate the whole clinical process, and to provide a simple and effective model for further research.Method:The establishment and evaluation of rabbit model with alkali burn related LSCD:(1) Model establishment:First Put an annular filter paper infiltrating lmol/L sodium hydroxide with10mm inner diameter and18mm external diameter on the corneal limb of a female rabbit for30seconds, and then remove. Drop1%sodium hydroxide in the conjunctival sac and the immediately wash it with500ml9%sodium chloride. Antibiotic ointment was applied every night after injury.(2) Model evaluation: Models were evaluated30days after injury when ocular surface inflammation is stable.①Observe, photograph and grade the model with slit lamp;②Impression cytology is applied to observe conjunctivalization;③Observe pathological changes in each layer of cornea and the limb by confocal microscopy;④Tear secreting funcation were test by Schirmer test Ⅱ;(3) Pathology:remove the corneal pannus, embed in the paraffin and stain with hematoxylin-eosin.Results:Cornea became opacity, edema and neovasculization30days after injury which were grade with clinical criteria.43of50eyes (86%) were scored as6～10,4out of50eyes (8%) were scored lower than6, and1of50eyes (2%) higher than10.1eye appeared corneal perforation and1eye lead to endophthalmitis. Goblet cells were found in all the cornea scored between6and10by impression cytology. By confocal microscopy were found that the vogt structures were disappeared, fibrous tissues covered the injured cornea, a number of new vessels deeply located in the epithelium and stroma, infiltrating with many inflammatory cells. Tear secreting were significantly decreased in the model eyes.Conclusion:Alkali burn is an effective method to establish limbal stem cell deficiency model, which can simulate the whole clinical process and provide provide a simple and effective model for further research. Part2Survival of Donor Cells after Ex-vivo Expanded Allogeneic Limbal Epithelial Cells on Amniotic Membrane TransplantationObjective:1. Ex-vivo expand allogeneic limbal epithelial cells on amniotic membrane, and transplant it to the LSCD models to reconstruct ocular surface.2. Evaluate the survival of donor cells by cell tracking technology, to resolve the long puzzled clinical problem.Method:1.(1) Cell culture:Limbal epithelial cells were digested by dispase Ⅱ from corneal limb of male rabbits, and then cultured on denuded amniotic membrane. When the epithelial cells overgrow and multi-layer on the denuded amniotic membrane, CM-Dil was applied to mark the cultured cells.(2) Cell status analysis:cell proliferation:Proliferative capacity of the cultured epithelial cell was detected by MTT1to8days after inoculation. Stem cells marker expression:ABCG2, p63and CK3were detected by immunofluorescent histochemical staining and PCR. Cell necrosis was detected by trypan blue and alizarin red staining. Cell apoptosis and cell senescence were respectively detected by TUNEL and beta-galactosidase kit.2. Cell transplantation and cell fate detection:(1) the ex-vivo expanded limbal epithelial cells were transplanted to the ocular surface of the LSCD models;(2) Survival of donor cells were detected by cell tracking technology at7,30,60,90days after transplantation with CM-DiI marking and SRY detecting;(3) Donor cell status analysis:cell differentiation, cell necrosis/apoptosis and cell senescence were detected at1,3,7,30,60,90days after transplantation.Results:1. The limbal epithelial cells can adhere, grow and form multi-layer on the amniotic membrane. Cell proliferative capacity increased significantly at the3rd day after inoculation, and increased to the top at the6th day, then decreased gradually. ABCG2, p63and CK3were all positive expressed in the ex-vivo expanded limbal epithelial cells on amniotic membrane. Only very few apoptotic and necrotic cells can be detected.2.20eyes scored between6and10received ex-vivo expanded limbal epithelial cells on amniotic membrane transplantation. Fluorescein sodium staining appeared positive in5eyes (25%) when we finished the surgeries. CM-Dil marked cells and SRY gene can be detected in the transplanted success corneas at90days after the transplantation, but both of them decreased gradually with time. Many apoptotic, necrotic and aging cells can be detected within7days after transplantation, most of which were detected as inflammatory cells. Regenerative corneal epithelium had been formed at30days after transplantation. Unlike normal corneal epithelium, the regenerative epithelium was thinner, and lack of cuboidal cells. Many inflammatory cells infiltrated under the amniotic membrane. Many of the corneas revascularized at60days after transplantation, many donor cells become apoptosis and necrosis. Only one cornea maintained transparent and avascular at90days after transplantation.Conclusion:Ex-vivo expanded allogeneic limbal epithelial cells on amniotic membrane can improve and reconstruct the ocular surface after alkali burn.7days after transplantation is the key period for donor cell survival. A few donor cells became apoptosis, necrosis and aging during7days after transplantation, but others can survive and form regenerated epithelium. But the regenerated epithelium is different from the normal epithelium with a poor resistentce. Many of them were injured60days after transplantation because of the inflammatory microenvironment in ocular surface. Part3Immunological Rejection after Ex-vivo Explanded Allogeneic Limbal Epithelial Cells TransplantationObjective:To observe the occurrence time, feature and the effect of anti-rejective treatment after ex-vivo expanded allogeneic limbal epithelial cells transplantation.Method:all the models were divided into4groups:A. immunosuppressive drug eye dropping group; B. immunosuppressive drug delivery system subconjunctival implanded group; C. no immunosuppressive drug applied group; D. negative control: autologous limbal epithelial cells transplantation. CD4+and CD8+T cells were detected in the cornea at14,30,60days after transplantation. The gene expression of CD4, CD8, were all examed by PCR.Results:Immunological rejection appeared at30～60days after transplantation. No immunological rejection was detected during14days postoperative.1case of epithelial rejection was found in A and B groups,3cases of rejection were found in C group. No rejection was found in D group. Variable degree of CD4+and CD8+T cells infiltration were detected in A, B, C group, but no CD8+T cells were found in D group. Gene expression of CD4and CD8were all detected in the four groups by PCR. CD4and CD8gene were significiantly higher in group C30days after transplantation. CD4and CD8gene expression in A group were significantly higher than that in B group at30day after transplantation.Conclusion:Although immunological rejection appeared later and milder in allogeneic limbal epithelial cells tansplantation than that in corneal or limbal transplantation, but it’s also an important factor in threatening donor cells survival. Immunosuppressive agents are benefit for the prognosis.14to90days after transplantation is the key period for immunosuppressive treatment. Subconjunctival implanted cyclosporine A drug delivery system is safty and effective for suppressing the rejection. But with the drug absorbing, the risk of rejection is higher. So it’s necessary to replenish more immunosuppressive durgs.