MHC Ⅱ Transactivator Promoter (CIITApⅣ) Regulation Of HLA-DR And Fas Ligand Expression On Retinal Pigment Epithelial (RPE) Cell In Vitro And In Vivo Study

Purpose: The retinal pigment epithelial(RPE) cell ,due to its unique anatomic location and particular physiologic function combined with neuron retina and choroidal microvascular ,give rise to its crucial role in maintaining the retinal vision potential,simutaniously,RPE cell increasingly displaying more immunologic characteristics that containing secrition of multi kinds of cytokines and expression of some cytokines receptors involves in keeping the neuron retina function ,reaction and healing to damage of retina.The normal subretinal space indicates certain immune privilege potential as antirior chamber and vitrous space,wherears RPE cell keep its characteristics through pathways including inducing immune tolerance, inhibiting even killing activited immune cells and secreting cytokines to modulate other cell activityby autocrine or paracrine mode.lt is well known that damaged RPE cell in retina detachment initially express some proinflammatory factors and other H2 alike cytokines such IFN-gamma and IL-2. MHC II molecular antigens are the major allograft antigen inducing transplantation rejection and simultaniously the APC associated episodes between the immune cell interactions. MHC II transactivator (CIITA) ,the major one of the modulation transcriptional factors for MHC II molecular antigen expression, holds a crucial role in promoting MHC II molecular expressions; recently studies indicate Fas ligand combinding to its recepter Fas mediate the Fas bearing cell apoptosis that mean the major mechanism to induce activated T cell death(ACID) for maintaining the immune system homeostasis . further study suggest that the CIITA derectly compete with the NFAT ,the transcriptional factor of Fas ligand , IL-2 and IL-4. Four kinds of CIITA promoters, CIITApI to pIV, selectively promote CIITA expressions in different histological cell.normal RPE cell in vivo is bare expression of MHCII but reversed under some conditions such as the blood eye barrier damage and infection as HIV. Another study showing Fas ligand on RPE cell basal memebrane involving in the choriod microvascular endothelial cell apoptosis in choriodal neovascularization(CNV) formation,while the CNV is the major cause of many blindness diseases.lt is reasonably infer that the RPE cell specific CIITAp may be pivatol in regulating the RPE cell immune reflection. So ,further understanding the RPE cell immune characteristics and its immunomodulation mechanism during various abnormalities contribute to both clinically early test or treatment of RPE cell associated diseases and establishment of RPE cell bank induced for tolerance in vitro , whichimplying double importances of principle study and clinical application .Ourpurpose is to observe the threee immuneassociated molecules expression of HLA-DR ,Fas ligand and Fas and to analyze their relatives under IFN-gamma stimulation in vitro and RPE damaged in vivo in rabbit models, simultaneously ascertain the RPE cell specific CIITAp and immunosuppressant FK506 and cystoporine A(CsA) effects on it activity. Methods:1. The human RPE cell culture.purification and observation reference as Wang previously described method and take a little modification.after information confirmed by family five accident cadaver eye balls within 12 hours were used to obtain the primary RPE cell as followed treatment as brief, the anterior segment of the eye ball were cut through serrata following the lens and vitreous body were mechanically removed .hylurodase combined with dispase and trypsine in sequence were used to dissociate the RPE cell, centrifuge the primary RPE cell through Ficoll gradient solution ,reseed RPE cell on human amnion pretreatmented with dispase for observation of the cultured RPE cell microstructure;inderect immunofluorescence staining for test of the three kinds of molecules expression through flowcytometry and confocal micrography.2. establish the rabbit RPE damaged model through clinically used treatment such as transcleral themotherapy ,...