| PurposeIris pigment epithelium cells have the same embryonic origin and similar cell morphology as RPE cells, and easy to harvest, so it is expectable to substitute RPE cells for autologous transplantation to treat age - related macular degeneration, retinitis pigmentosa, and macular dystrophy etc. We use%reverse transcription polymerase chain reaction (RT - PCR) technique to detect and compare the mRNA expression of VEGF and its receptor FLK -1 in rabbit iris pigment epithelium cells and retinal pigment epithelium cells in order to provide a theoretics for IPE cells instead of RPE cells to transplantation.MethodsAnimals: healthy adult pigmented rabbits.Isolation of IPE and RPE cells: rabbit eyes were enucleated, The eyes were opened along the edge of cornea under aseptic conditions. Then, we isolated the IPE and RPE cells with 0. 25% trypsin -0. 02% EDTA solution and collected the cells into eppendorf tubes. The tubes were thrown into dry ice immediately and stored at - 80℃ until use.Total RNA isolation of IPE and RPE cells; Total UNA was ex-traded from IPE and RPE cells using TRIzol reagent.PCR primers; target genes are rabbit VEGF( AC: AB020216) and FLK - 1 ( AC: AB017155) , house - keeping gene is rabbit β-actin(AC: AF309819).RT-PCR: RT: RNA of IPE and RPE cells are reverse transcribe to cDNA by using reverse transcriptase AMV. PCR: the products of reverse transcription are used as templates, and β- actin is used as house - keeping gene, PCR amplified each target gene.Electrophoresis and semi - quantitive analysis of PCR products; the products were electrophoresed in agarose gels, and the photograph were analyzed by software to caculate the relative contents of mRNA for target genes.All experiments were repeated for four times, and the results were checked up by t - test.ResultsExamination of RNA samples; extracted total RNA of our study is not be decomposed.Quality expression and semi - quantitive analysis of PCR products : both rabbit IPE and RPE cells expression VEGF and FLK - 1 mRNA . mRNA content of VEGF and FLK - 1 in IPE cells is lower than that of RPE cells. mRNA expression of VEGF in IPE cells is a-bout 44. 62% of that of RPE cells, and FLK - 1 about 52. 36%. Through the check up of statistics, the differences are significant.ConclusionsA principal risk for visual impairment in wet ARMD is the subret-inal neovasculars. Neovalcuar derived form choroids, and it invaded through Bruchs membrane and reached under the retina, and formed choroidal neovascular membranes ( CNVM) finally cause leakage and result in visual damage. VEGF is a important angiogenic growth factor in the development of CNVM. An increased expression of VEGF in the RPE could be involved in the pathogenesis of this disease. VEGF secreted by RPE cells may affact the course of CNVM. Short - time overexpression of VEGF in the rat RPE leads to the development of choroidal neovascularization( CNV).The cause of failure to transplant homologous RPE is immunore-jection, while harvest of autologous RPE is difficult and which come from the patient RPE cells may possess the genetic defects, so it is not ideal to transplant. The studies in vitro suggest that IPE cells possess characteristic and important functions maintaining visual acuity, such as retinol metabolism, phagocytosis of ROS, substance transfer. Autologous IPE cells are easy to obtain from iritoectomy, and it may be proper substitute for RPE cells. Recently few studies have reported the expression of angiogenic growth factors of IPE cells. Our study detect the the mRNA expression of VEGF and its receptor FLK -1 in rabbit IPE cells and RPE cells. The results show that both rabbit IPE and RPE cells expression VEGF and FLK -1 mRNA . mRNA content of VEGF and FLK -1 in IPE cells is significantly lower than that of RPE cells. Transplantation of IPE cells to the subretinal space might be less active in the stimulation of choroidal or retinal angiogenesis thanRPE cells , and might be less possible for the formation of epiretinal memb...
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