| Choroidal neovascularization (CNV), which derives from abnormal vessels of choriocapillaris, can progress to haemorrhage and disciform scarring, resulting in severe visual loss especially in age-related macular degeneration, pathologic myopia, centrol retinitis and so on. The retinal pigment epithelium (RPE) is a monolayer of neural ectoderm derived cells that is located between the photoreceptor layer of the retina and the choroid. The RPE fulfills several vital functions for the retina. It scavenges shed discs from the photoreceptor outer segments and recycles their components, particularly the retinoids that provide the visual pigment. It also transports nutrients from the copious blood supply of the choroid into the retina and transports waste in the opposite direction. Though the pathogenesis of CNV remains obscure, it has been postulated that retinal pigment epithelium (RPE) plays an important role in its occurrence. In this experiment, firstly, hRPE cells were isolated by trypsin enzyme-digesting technique and cultured in our laboratory. Next, Human primary RPE cells were successfully identified by the way of HE staining, Immunofluorescent staining and Microscopic examinations. The cultured hRPE cells formed monolayers showing typical polygonal morphology and pigmentation of scattered cells. Cells of epithelial origin express cytokeratin intermediate filaments, which are considered a characteristic marker of the epithelial phenotype. Primary hRPE cells showed positive immunoreactivity for a panel of anti-human cytokeratin antibodies. Lastly, we examined biological activities of hRPE. The data showed that the viability of hRPE cells under normal culturing condition was over 97.5 % and cryopreserved hRPE cells was 88.6%±3.2%. The purity of primary hRPE cells was over 95.6%. The population doubling time was 3.8 days and passage time was about 21 days. The primary hRPE cells were successfully cultured in this experiment, which established the basis for the study of CNV. Though the pathogenesis of CNV remains obscure, it is admitted that the dropout of choriocapillaris could cause RPE to become ischemic and produce growth factors that stimulate the formation of choroidal neovascularization (CNV). Hypoxia (or low O2 levels) affects acute or chronic pathologies. Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor that regulates cellular responses to physiological and pathological hypoxia by up-regulating the expression of genes involved in angiogenesis (e.g. vascular endothelial growth factor, VEGF). HIF-1 consists of two subunits, HIF-1 αand HIF-1 β. Undernormoxic conditions, HIF-1αsubunits are unstable, being rapidly targeted to the ubiquitin-proteasome pathway. Further study suggested that amino acids 401-603 of HIF-1 αconstituted an oxygen-dependent degradation domain (ODD), Oxygen-dependent instability can be conferred on other proteins by transfer of ODD from HIF-1α. To investigate the level of HIF-1αin hRPE induced by hypoxia on different time point and to study mechanism of choriodal neovascularization in the future, we established hRPE cells model of hypoxia. We detected the level of mRNA and the protein of HIF-1αin hRPE cells on different time point induced by hypoxia with the method of semi-quantitative RT-PCR and immunofluorescence technique. It showed that the level of HIF-1αrose obviously induced by hypoxia . After 8 hours induced by hypoxia, the level of HIF-1αin the hRPE reached a peak and sustained for more than 16 hours . But because of the long time effect of hypoxia , the morphosis of hPRE began to change and the level of HIF-1αdescended. The level of HIF-1αin hRPE rose obviously induced by hypoxia. It may be a important step in the process of choriodal neovascularization. RNA interference (RNAi) –the posttranscriptional gene silencing (PTGS) is one of the most exciting discoveries of the past decade in functional genomics.
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