| Retinal and optic nerve diseases caused by hypoxia and ischemiais the common blind eye diseases,including proliferative diabeticretinopathy,central/branch rentinal artery/vein occlusion, age-related macular degeneration,retinopathy of prematurity et al. latermost of diseases's complications is retinal neovascularization,causing vitreous repeating hemorrhage,proliferative retinopathy ,detachment of retinal,neovascular glaucoma,at last leading toserious vision disorder. At present, because of high incidence andlow cure rate ,it has attracted many scholar's concern , rapidlybecome a popular field of international researches, but in the worldwe are still lack of effective therapeutic means, it is most importantto find a suitable drug for the prevention and treatment.The pathologic changes of RPE cells is of very importance to thedevelopment prosess of retinal hypoxia and ischemia disease.Toobserve the pathologic changes of RPE cells of hypoxia maybe thetheoretical basis of pathogenesis,prevention and treatment of thediseases.EPO is one of th endocrine hormones which acts on bone marrowstromal cell and accelerats hyperplasia,differentiation andmaturation of erythroid progenitor cells. Recent studies has foundthat in the stress situation EPO protects the normal functions oforganisms.Recent studies think that EPO belongs to members of multifunctional cytokine superfamily which is induced by hypoxia-inducibhfactor.It protects the normal functions of organisms in the stresssituation.now the research of EPO has extended to retina and opticnerve which is the most vulnerable to hurt in hypoxia and ischemiasituation.At present most of the research of EPO focus on nerve protection,but the protection of retinal research is less.Cultured hRPE cellsin vitro were used in this experiment,the purpose of the experimentis to investigate the proliferation of cell by Cobalt Chlorlde-Simulated Hypoxia, detecte EPO mRNA expression and the relationshipbetween the quantities with various concentration and during timeof cocl2 ,which maybe establish basis for more animal experiment andclinical research and will be a profound research and applicationin ophthalmology.In this experiment cultured hRPE cells were treated with cocl2and investigated the expression of EPO.at first we established thehRPE cells line, MTT assay was used to determine the proliferationof cell of control group and experimental group.From the MTT assay result, control group cells were not be treatedwith cocl2 ,experimental group were be treated with cocl2 of variousconcentration,the concentration of cocl2 including 50μmol/L, 100μmol/L,150μmol/L,200mol/L,250μmol/L ,500μmol/L and 1000μmol/L,determined light absorption value(A value)of every hole with enzymelinked immunity instrument,when the the concentration of cocl2 is50μmol/L, A value between control group and experimental group werenot significantly different,t=1.372,P>0.05.When hRPE cells were treated with 100-500μmol/L,A value between control group and experimental group were ofapparently differences, t=12.13,t=94.11, t=87.92,t=63.21, t=100.12,P<0.01,but if the concentration was higher than 500μmol/L,thecells proliferation was apparently inhibited, in a certain range ofconcentration,hypoxia can promote the proliferation of hRPE cells.In this experiment we detect the expression of EPO in anoxic hRPEcells from two different points of view,first,semi-quantity RT-PCRmethod was used ,the electrophoretic mappings were analysed by imageanalysis system, accounted the grey ratio of EPO/β-actin of DNAladder pattern by electrophoresis, From the DNA agarose gelelectrophoresis of expression of EPO in hRPE cells treated with cocl2of various concentration,we confirmed that a small quantity ofpositive expression of EPO mRNA was found in primary hRPE cells,Whenthe concentration of cocl2 was 125μmol/ L, the expression of EPOincreased obviously, afterwards the expression of EPO was decreasedwhen concentration of cocl2 increased.When the concentration of cocl2was 1000μmol/ L,the expression was decreased obviously,in severehypoxia situation,hRPE cells were damaged,From the DNA agarose gelelectrophoresis of expression of EPO in hRPE cells treated with cocl2of same concentration (125μmol/L)on various during time. When theduring time of cocl2 was 4h ,the expression of EPO increased obviously,When the during time of cocl2 was 8h, the expression was decreasedseverely,maybe related with that cells were damaged in unintermittedhypoxia,the electrophoretic mappings were analysed by image analysissystem,accounted the grey ratio of EPO/β-actin of DNA ladder patternby electrophoresis,we also found that a small quantity of positiveexpression of EPO mRNA was found in primary hRPE cells,high levelexpression was observed When the concentration of cocl2 was125μmol/L, When the concentration of cocl2 was 1000μmol/L,hRPEcells were damaged and the expression of EPO was decreased .From theDNA agarose gel electrophoresis of expression of EPO in hRPE cellstreated with cocl2 of same concentration (125μmol/L)on variousduring time.at 4h,the expression of EPO reached its maximum,at8h,theexpression of EPO was not remarkable.second,By using the immuno-fluorescence method,the changes of the expression of EPO wasreflected sensitively,positive expression is red fluorescencesignal in the plasm of cells, using image analysis system,we randomlyselected three hRPE cells from different visual fields,accounted andanalyzed the fluorescence density average and standard error,Fromthe immunofluorescent photograph,when the concentration of cocl2 was125μmol/L,at 4h,high level expression was observed, when theconcentration of cocl2 was 500μmol/L,the expression of EPO decreased,when the concentration of cocl2 was 1000μmol/L, cellular morphologychanged,we found the expression was decreased severely, thefluorescence density average:control group 49±21;the concentrationof clcl2:125μmol/l group 123±23,250μmol/l group 114±30, 500μmol/l 93±26, 1000μmol/l 38±20,the result of immunofluorescencemethod accorded with semi-quantity RT-PCR method .a small quantity of positive expression of EPO was found in primaryhRPE cells, in a certain range of concentration,hypoxia can promotethe proliferation of hRPE cells. in severe hypoxia situation ,hRPEcells were damaged , the expression of EPO was not remarkable.wepresumed that a mass of the endogenous EPO generated by RPE cellsin hypoxia situation maybe protective mechanism of RPE cells, weshould study rentinal protective mechanism of EPO,established basisfor more animal experiment and clinical research, we thought thatEPO may provide a new therapeuticmethod for retinal and optic nervediseases caused by hypoxia and ischemia. |
PDF Full Text Request