The Study Of α-crystallin In Mice In Oxygen Induced Retinopathy

Objective:To establish the mice with oxygen induced retinopathy(OIR) and investigate the changes in retinal alphaA- and alphaB-crystallin protein mRNA expression and their location characteristics.The results will contribute to a further research of alphacrystallin's action mechanism and pathways in neovascular angiogenesis.Method:1.OIR mice model:Severn days(P7) wild type C57BL/6J mice chosen to put in a terrarium with oxygen density of 75%±5%,breeded for five days and return to normal atmosphere on P12.Expect for the oxygen density,there is no difference in the condition of breeding.2.On P13,P17 and P21,mice from OIR group and control group are taken for HE, to observe neovascular endothelial cells3;at the same time,mice from OIR group and control group are taken for FITC,under fluorescence microscope to observe shape and location of blood vessels3.3.Collectted retinal tissue from P13,P15,P17 and P21,3 mice in OIR and controlled groups.The alphaA- and alphaB-crystallin protein mRNA expression was studied by running Real time RT-PCR.and On P13,P15,P17 and P21,mice from OIR group and control group are taken for immunofluorescence test3 and to observe distribution and location of alphaA- as well as alphaB- crystallin protein.4.Statistic:the data are analyzed by 13.0 statistical software with methods of independent-sample T test and one-way ANOVA,P＜0.05 as the standard of significant difference in statistic.Results:1.HE staining:HE of the retina shows:Little lacuna vasorum founded on P17 of normal mice. There is more neovascular and lacuna vasorum,neovascular and neovascular clusters which break through internal limiting membrane or being near the crystal on P17 of OIR mice,among which founded the most retinal neovascular from P13 to P21.2.fluorescein isothiocyanate:FITC shows there is pedo-maturity vessles on P13 control mice,which eliminated on P17 of control group and none of normal vessles in peripheral retina, then in P21 there are normal retinal vessles.There are angioplerosis,distorted of vessles and nonperfusion in central retina of P13 OIR mice.On P17 OIR mice central retina vascular distend,few branch of proximal end,more nonperfusion with neovascular and leakage in peripheral retina.Then there are nonperfusion areas without leaking on P21 OIR mice.3.Expression of alphaA and alphaB crystallin protein mRNA:Expression of alphaA-crystallin protein mRNA of OIR mice is decreased and then increased and decreased again,decreased from P13 and down to a lower level on P15, Thereafter the express increased obviously and reached a peak on P17,There is a statistical difference between P17 and P13,P15(P＜0.05),and then decreased on P21.There is no statistical difference between P17 and P21(P＞0.05);OIR mice alphaB-crystallin protein mRNA express are stable on P 13,P 15 and P 17,There is no statistical difference between P17 and P13,P15(P＞0.05),then reached a peak on P21.There is a statistical difference between P21 and P13,P15,P17(P＜0.05).4.Immunofluorescence test of alpha-crystallin protein:In inner nuclear layer,the two proteins expresses both in cell membrane and cytoplast,but only in cell membrane in outer nuclear layer.The expression of alphaA-crystallin is the more on P17,the lower on P15,and there is no difference between the inner and outer nuclear layers;The expression of alphaA-crystall of OIR mice is more than control mice in retinal ganglion cell, inner and outer nuclear layers.The more in retinal ganglion cell layer on P21 and in the inner and outer nuclear layers on P17.The expression of alphaB-crystallin is existted in retinal ganglion cell layer of control mice at diffetent time,the more on P15,the lower on P21,The expression of alphaB-crystallin in inner and outer nuclear isn't obvious on P13,but they are obviouse on P15-P17,and decreased on P21;The expression of alphaB-crystallin manifest in retinal ganglion cell,inner and outer nuclear layers of OIR mice,increased on P 13 to P21.Conclusion:1.Authenticate the mice of Oxygen induced retinopathy,which can be modeling and progress of retinopathy of prematurity,and prove the retinal neovascular.2.Detect the retinal mRNA expression of alphaA- and alphaB-crystallin protein in OIR and control mice associated with time.3.Clear retinal location of alphaA- and alphaB-crystallin protein in OIR and control mice,which are associated with space.