The Effect Of Col4a3 Gene Knockout On Retina

Objective:Alport syndrome is an inherited disease characterized by progressive renal failure,hearing loss,and ocular abnormalities.Col4a3 knockout mice can be the disease model of Alport syndrome had been certified in the kidneys and ears,but there is little research in the eye.Our experiment used immunohistochemistry to assay the distribution of ? chains of type IV collagen in Bruch's membrane of wild type mice and col4a3 knockout mice and compared the differences between them.Then electron microscopy(TEM)and histology techniques were used to verify whether there were defects in the Bruch's membrane of col4a3 knockout mice.Also,to detect the damage of retinal pigment epithelial layer(RPE)and visual cells in col4a3 knockout mice.Our purpose is to verify whether col4a3 knockout mice can become the new animal model of Alport syndrome in retina or not.Methods:1.Immunohistochemistry:To compare the distribution of type IV collagen ? chains and other relative components in retina between wild type mice and col4a3 knockout mice.2.Electron microscopy(TEM)and histology techniques: To prove Bruch's membrane,RPE layer and optic cells had defects in col4a3 knockout mice.Results:1.Immunohistochemistry:Distribution of ?(IV)collagen chains in the retina basement membrane of col4a3 knockout mice is different from in wild type mice.Strong signals of ?1(IV)??2(IV)??3(IV)??4(IV)??5(IV)were shown in the Bruch's membrane of wild type mice;however there were only weaken signals of ?1(IV)and ?2(IV)in the Bruch's membrane of col4a3 knockout mice.2.Electron microscopy(TEM): Compared to wild type mice,col4a3 knockout mice's Microvilli structure was changed,basement membrane became thinner,the basal infoldings partly disappeared,the structure of RPE layer became confused,the conjunction of RPE cells became inattentive.3.Histology:The thickness of RPE layer in retina of wild type mice is obviously thicker than in col4a3 knockout mice(P<0.001).4.Immunohistochemistry:(1)Compared to wild type mice,the signals of RPE65 in col4a3 knockout mice were changed obviously: the distribution of RPE pigment granules was uneven and the number of pigment granules were decreased obviously.(2)Compared to wild type mice,the signals of Ezrin shown that in the RPE layer,the structure of microvilli in col4a3 knockout mice were changed obviously.(3)Compared to wild type mice,the signals of GFAP in col4a3 knockout mice were markedly enhanced from internal limiting membrane to external limiting membrane.(4)Compared to wild type mice,the signals of Fibronectin in col4a3 knockout mice were slightly enhanced.(5)Compared to wild type mice,the signals of Laminin5 in col4a3 knockout mice were markedly weakened.(6)Compared to wild type mice,the signals of Albumin in col4a3 knockout mice were changed obviously,signals were markedly enhanced and flooded with the full RPE layer.Conclusion:col4a3 gene knockout does produce pathological changes in the retina of mice.We can initially conclude that col4a3 knockout mice can be the animal model of Alport syndrome with ocular retinopathy.