The Study On The Characteristics And Mechanisms Of Keratopathy In Mycn Mutant Corneal Opacity Mice

lindness and impaired vision are serious public health,social and economic problems in the world.In China,corneal opacity caused by keratopathy is the most important cause of blindness,second only to cataract.Previously,a C57BL/6(B6)background heterozygous corneal opacity mouse was obtained with an N-ethyl-N-nitrosourea(ENU)mutagenesis strategy in our laboratory.In the Mycn gene,we identified a T1217C transition mutation,which is predicted to result in a V406A amino acid change in the open reading frame.Therefore,the mutant mouse was named Mycn V406A.In addition,genetic experiments found that the mutant homozygous mice were lethal.Therefore,MycnV406A mutant heterozygote(Mycn V406A/+)mice were studied in this paper from the following three parts:1.Genetic and corneal histological analysis on Mycn V406A corneal opacity miceThe MycnV406A/+mice were mated with wild-type B6 mice to observe the phenotype of offspring mice and their genotypes were identified.Among the 66 offspring mice,31 Mycn V406A/+mice were identified.Among the 31 Mycn V406A/+mice,20 showed corneal opacity,4 had obvious microphthalmia and 7 had normal phenotype.9 of the 20 mice identified as corneal opacity showed postnatal day 0(P0)eyelids open.Therefore,MycnV406A/+corneal opacity mice were divided into mice with eyelids open at birth(EOB)and mice with eyelids closed at birth(ECB).After H.E staining of eyeballs of 6-8 weeks(W)mice,it was found that:(1)Compared with the wild-type mice,the partial corneal epithelium of the mice with EOB was thickened,and corneal neovascularization and inflammatory cells were detected in the corneal stroma.(2)The corneal epithelium of the mice with ECB was thinned slightly,and corneal neovascularization was occasionally detected in the corneal stroma.2.The effect of MycnV406A mutation on the differentiation of mice corneal epithelial cellsWe performed staining for the corneal epithelial cells differentiation marker keratin 12(Krt12),the epithelial cell differentiation marker keratin 14(Krt14),and a specific epidermal differentiation marker,keratin 10(Krt10),by immunohistochemistry in EOB,ECB and control littermate mice at 6-8 weeks.Immunohistochemistry showed that:(1)In the control corneas,Krt12 was present in the corneal epithelium in the control corneas,Krt10 expression was not detected and Krt14 was present but weak in the basal layer of the corneal epithelium.(2)In EOB corneas,Krt12 staining of partial corneal epithelial cells in eyelid open mice were negative,Krt14 was expressed most prominently in the basal and suprabasal layer of the comeal epithelium,and Krt10 expression was detected in partial comeal epithelial cells.(3)In EOC corneas,no abnormalities were found in the staining of Krt12 and KrtlO corneal epithelial cells,while Krt14 was similar to the EOB corneas.In normal mice,eyelids closure occurres at embryonic 16.5 days(E16.5).In order to determine whether the phenotype of corneal opacity mice with ECB and abnormal differentiation of corneal epithelial cells were directly caused by delayed eyelid closure in embryonic mice,H.E and immunohistochemical staining of the ocular regions of MycnV406A/+E16.5 mice were continued.The results showed that:(1)Genetic statistics showed that the proportion of E16.5 mice with eyelids open was similar to that of corneal opacity mice,and higher than that of mice with EOB,suggesting that some mice had delayed eyelids closure and closed before birth.(2)Compared with wild-type mice,Mycn V406A/+E16.5 eyelids open ocular regions showed abnormal comeal epithelium thickening,abnormal Krt12 expression,and the expression level of Krt14 was significantly increased,while MycnV406A/+E16.5 eyelids closed ocular regions showed no abnormality,suggesting that eyelids opening in embryonic period might affect the differentiation of corneal epithelial cells.3.Preliminary analysis on the mechanism of MycnV406A mutation affecting the differentiation of corneal epithelial cellsThe 6-8 W corneal opacity mice with EOB and ECB were screened,and the wild-type mice in the same litter were set as the control group.The immunohistochemical staining was used to detect cyclin D1,integrin ?1,integrin ?4 and E-cadherin in mice corneal epithelial cells.The results showed that:(1)The positive cells of integrin ?1 and integrin ?4 in the corneal epithelium of wild-type mice were confined mainly to the basal layer and the expression of integrin ?1 was weakly;The expressions of integrin ?1 and integrin ?4 of comeal epithelial cells were strongly and the expression of corneal epithelial thickening areas was negative in the mice with EOB.The expressions of integrin ?1 and integrin ?4 of all the corneal epithelial cells of the mice with ECB were strongly.(2)Compared with wild-type mice,the expression level of positive cells of cyclin D1 in corneal epithelial cells of mice with EOB and ECB was decreased.(3)The immunohistochemical staining result of E-cadherin in the corneal epithelial cells of the three groups of mice was positive and there was no significant difference except that the expression of the corneal surface cells of mice with EOB were negativeResearch shows:we obtained a corneal opacity mouse in the early period,which was caused by Mycn gene mutation.On this basis,corneal opacities were divided into two types:one with EOB and the other with ECB.Genetic experiments and corneal epithelial histological analysis were conducted on mutant mice to know the characteristics of keratopathy caused by Mycn V406A mutation.Immunohistocheiical staining analysis showed abiormal differentiation of the corneal epithelial cells in the mutant mice,which might be related to the abnormal expressions of integrin ?,integrin ?4 and cyclin D1.