The Effect And Molecular Mechanism Of Amphiregulin On Binocular Lens Induced And Congential Myopia Eye Axial Growth

Objective: 1.To analyze the distribution of eyeball biological parameters in high myopia,we analyzed the associated factors of high myopia,by screening for pathogenic mutation sites in congenital high myopia and acquired high myopia,which would provide more therapeutic genes for myopia gene therapy.2.To examine the effect and molecular mechanism of amphiregulin on binocular lens induced and congential myopia eye axial growth,which would provide an effective basis for the prevention and treatment of myopia.Methods: The study included three parts.Part one: Clinical epidemiology and genetic study of high myopia.We collected 175 cases of high myopia patients who came to our hospital from June 2016 to April 2017,Collect the patient’s age,gender,age of onset,vision,diopter,axial length,corneal curvature,intraocular pressure,anterior chamber depth,fundus lesions and other basic data,and according to the age of onset were divided into congenital(n=61)and acquired(n=114),Epidemiological analysis of these basic data,And collecting peripheral blood anticoagulant EDTA 5-6ml,Objective to find the mutation sites in congenital and acquired high myopia by gene sequencing.Part two: The effect and molecular mechanism of Amphiregulin on binocular lens induced eyes axial growth 60 healthy 3-week-old guinea pigs were randomly divided into five groups: normal control group,myopia control group,low dose antibody myopia group,middle dose antibody myopia group and high dose antibody myopia group,with 12 mice in each group.Our study used binocular goggle induced method to construct myopia model,that is,both eyes of the guinea pigs were induced by-10 D lens,while the normal group did not make any treatment.Myopia control group continued wearing goggles.Wearing the goggles for 2 weeks,the right eyes of guinea pigs were given intraocular injections of amphiregulin antibody(doses: 5,10,or 20μg)for the low,medium and high dose antibody myopia groups,respectively.At the same time,the left eyes of these groups were given intraocular injections of the same dose of Ringer’s buffer.Continue wearing the goggles before and after injection of antibody.Before wearing goggles,wearing goggles for 2 weeks,5 weeks after the experiment(Three times after the injection of antibody)and other three time points with a retinoscopy and ophthalmology A-measure guinea pigs diopter and axial length.The thickness of retina,choroid and sclera in the posterior pole was detected by PAS staining.The expression of amphiregulin and EGFR m RNA and protein was detected by Real time quantitative PCR and immunofluorescence.Part three: The effect of amphiregulin on congential myopia eye axial growth.24 healthy 3-week-old congential myopia guinea pigs were randomly divided into 3 groups: low dose antibody myopia group,medium dose antibody myopia group,and high dose antibody myopia group,with 8 mice in each group.After feeding for 2 weeks,the experimental eyes of guinea pigs were given intraocular injections of amphiregulin antibody(doses: 5,10,or 20μg)for the low,medium and high dose antibody myopia groups,respectively.At the same time,the control eyes of these groups were given intraocular injections of the same dose of Ringer’s buffer.Before entering the group,After feeding for 2 weeks,Three times after the injection of antibody,and other three time points with a retinoscopy and ophthalmology A-measure guinea pigs diopter and axial length.The thickness of retina,choroid and sclera in the posterior pole was detected by PAS stainingResults:1.In this study,most of the people with high myopia were acquired myopia,and the age of onset was mostly in the adolescent period,and most of them are male.In congenital high myopia population,there is mostly familial myopia,while the acquired high myopia is mostly sporadic.Compared with acquired high myopia patients,in patients with congenital high myopia,the best-corrected visual acuity decreased(P<0.001),the refraction decreased more significantly(P<0.001),and the axial length was longer(P<0.05).Adolescents with high myopia are characterized by decreased vision,prolonged axial length,and deep anterior chamber and decreased intraocular pressure.The axial length of high myopia was significantly negatively correlated with the corneal curvature and spherical equivalent degree(P<0.01),and was significantly positively correlated with the anterior chamber depth(P<0.01).Genetic studies have found that amphiregulin(AREG),vasoactive intestinal peptide receptor 2(VIPR2),apolipoprotein(ACAN),α1-type I collagen gene(COL1A1),and transforming growth factor β-inducible gene 1(TGIF1)have a higher frequency of variation in the high myopia population.2.Binocular model myopia study: There was no significant difference in refraction and axial length between the guinea pigs before the entry(P>0.05).After 2 weeks of lens induction,compared with the normal control group,the axial length of the myopic model group increased and the refractive decreased.The difference was statistically significant(all P < 0.05).Compared with the myopia buffer group,after intravitreal injection of amphiregulin antibody,the axial length of the low,medium,and high dose groups decreased,the diopter increased,and the scleral thickness at the posterior pole increased significantly.The difference was statistically significant(all P<0.05)and in a dose-dependent manner.There was no significant change in retinal thickness in the posterior pole of each group.Real-time quantitative PCR and immunofluorescence showed increased expression of amphiregulin m RNA and protein in the retina of the myopic model group and buffer group,and decreased expression of amphiregulin m RNA and protein in the retina of the high,medium,and low antibody groups,compared with the buffer group,The expression of epidermal growth factor receptor m RNA in the retina of the low-dose group was decreased(t=2.606,P=0.022).There was no significant difference in other groups.3.Study of congenital myopia: There was no significant difference in the diopter and the axial length of the guinea pigs in the experimental group of congenital myopia at the beginning of the study(all P > 0.05).After the injection of amphiregulin antibody,compared with the myopic buffer group,the axial length of the low,medium,and high dose antibody groups decreased,the thickness of the posterior sclera thickened,and the diopter increased.The difference was statistically significant in a dose-dependent manner(all P < 0.05).Conclusion:1.Adolescent males are risk factors for high myopia;congenital myopia is a risk factor for fundus oculi.The prolongation of the axial length of high myopia is related to the decrease of the corneal curvature,the decrease of the equivalent spherical degree,and the deepening of the anterior chamber.The frequency of mutation sites on AREG,VIPR2,ACAN,COL1A1 and TGIF1 genes increased in high myopia group,suggesting that these genes may be closely related to high myopia.2.After intraocular injections of amphiregulin antibody into the eyes of bilateral lens induced myopic guinea pigs,Compared with the myopia buffer group(the left eye),In the myopic antibody group(right eye),the diopter of the guinea pig increased,the axial length was significantly shortened,and the posterior sclera thickened.Its mechanism may be related to a decrease in the expression of amphiregulin.3.After intraocular injections of amphiregulin antibody into the eyes of congenital myopic guinea pigs,compared with the myopia buffer group,in the myopic antibody group,the diopter of the guinea pig increased,the axial length was significantly shortened,and the posterior sclera thickened.This shows that amphiregulin antibody can also delay the growth of the axial of congenital myopia.