The Study On The Effect Of Retinoic Acid To The RPE-choroid Complex In Guinea Pigs With Lens-induced Myopia

Chapter1The Expression of RA and Tight-junction protein ZO-1、 Occludin in the Lens-induced Myopia RPE-Choroid Complex of Guinea PigsPurpose:To observe the dynamic change of RA, connexin ZO-1and Occludin’s expression in RPE-choroid complex, and investigate the possible control performed by RA, ZO-1, and Occludin in myopia, by establishing guinea pig’s defocused myopia model.Method:Randomly divided30three-color guinea pigs with the age of three weeks (each weigh about150-180g, male or female) into Group A: Normal Group (n=15) and Group B(n=15):Lens-induced Group. Guinea pigs of Group A wear no glasses. In Group B, each guinea pig weared-6.00D concave lens on its right eye and no lens on its left eye. Right eye was used as the experiment eye and left eye is used as the self control eye. We inspected their diopter and axials on Day0, Day3, Day7and Day14, respectively.We also examed the content of RA in RPE-choroid complex using high performance liquid chromatography, examed the content of mRNA in ZO-1and Occludin using Real-time PCR, examed the content of ZO-1and Occludin using Western blot technique,and observed their expression locations using Immunofluorescence technique.Results:1. Change of diopter and axial:with change of time, the diopter of both eyes of the guinea pigs in Group A were emmetropized, the axials were increased in normal speed. There was no statistical difference in the diopter and axial length of both eyes (P>0.05) on each of experiment time. In Group B, the experiment eye shifted to myopia. On Day14, the relative myopic diopter was about4.7D. There was statistical difference between the diopter of the experiment eye and that of the self control eye (P<0.05). The increase speed of the axial of the experiment eye was faster than that of the self control eye. On Day14, the difference in axial length between the experiment eye (8.18±0.09mm) and the self control eye (7.68±0.06mm) was the largest. There was statistical difference between the axial length of the experiment eye and that of the self control eye (P<0.05). On each of the Day0, Day3, Day7and Day14, there was no statistical difference between normal eye and self control eye (P>0.05).2.Change of RA content:The RA content of RPE-choroid complex upregulated along with the time ago, and the experimental eye occured the highest expression on day14(137.85±1.02ng/100mg), There was statistical difference which compared to the self control eye (12.67±0.40ng/100mg)(P<0.05). On each of the Day0, Day3, Day7 and Day14, there was no statistical difference between normal eye and self control eye (P>0.05)..3.Change of tight junction proteins:ZO-1、Occludin mRNA and protein were upregulated in RPE-complex of myopia eyes along with the time ago,and expressed highest on day14, There was statistical difference which compared to the normal eye and contrast eye (P<0.05).On each of the Day3, Day7and Day14, there was no statistical difference between Group A and Group B (P>0.05).The result of immunofluorescence technique was according to the Real-time PCR and Western blot.Conclusion:Guinea pig wears-6.00D concave lens can establish lens-induced myopia model successfully. RA was upregulated in the lens-induced myopia of guinea pig, which may participate in the development of myopia; also ZO-1and Occludin which upregulated may participate in the development of myopia. Chapter2The Effect of LE540on the RA and Tight-junction protein ZO-1、Occludin in the Lens-induced Myopia RPE-Choroid Complex of Guinea PigsPurpose:To observe the effect of LE540on the RA and tight-junction protein ZO-1、Occludin in the lens-induced myopia RPE-choroid complex of guinea pigs, to investigate the regulation relationship of RA and ZO-1、Occludin.Method:Randomly divided90three-color guinea pigs with the age of three weeks (each weigh about150-180g, male or female) into Group A:Normal Group (n=15),guinea pigs weared no glasses, with no drug injected into each of eye; Group B:Lens-induced Group(n=15), each guinea pig weared-6.00D concave lens on its right eye and no lens on its left eye, with no drug injected into each of eye; Group C:Negative control Group(n=15):each guinea pig wears-6.00D concave lens on its right eye and no lens on its left eye, with the PBS injected into each right eye;Group D:Drug injection Group(n=15):each guinea pig wears-6.00D concave lens on its right eye and no lens on its left eye,with LE540injected into each right eye.The Group D would be divided into3groups by different concentration:Group D1(10μM),Group D2(50μM), Group D3(100μM). Left eye is used as the contrast eye in each group. We inspected their diopter and axials on Day0,Day3, Day7and Day14, respectively. We picked out the Group D among the Group D1、D2and D3by their effect to the myopia.Then on Day3, Day7and Day14, we also examed the content of RA in RPE-choroid complex using high performance liquid chromatography, examed the content of mRNA in ZO-1and Occludin using Real-time technique, examed the content of connexin ZO-1and Occludin using Western blot technique, and observed their expression locations using Immunofluorescence technique.Results:1. Change of diopter and axial:Group A and Group B had the same change trend as the development of chapter1.Group C had the same change trend as the Group B.In Group D,the trend of diopter shifted to myopia and the increased axial length were inhibited by LE540injected into vitreous cavity.The most effection to the myopia was Group D2(50μM). On each of the Day0, Day3, Day7and Day14, there was no statistical difference between normal eye and self control eye (P>0.05).2.The effect to the RA in the RPE-choroid complex by LE540injection: There was no apparent change in double eyes of Group A.The RA was upregulated in right eyes of Group B.The Group C had the same change trend as Group B,there was no statistical difference between tow groups(P<0.05).In Group D,the upregulation trend of RA was inhibited by LE540, the Group D2((50μM) had the most effective inhibition.There was no statistical difference between normal eye and self control eye (P>0.05).3.The effect to the tight junction proteins in the RPE-choroid complex by LE540injection:There was no apparent change In Group A. The tight junction proteis were upregulated in Group B.The Group C had the same change trend as Group B.In Group D,the upregulated trend of the ZO-1and Occludin’s mRNA were inhibit by LE540injection. The expression of ZO-1and Occludin were expressed mainly in RPE layer of RPE/choroid complex. The result of immunofluorescence technique was according to the Real-time PCR and Western blot.Conclusion:The development of lens-induced myopia in guinea pig can be inhibited effectively by LE540injected into the vitreous cavity. RA may participate in regulation of myopia by modulate the tight junction protein protein ZO-1and Occludin.