玻璃体内注射双调蛋白抗体对透镜诱导型近视豚鼠屈光度、眼轴长度及视网膜中双调蛋白表达的影响

Effects of amphiregulin antibody on eyeball biological parameters in lens-induced myopic guinea pigs

目的探讨双调蛋白(amphiregulin,AREG)对透镜诱导型近视豚鼠屈光度、眼轴长度以及视网膜中AREG蛋白和mRNA表达的影响。方法选取2周龄健康英国三色短毛豚鼠30只,随机分为正常对照组、近视组、AREG低剂量组、中剂量组、高剂量组,每组各6只。除正常对照组外,其余组均右眼配戴-10.0 D透镜,左眼作为自身对照与正常对照组均不做任何处理,戴镜2周后分别测量各组的屈光度及眼轴长度。第3周开始,近视组豚鼠右眼玻璃体内注射Linger注射液3μL,低剂量组、中剂量组、高剂量组豚鼠右眼分别注射500 mg·L-1AREG抗体(R&D:MAB989-500)1μL、2μL、3μL,对侧眼作为自身对照,不做任何处理,每周注射1次,连续注射3周,期间继续配戴眼镜,3周后分别测量各组豚鼠的屈光度和眼轴长度。采用免疫荧光标记法检测豚鼠视网膜AREG蛋白的表达,实时荧光定量PCR法检测豚鼠视网膜AREG mRNA的表达。结果入组前各组豚鼠的初始屈光度及眼轴长度差异均无统计学意义(均为P>0.05);与正常对照组相比,透镜诱导2周后近视组、低剂量组、中剂量组、高剂量组豚鼠右眼屈光度下降,眼轴延长(均为P<0.05);各组左眼屈光度和眼轴长度差异均无统计学意义(均为P>0.05)。玻璃体内注射AREG抗体3次后,与近视组相比,低剂量组屈光度和眼轴长度差异均无统计学意义(均为P>0.05);中剂量组屈光度差异无统计学意义(P>0.05),但眼轴缩短,差异有统计学意义(P<0.01);高剂量组屈光度上升且眼轴缩短,差异均有统计学意义(均为P<0.05);各组对侧眼的屈光度和眼轴长度差异均无统计学意义(均为P>0.05)。免疫荧光标记法在各组豚鼠眼球后极部、赤道部以及后极部与赤道部之间的视网膜均检测到AREG蛋白,说明AREG主要分布在眼球视网膜组织,且无部位特异性。AREG蛋白在近视组豚鼠视网膜高表达,正常对照组低表达,玻璃体内注射AREG抗体后低剂量组、中剂量组和高剂量组AREG蛋白表达逐渐下降。将正常对照组视网膜AREG mRNA表达量标准化为100%,近视组、低剂量组、中剂量组、高剂量组右眼AREG mRNA表达量分别与标准化后的正常对照组比较得到目的基因的相对表达量。近视组AREG mRNA相对表达量最高,正常对照组其次,低剂量组、中剂量组和高剂量组AREG mRNA的表达呈剂量依赖性下降。结论玻璃体内注射AREG抗体后,豚鼠眼轴缩短,屈光度增加,视网膜中AREG蛋白和mRNA呈剂量依赖性表达下降。AREG在近视发生发展过程中起重要作用。

Objective To investigate the effect of Amphiregulin(AREG)antibody on refraction,axial length and retinal AREG mRNA and protein expression in lens-induced myopic guinea pigs.Methods Thirty two-week-old pigmented guinea pigs were randomly divided into five groups:normal group,myopia group,low-dosage group,medium-dosage group,high-dosage group,with six animals in each group.Right eyes of other four groups were covered with-10.00 D lens,while the left eyes and the normal group were not treated.After two weeks,the refraction,axial length were measured by Ribbon retinoscope and A/B scan of ophthalmology respectively.From the third week,the injection of Ringer’s solution and AREG antibody for right eyes were performed once a week and continue to wear glasses lasted for 3 weeks.Then the refraction and axial length were measured again.The expression of mRNA and protein of AREG were detected by quantitative real-time PCR and immunofluorescence staining.Results There was no statistically significant difference in the initial refraction and axial length of the guinea pigs before the experiment(all P>0.05).Two weeks after wearing-10.00 D lens,the rats’refraction decreased but axial length increased in right eyes of myopia group,low-dosage group,medium-dosage group and high-dose group when compared with normal group(all P<0.05),but there was no statistical significance in left eyes in each groups.After intravitreal injection of AREG antibody for three weeks,compared with myopia group,the difference of refraction and axial length of the low-dosage group approached no statistical significance(all P>0.05);the change of refraction in medium-dosage group had no statistical significance(P>0.05),but the axial length was decrease statistically significant(P<0.01);the axial length of right eyes in high dosage-group reduced distinctly and refraction increased statistically significant(all P<0.05);otherwise there were no statistically significant difference the refraction and axial length of left eyes in each groups(all P>0.05).The protein expression of AREG were detected through immunofluorescence in retinal posterior pole,equator and the midpoint between equator and posterior pole in retina in each group,which suggested that the protein expression of AREG were distributed in retina and without location specificity.After intravitreal injection of AREG antibody,the protein expression of AREG in low-dosage group,medium-dosage group and high-dosage group were decrease.The normal group retinal AREG mRNA levels were normalized to 100%,other groups compared with the standardized group respectively to obtain a relative expression of AREG mRNA.The mRNA expression of myopia group is the highest in the eyes,followed by eyes in normal group,the low-dosage group,medium-dosage group,high-dosagegroup were decreased with the increasing dose of AREG antibody.Conclusion In guinea pigs with lens-induced myopia,the axial length is shortened and refraction is elevated obviously after intraocular injection of AREG antibody,and meanwhile the expression of AREG mRNA and protein in the retina is down-regulated in a dose-dependent manner,thereby indicating that AREG plays a certain role in the development of myopia.