玻璃体内注射腺相关病毒介导原纤维蛋白-2基因干扰对小鼠视网膜的影响

Effect of intervening the fibrillin-2 gene via intravitreal injection of adeno-associated virus on mouse retina

目的探讨玻璃体内注射腺相关病毒(AAV)介导原纤维蛋白-2(FBN2)基因干扰进行基因敲低对小鼠视网膜的影响。方法54只8周龄C57BL/6J小鼠随机分为3组正常对照组、阴性对照组、AAV组。正常对照组小鼠正常饲养,阴性对照组和AAV组小鼠右眼玻璃体内分别注射3μL阴性病毒或AAV。于注射后2周、3周、4周利用共焦扫描激光检眼镜(SLO)、视网膜电图(ERG)和光学相干断层扫描(OCT)分别检测各组小鼠眼底变化、视功能变化,并测量视网膜外核层(ONL)厚度,同时使用实时荧光定量PCR(RT-PCR)、ELISA检测各组小鼠视网膜中FBN2 mRNA和蛋白表达水平。结果SLO检测结果显示,AAV组小鼠玻璃体内注射后2周视网膜开始出现黄白色沉积物,并保持至实验结束。OCT检测结果显示,AAV组小鼠出现视网膜色素上皮层光带不规则、点状高密度反射区;注射后2周、3周、4周,AAV组小鼠视网膜ONL厚度均明显小于阴性对照组和正常对照组(均为P<0.05)。ERG检测结果显示,注射后2周、3周、4周与阴性对照组和正常对照组比较,AAV组小鼠视网膜Rod-b、Max-a波振幅均明显变小(均为P<0.05)。RT-PCR、ELISA检测结果显示,注射后2周、3周、4周与阴性对照组和正常对照组比较,AAV组小鼠视网膜中FBN2 mRNA和蛋白表达均明显降低(均为P<0.05)。结论玻璃体内注射AAV介导FBN2基因干扰进行基因敲低可以引发小鼠视网膜病变。

Objective To investigate the effect of intervening the fibrillin-2(FBN2)gene via intravitreal injection of adeno-associated virus(AAV)on mouse retina.Methods Fifty-four 8-week-old C57BL/6J mice were randomly divided into the normal control group,negative control group and AAV group,with 18 mice in each group.Mice in the normal control group were normally fed,while those in the latter two groups received an intravitreal injection of 3μL of negative virus or AAV in the right eye,respectively.At 2,3 and 4 weeks after injection,the changes of fundus and visual function,the thickness of outer nuclear layer(ONL)of retina were examined by the scanning laser ophthalmoscope(SLO),electroretinogram(ERG)and optical coherence tomography(OCT).In addition,mRNA and protein levels of FBN2 in mouse retina were detected by real-time quantitative PCR(RT-PCR)and enzyme-linked immunosorbent assay(ELISA),respectively.Results SLO results showed the presence of drusen-like deposits in mouse ocular fundus of AAV group after 2 weeks of intravitreal injection,which remained until the end of the experiment.OCT results showed irregular and punctate high-density reflex area in the retinal pigment epithelium of AAV group.After 2,3 and 4 weeks of injection,the thickness of ONL in AAV group was significantly smaller than that of normal control group and negative control group(both P<0.05).ERG results showed that the amplitudes of Rod-b and Max-a in AAV group after 2,3 and 4 weeks of injection were significantly lower than those of negative control group and normal control group(all P<0.05).RT-PCR and ELISA results showed that mRNA and protein levels of FBN2 in AAV group were significantly lower than those in negative control group and normal control group at 2,3 and 4 weeks after injection(all P<0.05).Conclusion Intervene of FBN2 by intravitreal injection of AAV can induce retinopathy in mice.