目的:探索定向转染内源性光感受蛋白黑视素(melanopsin/opsin 4,Opn4)基因进入给光型双极细胞后,视网膜变性小鼠模型中视网膜神经元的光反应以及整体视觉行为的改变.方法:选用由甲基亚硝基脲(N-methyl-N-nitrosourea,MNU)诱导的成年CD1小鼠作为视网膜变性模型.于P0～P1 CD1乳鼠视网膜底注射Grm6-Opn4-GFP质粒,Grm6-GFP作为阴性对照.通过电转进行基因转染.术后5周对基因转染小鼠腹腔注射MNU诱导视网膜感光细胞变性,对照组注射生理盐水,共设计5个实验组:正常对照组(normal)、生理盐水Grm6-Opn4-GFP对照组(NS+ melanopsin)、MNU诱导模型Grm6-Opn4-GFP治疗组(MNU+ melanopsin)、MNU诱导模型Grm6-GFP对照组(MNU+ GFP)和MNU诱导组(MNU).诱导后连续7d进行明暗箱测试,统计动物在暗箱中的活动时间比.随后进行视网膜电图(electroretinogram,ERG)测试,计算体现给光双极细胞光反应的b波峰值、潜伏期和反映视网膜神经节细胞(retinal ganglion cells,RGCs)光反应的明视负波反应(photopic negative response,PhNR).利用免疫荧光法检测动物视网膜黑视素基因转导效果.结果:黑视素可以被定向转染进入视网膜给光双极细胞.明暗箱实验显示MNU诱导7d后Grm6-Opn4-GFP转染的CD1小鼠滞留在黑箱的时间显著长于未转染组(P＜0.05),ERG测试显示Grm6-Opn4-GFP转染的CD1小鼠的b波也有明显恢复(P＜0.05).结论:定向转染内源性光感受蛋白黑视素基因进入给光型双极细胞可部分恢复视网膜变性模型动物视觉.%AIM:To investigate the changes of light response and visual behavior after transfecting intrinsic photosensitive protein melanopsin (opsin 4,Opn4) into ON-bipolar cells in.a mouse retinal degeneration model.METHODS:N-methyl-N-nitrosourea (MNU)-induced CD1 mice served as the retinal degeneration model.CD1 mice on postnatal day 0 (P0) and postnatal day 1 (P1) were subretinally injected with Grm6-Opn4-GFP plasmid,and those injected with Grm6-GFP plasmid served as negative control group.Electroporation was applied to transfect the plasmids into cells.Five weeks later,the transfected mice received intraperitoneal injection of MNU to induce the apoptosis of photosensory cells in 7 days.The mice were divided into 5 groups:normal control group,normal saline (NS) intraperitoneal injection with Grm6-Opn4-GFP transfection (NS + melanopsin) group,MNU intraperitoneal injection with Grm6-Opn4-GFP transfection (MNU + meanopsin) group,MNU intraperitoneal injection with Grm6-GFP transfection (MNU + GFP) group and MNU intraperitoneal injection (MNU) group.Visual behavior was tested by Dark/Light Box test for 7 days after MNU injection,in which the time spending in each area of the box was measured every day for each animal.Electroretinagram was applied to measure the peak amplitude and the latency time of b-wave,and photopic negative response (PhNR),which stand for the functions of ON-bipolar cells and retinal ganglion cells,respectively.Immunohistochemistry was performed to check the expression of melanopsin in the ON-bipolar cells in the transfected retina.RFSULTS:Melanopsin was specifically transfected into ON-bipolar cells and was expressed in the transfected cells.The dark/light box test showed that the mice in MNU + meanopsin group stayed significantly longer in the dark zone than those in negative control group (P ＜ 0.05).Their b-waves tended to recover as well (P ＜ 0.05 vs negative control group).CONCLUSION:Specific transfection of melanopsin into ON-bipolar cells can partially restore the visual function of mice.
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