rd11小鼠视锥细胞变性过程中视蛋白的变化特点

背景视网膜变性11（rd11）小鼠是近年来新发现的一种自发突变的视网膜变性小鼠。研究证实，rd11小鼠出生后随着鼠龄的增长出现快速的光感受器变性，且视杆细胞变性早于视锥细胞变性，但对于视网膜不同区域视锥细胞变性的特点还不十分清楚。目的应用视网膜铺片免疫荧光染色技术观察不同鼠龄rd11小鼠M-视蛋白和S-视蛋白在视网膜的表达分布及变化特点，为相关疾病的基因治疗研究提供实验依据。方法取出生后14、28、42d的rd11小鼠各5只，制备视网膜铺片，采用免疫荧光组织化学法分别标记小鼠视网膜后极部颞上、颞下、鼻上和鼻下象限M-视蛋白和S-视蛋白的表达，观察随rd11小鼠鼠龄的变化视网膜各区域M-视蛋白和S-视蛋白的荧光形态和密度，并与相应鼠龄的C57BL／6J小鼠进行比较。结果出生14d的rd11小鼠视网膜M-视蛋白和S-视蛋白的红色荧光形态和密度与C57BL／6J小鼠接近，但出生28d的rd11小鼠视网膜后极部颞上、颞下、鼻上、鼻下4个区域M-视蛋白和S-视蛋白表达密度均明显降低，荧光形态由纺锤形逐渐变为点状，出生42d的rd11小鼠视网膜部分区域M-视蛋白和S-视蛋白表达消失。出生28d的rd11小鼠视网膜后极部颞上、颞下、鼻上、鼻下区域M-视蛋白的表达密度分别为（414±32）、（300±8）、（324±22）和（250±20）个／0．037mm^2，明显低于同龄C57BL／6J小鼠的（484±21）、（442±19）、（459±34）和（436±12）个／0．037mm^2，差异均有统计学意义（t=4．114、15．225、7．505、17．990，均P〈0．05）；上述4个区域S-视蛋白的表达密度下降更明显，分别为（8±4）、（175±16）、（74±13）、（315±20）个／0．037mm^2，明显低于C57BL／6J小鼠的（73±16）、（436±30）、（393±30）和（480±19）个／0．037mm^2，差异均有统计学意义（t=8．555、17．076、21．637、13．498，均P〈0．05）。结论rd11小鼠视锥细胞中的M-视蛋白和S-视蛋白均随着鼠龄的增长而急剧减少，以S-视蛋白更为明显。随着鼠龄的增长，rd11小鼠M-视蛋白变性从视神经周围区向鼻下方，再向颞上方逐渐进展，而S-视蛋白变性由颞上方向鼻下方逐渐进展。

The distribution and degeneration pattern of the cone opsins in rd11 mice

Background The retinal degeneration 11 （rd11） mouse is a newly discovered naturally occurring recessive animal model with lysophosphatidylcholine acyltransferase 1 （ Lpcatl ） mutation. Previous studies showed that the photoreceptor cells are characterized by typical rod-cone degeneration pattern in rdl 1 mice, while cone degeneration pattern in rd11 mice is unclear. Objective Using immunofluorescence staining techniques with retinal wholemount,we aim to clarify the degeneration patterns of cone-function related M-opsin or S-opsin in different ages of rdll mice. Methods A total of thirty rdll and C57BL/6J mice at postnatal （P） day 14,28,42 （five in each age group） were sacrificed and retinal wholemounts were prepared. Immunohistochemistry was performed to identify the expression of M-opsin or S-opsin in retinal wholemounts, which were photographed with a fluorescent microscope. Cone opsins were compared between rd11 retinas and age-matched normal C57BL/6J retinas by manually counting the opsin positive cone cells in different quadrants of the retinas. Results The number of M-opsin or S-opsin positive fluorescent dots in each quadrant was similar at all ages of normal C57BL/6J retina. M-opsin positive fluorescent dots in dorsal/temporal, ventral/temporal, dorsal/nasal and ventral/nasal quadrants of rd11 retina at P28 were （414±32）, （300±8）, （ 324±22 ） and （ 250±20）/0. 037 mm^2 , which were lower than the age-matched normal C57BL/6J mice （ t = 4. 114,15. 225,7. 505,17. 990, all at P〈0. 05 ）. At the same time the S-opsin positive fluorescent dots in P28 rd11 were （8±4）,（175±16）,（74±13） and （315±20）/0.037 mm^2,with significant decrease in comparison with those in the age-matched normal C57BL/6J mice （t = 8. 555,17. 076,21. 637,13. 498, all at P〈 0.05）. With the development of retinal degeneration in rdl i mice, the M-opsin degeneration spread from central to ventral,nasal and then to temporal and dorsal peripheral retina;and the S-opsin loss started from dorsal/temporal to ventral/nasal retina. Coneluslons Most of the M-opsin and S-opsins, especially the S-opsins in rdll mice, degenerate in 6 weeks. Retinal wholemount and cone opsin immunofluorescent staining provide a useful tool to show the cone degeneration pattern and to evaluate the therapeutic efficiency in ongoing gene therapy study.