出 处:《中华实验眼科杂志》2014年第1期12-17,共6页Chinese Journal Of Experimental Ophthalmology
基 金:国家自然科学基金项目(81371060)
摘 要: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〈0.0Background 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 f
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