摘要背景 视网膜色素变性是先天性盲的重要原因之一,由于视网膜色素变性患者视杆细胞为原发性变性,因此无法了解视细胞变性的机制和过程.研究发现Leber先天性黑矇(LCA)自发突变小鼠具有与视网膜色素变性类似的自然过程,有助于深入探讨视网膜色素变性的发病机制及确定疾病基因治疗靶点.目的研究视网膜色素变性模型LCA自发突变小鼠视网膜短波长敏感的视锥细胞自然变性过程. 方法 按出生后天数(P)取P14、P21、P35、P90的遗传性视网膜疾病LCA的自发突变动物模型Rpe65rd12(B6[A]-Rpe65 rd1 2/J,rd12)小鼠各5只,同时选择鼠龄匹配的野生型C57BL/6J(B6)小鼠作为对照.采用RolandQ450SC UV视觉电生理检测仪行小鼠视网膜电图(ERG)检查,采用单次白色发光二极管(LED)闪光刺激记录总视锥细胞反应,刺激强度分别为1.00 cds/m2和1.96 cds/m2;采用紫外光闪光刺激记录短波长紫外光(UV)敏感的视锥细胞反应,刺激波长为(363±6)nm,刺激强度分别为2.0 mWs/m2和3.0 mWs/m2.ERG记录后次日处死小鼠并制备视网膜铺片,分别采用FITC荧光标记的花生凝集素(FITC-PNA)和Cy3荧光染色法检测2个组不同鼠龄小鼠视网膜中总视锥细胞和UV敏感的视锥细胞的分布和数量变化. 结果 P14 rd12小鼠明适应ERG反应为负波型,各波潜伏期延长,UV敏感的视锥细胞ERG各波记录不到.P21 rd12小鼠视锥细胞ERG b波振幅较野生型B6小鼠下降了约75％,b波潜伏期为(102.80±11.39)ms,较B6小鼠的(43.40±5.60) ms明显延长,差异有统计学意义(t=-8.106,P=0.001);P21、P35和P90野生型B6小鼠UV敏感的视锥细胞ERG b波振幅分别为(59.60±36.00)、(82.40±12.22)和(68.43±17.63) μV,P21、P35和P90 rd12小鼠UV敏感的视锥细胞反应均记录不到.免疫荧光检测显示,P14野生型B6小鼠视网膜视锥细胞较多,呈绿色荧光,大量的视蛋白主要分布在视网膜腹部鼻侧象限UV敏感的视锥细胞中,呈红色荧光,P14、P21、P35 rd12小鼠视网膜中视锥细胞逐渐减少,绿色荧光逐渐减弱,UV敏感的视锥细胞呈散在表达.结论 rd12小鼠出生后早期即出现视锥细胞的数量减少和功能异常,短波长敏感的视锥细胞的丢失和功能异常更早于中长波长的视锥细胞.

abstractsBackground Retinitis pigmentosa (RP) is one of the causes of congenital blindness.It is well known that the degeneration process of rod cells is difficult to detect in RP.Retinal degeneration 12 (rd12) mice is a new,spontaneously arising mouse model for human Leber congenital amaurosis (LCA),and it is helpful for us to explore the pathogenesis and determine the treating target of RP.Objective This study was to investigate the natural disease process of short-length sensitive cone cells in rd12 mice,a LCA Rpe65rd12 (B6 [A]-Rpe65rd12/J) mouse.Methods The rd12 mice at postnatal (P) 14,P21,P35 and P90 were selected (5 mice for each),and the wild-type C57BL/6J (B6) mice with matched ages were included as controls.Photopic full-field electroretinogram (ERG) was recorded with Roland Q450SC UV visual physiology instrument.Cone response was recorded using single white light-emitting diode (LED) stimulation with the flash intensity of 1.00 cds/m2 and 1.96 cds/m2,and short wave-length sensitive cone response was recorded using ultraviolet light ([363 ±6] nm) stimulation with the flash intensity of 2.0 mWs/m2 and 3.0 mW/m2.The mice were sacrificed and retinal whole-mounts were prepared.The distribution and number of cone cells and UV-sensitive cone cells were detected by FITC-peanut agglutinin (FITC-PNA) and Cy3 immunofluorescence stainning,respectively.Results In P14 rd12 mice,the ERG responses of overall cone cells presented the negative waveform and the latency was delayed,and UV-sensitive cone response was unrecordable.The b-wave amplitude of overall cone cells reduced by 75％ in P21 rd12 mice compared with wild-type B6 mice,and the mean latency of b-wave in the P21 rd12 mice was significantly longer than that in the wild-type B6 mice ([102.80± 11.39] ms vs.[43.40± 5.60] ms) (t =-8.106,P =0.001).The mean b-wave amplitudes of U Vsensitive cone cells were (59.60± 36.00),(82.40± 12.22) and (68.43 ± 17.63) μV in the wild-type B6 mice,andthose in the rd12 mice were unrecordable.Immunofluorescence showed that a lager number of cone cells with green fluorescence were seen,and the expression of opsin with red fluorescence was displayed in the UV-sensitive cone cells of nasal lateral on retinal ventral side in P14 wild-type B6 mice;while only a few opsin positive-response cells were seen in P14,P21 and P35 rd12 mice.Conclusions In rd12 mice,the functional abnormality and quantitative reduction of cone cells appear in the early postnatal days,and the loss of UV-sensitive cone cells is earlier and more obvious.