光照节律改变对大鼠视网膜中Cry2表达的影响

Effect of light rhythm on the expression of cryptochrom 2 in retina

背景Cry2存在于哺乳动物视网膜上，是生物钟振荡器环路中重要的调控因子。目的探讨光照节律改变对大鼠视网膜中Cry2表达的影响。方法将清洁级健康无眼疾SD大鼠30只按随机数字表法分成2个组，正常对照组大鼠在自然昼夜光线照明下喂养，实验组大鼠饲养在光照控制室内，大鼠活动平面光照度为（533±16）lx，设定每日光照明（6：00～24：00）、暗（24：00～6：00）循环交替时间为18h／6h，持续时间为3个月。于实验后3个月时在光学显微镜及透射电子显微镜下观察大鼠视网膜组织结构和超微结构的改变，采用免疫组织化学法检测各组大鼠视网膜中Cry2蛋白的表达，采用实时荧光定量PER法检测Cry2mRNA在视网膜组织中的表达变化。结果光学显微镜下可见，正常对照组大鼠喂养3个月时视网膜各层结构清晰，排列整齐，而实验组大鼠视网膜萎缩变薄，排列紊乱。透射电子显微镜下观察表明，正常对照组大鼠光感受器细胞外节膜盘结构清晰，内节线粒体嵴连续，外核层细胞核形态规则，但实验组大鼠光感受器内外节结构紊乱，外节膜盘间隙增大，出现溶解、空泡变，光感受器内节线粒体空泡变，视网膜外核层细胞核染色质浓集，部分出现核碎裂、边集，核膜皱缩、内陷。免疫组织化学检测显示，2个组大鼠Cry2蛋白均在视网膜节细胞及内核层部分细胞的细胞质和核膜阳性表达，实验组大鼠Cry2蛋白表达评分为（0．833±O．197）分，明显低于正常对照组的（1．700±0．245）分，差异有统计学意义（P=0．009）。实时荧光定量PCR定量检测结果显示，正常对照组大鼠视网膜中Cry2mRNA表达量为0．962±0．125，实验组为0．615±0．100，差异有统计学意义（P=0．006）。结论533lx的光照强度长期节律性照射可导致大鼠视网膜组织结构损伤，其机制可能与视网膜中Cry2的表达下调有关，调整Cry2的表达是否是临床上稳定生物节律的一个重要环节值得探讨。

Background The cryptochrom 2 （Cry2）in mammalian retina is a main influential factor of circadian clock. Objective Purpose of this study was to investigate the effect of light exposure rhythm on expression of Cry2 in retina. Methods Thirty clean healthy Sprague Dawley（SD）rats were collected and divided into two groups randomly. The rats of the control group exposed to natural light with the normal rhythm for 30 days, but rats of the experimental group exposed to the artificial light （light：dark = 18 hours：6 hours）for 3 months with the light intensity of（533~16）Ix. The histopathological change and ultrastructural alteration of rat retina in both groups were examined under the light microscope and transmission electron microscope at the end of the experiment. Expressions of Cry2 protein and its mRNA were assayed by immunohistochemistry and quantitative PCR（Q-PCR）. Results The rat retinal morphology and ultrastructure were clear and order-arranged under the light microscope and transmission electron microscope in the control group. However, atrophy and disorganization of retina were found under the light microscope, and liquefaction and vacuole of outer segments of photoreceptors were observed. The vacuolar degeneration of mitochondria in the inner segments of photoreeeptors, cellular nuclear shrinkage, chromatin margination, nuclear notch and destruction were seen in the outer nuclear layer under the transmission electron microscope. Immunohistochemistry showed that the expression of Cry2 protein located in cytoplasm and nuclei membrane of the retinal ganglion cell layer and inner nuclear layer in both normal rats and experimental rats. The scores of Cry2 protein expression were 0. 833±0. 197 in the experimental group, and I. 700±0. 245 in the control group, with a significant difference between them（P=0. 009）. The quantities of Cry2 mRNA were 0. 962±0. 125 in the control group and 0. 615±0. 100 in the experimental group, showing a significant difference between the two groups （P = 0. 006 ）. Conclusions Long-term light exposure under the 533 lx leads to retinal structural and functional damage probably by down-regulating Cry2 expression in retina. Whether the regulation of Cry2 expression is helpful for stabilizing the biorhythm or not is a worthy question to explore.