【作者】 王飞；

【导师】 卢奕；

【作者基本信息】 复旦大学 ， 眼科学， 2008， 博士

【摘要】 第一部分不同波长光线日间照明对豚鼠生物节律的影响目的:探讨绿光、蓝光日间照明对豚鼠生物节律分子褪黑激素的影响。方法:选取10天龄豚鼠48只,分为三组分别饲养于绿光照明(波长530nm)、蓝光照明(波长480nm)、白光照明(各波长混合)的环境中,每天照明时间8:00am至8:00pm,共10天。分别于10:00am和10:00pm取各组豚鼠脑松果体组织,以高效液相色谱方法检测褪黑激素的含量。以白光组的褪黑激素含量作为基线值,比较绿光组、蓝光组褪黑激素的含量的改变。统计学分析采用Stata软件作单因素方差分析和配对t检验,P＜0.05为具有统计学意义。结果:10:00am时绿光照明组、蓝光照明组和白光照明组豚鼠脑松果体组织内褪黑激素的含量分别是85.2±2.79 pg/ml,45.92±1.74 pg/ml,53.32±2.51pg/ml;10:00pm时三组豚鼠脑松果体组织内褪黑激素的含量分别是118.15±4.23 pg/ml,63.27±1.43pg/ml,73.12±2.72 pg/ml。与白光照明组相比,绿光照明组豚鼠日间、夜间褪黑激素的含量均上升(P＜0.05),而蓝光组下降(P＜0.05)。结论:绿光和蓝光照明日间与夜间均可引起豚鼠生物节律改变,绿光照明组豚鼠脑松果体组织内褪黑激素含量较蓝光照明组高,说明绿光照明可以减轻光刺激导致的褪黑激素分泌抑制,而蓝光照明可以加剧光刺激导致的褪黑分泌抑制。第二部分单色光诱导豚鼠近视眼中褪黑激素受体-1A和黑视素的表达变化目的:观察530nm绿光诱导的豚鼠近视眼球内黑视素和褪黑激素受体-1A的表达变化,并初步探讨两种蛋白表达与色光诱导近视之间的关系。方法:30只十天龄英国短毛豚鼠随机分为3组,分别以绿光(530nm)、蓝光(480nm)和混合白光作为照明光饲养8周。实验前及实验后,各组经散瞳联合带状光检影法测定其屈光不正状态,A超测量玻璃体腔深度和眼轴长度。实验结束时各组于10:00pm摘除眼球,分别行透射电镜检查观察巩膜增殖状态;免疫荧光法观察黑视素和褪黑激素受体-1A在视网膜的分布、表达;实时荧光定量-聚合酶链反应法观察黑视素和褪黑激素受体-1A mRNA的变化;Western-blot观察蛋白的变化。结果:1)实验前的屈光状态、玻璃体腔深度和眼轴长度,各组之间的差异无统计学意义。经不同色光照明8周后,各组均发生玻璃体腔深度增加、眼轴延长。实验后绿光组、白光组和蓝光组屈光不正值分别为1.37±0.75D,3.10±0.84D.3.25±0.78D(F=17.46,P=0.0032);玻璃体腔深度分别为3.70mm±0.16mm,3.24mm±0.21mm,3.21mm±0.19mm(F=11.04,P=0.0098);眼轴长度8.46±0.13mm,7.62±0.17mm,7.56±0.18mm(F=20.86,P=0.0020);绿光组豚鼠明显趋于近视化。2)经免疫荧光法证实,与蓝光组相比,绿光组视网膜内黑视素蛋白密度下降,而褪黑激素受体-1A表达增多。3)绿光照明组黑视素mRNA较蓝光组显著减少(F=3.61,p=0.016),蛋白表达也明显降低。同时,绿光组豚鼠巩膜和视网膜中褪黑激素受体-1A mRNA与蓝光组相比均显著增加(分别为F=0.7945,p=0.000;F=5.3987,p=0.000),绿光照明组豚鼠巩膜和视网膜中褪黑激素受体-1A蛋白表达也增加。结论:530nm单色光(绿光)照明可以相对促使豚鼠玻璃体腔深度增加,眼轴延长,改变其屈光状态、加速眼球近视的发生。530nm单色光照明时豚鼠眼内黑视素表达下降、褪黑激素受体-1A表达上升。褪黑激素受体-1A和黑视素的变化可能在530nm单色光照明诱导的近视中起相应作用。第三部分褪黑激素球旁注射对豚鼠近视的影响目的:探讨褪黑激素球旁注射对豚鼠正常的眼球生长及屈光状态的影响。方法:30只四周龄英国短毛豚鼠随机分为2组,饲养于12小时明暗周期环境中。实验组随机选取一眼每天8:00pm球旁注射溶于2%乙醇中的500pg/ml浓度褪黑激素0.2ml,对照组每天球旁注射溶剂2%乙醇0.2ml,共两周。实验前及实验后行散瞳联合带状光检影法测定屈光不正值,A超测定玻璃体腔深度和眼轴长度,并取眼球做病理切片,测量相同部位视网膜、脉络膜和巩膜的厚度。结果:实验组处理前随机选择眼和对照组随机选择眼的屈光不正值、玻璃体腔深度和眼轴长度三者组间比较均不具有统计学意义(均P＞0.05)。2周后对照组屈光不正值为3.65D±0.81D,玻璃体腔深度为3.28mm±0.49mm,眼轴长度为7.6mm±0.29mm,视网膜厚度0.1270mm±0.0057mm,脉络膜厚度0.0473mm±0.0016mm,巩膜厚度0.1181mm±0.0022mm;实验处理眼屈光不正值为2.76D±0.45D,玻璃体腔深度为3.59mm±0.86mm,眼轴长为8.7mm±0.38mm,视网膜厚度0.1028mm±0.0061mm,脉络膜厚度0.0449mm±0.0023mm,巩膜厚度0.0901mm±0.0012mm。各项指标两组间比较,差异均具有显著统计学意义(P＜0.05)。结论:褪黑激素球旁注射可改变豚鼠眼球组织生长时的多项生物参数,其中玻璃体腔深度增加、眼轴延长,视网膜、脉络膜和巩膜厚度降低,结果导致近视发生。提示褪黑激素可能参与调节眼球的节律性生长。第四部分褪黑激素对大鼠巩膜成纤维细胞增殖的影响目的:研究褪黑激素在体外对大鼠后巩膜成纤维细胞生长的影响,并探讨其可能的调控机制。方法:采用植块法培养大鼠后巩膜成纤维细胞,细胞经传代纯化鉴定后,取第3～4代细胞分为6组,加入不同浓度的褪黑激素(分为空白对照、溶剂对照、50pg/ml、100pg/ml、500pg/ml和1000pg/ml组),分别培养于24、48、72h后采用四甲基偶氮唑盐法检测细胞的增殖密度、氯胺T法检测胶原蛋白的合成;培养72h后采用免疫荧光法观察褪黑激素受体-1A蛋白在细胞内的表达;采用实时定量聚合酶链反应法测定褪黑激素受体-1A的mRNA表达水平。结果:褪黑激素在体外对大鼠巩膜成纤维细胞生长具有明显的抑制增殖作用,最佳浓度为500pg/ml;褪黑激素在100～500pg/ml浓度范围内可以抑制胶原蛋白的合成,且该抑制作用呈剂量相关性。在褪黑激素培养的条件下,褪黑激素受体-1AmRNA表达明显增加,蛋白浓度上升。结论:一定浓度的褪黑激素对体外培养的大鼠巩膜成纤维细胞的生长及其胶原蛋白合成均具有抑制作用,褪黑激素受体的表达可能在其中发挥重要作用。更多还原

【Abstract】 Part 1 The effect of blue and green light in daytime on the the light-induced melatonin suppression in guinea pigObjective:To investigate the effect of blue and green light in daytime on the the light-induced melatonin suppression in guinea pigs.Method:48 guinea pigs aged 10 days were exposed to blue light(wavelength 480nm)、green light(wavelength 530nm) and white light(mixed wavelength) respectively,between 08:00am and 08:00pm for 10 days.The amounts of pineal gland melatonin were measured before and after the light pulse at 10:00am and 10:00pm.Changes observed between before and after the light pulse was compared with a melatonin baseline obtained in the white light group.Results:The amount of pineal gland melatonin was 45.92±1.74pg/ml at 10:00am and 63.27±1.43pg/ml at 10:00pm in blue light group,53.32±2.51 pg/ml and 73.12±2.72 pg/ml in white light group,85.2±2.79 pg/ml and 118.15±4.23 pg/ml in green light group.A significant increase was observed in the red light group(P＜0.05) whereas a significant reduction was observed in the blue light group at night(P＜0.05).Conclusions:Green light means to prevent the light-induced melatonin suppression, blue light enhances the light-induced melatonin suppression,they all change biological rhythms.Part 2 The changes of expression of melanopsin and melatonin receptor -1A for myopia induced by 530nm monochromatic light in guinea pigObjective:To investigate the expression of melanopsin and melatonin receptor 1-A in myopia induced by 530nm monochromatic light in guinea pig and the relationship between the expression of melanopsin and melatonin receptor 1-A and myopia induced by 530nm monochromatic light.Method:Thirty 10-day-old guinea pigs were randomized into3 groups and exposed to green light(530nm),blue light(480nm) and white light for 8 weeks. Measurement of the refractive status was taken after cycloplegia with streak retinoscopy,ocular length and vitreous depth detected by ultrasound biometry were examined before and after the experiment in guinea pigs.Eyes were dissected at 10:00pm,and the ultramicro-structures of the sclear were observed through electromicroscope.The expression of melanopsin and melatonin receptor 1-A were observed by immunocytochemisty.The level of melanopsin and melatonin receptor 1-A and their mRNA were observed through Western-blot and real-time PCR respectively.Result:1) There were no statistically significantly variance of the refractive error,ocular length nd vitreous depth among green light group,white light group and blue light group before experiment.Eye elongation and vitreous depth gain could be observed after 8 weeks of light treatment.The refractive status was 1.37±0.75D in green light group,3.10±0.84D in white light group and 3.25±0.78D in blue light group(F= 17.46,P=0.0032);The vitreous depth was 3.70mm±0.16mm in green light group, 3.24mm±0.21mm in white light group and 3.21mm±0.19mm in blue light group respectively(F=11.04,P=0.0098).Ocular length was 8.46±0.13mm,7.62±0.17mm and 7.56±0.18mm respectively(F=20.86,P=0.0020).Statistical analysis of the three parameter mentioned above indicate statistically significance among them. Guinea pigs in 530nm green light group were in relatively myopic refractive status.2) Compared with those in retina of blue light group,density of melanopsin in retina of green light group decreased,but melatonin receptor 1-A was more abundant in the retina of green light group by immunocytochemisty.3) There was significant difference in expression of melanopsin mRNA and protein between the green light group and blue light group(F=3.61,p=0.016).There was also significant difference in expression of melatonin receptor 1-A mRNA and protein in retina and sclea between the green light group and blue light group(F=0.7945,p= 0.000;F=5.3987,p=0.000)).Green light group expressed higher level of melatonin receptor 1-A mRNA and protein in retina and sclea,while blue light group expressed lower level.Conclusion:Green light(530nm) alter the refractive status of guinea pig,may accelerate myopic development.Monochromatic light with the long-wavelength of 530nm induces the increment of melatonin receptor 1-A and decreasement of melanopsin in myopic guinea pigs.melatonin receptor 1-A and melanopsin might play a role in the forming of 530nm monochromatic induced guinea pig myopia.Part 3 The effect of melatonin injecting periglomerularly on guinea pig myopic shift.Objective:To examine the role of the circadian signaling molecule melatonin in normal ocular growth and the exaggerated ocular growth associated with the development of myopia in guinea pig.Method:30 pigmented guinea pigs aged 4weeks were maintained on a 12-hour lightdark cycle for 14 days.During the 14-day treatment period,guinea pigs were injected periglomerularly on one randomly selected eye during the early dark period(8:00pm) with melatonin 0.2ml(500pg/ml) or 2%ethanol vehicle control 0.2ml each day,and the fellow eye was taken as control. Measurement of the refractive status after cycloplegia with streak retinoscopy,ocular length and vitreous depth detected by ultrasound biometry were examined before experiment and 2 weeks after the treatment of MLT or 2%ethanol in guinea pigs. The eyes were conducted electron microscopy examination to evaluate the pathological changes of the melatonin treated eye and the control eye.Sclera thickness,choroidal thickness and retina thickness were measured in the same site by HE bioscopy.Results:There was no statistically significantly variance of the refractive error,ocular length and vitreous depth between control group and melatonin treated group before treatment.Two weeks later,Eye elongation and vitreous depth gain could be observed.In control group,the refractive status was 3.65D±0.81D,the vitreous depth was 3.28mm±0.49mm,ocular length was 7.6mm±0.29mm,retina thickness was 0.1270mm±0.0057mm,Choroidal thickness was 0.0473mm±0.0016mm,Sclera thickness was 0.1181mm±0.0022mm;In melatonin treated group,the refractive status was 2.76D±0.45D,the vitreous depth was 3.59mm±0.86mm,ocular length was 8.7mm±0.38mm,retina thickness was 0.1028mm±0.0061mm,Choroidal thickness was 0.0449mm±0.0023mm,Sclera thickness was 0.0901mm±0.0012mm respectively.There were statistically significantly variance of the refractive error,ocular length,vitreous depth,retina thickness,choroidal thickness and sclera thickness between control group and melatonin treated group(P＜0.05).Conclusions:The finding that administration of melatonin alters the growth of several ocular tissues in pigmented guinea pig’s eyes suggests that melatonin plays a role in ocular growth and myopic development.This conclusion suggests that the action of melatonin is involved in the regulation of the diurnal rhythm of ocular growth。Part 4 Effect of melatonin on rat scleral fibroblasts proliferation in vitroObjective:To investigate the effects of melatonin on rat scleral fibroblasts proliferation in vitro and its mechanisms.Methods:The rat scleral fibroblasts were grown in tissue culture,then were treated with different doses of melatonin((0 vacuity matched control,0 solvent matched control,50pg/ml、100pg/ml、500pg/ml and 1000pg/ml) for 24h、48h、72h individually.The effects of melatonin on scleral fibroblasts proliferation were measured by MTT assay,synthesis of collagen was observed by Chlorozone T Results.After 72h,the expression of melatonin receptor-1A mRNA and protein was detected by real time PCR and immunofluorescent staining respectively.Results melatonin inhibited rat scleral fibroblasts proliferation in vitro significantly,the most efficient concentrations was 500pg/ml;melatonin could inhibit collagen synthesis of scleral fibroblast in concentrations from 100～500pg/ml; Cultured with melatonin,the expression of melatonin receptor-1A mRNA and protein increased.Conclusions:The growth of rat scleral fibroblasts in vitro and collagen synthesis could be inhibited by melatonin.Increased melatonin receptor 1A expression might be involved.更多还原