视黄酸在形觉剥夺性近视形成中作用的研究
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摘要
探讨视黄酸(Retinoic Acid，RA)在新生小鸡形觉剥夺性近视(FormDeprivation Myopia，FDM)形成中的作用。实验性的形觉剥夺性近视是近30年来作为研究人类近视的动物模型。所谓形觉剥夺性近视，即剥夺新生动物视网膜正常成像可导致轴性近视的发生。因其结构和屈光特点与人类轴性近视极为相似，即都伴有眼轴的增长及纤维巩膜的变薄、延伸。因此，对动物FDM机制的研究有利于人类近视眼机制的探讨。
有研究指出，眼球的生长除受发育机制的调节外，也受到视觉输入所引起的反馈机制的调节。大量研究表明，视网膜上的视觉活动能够引起眼轴的变化，而这种眼轴的变化机制目前认为主要是巩膜细胞外基质的主动重塑机制。实验性近视的形成和恢复中存在巩膜细胞外基质的合成和降解的变化，其中蛋白酶在其合成和降解的变化中起主要作用。基质金属蛋白酶2(Matrix metalloproteinase 2，MMP-2)是能降解多种大分子的酶，MMP-2与其抑制因子组织金属蛋白酶抑制剂2(Tis-sue inhibitor of matrix metalloproteinase 2，TIMP-2)之间的失衡在眼巩膜塑形中起重要作用。但目前对造成这种平衡失调的内在机制尚不清楚。
有研究表明，实验性近视形成过程中，视网膜中的某些因子，包括一些神经递质如多巴胺、乙酰胆碱、胰高血糖素等及其它一些分子或生长因子都参与了近视的形成。能在视网膜细胞内合成的视黄酸可能与之有关。视黄酸，又称维生素A酸，是维生素A最具活性的代谢产物。研究表明，视黄酸对于巩膜的发育和特定细胞类型(如光感受器细胞、无长突细胞)的分化和成熟具有重要影响。已知内层视网膜的无长突细胞参与了视黄酸的代谢，而视网膜无长突细胞位于视网膜图象加工过程的输出端。因此，这种加工过程的输出是否就与视觉控制眼球的生长有关?视网膜能对外界视环境的改变发生反应，继而释放一种(或几种)信使分子控制其下方组织的生长速率。视黄酸可能就是调节实验性近视眼球生长的一个信使分子。另外视黄酸作为体内的一种非甾体类激素，可以直接与核受体相结合来参与不同目标基因的转录，诱导一些生长因子和它们的受体表达，进而诱导特异环境下的细胞分化过程。因此，视黄酸也可称为是一种重要的分化及调节因子。同时由于巩膜细胞中有RA受体的存在，所以，我们有理由相信，RA对巩膜细胞的生长分化具有调节作用。Seko Y等报道，在生理状态下，视黄酸对巩膜软骨细胞具有刺激作用，而对巩膜成纤维细胞具有抑制作用。而巩膜细胞能合成有活性的MMP-2，这提示RA极可能在小鸡FDM巩膜重塑调控过程中起重要作用。
视黄醛脱氢酶2(Retinaldehyde Dehydrogenase-2，RALDH2)是合成RA的关键酶，其表达反映RA的合成部位及合成水平的高低。通过对形觉剥夺眼及形觉剥夺恢复眼中RA及RALDH2在视网膜、脉络膜及巩膜中表达的分析，来判定RA的合成部位及RA是否参与了FDM的形成。视黄酸受体-β(Retinoie Acid Receptor-β，RARβ)是细胞生长分化中具有重要作用的RA的受体。它属核受体超家族成员，与其配体RA相结合后，能直接参与基因转录，调节细胞的生长、分化及代谢。通过对后极部巩膜中RARβ的检测来进一步明确RA对FDM的作用表现为RA对巩膜的作用。通过对形觉剥夺眼及形觉剥夺恢复眼后极部巩膜中MMP-2和TIMP-2表达的分析，及其与RARβ表达相关性的分析来明确视环境的变化与巩膜的重新塑形的关系，且这种巩膜的重新塑形与RA有关。
由此推断：视网膜的视环境决定视网膜(和／或脉络膜，和／或巩膜)合成和释放一定水平的视黄酸，这种视黄酸能引起后极部巩膜MMP-2／TIMP-2的表达失衡，促使巩膜细胞外基质重新塑形，进而引起眼轴的过度延长，形成轴性近视眼。
方法
本研究分四部分：
实验一：观察视环境的改变与眼屈光状态、眼轴长度及巩膜形态学之间的关系。
选用新孵出的普通肉食家鸡25只，体重30-45g。孵出后2天，采用半透明薄膜眼罩遮盖的方法对单眼(左眼)进行形觉剥夺，分为两个实验组：形觉剥夺组：遮盖时间为14天；形觉剥夺恢复组：遮盖11天后，将薄膜取下，恢复3天正常视力。两组的对侧眼(右眼)分别作为对照眼。分别对两实验组进行检影验光及A超测量眼轴长度。之后，各组分别处死小鸡10只，取出眼球，4％多聚甲醛固定，于眼球中央部做矢状面的纵向切开，石蜡包埋，切片，HE染色，光镜下观察巩膜形态学改变，采用Metamorph图像分析软件测量巩膜软骨层和纤维层厚度。
实验二：形觉剥夺眼和形觉剥夺恢复眼视网膜、脉络膜和巩膜中RA含量的测定。
标本制作：两实验组各30只及空白对照组15只小鸡，处死后摘出眼球，冰台上锯齿缘处对半切开眼球，去掉前段眼组织和玻璃体，用角膜钻于统一部位取直径8mm的后极部眼组织块，分离出视网膜、脉络膜及巩膜组织，进一步在显微镜下用手术刀片将巩膜纤维层和软骨层分离开。将上述组织快速置入灭菌的Eppendorf管中，外包金属铝箔纸，标号后放入液氮中保存。
检测方法：应用高效液相色谱技术检测形觉剥夺眼和形觉剥夺恢复眼视网膜、脉络膜和巩膜(包括纤维巩膜和软骨巩膜)中RA的含量。
实验三：形觉剥夺眼和形觉剥夺恢复眼视网膜、脉络膜和巩膜中RALDH2表达的测定。
按实验二标本制作方法取两实验组(各10只小鸡)及空白对照组(5只小鸡)的视网膜、脉络膜、纤维巩膜和软骨巩膜的组织标本后，采用RT-PCR技术检测RALDH2的表达，计算mRNA相对含量，作统计学分析。
实验四：形觉剥夺眼和形觉剥夺恢复眼后极部巩膜中RARβ、MMP-2及TIMP-2表达的测定。
按实验二方法制作出后极部软骨巩膜和纤维巩膜的标本后，(1)应用RT-PCR技术检测PARβ在形觉剥夺眼和形觉剥夺恢复眼两层巩膜组织中的表达，计算mRNA相对含量。(2)应用RT-PCR技术检测两实验组后极部纤维巩膜和软骨巩膜中MMP-2和TIMP-2的表达，计算mRNA相对含量。(3)分别将MMP-2的mRNA水平及TIMP-2的mRNA水平与RARβ的mRNA水平作线性相关性分析。
统计学处理：采用均数差异t检验的方法对各组实验结果进行差异的显著性分析，线性相关性结果作相关性检验。统计软件为SPSS10.0软件。
结果
实验一：屈光检查、眼轴测定及巩膜形态学变化结果
1．形觉剥夺14天后，剥夺眼形成了高度近视(-12.1±4.3D)，眼轴增长(9.86±0.38mm)，与对侧对照眼的屈光(+2.7±0.5D)和眼轴(8.71±0.28mm)相比，差异具有显著性(p＜0.01)；形觉剥夺恢复3天后，与剥夺14天的实验眼相比，其实验眼的屈光度(-5.5±1.2D)减低(p＜0.05)，眼轴长度虽无明显变化(p＞0.05)，但眼轴增长速度明显减慢(0.003mm／天VS 0.196mm／天)，甚至比对侧对照眼的增长速度(0.116mm／天)还要慢。而各组前房深度、晶体厚度无明显变化(p＞0.05)。说明剥夺眼的眼轴增长，主要是眼球后段的增长。
2．巩膜HE染色及厚度测量可见，剥夺眼的巩膜软骨层增厚(144.3±4.78 VS 128.5±3.84μm)，巩膜纤维层变薄(12.1±0.9 VS 26.9±1.7μm)；剥夺恢复眼与剥夺眼相比，软骨层厚度明显减低(135.4±3.32 VS144.3±4.78μm)，纤维层厚度增加(20.6±1.2 VS 12.1±0.9μm)，分别接近对侧对照眼的软骨层和纤维层厚度。
实验二：形觉剥夺眼及形觉剥夺恢复眼视网膜、脉络膜和巩膜中RA含量的变化
1．RA在视网膜中的变化。在形觉剥夺14天后，视网膜中RA含量明显增高(0.769±0.051 VS 0.249±0.031 ng／mg WW)(P＜0.01)，去掉形觉剥夺3天后，RA含量明显下降(0.125±0.038 VS 0.244±0.030 ng／nag WW)(P＜0.05)。各对照组间无明显变化(P＞0.05)。
2．RA在脉络膜中的变化。形觉剥夺14天后，脉络膜中RA水平明显下降(0.385±0.010 VS 1.306±0.273 ng／mg WW)(P＜0.01)，而形觉剥夺除去3天后，RA水平明显升高(2.725±0.188 ng／mg WW)，可达剥夺眼RA水平(0.385±0.010 ng／mg WW)的7倍，甚至是对照眼(1.312±0.299ng／mg WW)的2倍。各对照组间无明显变化。
3．RA在巩膜中的变化。形觉剥夺14天后，巩膜软骨层RA(0.122±0.005 VS 0.403±0.011 ng／mg WW)及巩膜纤维层RA(0.085±0.001 VS0.507±0.009 ng／mg WW)均明显下降(P＜0.01)。其中，巩膜纤维层中RA水平下降更为明显。去除形觉剥夺3天后，巩膜软骨层(0.521±0.021ng／mg WW)和纤维巩膜中的RA水平(0.623±0.019 ng／mg WW)均明显升高(P＜0.01)。由此可见，视环境的改变使巩膜软骨层和纤维层中的RA水平都发生了明显变化，其中纤维层中RA的变化要比软骨层中的明显。
实验三：RALDH2在形觉剥夺眼及形觉剥夺恢复眼视网膜、脉络膜及巩膜中的表达
1．RALDH2在视网膜和脉络膜中均有表达，而在巩膜中没有表达。形觉剥夺14天后，视网膜中RALDH2的表达明显增高(mRNA相对含量：64.52±0.27 VS 38.71±0.19)(p＜0.01)，而脉络膜中的表达明显下降(mR-NA相对含量：36.56±0.48 VS 85.98±0.53)(P＜0.01)。视网膜和脉络膜组织中的对照眼与各自的空白对照眼相比无明显差异(P＞0.05)。
2．视网膜中RALDH2的表达在形视剥夺14天后明显增强(mRNA相对含量：67.23±0.32 VS 37.34±0.25)(P＜0.01)，而去除剥夺3天后，其表达下降(30.74±0.33 VS 37.06±0.19)(P＜0.05)。说明视网膜中RALDH2的表达随着视环境的改变而发生了明显的变化。
3．脉络膜中RALDH2的表达在形觉剥夺14天后明显下降(mRNA相对含量：35.30±0.39 VS 86.74±0.55)(P＜0.01)，而去除剥夺3天后，其表达增强(mRNA相对含量：99.86±0.46 VS 87.02±0.62)(P＜0.01)。说明，脉络膜中RALDH2的表达随着视环境的改变而发生了明显的变化。
实验四：形觉剥夺眼和形觉剥夺恢复眼后极部巩膜中RARβ、MMP-2及TIMP-2表达
1．正常后极部巩膜中的软骨层和纤维层中均有RARβ的存在，其表达程度相似(mRNA相对含量：36.64±0.43，36.75±0.31)，差异没有显著性(P＞0.05)。形觉剥夺14天后，巩膜软骨层和纤维层中，RARβ的表达均增强(p＜0.01)，但巩膜纤维层中的表达比软骨层表达更为强烈(mRNA相对含量：81.08±0.42 VS 60.21±0.43)(p＜0.01)。巩膜软骨层中RARβ的表达在形觉剥夺14天后明显增强(mRNA相对含量：62.34±0.39 VS 38.43±0.30)(P＜0.01)，去除剥夺3天后，其表达与对侧对照眼的表达(mRNA相对含量：39.05±0.26 VS 36.98±0.35)无明显差异(P＞0.05)。巩膜纤维层中RARβ的表达在形觉剥夺14天后明显增强(mRNA相对含量：72.32±0.43 VS 37.96±0.40)(P＜0.01)，去除剥夺3天后，其表达与对侧对照眼的表达(mRNA相对含量：39.21±0.33 VS 37.31±0.27)无明显差异(P＞0.05)。
2．MMP-2和TIMP-2在巩膜纤维层中均有较强的表达(mRNA相对含量分别为：46.69±0.28，36.74±0.22，而在巩膜软骨层中呈极弱的表达而难以测出。形觉剥夺及形觉剥夺恢复后，软骨层中的表达没有明显变化，均呈极弱的表达，而纤维层中的表达变化较为明显。在纤维层中，MMP-2的表达在形觉剥夺14天后明显增强(mRNA相对含量：60.51±0.36 VS46.36±0.24)(p＜0.01)；形觉剥夺恢复后其表达水平下降(mRNA相对含量：36.90±0.21 VS 47.05±0.29)(p＜0.01)。TIMP-2的表达在形觉剥夺14天后明显减弱(mRNA相对含量：19.24±0.16 VS 35.93±0.20)(p＜0.05)；形觉剥夺恢复后其表达水平与对侧对照眼的表达(mRNA相对含量：37.01±0.21 VS 36.21±0.23)无明显差异(p＞0.05)。
3．FDM中，后极部巩膜纤维层中MMP-2和RARβ的mRNA相对含量呈正相关(r=0.944，p＜0.05)，TIMP-2和RARβ的mRNA相对含量呈负相关(r=-0.863，p＜0.05)。
结论
1．RA在新生小鸡视网膜、脉络膜、巩膜中均有表达，其中脉络膜含量最高，其次为巩膜和视网膜。形觉剥夺及去剥夺所致视环境改变后，视网膜、脉络膜、巩膜中的视黄酸表达均发生了明显的变化，说明视黄酸参与了形觉剥夺性近视的形成。
2．小鸡后极部巩膜中有视黄酸受体RARβ的表达。形觉剥夺后，其表达明显增强，而且巩膜纤维层的表达水平比软骨层更为强烈。因此，视黄酸在形觉剥夺性近视中对巩膜有直接的作用。
3．FDM中，后极部巩膜纤维层中MMP-2表达增强，而TIMP-2表达减弱，表明巩膜纤维层中细胞外基质的降解增加，说明形觉剥夺性近视中巩膜纤维层的变薄为胞外基质的降解所致。
4．FDM中，巩膜纤维层中MMP-2的表达与RARβ的表达呈正相关，而TIMP-2的表达与RARβ的表达呈负相关，说明RA对巩膜纤维层细胞外基质具有调节作用。
Purpose
To Discuss the effect of retinoic acid on the formation of FDM in chicken.Experimental FDM has been used as the animal model for the research of humanmyopia. FDM is induced by the deprivation of normal imagination on theretina in newborn animal. Its structure and refractive feature is very similar tothat of spontaneous human myopia.
The growth of eyeball is regulated by the developmental mechanism, andthe feedback mechanism of vision input signal as well. Many research haveshown that vision on the retina leads to the change in eye length, which is inthe form of sclera growth. So, it is reasonable to say, that there are certainfactors secreted by retain, changed with the environment, which may serve assignal to regulate the growth and metabolism of sclera cells.
RA is thought to be related to it. RA, also know as VA acid, is the mostactive metabolite of Vitamin. A. It is already well known that RA has importanteffect on the cell proliferation. Difference and maintenance of cellular pheno-type. For example, amacrine cell on inner retina take a port in the metabolismof RA, which is located at the output end of retinal image processing. So isthis procession related to the control of eyeball growth? RA may be a messengerof experimental eyeball growth regulation. Whatsmore, RA, a nonsteroid in vivohormone, can play a role in the transcription of different gene by combined withnuclear receptors directly, activate the cellular difference. Because thereare RA receptors in sclera cells, RA may has effect on the regulation of sclera cell growth and difference.
So it has theoretical and experimental basis to think that RA is a signalmolecule and growth regulator of eyeball axis changes in FDM.
RALDH2 is the key enzyme in RA synthesis. The analysis of expression ofRA and RALDH2 in retina. Choroids and sclera can be used to judge the sit ofRA synthesis and whether RA has taken a part in the formation of FDM. RARβ,a nuclear receptor, comhined with RA, Can regulate the transcription of gene,cell growth difference and metabolism. The effect of RA on formation of FDMcan be further determined by the detection of RARβin posterior pole sclera. Thesclera active play on important role in the formation and recovery of FDM. Thereis delicate balance between the matrix synthesis and degeneration in sclera remodeling ting. MMP-2 and its inhibitor TIMP-2 are important in the synthesisand degeneration of matrix. So the expression of these enzymes, and their relation to RARβmay provide a clue to understand the relation of change in visualenvironment and sclera remodeling.
Methods
Experiment 1:To observe the relation of change in visual environment to therefraction, axis length, sclera morphology.
Newborn children 25, weight 30～40g. Left eye covered by semi-trans-parent eye pack at 2 days. FDM group: covered 14days. FDM recovery group:covered days, 3 day in normal vision condition. Control group: The other eye.The refraction and axis length were examined. Then the eyeballs were recov-ered. Prepared for pathology exam. The morphological change was observed byHE staining. The thickness of sclera cartilage and fiber were measured by meta-morph software.
Experiment 2: Detection of RA in retain. Choroid and sclera. Specimenpreparation a tissue block in diameter 8 mm was removed from the posteriorpole, retina, choroid, and sclera were separated. The fiber and cartilage layerof sclera were separated under microscope. Then the tissue were put into Eppen-dorf tube, put in fluid nitrogen. Detection Method: The RA level in retina, cho-roids and sclera was detected by HPLC. Experiment 3: Detection of RALDH2 in Retina, Choroid, and Sclera.
RT-PCR technique was used to detect the expression of RALDH2, mRNAlevel was calculated for statistical analysis.
Experiment 4: Detection of RARβ, MMP-2 and TIMP-2 in posteriorpole sclera. The specimen were same as that in ExperimentⅡ. (1)RT-PCRwas used to detect the level of RARβin sclera tissue, calculate mRNA. (2)RT-PCR was used to detect the level of MMP-2. TIMP-2 in fiber and cartilagelayer of sclera. Calculate the mRNA. (3) Analysis the relatively between mRANof MMP-2 and mRNA of RARβ. MRNA of RARβin posterior pole sclera andRA in retina and choroids respectively.
Statistical Analysis: t Test was used to determine the difference betweengroups, linear relativity was analyzed. Statistical soft are: SPSS 10.0.
Results
Refraction, axis length and morphorlogical change in sclera. (1)Ai 14days of form deprivation, the refraction was 12.1+-4.3D,axis length was 9.86+-0.38 mm, compared with the control group, which was +2.7+-0.5Dand 8.71+-0.28 mm, the difference was significant (p＜0.01);days afterrecovery from FDM, the refraction was less (-5.5+- 1.2D, p＜0.05), whilethe axis length no difference (p＜0.05), but the increase rate of axis lengthwas slower (0.003 mm/d Vs 0.196 mm/d), even slower than the posterior partof eye. (2) By HE staining, the cartilage sclera of FDM was thicker (p＜0.05), fibrous sclera thinner (p＜0.05); Compared with FDM eyes, the recovery group has thinner cartilage sclera (p＜0.05), fibrous sclera thicker (p＜0.05), which was close to that of the control group.
Chang of RA in retina choroids and sclera:
In retina: Ai 14 days, RA of FDM eyes increased markedly (0.769+-0.051 Vs 0.249+-0.031 ng/mg ww) (p＜0.01), Ai 3 days afar recovery fromFDM, RA decreased (p＜0.01,0.125+-0.038 Vs 0.769+-0.051 ng/mgww), even lower than that of control group (0.244+- 0.030 ng/mg ww). Nosignificant change between control groups.
In choroids: Ai 14days, RA of FDM eyes decreased markedly (0.385+-0.01 Vs 1.306+-0.273, p＜0.01), at 3 days after recovery from FDM, RAincreased (2.725+-0.108), which was t times of that of FDM eyes and 2times of the control group. No significant change between control groups.
In sclera: Ai 14 days, RA in cartilaginous sclera and fibrous sclera werebeth detected (p＜0.01). In which, RA in fibrous sclera were both increasedagain (p＜0.01), and higher than the control eyes. So the change in visual en-vironment can lead to the change of RA in sclera, in which, the change of RAin fibrous sclera was more obvious.
Expression of RALDH2 in retina, choroids and sclera.
There were expression of RACDH2 in retina and choroids, none in sclera.Ai 14 days of FDM, RALDH2 in retina increased (mRNA: 64.52+-0.27 Vs38.71+-0.19. p＜0.01) while RALDH2 in choroids (p＜0.01). No signif-icant difference between the control groups.
RALDH2 in FDM retina was increased (p＜0.01), at 3 days after recov-ery from FDM, it decreased (p＜0.01), even lower than the control eyes. Thisindicated that the change in visual environment lead to the change in RALDH2in retina.
RALDH2 in FDM choroids was decreased (p＜0.01), at 3 days after re-covery from FDM, it increased (p＜0.01), even higher than control eyes, thisindicated that the change in visual environment lead to the change in RALDH2in choroids.
Expression of RARβ, MMP-2 and TIMP-2 in the posterior sclera.
There was RARβin normal posterior sclera, the content was same in fibrous and cartilage sclera. At 14 days of FDM, RARβincreased (p＜0.01),more obvious in the fibrous sclera (mRNA: 81.08+-0.42 Vs 60.21+-0.43, p＜0.01). In cartilage sclera, [RARβincrease markedly ay 14 days, (p＜0.01), decreased at 3 days after recovery from FDM, (p＜0.01), no signif-icant difference compared with control group.] In fibrous sclera, the same as a-bove.
There was strong expression of MMP-2 to TIMP-2 in fibrous selera,while none in cartilaginous sclera. While in fibrous sclera, MMP-2 was in- creased, TIMP-2 was decreased at 14 days of FDM; Ai 3 days after recovery from FDM, MMP-2 was decreased (p＜0.01), even lower than that of control group (p＜0.01) and TIMP - 2 was increased ( p＜0.05), but no difference with the control group.
There was positive relatively between mRNA of RARβand MMP-2 in posterior fibrous sclera(r=0.944). There was negative relatively between mRNA of RARβand TIMP-2 (r=-0.863 ).
Conclusions
1. After from deprivation, the RA level in retina, choroids and sclera were changed markedly. So RA is related to the formation of FDM.
2. There was the expression of RA receptor-RARβin posterior sclera. Its expression had close relation to the change of RA level in retina and choroids. So the effect of RA on FDM manifested as the function on sclera.
3. In FDM, the expression of MMP-2 in posterior fibrous sclera increase, TIMP-2 decreased, which indicated the extracellular matrix degeneration increased in fibrous sclera. So the matrix degeneration was the cause of fibrous sclera thinning in FDH.
4. There was positive relativity between RARβand MMP-2, and negative relativity between RARβand IMMP-2 in posterior fibrous sclera, which indicated that RA had regulation affect on extra cellular matrix of fibrous sclera.
有研究指出，眼球的生长除受发育机制的调节外，也受到视觉输入所引起的反馈机制的调节。大量研究表明，视网膜上的视觉活动能够引起眼轴的变化，而这种眼轴的变化机制目前认为主要是巩膜细胞外基质的主动重塑机制。实验性近视的形成和恢复中存在巩膜细胞外基质的合成和降解的变化，其中蛋白酶在其合成和降解的变化中起主要作用。基质金属蛋白酶2(Matrix metalloproteinase 2，MMP-2)是能降解多种大分子的酶，MMP-2与其抑制因子组织金属蛋白酶抑制剂2(Tis-sue inhibitor of matrix metalloproteinase 2，TIMP-2)之间的失衡在眼巩膜塑形中起重要作用。但目前对造成这种平衡失调的内在机制尚不清楚。
有研究表明，实验性近视形成过程中，视网膜中的某些因子，包括一些神经递质如多巴胺、乙酰胆碱、胰高血糖素等及其它一些分子或生长因子都参与了近视的形成。能在视网膜细胞内合成的视黄酸可能与之有关。视黄酸，又称维生素A酸，是维生素A最具活性的代谢产物。研究表明，视黄酸对于巩膜的发育和特定细胞类型(如光感受器细胞、无长突细胞)的分化和成熟具有重要影响。已知内层视网膜的无长突细胞参与了视黄酸的代谢，而视网膜无长突细胞位于视网膜图象加工过程的输出端。因此，这种加工过程的输出是否就与视觉控制眼球的生长有关?视网膜能对外界视环境的改变发生反应，继而释放一种(或几种)信使分子控制其下方组织的生长速率。视黄酸可能就是调节实验性近视眼球生长的一个信使分子。另外视黄酸作为体内的一种非甾体类激素，可以直接与核受体相结合来参与不同目标基因的转录，诱导一些生长因子和它们的受体表达，进而诱导特异环境下的细胞分化过程。因此，视黄酸也可称为是一种重要的分化及调节因子。同时由于巩膜细胞中有RA受体的存在，所以，我们有理由相信，RA对巩膜细胞的生长分化具有调节作用。Seko Y等报道，在生理状态下，视黄酸对巩膜软骨细胞具有刺激作用，而对巩膜成纤维细胞具有抑制作用。而巩膜细胞能合成有活性的MMP-2，这提示RA极可能在小鸡FDM巩膜重塑调控过程中起重要作用。
视黄醛脱氢酶2(Retinaldehyde Dehydrogenase-2，RALDH2)是合成RA的关键酶，其表达反映RA的合成部位及合成水平的高低。通过对形觉剥夺眼及形觉剥夺恢复眼中RA及RALDH2在视网膜、脉络膜及巩膜中表达的分析，来判定RA的合成部位及RA是否参与了FDM的形成。视黄酸受体-β(Retinoie Acid Receptor-β，RARβ)是细胞生长分化中具有重要作用的RA的受体。它属核受体超家族成员，与其配体RA相结合后，能直接参与基因转录，调节细胞的生长、分化及代谢。通过对后极部巩膜中RARβ的检测来进一步明确RA对FDM的作用表现为RA对巩膜的作用。通过对形觉剥夺眼及形觉剥夺恢复眼后极部巩膜中MMP-2和TIMP-2表达的分析，及其与RARβ表达相关性的分析来明确视环境的变化与巩膜的重新塑形的关系，且这种巩膜的重新塑形与RA有关。
由此推断：视网膜的视环境决定视网膜(和／或脉络膜，和／或巩膜)合成和释放一定水平的视黄酸，这种视黄酸能引起后极部巩膜MMP-2／TIMP-2的表达失衡，促使巩膜细胞外基质重新塑形，进而引起眼轴的过度延长，形成轴性近视眼。
方法
本研究分四部分：
实验一：观察视环境的改变与眼屈光状态、眼轴长度及巩膜形态学之间的关系。
选用新孵出的普通肉食家鸡25只，体重30-45g。孵出后2天，采用半透明薄膜眼罩遮盖的方法对单眼(左眼)进行形觉剥夺，分为两个实验组：形觉剥夺组：遮盖时间为14天；形觉剥夺恢复组：遮盖11天后，将薄膜取下，恢复3天正常视力。两组的对侧眼(右眼)分别作为对照眼。分别对两实验组进行检影验光及A超测量眼轴长度。之后，各组分别处死小鸡10只，取出眼球，4％多聚甲醛固定，于眼球中央部做矢状面的纵向切开，石蜡包埋，切片，HE染色，光镜下观察巩膜形态学改变，采用Metamorph图像分析软件测量巩膜软骨层和纤维层厚度。
实验二：形觉剥夺眼和形觉剥夺恢复眼视网膜、脉络膜和巩膜中RA含量的测定。
标本制作：两实验组各30只及空白对照组15只小鸡，处死后摘出眼球，冰台上锯齿缘处对半切开眼球，去掉前段眼组织和玻璃体，用角膜钻于统一部位取直径8mm的后极部眼组织块，分离出视网膜、脉络膜及巩膜组织，进一步在显微镜下用手术刀片将巩膜纤维层和软骨层分离开。将上述组织快速置入灭菌的Eppendorf管中，外包金属铝箔纸，标号后放入液氮中保存。
检测方法：应用高效液相色谱技术检测形觉剥夺眼和形觉剥夺恢复眼视网膜、脉络膜和巩膜(包括纤维巩膜和软骨巩膜)中RA的含量。
实验三：形觉剥夺眼和形觉剥夺恢复眼视网膜、脉络膜和巩膜中RALDH2表达的测定。
按实验二标本制作方法取两实验组(各10只小鸡)及空白对照组(5只小鸡)的视网膜、脉络膜、纤维巩膜和软骨巩膜的组织标本后，采用RT-PCR技术检测RALDH2的表达，计算mRNA相对含量，作统计学分析。
实验四：形觉剥夺眼和形觉剥夺恢复眼后极部巩膜中RARβ、MMP-2及TIMP-2表达的测定。
按实验二方法制作出后极部软骨巩膜和纤维巩膜的标本后，(1)应用RT-PCR技术检测PARβ在形觉剥夺眼和形觉剥夺恢复眼两层巩膜组织中的表达，计算mRNA相对含量。(2)应用RT-PCR技术检测两实验组后极部纤维巩膜和软骨巩膜中MMP-2和TIMP-2的表达，计算mRNA相对含量。(3)分别将MMP-2的mRNA水平及TIMP-2的mRNA水平与RARβ的mRNA水平作线性相关性分析。
统计学处理：采用均数差异t检验的方法对各组实验结果进行差异的显著性分析，线性相关性结果作相关性检验。统计软件为SPSS10.0软件。
结果
实验一：屈光检查、眼轴测定及巩膜形态学变化结果
1．形觉剥夺14天后，剥夺眼形成了高度近视(-12.1±4.3D)，眼轴增长(9.86±0.38mm)，与对侧对照眼的屈光(+2.7±0.5D)和眼轴(8.71±0.28mm)相比，差异具有显著性(p＜0.01)；形觉剥夺恢复3天后，与剥夺14天的实验眼相比，其实验眼的屈光度(-5.5±1.2D)减低(p＜0.05)，眼轴长度虽无明显变化(p＞0.05)，但眼轴增长速度明显减慢(0.003mm／天VS 0.196mm／天)，甚至比对侧对照眼的增长速度(0.116mm／天)还要慢。而各组前房深度、晶体厚度无明显变化(p＞0.05)。说明剥夺眼的眼轴增长，主要是眼球后段的增长。
2．巩膜HE染色及厚度测量可见，剥夺眼的巩膜软骨层增厚(144.3±4.78 VS 128.5±3.84μm)，巩膜纤维层变薄(12.1±0.9 VS 26.9±1.7μm)；剥夺恢复眼与剥夺眼相比，软骨层厚度明显减低(135.4±3.32 VS144.3±4.78μm)，纤维层厚度增加(20.6±1.2 VS 12.1±0.9μm)，分别接近对侧对照眼的软骨层和纤维层厚度。
实验二：形觉剥夺眼及形觉剥夺恢复眼视网膜、脉络膜和巩膜中RA含量的变化
1．RA在视网膜中的变化。在形觉剥夺14天后，视网膜中RA含量明显增高(0.769±0.051 VS 0.249±0.031 ng／mg WW)(P＜0.01)，去掉形觉剥夺3天后，RA含量明显下降(0.125±0.038 VS 0.244±0.030 ng／nag WW)(P＜0.05)。各对照组间无明显变化(P＞0.05)。
2．RA在脉络膜中的变化。形觉剥夺14天后，脉络膜中RA水平明显下降(0.385±0.010 VS 1.306±0.273 ng／mg WW)(P＜0.01)，而形觉剥夺除去3天后，RA水平明显升高(2.725±0.188 ng／mg WW)，可达剥夺眼RA水平(0.385±0.010 ng／mg WW)的7倍，甚至是对照眼(1.312±0.299ng／mg WW)的2倍。各对照组间无明显变化。
3．RA在巩膜中的变化。形觉剥夺14天后，巩膜软骨层RA(0.122±0.005 VS 0.403±0.011 ng／mg WW)及巩膜纤维层RA(0.085±0.001 VS0.507±0.009 ng／mg WW)均明显下降(P＜0.01)。其中，巩膜纤维层中RA水平下降更为明显。去除形觉剥夺3天后，巩膜软骨层(0.521±0.021ng／mg WW)和纤维巩膜中的RA水平(0.623±0.019 ng／mg WW)均明显升高(P＜0.01)。由此可见，视环境的改变使巩膜软骨层和纤维层中的RA水平都发生了明显变化，其中纤维层中RA的变化要比软骨层中的明显。
实验三：RALDH2在形觉剥夺眼及形觉剥夺恢复眼视网膜、脉络膜及巩膜中的表达
1．RALDH2在视网膜和脉络膜中均有表达，而在巩膜中没有表达。形觉剥夺14天后，视网膜中RALDH2的表达明显增高(mRNA相对含量：64.52±0.27 VS 38.71±0.19)(p＜0.01)，而脉络膜中的表达明显下降(mR-NA相对含量：36.56±0.48 VS 85.98±0.53)(P＜0.01)。视网膜和脉络膜组织中的对照眼与各自的空白对照眼相比无明显差异(P＞0.05)。
2．视网膜中RALDH2的表达在形视剥夺14天后明显增强(mRNA相对含量：67.23±0.32 VS 37.34±0.25)(P＜0.01)，而去除剥夺3天后，其表达下降(30.74±0.33 VS 37.06±0.19)(P＜0.05)。说明视网膜中RALDH2的表达随着视环境的改变而发生了明显的变化。
3．脉络膜中RALDH2的表达在形觉剥夺14天后明显下降(mRNA相对含量：35.30±0.39 VS 86.74±0.55)(P＜0.01)，而去除剥夺3天后，其表达增强(mRNA相对含量：99.86±0.46 VS 87.02±0.62)(P＜0.01)。说明，脉络膜中RALDH2的表达随着视环境的改变而发生了明显的变化。
实验四：形觉剥夺眼和形觉剥夺恢复眼后极部巩膜中RARβ、MMP-2及TIMP-2表达
1．正常后极部巩膜中的软骨层和纤维层中均有RARβ的存在，其表达程度相似(mRNA相对含量：36.64±0.43，36.75±0.31)，差异没有显著性(P＞0.05)。形觉剥夺14天后，巩膜软骨层和纤维层中，RARβ的表达均增强(p＜0.01)，但巩膜纤维层中的表达比软骨层表达更为强烈(mRNA相对含量：81.08±0.42 VS 60.21±0.43)(p＜0.01)。巩膜软骨层中RARβ的表达在形觉剥夺14天后明显增强(mRNA相对含量：62.34±0.39 VS 38.43±0.30)(P＜0.01)，去除剥夺3天后，其表达与对侧对照眼的表达(mRNA相对含量：39.05±0.26 VS 36.98±0.35)无明显差异(P＞0.05)。巩膜纤维层中RARβ的表达在形觉剥夺14天后明显增强(mRNA相对含量：72.32±0.43 VS 37.96±0.40)(P＜0.01)，去除剥夺3天后，其表达与对侧对照眼的表达(mRNA相对含量：39.21±0.33 VS 37.31±0.27)无明显差异(P＞0.05)。
2．MMP-2和TIMP-2在巩膜纤维层中均有较强的表达(mRNA相对含量分别为：46.69±0.28，36.74±0.22，而在巩膜软骨层中呈极弱的表达而难以测出。形觉剥夺及形觉剥夺恢复后，软骨层中的表达没有明显变化，均呈极弱的表达，而纤维层中的表达变化较为明显。在纤维层中，MMP-2的表达在形觉剥夺14天后明显增强(mRNA相对含量：60.51±0.36 VS46.36±0.24)(p＜0.01)；形觉剥夺恢复后其表达水平下降(mRNA相对含量：36.90±0.21 VS 47.05±0.29)(p＜0.01)。TIMP-2的表达在形觉剥夺14天后明显减弱(mRNA相对含量：19.24±0.16 VS 35.93±0.20)(p＜0.05)；形觉剥夺恢复后其表达水平与对侧对照眼的表达(mRNA相对含量：37.01±0.21 VS 36.21±0.23)无明显差异(p＞0.05)。
3．FDM中，后极部巩膜纤维层中MMP-2和RARβ的mRNA相对含量呈正相关(r=0.944，p＜0.05)，TIMP-2和RARβ的mRNA相对含量呈负相关(r=-0.863，p＜0.05)。
结论
1．RA在新生小鸡视网膜、脉络膜、巩膜中均有表达，其中脉络膜含量最高，其次为巩膜和视网膜。形觉剥夺及去剥夺所致视环境改变后，视网膜、脉络膜、巩膜中的视黄酸表达均发生了明显的变化，说明视黄酸参与了形觉剥夺性近视的形成。
2．小鸡后极部巩膜中有视黄酸受体RARβ的表达。形觉剥夺后，其表达明显增强，而且巩膜纤维层的表达水平比软骨层更为强烈。因此，视黄酸在形觉剥夺性近视中对巩膜有直接的作用。
3．FDM中，后极部巩膜纤维层中MMP-2表达增强，而TIMP-2表达减弱，表明巩膜纤维层中细胞外基质的降解增加，说明形觉剥夺性近视中巩膜纤维层的变薄为胞外基质的降解所致。
4．FDM中，巩膜纤维层中MMP-2的表达与RARβ的表达呈正相关，而TIMP-2的表达与RARβ的表达呈负相关，说明RA对巩膜纤维层细胞外基质具有调节作用。
Purpose
To Discuss the effect of retinoic acid on the formation of FDM in chicken.Experimental FDM has been used as the animal model for the research of humanmyopia. FDM is induced by the deprivation of normal imagination on theretina in newborn animal. Its structure and refractive feature is very similar tothat of spontaneous human myopia.
The growth of eyeball is regulated by the developmental mechanism, andthe feedback mechanism of vision input signal as well. Many research haveshown that vision on the retina leads to the change in eye length, which is inthe form of sclera growth. So, it is reasonable to say, that there are certainfactors secreted by retain, changed with the environment, which may serve assignal to regulate the growth and metabolism of sclera cells.
RA is thought to be related to it. RA, also know as VA acid, is the mostactive metabolite of Vitamin. A. It is already well known that RA has importanteffect on the cell proliferation. Difference and maintenance of cellular pheno-type. For example, amacrine cell on inner retina take a port in the metabolismof RA, which is located at the output end of retinal image processing. So isthis procession related to the control of eyeball growth? RA may be a messengerof experimental eyeball growth regulation. Whatsmore, RA, a nonsteroid in vivohormone, can play a role in the transcription of different gene by combined withnuclear receptors directly, activate the cellular difference. Because thereare RA receptors in sclera cells, RA may has effect on the regulation of sclera cell growth and difference.
So it has theoretical and experimental basis to think that RA is a signalmolecule and growth regulator of eyeball axis changes in FDM.
RALDH2 is the key enzyme in RA synthesis. The analysis of expression ofRA and RALDH2 in retina. Choroids and sclera can be used to judge the sit ofRA synthesis and whether RA has taken a part in the formation of FDM. RARβ,a nuclear receptor, comhined with RA, Can regulate the transcription of gene,cell growth difference and metabolism. The effect of RA on formation of FDMcan be further determined by the detection of RARβin posterior pole sclera. Thesclera active play on important role in the formation and recovery of FDM. Thereis delicate balance between the matrix synthesis and degeneration in sclera remodeling ting. MMP-2 and its inhibitor TIMP-2 are important in the synthesisand degeneration of matrix. So the expression of these enzymes, and their relation to RARβmay provide a clue to understand the relation of change in visualenvironment and sclera remodeling.
Methods
Experiment 1:To observe the relation of change in visual environment to therefraction, axis length, sclera morphology.
Newborn children 25, weight 30～40g. Left eye covered by semi-trans-parent eye pack at 2 days. FDM group: covered 14days. FDM recovery group:covered days, 3 day in normal vision condition. Control group: The other eye.The refraction and axis length were examined. Then the eyeballs were recov-ered. Prepared for pathology exam. The morphological change was observed byHE staining. The thickness of sclera cartilage and fiber were measured by meta-morph software.
Experiment 2: Detection of RA in retain. Choroid and sclera. Specimenpreparation a tissue block in diameter 8 mm was removed from the posteriorpole, retina, choroid, and sclera were separated. The fiber and cartilage layerof sclera were separated under microscope. Then the tissue were put into Eppen-dorf tube, put in fluid nitrogen. Detection Method: The RA level in retina, cho-roids and sclera was detected by HPLC. Experiment 3: Detection of RALDH2 in Retina, Choroid, and Sclera.
RT-PCR technique was used to detect the expression of RALDH2, mRNAlevel was calculated for statistical analysis.
Experiment 4: Detection of RARβ, MMP-2 and TIMP-2 in posteriorpole sclera. The specimen were same as that in ExperimentⅡ. (1)RT-PCRwas used to detect the level of RARβin sclera tissue, calculate mRNA. (2)RT-PCR was used to detect the level of MMP-2. TIMP-2 in fiber and cartilagelayer of sclera. Calculate the mRNA. (3) Analysis the relatively between mRANof MMP-2 and mRNA of RARβ. MRNA of RARβin posterior pole sclera andRA in retina and choroids respectively.
Statistical Analysis: t Test was used to determine the difference betweengroups, linear relativity was analyzed. Statistical soft are: SPSS 10.0.
Results
Refraction, axis length and morphorlogical change in sclera. (1)Ai 14days of form deprivation, the refraction was 12.1+-4.3D,axis length was 9.86+-0.38 mm, compared with the control group, which was +2.7+-0.5Dand 8.71+-0.28 mm, the difference was significant (p＜0.01);days afterrecovery from FDM, the refraction was less (-5.5+- 1.2D, p＜0.05), whilethe axis length no difference (p＜0.05), but the increase rate of axis lengthwas slower (0.003 mm/d Vs 0.196 mm/d), even slower than the posterior partof eye. (2) By HE staining, the cartilage sclera of FDM was thicker (p＜0.05), fibrous sclera thinner (p＜0.05); Compared with FDM eyes, the recovery group has thinner cartilage sclera (p＜0.05), fibrous sclera thicker (p＜0.05), which was close to that of the control group.
Chang of RA in retina choroids and sclera:
In retina: Ai 14 days, RA of FDM eyes increased markedly (0.769+-0.051 Vs 0.249+-0.031 ng/mg ww) (p＜0.01), Ai 3 days afar recovery fromFDM, RA decreased (p＜0.01,0.125+-0.038 Vs 0.769+-0.051 ng/mgww), even lower than that of control group (0.244+- 0.030 ng/mg ww). Nosignificant change between control groups.
In choroids: Ai 14days, RA of FDM eyes decreased markedly (0.385+-0.01 Vs 1.306+-0.273, p＜0.01), at 3 days after recovery from FDM, RAincreased (2.725+-0.108), which was t times of that of FDM eyes and 2times of the control group. No significant change between control groups.
In sclera: Ai 14 days, RA in cartilaginous sclera and fibrous sclera werebeth detected (p＜0.01). In which, RA in fibrous sclera were both increasedagain (p＜0.01), and higher than the control eyes. So the change in visual en-vironment can lead to the change of RA in sclera, in which, the change of RAin fibrous sclera was more obvious.
Expression of RALDH2 in retina, choroids and sclera.
There were expression of RACDH2 in retina and choroids, none in sclera.Ai 14 days of FDM, RALDH2 in retina increased (mRNA: 64.52+-0.27 Vs38.71+-0.19. p＜0.01) while RALDH2 in choroids (p＜0.01). No signif-icant difference between the control groups.
RALDH2 in FDM retina was increased (p＜0.01), at 3 days after recov-ery from FDM, it decreased (p＜0.01), even lower than the control eyes. Thisindicated that the change in visual environment lead to the change in RALDH2in retina.
RALDH2 in FDM choroids was decreased (p＜0.01), at 3 days after re-covery from FDM, it increased (p＜0.01), even higher than control eyes, thisindicated that the change in visual environment lead to the change in RALDH2in choroids.
Expression of RARβ, MMP-2 and TIMP-2 in the posterior sclera.
There was RARβin normal posterior sclera, the content was same in fibrous and cartilage sclera. At 14 days of FDM, RARβincreased (p＜0.01),more obvious in the fibrous sclera (mRNA: 81.08+-0.42 Vs 60.21+-0.43, p＜0.01). In cartilage sclera, [RARβincrease markedly ay 14 days, (p＜0.01), decreased at 3 days after recovery from FDM, (p＜0.01), no signif-icant difference compared with control group.] In fibrous sclera, the same as a-bove.
There was strong expression of MMP-2 to TIMP-2 in fibrous selera,while none in cartilaginous sclera. While in fibrous sclera, MMP-2 was in- creased, TIMP-2 was decreased at 14 days of FDM; Ai 3 days after recovery from FDM, MMP-2 was decreased (p＜0.01), even lower than that of control group (p＜0.01) and TIMP - 2 was increased ( p＜0.05), but no difference with the control group.
There was positive relatively between mRNA of RARβand MMP-2 in posterior fibrous sclera(r=0.944). There was negative relatively between mRNA of RARβand TIMP-2 (r=-0.863 ).
Conclusions
1. After from deprivation, the RA level in retina, choroids and sclera were changed markedly. So RA is related to the formation of FDM.
2. There was the expression of RA receptor-RARβin posterior sclera. Its expression had close relation to the change of RA level in retina and choroids. So the effect of RA on FDM manifested as the function on sclera.
3. In FDM, the expression of MMP-2 in posterior fibrous sclera increase, TIMP-2 decreased, which indicated the extracellular matrix degeneration increased in fibrous sclera. So the matrix degeneration was the cause of fibrous sclera thinning in FDH.
4. There was positive relativity between RARβand MMP-2, and negative relativity between RARβand IMMP-2 in posterior fibrous sclera, which indicated that RA had regulation affect on extra cellular matrix of fibrous sclera.
引文
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