形觉剥夺性近视形成过程中巩膜的重新塑形
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摘要
近视是眼科的常见病、多发病，关于其发病机制尚无定论。形觉剥夺学说认为多种影响视网膜正常成像的因素，均可导致眼轴延长，形成近视，该学说的提出对近视发生机制的研究起到重要的推动作用。近年来，国外有报道形觉剥夺性近视可能是巩膜重新塑形的结果，但其机制尚不清楚；国内无相关报道。本实验拟通过成功建立形觉剥夺性近视动物模型；采用常规HE染色、链酶亲和素—生物素—过氧化物酶复合物免疫组化(SABC)染色，检测鸡雏形觉剥夺性近视眼巩膜胶原型表达的改变，并采用十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western印迹法(Western-blot)检测巩膜基质金属蛋白酶Ⅱ(MMP-2)和基质金属蛋白酶Ⅱ抑制剂(TIMP-2)表达的变化，来说明在形觉剥夺性近视形成过程中巩膜的重新塑形，以探讨形觉剥夺性近视的发生机制。
1．形觉剥夺性近视形成过程中巩膜胶原型表达的改变
材料和方法：选用健康雄性海兰鸡雏40只，于出生后第2天，将右眼以半透明眼罩遮盖作为遮盖眼即形觉剥夺眼，左眼为开放对照眼。分别于遮盖第1、3、5、7、14天后去除遮盖，采用带状光检影镜在暗室中检影验光后，处死鸡雏并迅速摘出两侧眼球，用游标卡尺测量眼轴长；然后常规固定、脱水、透明、浸
形觉剥夺性近视形成过程中巩膜的重若，望
蜡、包埋制成蜡块，作3林m厚石蜡切片，一部分常规HE染色光学显微镜下观
察巩膜的形态学改变;另一部分采用SABC染色，检测鸡眼巩膜胶原型表达的
改变。实验设阳性对照和阴性对照，数据经统计学处理。
结果:(l)形觉剥夺导致了鸡眼近视性屈光异常;且近视程度随遮盖时间的
延长而加深。遮盖眼平均屈光度1天为+l .937切.079D、3天为一2.78士0.27D、5天
为一9.17士0.22n、7天为一14.275士o.609D、14天为一20.5肚0.slD;对照眼平均屈光
度1天为+1.90肚0.078D、3天为+1.725士0.20D、5天为+1.425士0.175D、7天为
+1 .2625士o.122D和14天为+0.7升0.250，从第3天起，两眼屈光度相比较差异有
显著性(尸<。.01)。遮盖眼眼轴长l天为8.265士0.15mm、3天为8.995士0.069mm、
5天为9.33切.051nnl、7天为9.肚0.11~和14天为9.78切.44~;对照眼眼轴
长l天为8.1牡0.25mm、3天为8.12士0.062mm、5天为8.53士0.llmm、7天为
8.61士0.064~和14天为8.7处0.18~，两眼眼轴长相比较差异有显著性
(尸<0 .01)。(2) HE染色显示;形觉剥夺造成遮盖眼后极部巩膜软骨层变厚、纤
维层变薄，且软骨细胞和双核细胞数较对照眼增多。(3)免疫组化染色显示:遮
盖眼巩膜胶原表达主要为I型胶原，对照眼巩膜胶原表达主要为111型胶原。
结论:形觉剥夺导致鸡眼形成形觉剥夺性近视，且近视程度随遮盖时间的延
长而加深。遮盖眼巩膜形态学改变伴随软骨层厚度的增加和纤维层厚度的减少，
遮盖眼巩膜胶原型表达发生改变是由于I型胶原增加和川型胶原减少的原因。
2.形觉剥夺性近视形成过程中巩膜基质金属蛋白酶n(姗P一2)及基
质金属蛋白酶n抑制剂(TIMP一2)表达的改变
材料和方法:选用健康雄性海兰鸡雏80只，于出生后第2天遮盖右眼，左
眼开放对照。饲养2周后去除遮盖，随机分为实验组和恢复组。实验组在暗室检
郑州大学2003年研究生毕业论文
形觉剥夺性近视形成过程中巩膜的重新塑形
影验光，确定遮盖眼及对照眼的屈光状态后，处死鸡雏并迅速摘出两侧眼球，在
冰台上用直径8~的环钻钻取后极部巩膜组织，并在显微镜下去除视网膜、脉
络膜等眼组织，每5个眼球巩膜组织为一组，剪碎、匀浆、低温离心机10，000 rpm
离心20分钟，取上清液为待测样本，一70℃低温冰箱保存;恢复组于白然条件一下
再饲养1周，用同样的方法制备待测样本。采用i一二烷基磺酸钠聚丙烯酞胺凝胶
电泳(SDS一PAGE)和westem印迹法(westem-blot)测定实验组遮盖眼和对照眼、恢
复组去遮盖眼(即恢复眼)和对照眼基质金属蛋白酶11和基质金属蛋白酶H抑制
剂表达的改变。实验设空白对照，数据经统计学处理。
结果:(l)实验组遮盖眼和对照眼MMP一2活性平均值分别为5511.18士621.77
和2949.39士795.69，两者相比较遮盖眼MMP一2活性较对照眼有显著性提高
(P<0.ol);遮盖眼和对照眼TIMP一2活性平均值分别为2738.01士62.67和4541.65
士389.39，两者相比较遮盖眼TIMP一2活性较对照眼有显著性下降(尸<0.01)。(2)
恢复组恢复眼和对照眼MMP一2活性平均值分别为2936.96士1 13.22和2932.71士
70.85，两者相比较尚不能说差异有显著性(乃0.5);恢复眼和对照眼TIMP一2活性
平均值分别为4601 .4士95 .87和4608.47士48.28，两者相比较尚不能说差异有显
著性(外0.5)。(3)实验组遮盖眼和恢复组恢复眼的MMP一2活性相比较差异有显
著性(P<0.OOI)。
结论:形觉剥夺性近视的形成是与实验组遮盖眼MMP一2活性的增加及
TIMP一2活性的减少相关的，所以，这种MMP一2和TIMP一2之间的失衡可能就是
FDM形成过程中巩膜重新塑形的原因之一。
Myopia or nearsightedness is a commonly and frequently encountered disease in ophthalmology. It is not clear about its pathogenesis, but form deprivation (FM) theory has played an important role in the study of myopia pathogenesis. FM, which means using multiple methods to effect the normal formation in the retina can result in abnormal growth of ocular globe, showing longer axial length and leading to myopia.
There were some foreign reports about relationship of MMP, TIMP and FDM, but the mechanism is not clear yet. In my experiment, myopia is characterized by excessive axial lengthening of the ocular globe and morphological changes. Using HE staining, SABC staining, SDS-PAGE and WB to study the express changes of scleral morphology, collagen types, MMP-2 and TIMP-2 in FDM. We hope study and understand the pathogenesis of FDM.
1. The express changes of scleral collagen types in the deprived eyes of FDM
Materials and methods: Forty healthy male hailan chickens were selected after
born 2 days. All the right eyes underwent form deprivation with translucent goggles, and the left eyes were used as the control. After deprived for 1, 3, 5, 7 and 14 days, the refractive state of the deprived eyes were characterized by means of streak retinoscopy (without cycloplegia), then the eye globes were removed and its axial length were measured by sliding gage. Routine fixation, dehydration, diaphanous, paraffin and embedding were done. Sum paraffin sections were taken. We used routine hematoxylin-eosin (HE) staining and observed the morphological changes of deprived sclera under light microscopy. Streptaridin-biotin-peroxidase complex (SABC) immunohistochemistry method was used to study the express changes of collagen types.
Results: (1). Form deprivation leads to myopia refractive abnormality. After deprived for 1, 3, 5, 7 and 14 days, the average dipoter of the deprived eyes were +1.937?.079 D, -2.78?.27 D, -9.17?.22 D, -14.275?.609 D, -20.56?.51 D, and the axial length of the deprived eyes were +8.265?.15 mm, +8.995?.069 mm, +9.33?.05 mm, +9.6?.106 mm, +9.78?.44 mm. Significant differences were detected in axial length and diopter between deprived and control eyes (P<0.01). (2) HE staining shows that the deprived posterior cartilaginous sclera was thicker and the fibrous sclera was thinner than control sclera. The number of cells in the deprived cartilaginous sclera was more than control sclera. (3) Immunohistrochemistry staining shows that there were collagen type I mainly in deprived eyes and collagen type III in control eyes.
Conclusions: Form deprivation leads to myopia refractive abnormality, and the
longer the eyes were deprived, the more severe the myopia refractive state was. Morphological changes of sclera were associated with an increased thickness of cartilaginous and a decreased thickness of fibrous within the posterior sclera. The express changes of scleral collagen types were related with an increased amount of collagen type I and a decreased amount of collagen type III.
2. The express changes of MMP-2 and TIMP-2 in deprived and recovering eyes
Materials and methods: 80 healthy male hailan chickens were selected. The right eyes were deprived after born 2 days and the left eyes were control. Two groups were divided at random. One was experimental group and the other was recovering group. The deprived eyes were discovered after being deprived 2 weeks. The refractive state was characterized by means of streak retinoscopy. Then killed the chickens and removed the eye globes. Using a surgical trephine, 8mm diameter tissue buttons of posterior sclera were excised. The attached ocular tissues were separated. The five tissue-buttons form each group were homogenized together in 0.01M Tris ?HC1, ph 8.0. Insoluble material was removed from the homogenized material by centrifugation at 10,000 rpm for 20 minutes. The supernatant was used for measurement. The recovering group was discovered after 2 weeks and feed normally for another 1 week. The specimens were made by the same method. The supern
1．形觉剥夺性近视形成过程中巩膜胶原型表达的改变
材料和方法：选用健康雄性海兰鸡雏40只，于出生后第2天，将右眼以半透明眼罩遮盖作为遮盖眼即形觉剥夺眼，左眼为开放对照眼。分别于遮盖第1、3、5、7、14天后去除遮盖，采用带状光检影镜在暗室中检影验光后，处死鸡雏并迅速摘出两侧眼球，用游标卡尺测量眼轴长；然后常规固定、脱水、透明、浸
形觉剥夺性近视形成过程中巩膜的重若，望
蜡、包埋制成蜡块，作3林m厚石蜡切片，一部分常规HE染色光学显微镜下观
察巩膜的形态学改变;另一部分采用SABC染色，检测鸡眼巩膜胶原型表达的
改变。实验设阳性对照和阴性对照，数据经统计学处理。
结果:(l)形觉剥夺导致了鸡眼近视性屈光异常;且近视程度随遮盖时间的
延长而加深。遮盖眼平均屈光度1天为+l .937切.079D、3天为一2.78士0.27D、5天
为一9.17士0.22n、7天为一14.275士o.609D、14天为一20.5肚0.slD;对照眼平均屈光
度1天为+1.90肚0.078D、3天为+1.725士0.20D、5天为+1.425士0.175D、7天为
+1 .2625士o.122D和14天为+0.7升0.250，从第3天起，两眼屈光度相比较差异有
显著性(尸<。.01)。遮盖眼眼轴长l天为8.265士0.15mm、3天为8.995士0.069mm、
5天为9.33切.051nnl、7天为9.肚0.11~和14天为9.78切.44~;对照眼眼轴
长l天为8.1牡0.25mm、3天为8.12士0.062mm、5天为8.53士0.llmm、7天为
8.61士0.064~和14天为8.7处0.18~，两眼眼轴长相比较差异有显著性
(尸<0 .01)。(2) HE染色显示;形觉剥夺造成遮盖眼后极部巩膜软骨层变厚、纤
维层变薄，且软骨细胞和双核细胞数较对照眼增多。(3)免疫组化染色显示:遮
盖眼巩膜胶原表达主要为I型胶原，对照眼巩膜胶原表达主要为111型胶原。
结论:形觉剥夺导致鸡眼形成形觉剥夺性近视，且近视程度随遮盖时间的延
长而加深。遮盖眼巩膜形态学改变伴随软骨层厚度的增加和纤维层厚度的减少，
遮盖眼巩膜胶原型表达发生改变是由于I型胶原增加和川型胶原减少的原因。
2.形觉剥夺性近视形成过程中巩膜基质金属蛋白酶n(姗P一2)及基
质金属蛋白酶n抑制剂(TIMP一2)表达的改变
材料和方法:选用健康雄性海兰鸡雏80只，于出生后第2天遮盖右眼，左
眼开放对照。饲养2周后去除遮盖，随机分为实验组和恢复组。实验组在暗室检
郑州大学2003年研究生毕业论文
形觉剥夺性近视形成过程中巩膜的重新塑形
影验光，确定遮盖眼及对照眼的屈光状态后，处死鸡雏并迅速摘出两侧眼球，在
冰台上用直径8~的环钻钻取后极部巩膜组织，并在显微镜下去除视网膜、脉
络膜等眼组织，每5个眼球巩膜组织为一组，剪碎、匀浆、低温离心机10，000 rpm
离心20分钟，取上清液为待测样本，一70℃低温冰箱保存;恢复组于白然条件一下
再饲养1周，用同样的方法制备待测样本。采用i一二烷基磺酸钠聚丙烯酞胺凝胶
电泳(SDS一PAGE)和westem印迹法(westem-blot)测定实验组遮盖眼和对照眼、恢
复组去遮盖眼(即恢复眼)和对照眼基质金属蛋白酶11和基质金属蛋白酶H抑制
剂表达的改变。实验设空白对照，数据经统计学处理。
结果:(l)实验组遮盖眼和对照眼MMP一2活性平均值分别为5511.18士621.77
和2949.39士795.69，两者相比较遮盖眼MMP一2活性较对照眼有显著性提高
(P<0.ol);遮盖眼和对照眼TIMP一2活性平均值分别为2738.01士62.67和4541.65
士389.39，两者相比较遮盖眼TIMP一2活性较对照眼有显著性下降(尸<0.01)。(2)
恢复组恢复眼和对照眼MMP一2活性平均值分别为2936.96士1 13.22和2932.71士
70.85，两者相比较尚不能说差异有显著性(乃0.5);恢复眼和对照眼TIMP一2活性
平均值分别为4601 .4士95 .87和4608.47士48.28，两者相比较尚不能说差异有显
著性(外0.5)。(3)实验组遮盖眼和恢复组恢复眼的MMP一2活性相比较差异有显
著性(P<0.OOI)。
结论:形觉剥夺性近视的形成是与实验组遮盖眼MMP一2活性的增加及
TIMP一2活性的减少相关的，所以，这种MMP一2和TIMP一2之间的失衡可能就是
FDM形成过程中巩膜重新塑形的原因之一。
Myopia or nearsightedness is a commonly and frequently encountered disease in ophthalmology. It is not clear about its pathogenesis, but form deprivation (FM) theory has played an important role in the study of myopia pathogenesis. FM, which means using multiple methods to effect the normal formation in the retina can result in abnormal growth of ocular globe, showing longer axial length and leading to myopia.
There were some foreign reports about relationship of MMP, TIMP and FDM, but the mechanism is not clear yet. In my experiment, myopia is characterized by excessive axial lengthening of the ocular globe and morphological changes. Using HE staining, SABC staining, SDS-PAGE and WB to study the express changes of scleral morphology, collagen types, MMP-2 and TIMP-2 in FDM. We hope study and understand the pathogenesis of FDM.
1. The express changes of scleral collagen types in the deprived eyes of FDM
Materials and methods: Forty healthy male hailan chickens were selected after
born 2 days. All the right eyes underwent form deprivation with translucent goggles, and the left eyes were used as the control. After deprived for 1, 3, 5, 7 and 14 days, the refractive state of the deprived eyes were characterized by means of streak retinoscopy (without cycloplegia), then the eye globes were removed and its axial length were measured by sliding gage. Routine fixation, dehydration, diaphanous, paraffin and embedding were done. Sum paraffin sections were taken. We used routine hematoxylin-eosin (HE) staining and observed the morphological changes of deprived sclera under light microscopy. Streptaridin-biotin-peroxidase complex (SABC) immunohistochemistry method was used to study the express changes of collagen types.
Results: (1). Form deprivation leads to myopia refractive abnormality. After deprived for 1, 3, 5, 7 and 14 days, the average dipoter of the deprived eyes were +1.937?.079 D, -2.78?.27 D, -9.17?.22 D, -14.275?.609 D, -20.56?.51 D, and the axial length of the deprived eyes were +8.265?.15 mm, +8.995?.069 mm, +9.33?.05 mm, +9.6?.106 mm, +9.78?.44 mm. Significant differences were detected in axial length and diopter between deprived and control eyes (P<0.01). (2) HE staining shows that the deprived posterior cartilaginous sclera was thicker and the fibrous sclera was thinner than control sclera. The number of cells in the deprived cartilaginous sclera was more than control sclera. (3) Immunohistrochemistry staining shows that there were collagen type I mainly in deprived eyes and collagen type III in control eyes.
Conclusions: Form deprivation leads to myopia refractive abnormality, and the
longer the eyes were deprived, the more severe the myopia refractive state was. Morphological changes of sclera were associated with an increased thickness of cartilaginous and a decreased thickness of fibrous within the posterior sclera. The express changes of scleral collagen types were related with an increased amount of collagen type I and a decreased amount of collagen type III.
2. The express changes of MMP-2 and TIMP-2 in deprived and recovering eyes
Materials and methods: 80 healthy male hailan chickens were selected. The right eyes were deprived after born 2 days and the left eyes were control. Two groups were divided at random. One was experimental group and the other was recovering group. The deprived eyes were discovered after being deprived 2 weeks. The refractive state was characterized by means of streak retinoscopy. Then killed the chickens and removed the eye globes. Using a surgical trephine, 8mm diameter tissue buttons of posterior sclera were excised. The attached ocular tissues were separated. The five tissue-buttons form each group were homogenized together in 0.01M Tris ?HC1, ph 8.0. Insoluble material was removed from the homogenized material by centrifugation at 10,000 rpm for 20 minutes. The supernatant was used for measurement. The recovering group was discovered after 2 weeks and feed normally for another 1 week. The specimens were made by the same method. The supern
引文
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[25] .Prockop DJ,Kivirikko KI.Collagen:molecular biology diseases and potentials for therapy.Annu Rev Biochem,1995,64:403-434
[26] .Watanabe K,Bruder SP,Caplan Al.Transient expression of type collagen and tissue mobilization during development of the sclera ossicle,a memberanous bone,in the chick embryo.Dev Dyn,1994,200:212-216
[27] ．朱小松，刘家琦，陈瑞英等．实验性近视鸡模型眼的形态学和病理学研究． 中国斜视与小儿眼科，1994，2(1) ：24～28
[28] .Snead MP,Yates JRW.Clinical and molecular genetics of stickler syndrome.J Med Genet,1999,36(5) :353-359
[29] .Baramora E,Foiolart JM.Matrix metalloproteinase family.Cell Biol Int,1995,19(3) :239-242
[30] .Guggenheim JA,McBrien NA.Form-deprivation myopia induces activation of scleral matrix metalloproteinase-2 in tree shrew.Invest Ophthalmol Vis Sci,1996,37(7) :1380-1395
[31] .Rada JA,Perry CA,Slover ML,et al.Gelatinase A and TIMP-2 expression in the fibrous sclera of myopic and recovering chick eyes,Invest Ophthalmol Vis Sci,1999,40:3091-3099
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