视网膜Sonic hedgehog信号通路在豚鼠形觉剥夺性近视眼模型中的作用机制研究

发布时间：2018-04-04 06:13

本文选题：Sonic　切入点：hedgehog　出处：《复旦大学》2012年博士论文

【摘要】：前言近视发病机制至今未完全明确,目前公认的近视发病机制之一是外界因素刺激了视网膜脉络膜中的某些调控因子,影响了转化生长因子β (trans-forming growth factor-β, TGF-β)等生物活性物质的平衡状态,导致基质金属蛋白酶(matrix metalloproteinases, MMP)活性增加,造成巩膜组织松解、眼轴增长而产生近视。因此寻找确切的信号调控因子已成为近视发病机制研究的热点。已有研究和我们的前期实验均提示Sonic hedgehog (Shh)可能作为一种视网膜重要信号因子参与了眼轴的生长导致近视形成。同时眼外研究中发现Shh信号通路可调控MMPs、TGF-β等活性因子的表达,亦提示上游视网膜Shh信号通路在近视发展过程中可能的下游通路。因此我们采用豚鼠的形觉剥夺性近视模型,通过外源性Shh-N玻璃体腔注射上调及特异性抑制剂cyclopamine抑制Shh表达,检测Shh表达与TGF-β、MMP-2等生物活性因子改变的关系,探索Shh信号因子对近视调控的作用和具体调控途径,为近视眼防治提供依据。第一部分：豚鼠形觉剥夺性近视眼视网膜Shh信-号通路和巩膜MMP—2、TGF-β表达变化目的：在豚鼠形觉剥夺性近视模型(form-deprivation myopia, FDM)中检测视网膜Shh及相关下游因子的表达变化,观察巩膜中MMP-2和TGF-β含量的改变。方法：56只2-3周龄三色健康豚鼠随机分为FDM组(n=40)和空白对照组(control组)(n=16)。FDM组右眼眼周黏贴半透明薄膜,左眼及control组不作任何处理。FDM组和control组分别于形觉剥夺0周、1周、2周及4周取10只和4只豚鼠进行检影验光及眼轴长度等生物学测量后,处死取眼球行免疫荧光染色检测视网膜Shh、受体Ptc-1以及核转录因子Gli-3表达水平,实时荧光定量逆转录聚合酶链反应(real-time fluorescent quantitative reverse transcription polymerase chain reaction,real-time PCR)检测视网膜Shh、Ptc-1以及Gli-3mRNA水平变化,Western-blot检测视网膜Shh蛋白表达水平及巩膜组织中MMP-2、TGF-β的表达变化。结果：形觉剥夺1周、2周及4周后,FDM组遮盖眼较对侧眼分别诱导出-2.35±1.10D、-5.13±1.73D及-6.78±1.04D的相对近视,且随着剥夺时间的延长,近视程度加剧。形觉剥夺1周、2周及4周后,FDM组诱导出的相对眼轴分别延长0.1±0.05mmm、0.11±0.04mmm和0.37±0.1mm,差异均有统计学意义。形觉剥夺1周、2周及4周后,FDM组诱导出的相对玻璃体腔长度分别延长0.07±0.04mm、0.10±0.07mm和0.36±0.18mm,差异均有统计学意义。免疫荧光染色显示,Shh弥漫表达于豚鼠视网膜各层；Ptc-1和G1i-3均表达于豚鼠视网膜的神经节细胞胞浆内。形觉剥夺4周时,FDM组中遮盖眼较对侧眼ShhmRNA和Ptc-lmRNA表达水平明显增加(P值分别为0.043和0.024)。Western结果显示,形觉剥夺2周开始,遮盖眼的Shh蛋白表达水平开始较左眼升高；遮盖眼巩膜MMP-2在剥夺2周后开始明显升高,而TGF-β表达水平下降,并持续至4周。结论：在半透明薄膜法诱导的豚鼠FDM中,伴随视网膜组织Shh及Ptc-1表达水平升高,以及巩膜MMP-2表达上升和TGF-β表达下降,提示Shh信号通路可能参与了豚鼠FDM的调控过程。第二部分：玻璃体腔内注射Sonic hedgehog溶液对豚鼠近视发生发展的作用及相关机制研究目的：通过外源性玻璃体腔内注射Shh溶液来观察豚鼠近视发生发展及巩膜组织中MMP-2、TGF-β表达水平的变化。方法：将出生2-3周龄三色健康豚鼠40只随机分为2组,每组20只。每组的右眼进行Shh-N/0.1%牛血清蛋白(bovine serum albumin, BSA)注射,左眼进行0.1%BSA注射。双眼依次进行,隔2天注射一次,每次每只眼球注射10μ1,共注射4次。两组的Shh-N注药浓度分别为20μ g/ml(Shh20组)和50μ g/ml(Shh50组)。在注药的第14天(2周)进行验光和眼轴等生物学测量后,处死取眼球行石蜡切片HE染色观察视网膜形态变化,取巩膜组织进行Western-Blot检测MMP-2和TGF-β蛋白表达水平。结果与第一部分FDM组2周(FDM2W)和Conrtrol组2周(Control2W)进行统计比较。结果：豚鼠玻璃体腔内注射Shh-N20μg/ml及50μ g/ml分别诱导豚鼠出现-1.54±0.75D和-4.04±1.48D的相对近视；0.11±0.09mm和0.14±0.03mm的相对眼轴延长；以及0.1±0.09mm和0.13±0.03mm的相对玻璃体腔长度延长；差异均有统计学意义。Shh50组较Shh20注药组诱导出更深的相对近视度数(P0.001)及眼轴长度(P=0.0019)。Western-blot结果显示,璃体腔内注射Shh-N可使巩膜组织MMP-2表达水平明显上升,但Shh20组和Shh50组间表达未表现出明显的差异；TGF-β表达水平未见明显差异。结论：豚鼠玻璃体腔内注射Shh-N可促进眼球向近视方向发展及眼轴延长,并且促进近视程度与注射的药物呈一定浓度梯度；Shh-N注射后可导致巩膜组织中MMP-2表达明显上调,提示Shh信号通路通过上调MMP-2表达水平,参与巩膜重塑,眼轴增长,最终出现近视。第三部分：玻璃体腔内注射Shh特异性抑制剂Cyclopamine对豚鼠形觉剥夺性近视发生发展的作用及相关机制研究目的：在FDM豚鼠玻璃体腔注射Cyclopamine以观察其对近视发展抑制的作用,同时观察下游巩膜中MMP-2及TGF-β表达水平的变化,进一步探究Shh作用的下游巩膜机制方法：将出生2-3周龄三色健康豚鼠60只随机分为3组,每组20只。每组的右眼进行半透明薄膜遮盖,同时双眼进行相同浓度cyclopamine注射。双眼依次进行玻璃体腔内注射,隔2天注射一次,每次每只眼球注射10μ1,共注射4次。3组的cyclopamine注药浓度分别为50μg/ml(FDM50组)、100μg/ml(FDM100组)及200μg/ml(FDM200组)。在注药的第14天(2周)进行验光和眼轴等生物学测量后,处死取眼球行石蜡切片HE染色观察视网膜形态变化,取巩膜组织进行Western-Blot检测MMP-2和TGF-β蛋白表达水平。结果与第一部分FDM组2周(FDM2W)和Conrtrol组2周(Control2W)进行统计比较。结果：FDM50组、FDM100组及FDM200组遮盖眼较对侧眼分别出现了-2.69±1.15D、-1.46±0.91D和-0.38±0.81D的相对近视；豚鼠玻璃体腔内注射cyclopamine均能抑制豚鼠FDM的形成,FDM200组较FDM50组在程度上更能抑制形觉剥夺性近视的形成(P0.0001)。FDM50组、FDM100组及FDM200组遮盖眼较对侧眼分别出现了0.09±0.05mm、0.06±0.04mm和0.04±0.02mm的相对眼轴延长；FDM100组和FDM200组均抑制了部分形觉剥夺引起的相对眼轴延长(PFDM100=0.044,PFDM200=0.001)。FDM50组、FDM100组及FDM200组遮盖眼较对侧眼分别出现了0.09±0.05mm、0.07±0.05mm和0.04±0.05mm的相对玻璃体腔延长。FDM200组较FDM2W组和FDM50组更能抑制部分形觉剥夺引起的相对玻璃体腔长度延长(P值分别为0.0446和0.0388)。巩膜组织western-blot结果显示,右眼的FDM2W组和FDM50组的MMP-2水平表达相当,均较对侧眼明显上调,但FDM100组和FDM200组的MMP-2水平较FDM2W组和FDM50组明显下调。结论：玻璃体腔内注射Shh特异性抑制剂cyclopamine可阻滞豚鼠形觉剥夺性近视的发展及眼轴延长,并伴随巩膜组织中的MMP-2表达明显下调。提示Shh通路阻断(如cyclopamine)可能会成为近视眼预防及治疗的新途径。
[Abstract]:The pathogenesis of myopia is not completely clear . One of the most commonly accepted pathogenesis of myopia is that the external factors stimulate certain regulatory factors in the retina ' s choroidochoroiditis . It also suggests that the activity of matrix metalloproteinases ( MMP ) is increased , which results in the formation of myopia .

Part I : Changes in the expression of MMP - 2 and TGF - 尾 in the retina of guinea pig - shaped deprivation myopia

Objective : To detect the changes of the expression of MMP - 2 and TGF - 尾 in the sclera by detecting the changes of the expression of the retinal and related downstream factors in the guinea pig form - deprivation myopia model ( FDM ) .

Methods : Fifty - six healthy guinea pigs aged 2 - 3 weeks were randomly divided into FDM group ( n = 40 ) and control group ( n = 16 ) .

Results : After 1 week , 2 weeks and 4 weeks after deprivation , the relative myopia of FDM group was increased by 0 . 1 卤 0 . 05 mm , - 5 . 13 卤 1 . 73D and - 6.78 卤 1 . 04D .
The expression levels of ptc - 1 and G1i - 3 were all expressed in the cytoplasm of ganglion cells in the retina of guinea pigs . The expression levels of ShhmRNA and Ptc - lmRNA were significantly increased in FDM group at 4 weeks ( P < 0.01 ) .
The expression level of MMP - 2 increased significantly after 2 weeks of deprivation , while TGF - 尾 expression level decreased and lasted to 4 weeks .

Conclusion : In the guinea pig FDM induced by the semi - transparent film method , the expression level and the expression of MMP - 2 and TGF - 尾 in the scleral MMP - 2 increased and the expression of TGF - 尾 in the scleral MMP - 2 was decreased , suggesting that the pathway might be involved in the regulation of FDM in guinea pig .

The second part : The effect of intravenous injection of Sonic ' s solution in vitreous cavity on the development of myopia in guinea pig and its related mechanism

Objective : To observe the changes of MMP - 2 and TGF - 尾 expression level in guinea pigs by injection of an in vitro vitreous cavity .

Methods : Forty - four healthy guinea pigs were randomly divided into 2 groups ( 20 rats in each group ) . The left eyes were injected intravenously with a total of 20 渭g / ml ( Shh20 group ) and 50 渭g / ml ( Shh50 group ) . The results were compared with two weeks ( control 2W ) of the first FDM group and 2 weeks ( Control2W ) of the Conrtrol group .

Results : In the vitreous cavity of guinea pigs , the relative myopia was induced in guinea pigs - 1.54 卤 0.75D and - 4.04 卤 1.48D , respectively .
0 . 11 卤 0 . 09mm and 0 . 14 卤 0 . 03mm relative eye - axis extension ;
and a relative vitreous cavity length of 0.1 . + - . 0.09 mm and 0.13 & # xb1 ; 0.03 mm is prolonged ;
The results of Western - blot showed that the expression level of MMP - 2 in scleral tissue was significantly increased , but there was no significant difference between Shh20 and Shh50 groups .
There was no significant difference in TGF - 尾 expression level .

Conclusion : In the vitreous cavity of guinea pig , it can promote the development of eyeball to myopia and prolong the ocular axis , and promote myopia to a certain concentration gradient .
The expression of MMP - 2 in the scleral tissue can be increased significantly after the injection of H _ 2 - N . It is suggested that the pathway can increase the level of MMP - 2 expression , participate in the reconstruction of the sclera , increase the ocular axis , and eventually appear myopia .

The third part : The effect of Cyclopamine on the development of guinea pig form deprivation myopia and its related mechanism .

Objective : To observe the effect of Cyclopamine on the inhibition of myopia progression in FDM guinea pig vitreous cavity , and observe the changes of MMP - 2 and TGF - 尾 expression in the downstream sclera .

Methods : 60 rabbits were randomly divided into 3 groups , 20 rats in each group . The right eyes were covered with semi - transparent films , and the eyes were injected with cyclopamine . The concentration of cyclopamine was 50 渭g / ml ( FDM50 group ) , 100 渭g / ml ( FD100 group ) and 200 渭g / ml ( FDM200 group ) . The results were compared with 2 weeks ( FDM2W ) and Conrtrol group ( 2 weeks ) of the first FDM group .

Results : Compared with those of the FDM50 group , FD100 group and FDM200 group , the relative myopia was - 2.69 卤 1.16 , - 1.46 卤 0.335 and - 0.38 卤 0.81D , respectively .
The results showed that cyclopamine could inhibit FDM formation in guinea pigs . FDM200 and FDM50 could inhibit the formation of myopia in guinea pigs ( P < 0.01 ) . The relative eye axes of FDM50 , FD100 and FDM200 were 0.09 卤 0.05mm , 0.06 卤 0.04 mm and 0.04 卤 0.02mm respectively .
The relative eye - axis extension caused by partial - form deprivation was inhibited in both FD100 and FDM200 groups ( PFD100 = 0.044 , PFDM200 = 0.001 ) . Compared with FDM2W group and FDM50 group , the expression of MMP - 2 in FDM200 group and FDM50 group was significantly higher than that in FDM2W group and FDM50 group , but the levels of MMP - 2 in FDM200 group and FDM200 group were significantly lower than those in FDM2W group and FDM200 group .

Conclusion : cyclopamine , a specific inhibitor cyclopamine , can block the development of guinea pig form deprivation myopia and prolong the ocular axis and decrease the expression of MMP - 2 in scleral tissues . It is suggested that cyclopamine may become a new way to prevent and treat myopia .

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R778.11

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