發(fā)布時(shí)間：2018-04-04 06:13

  本文選題：Sonic　切入點(diǎn)：hedgehog　出處：《復(fù)旦大學(xué)》2012年博士論文

【摘要】：前言近視發(fā)病機(jī)制至今未完全明確,目前公認(rèn)的近視發(fā)病機(jī)制之一是外界因素刺激了視網(wǎng)膜脈絡(luò)膜中的某些調(diào)控因子,影響了轉(zhuǎn)化生長因子β (trans-forming growth factor-β, TGF-β)等生物活性物質(zhì)的平衡狀態(tài),導(dǎo)致基質(zhì)金屬蛋白酶(matrix metalloproteinases, MMP)活性增加,造成鞏膜組織松解、眼軸增長而產(chǎn)生近視。因此尋找確切的信號(hào)調(diào)控因子已成為近視發(fā)病機(jī)制研究的熱點(diǎn)。已有研究和我們的前期實(shí)驗(yàn)均提示Sonic hedgehog (Shh)可能作為一種視網(wǎng)膜重要信號(hào)因子參與了眼軸的生長導(dǎo)致近視形成。同時(shí)眼外研究中發(fā)現(xiàn)Shh信號(hào)通路可調(diào)控MMPs、TGF-β等活性因子的表達(dá),亦提示上游視網(wǎng)膜Shh信號(hào)通路在近視發(fā)展過程中可能的下游通路。因此我們采用豚鼠的形覺剝奪性近視模型,通過外源性Shh-N玻璃體腔注射上調(diào)及特異性抑制劑cyclopamine抑制Shh表達(dá),檢測Shh表達(dá)與TGF-β、MMP-2等生物活性因子改變的關(guān)系,探索Shh信號(hào)因子對(duì)近視調(diào)控的作用和具體調(diào)控途徑,為近視眼防治提供依據(jù)。 第一部分：豚鼠形覺剝奪性近視眼視網(wǎng)膜Shh信-號(hào)通路和鞏膜MMP—2、TGF-β表達(dá)變化 目的：在豚鼠形覺剝奪性近視模型(form-deprivation myopia, FDM)中檢測視網(wǎng)膜Shh及相關(guān)下游因子的表達(dá)變化,觀察鞏膜中MMP-2和TGF-β含量的改變。 方法：56只2-3周齡三色健康豚鼠隨機(jī)分為FDM組(n=40)和空白對(duì)照組(control組)(n=16)。FDM組右眼眼周黏貼半透明薄膜,左眼及control組不作任何處理。FDM組和control組分別于形覺剝奪0周、1周、2周及4周取10只和4只豚鼠進(jìn)行檢影驗(yàn)光及眼軸長度等生物學(xué)測量后,處死取眼球行免疫熒光染色檢測視網(wǎng)膜Shh、受體Ptc-1以及核轉(zhuǎn)錄因子Gli-3表達(dá)水平,實(shí)時(shí)熒光定量逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(real-time fluorescent quantitative reverse transcription polymerase chain reaction,real-time PCR)檢測視網(wǎng)膜Shh、Ptc-1以及Gli-3mRNA水平變化,Western-blot檢測視網(wǎng)膜Shh蛋白表達(dá)水平及鞏膜組織中MMP-2、TGF-β的表達(dá)變化。 結(jié)果：形覺剝奪1周、2周及4周后,FDM組遮蓋眼較對(duì)側(cè)眼分別誘導(dǎo)出-2.35±1.10D、-5.13±1.73D及-6.78±1.04D的相對(duì)近視,且隨著剝奪時(shí)間的延長,近視程度加劇。形覺剝奪1周、2周及4周后,FDM組誘導(dǎo)出的相對(duì)眼軸分別延長0.1±0.05mmm、0.11±0.04mmm和0.37±0.1mm,差異均有統(tǒng)計(jì)學(xué)意義。形覺剝奪1周、2周及4周后,FDM組誘導(dǎo)出的相對(duì)玻璃體腔長度分別延長0.07±0.04mm、0.10±0.07mm和0.36±0.18mm,差異均有統(tǒng)計(jì)學(xué)意義。免疫熒光染色顯示,Shh彌漫表達(dá)于豚鼠視網(wǎng)膜各層；Ptc-1和G1i-3均表達(dá)于豚鼠視網(wǎng)膜的神經(jīng)節(jié)細(xì)胞胞漿內(nèi)。形覺剝奪4周時(shí),FDM組中遮蓋眼較對(duì)側(cè)眼ShhmRNA和Ptc-lmRNA表達(dá)水平明顯增加(P值分別為0.043和0.024)。Western結(jié)果顯示,形覺剝奪2周開始,遮蓋眼的Shh蛋白表達(dá)水平開始較左眼升高；遮蓋眼鞏膜MMP-2在剝奪2周后開始明顯升高,而TGF-β表達(dá)水平下降,并持續(xù)至4周。 結(jié)論：在半透明薄膜法誘導(dǎo)的豚鼠FDM中,伴隨視網(wǎng)膜組織Shh及Ptc-1表達(dá)水平升高,以及鞏膜MMP-2表達(dá)上升和TGF-β表達(dá)下降,提示Shh信號(hào)通路可能參與了豚鼠FDM的調(diào)控過程。 第二部分：玻璃體腔內(nèi)注射Sonic hedgehog溶液對(duì)豚鼠近視發(fā)生發(fā)展的作用及相關(guān)機(jī)制研究 目的：通過外源性玻璃體腔內(nèi)注射Shh溶液來觀察豚鼠近視發(fā)生發(fā)展及鞏膜組織中MMP-2、TGF-β表達(dá)水平的變化。 方法：將出生2-3周齡三色健康豚鼠40只隨機(jī)分為2組,每組20只。每組的右眼進(jìn)行Shh-N/0.1%牛血清蛋白(bovine serum albumin, BSA)注射,左眼進(jìn)行0.1%BSA注射。雙眼依次進(jìn)行,隔2天注射一次,每次每只眼球注射10μ1,共注射4次。兩組的Shh-N注藥濃度分別為20μ g/ml(Shh20組)和50μ g/ml(Shh50組)。在注藥的第14天(2周)進(jìn)行驗(yàn)光和眼軸等生物學(xué)測量后,處死取眼球行石蠟切片HE染色觀察視網(wǎng)膜形態(tài)變化,取鞏膜組織進(jìn)行Western-Blot檢測MMP-2和TGF-β蛋白表達(dá)水平。結(jié)果與第一部分FDM組2周(FDM2W)和Conrtrol組2周(Control2W)進(jìn)行統(tǒng)計(jì)比較。 結(jié)果：豚鼠玻璃體腔內(nèi)注射Shh-N20μg/ml及50μ g/ml分別誘導(dǎo)豚鼠出現(xiàn)-1.54±0.75D和-4.04±1.48D的相對(duì)近視；0.11±0.09mm和0.14±0.03mm的相對(duì)眼軸延長；以及0.1±0.09mm和0.13±0.03mm的相對(duì)玻璃體腔長度延長；差異均有統(tǒng)計(jì)學(xué)意義。Shh50組較Shh20注藥組誘導(dǎo)出更深的相對(duì)近視度數(shù)(P0.001)及眼軸長度(P=0.0019)。Western-blot結(jié)果顯示,璃體腔內(nèi)注射Shh-N可使鞏膜組織MMP-2表達(dá)水平明顯上升,但Shh20組和Shh50組間表達(dá)未表現(xiàn)出明顯的差異；TGF-β表達(dá)水平未見明顯差異。 結(jié)論：豚鼠玻璃體腔內(nèi)注射Shh-N可促進(jìn)眼球向近視方向發(fā)展及眼軸延長,并且促進(jìn)近視程度與注射的藥物呈一定濃度梯度；Shh-N注射后可導(dǎo)致鞏膜組織中MMP-2表達(dá)明顯上調(diào),提示Shh信號(hào)通路通過上調(diào)MMP-2表達(dá)水平,參與鞏膜重塑,眼軸增長,最終出現(xiàn)近視。 第三部分：玻璃體腔內(nèi)注射Shh特異性抑制劑Cyclopamine對(duì)豚鼠形覺剝奪性近視發(fā)生發(fā)展的作用及相關(guān)機(jī)制研究 目的：在FDM豚鼠玻璃體腔注射Cyclopamine以觀察其對(duì)近視發(fā)展抑制的作用,同時(shí)觀察下游鞏膜中MMP-2及TGF-β表達(dá)水平的變化,進(jìn)一步探究Shh作用的下游鞏膜機(jī)制 方法：將出生2-3周齡三色健康豚鼠60只隨機(jī)分為3組,每組20只。每組的右眼進(jìn)行半透明薄膜遮蓋,同時(shí)雙眼進(jìn)行相同濃度cyclopamine注射。雙眼依次進(jìn)行玻璃體腔內(nèi)注射,隔2天注射一次,每次每只眼球注射10μ1,共注射4次。3組的cyclopamine注藥濃度分別為50μg/ml(FDM50組)、100μg/ml(FDM100組)及200μg/ml(FDM200組)。在注藥的第14天(2周)進(jìn)行驗(yàn)光和眼軸等生物學(xué)測量后,處死取眼球行石蠟切片HE染色觀察視網(wǎng)膜形態(tài)變化,取鞏膜組織進(jìn)行Western-Blot檢測MMP-2和TGF-β蛋白表達(dá)水平。結(jié)果與第一部分FDM組2周(FDM2W)和Conrtrol組2周(Control2W)進(jìn)行統(tǒng)計(jì)比較。 結(jié)果：FDM50組、FDM100組及FDM200組遮蓋眼較對(duì)側(cè)眼分別出現(xiàn)了-2.69±1.15D、-1.46±0.91D和-0.38±0.81D的相對(duì)近視；豚鼠玻璃體腔內(nèi)注射cyclopamine均能抑制豚鼠FDM的形成,FDM200組較FDM50組在程度上更能抑制形覺剝奪性近視的形成(P0.0001)。FDM50組、FDM100組及FDM200組遮蓋眼較對(duì)側(cè)眼分別出現(xiàn)了0.09±0.05mm、0.06±0.04mm和0.04±0.02mm的相對(duì)眼軸延長；FDM100組和FDM200組均抑制了部分形覺剝奪引起的相對(duì)眼軸延長(PFDM100=0.044,PFDM200=0.001)。FDM50組、FDM100組及FDM200組遮蓋眼較對(duì)側(cè)眼分別出現(xiàn)了0.09±0.05mm、0.07±0.05mm和0.04±0.05mm的相對(duì)玻璃體腔延長。FDM200組較FDM2W組和FDM50組更能抑制部分形覺剝奪引起的相對(duì)玻璃體腔長度延長(P值分別為0.0446和0.0388)。鞏膜組織western-blot結(jié)果顯示,右眼的FDM2W組和FDM50組的MMP-2水平表達(dá)相當(dāng),均較對(duì)側(cè)眼明顯上調(diào),但FDM100組和FDM200組的MMP-2水平較FDM2W組和FDM50組明顯下調(diào)。 結(jié)論：玻璃體腔內(nèi)注射Shh特異性抑制劑cyclopamine可阻滯豚鼠形覺剝奪性近視的發(fā)展及眼軸延長,并伴隨鞏膜組織中的MMP-2表達(dá)明顯下調(diào)。提示Shh通路阻斷(如cyclopamine)可能會(huì)成為近視眼預(yù)防及治療的新途徑。
[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 鹵 0 . 05 mm , - 5 . 13 鹵 1 . 73D and - 6.78 鹵 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 鹵 0.75D and - 4.04 鹵 1.48D , respectively .
0 . 11 鹵 0 . 09mm and 0 . 14 鹵 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 鹵 1.16 , - 1.46 鹵 0.335 and - 0.38 鹵 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 鹵 0.05mm , 0.06 鹵 0.04 mm and 0.04 鹵 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 .

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R778.11

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