本文選題：形覺剝奪性近視　切入點(diǎn)：豚鼠　出處：《天津醫(yī)科大學(xué)》2012年博士論文

【摘要】：研究目的 病理性近視(Pathologic myopia, PM)是成年人重要的致盲性眼病之一,目前尚無良好治療對(duì)策。近視發(fā)病機(jī)制的研究提示：鞏膜是各種致病因素作用的主要靶器官,鞏膜病理性的薄弱是PM作用的結(jié)果,也是導(dǎo)致其它相關(guān)病變的誘因,而針對(duì)鞏膜生物力學(xué)強(qiáng)度變化的干預(yù)有可能是阻斷PM進(jìn)展的一條可行途徑。本研究通過建立豚鼠形覺剝奪性近視(form deprivation myopia, FDM)模型,應(yīng)用甘油醛促進(jìn)鞏膜交聯(lián),探討甘油醛對(duì)鞏膜生物力學(xué)特征的影響及其對(duì)實(shí)驗(yàn)性近視的抑制作用,從控制鞏膜重塑并增加其強(qiáng)度的角度,為防治PM提供新思路。 研究內(nèi)容和方法 1.甘油醛對(duì)離體鞏膜條帶的交聯(lián)作用：選用15只三周齡三色豚鼠,按隨機(jī)數(shù)字表法分成3組,每組5只。均取右眼為實(shí)驗(yàn)眼。矢狀位取2mm×6mm的鞏膜條帶,給予0.005mol/L甘油醛組,0.05mol/L甘油醛組和空白對(duì)照液室溫下浸泡4天。應(yīng)用微材料力學(xué)性能測試系統(tǒng),對(duì)各組鞏膜條帶的極限應(yīng)力(σmax (MPa)、極限應(yīng)變?chǔ)舖ax (%)、6%彈性模量E(MPs)進(jìn)行統(tǒng)計(jì)分析。 2.豚鼠FDM模型的建立：用6號(hào)乳白色半透明乳膠氣球,依照豚鼠頭部形狀剪制成頭套,暴露出動(dòng)物的左眼,雙耳及口鼻。制作動(dòng)物右眼形覺剝奪模型。室溫下飼養(yǎng),正常晝夜節(jié)律。分別于實(shí)驗(yàn)開始前、形覺剝奪7、14、21天時(shí)測量眼軸長度、玻璃體腔長度及屈光度,進(jìn)行統(tǒng)計(jì)學(xué)分析。 3.甘油醛后鞏膜交聯(lián)：選用35只三周齡三色豚鼠,將動(dòng)物按隨機(jī)數(shù)字表法分成5組,每組7只,右眼為實(shí)驗(yàn)眼,左眼為對(duì)照眼。A組：右眼遮蓋7天；B組：右眼遮蓋21天,0.9%生理鹽水第1、8、15天Tenon's囊下球后注射；C組：右眼遮蓋21天,0.05mol/L甘油醛第1、8、15天Tenon's囊下球后注射；D組：右眼遮蓋21天,0.5mol/L甘油醛第1、8、15天Tenon's囊下球后注射；E組：正常對(duì)照組。并于實(shí)驗(yàn)開始前、形覺剝奪7、14、21天時(shí)測量眼軸長度、玻璃體腔長度及屈光度。在實(shí)驗(yàn)結(jié)束時(shí),應(yīng)用微材料力學(xué)性能測試系統(tǒng)測量5個(gè)組鞏膜的極限應(yīng)力σmax (MPa)、極限應(yīng)變?chǔ)舖ax (%)、6%彈性模量E(MPa),并與對(duì)側(cè)眼比較,進(jìn)行統(tǒng)計(jì)學(xué)分析。繪制5個(gè)組的應(yīng)力-應(yīng)變曲線,比較甘油醛交聯(lián)對(duì)豚鼠鞏膜生物力學(xué)強(qiáng)度的影響。 4.后鞏膜交聯(lián)術(shù)對(duì)豚鼠FDM眼超微結(jié)構(gòu)影響的觀察：選用18只三周齡三色豚鼠,隨機(jī)抽取其中的15只豚鼠,按隨機(jī)數(shù)字表法分成5組。右眼為實(shí)驗(yàn)眼,左眼為對(duì)照眼。實(shí)驗(yàn)方法同3。實(shí)驗(yàn)結(jié)束后處死豚鼠,在視神經(jīng)顳側(cè)連續(xù)切取5片組織,厚度0.5μm,2片行蘇木精-伊紅染色,3片行免疫組化染色,檢測后極部鞏膜、脈絡(luò)膜、視網(wǎng)膜MMP-2表達(dá),光學(xué)顯微鏡下觀察并照相。余下的3只豚鼠給予0.5mol/L的甘油醛Tenon's囊下球后注射,分別在第3、7、14天處死。取注射相鄰部位的角膜緣、眼外肌和視神經(jīng)篩板做切片,行蘇木精-伊紅染色。判斷甘油醛對(duì)視網(wǎng)膜、視神經(jīng)是否有毒性。 結(jié)果 1.離體鞏膜實(shí)驗(yàn)：不同濃度甘油醛組與對(duì)照組實(shí)驗(yàn)眼鞏膜的應(yīng)力-應(yīng)變曲線有明顯的梯度變化�？瞻讓�(duì)照組鞏膜的極限應(yīng)力σmaax、極限應(yīng)變smax、6%彈性模量E分別為(7.198±0.991)MPa、(21.480±0.853)%和(22.808±2.159)MPa。甘油醛交聯(lián)組中,6%彈性模量E0.005mol/L甘油醛組、0.05mo/L甘油醛組分別為(25.903±0.892) MPa (P=0.019)和(36.156±2.026) MPa (P=0.000),較空白對(duì)照組分別增加了13.57%和58.52%。0.005mol/L甘油醛組、0.05mo/L甘油醛極限應(yīng)力σmax分別為(10.386±1.023)MPa(P=0.0012)和(14.851±1.602)MPa (P=0.000),較空白對(duì)照組分別增加了44.30%和106.32%；0.005mol/L甘油醛組、0.05mo/L甘油醛組極限應(yīng)變smax分別為(19.320±±0.672)%(P=0.002)和(14.140±±1.001)%(P=0.000),較空白對(duì)照組分別降低10.06%和34.17%。 2.形覺剝奪后,實(shí)驗(yàn)眼均出現(xiàn)了玻璃體腔長度和眼軸長度的增加以及相對(duì)近視度數(shù)的增加,在A組、B組和C組中,玻璃體腔長度、眼軸長度和屈光度與對(duì)側(cè)眼相比,差異有統(tǒng)計(jì)學(xué)意義(P玻璃體腔=0.018,0.002,0.001；P眼軸=0.019,0.002,0.000；P屈光=0.000,0.005,0.001)。D組、E組玻璃體腔長度、眼軸長度與對(duì)側(cè)眼相比,差異無統(tǒng)計(jì)學(xué)意義(P玻璃體腔=0.607,0.539；P眼軸=0.607,0.539)D組實(shí)驗(yàn)眼與對(duì)側(cè)眼屈光度相比差異有統(tǒng)計(jì)學(xué)意義(P屈光=0.020)。 3.在實(shí)驗(yàn)第21天結(jié)束時(shí),B、C、D、E組實(shí)驗(yàn)眼的屈光度變化值分別為(8.800±0.616)D,(7.236±2.198)D,(6.271±1.112)D和(0.934±0.158)D,總體差異有統(tǒng)計(jì)學(xué)意義(F=61.249,P=0.000),B、C、D組的屈光度變化值較正常對(duì)照組大,差異有統(tǒng)計(jì)學(xué)意義(PB=0.000,PC=0.000,PD=0.000)；B、C、D組的眼軸變化值較正常對(duì)照組大,但差異無統(tǒng)計(jì)學(xué)意義(PB=0.430,PC=0.840,PD=0.386) 4.在實(shí)驗(yàn)第21天結(jié)束時(shí),B組和E組對(duì)側(cè)眼比較,B組玻璃體腔長度的增加值為(0.198±0.038)mm,E組為(0.139±0.026)mm,差異有統(tǒng)計(jì)學(xué)意義(t=-3.689,P=0.002)；B組眼軸長度的增加值為(0.481±0.062)mm,E組為(0.438±0.068)mm,差異無統(tǒng)計(jì)學(xué)意義(t=-1.295,P=0.216) 5.B組實(shí)驗(yàn)眼眼軸長度與屈光呈高度負(fù)相關(guān)(r=-0.832,P=0.000)；B組實(shí)驗(yàn)眼玻璃體腔長度與屈光度呈高度負(fù)相關(guān)(r=-0.804,P=0.000)；E組右眼的眼軸長度和屈光度呈中度負(fù)相關(guān)(r=-0.604,P=0.000)；E組雙眼的眼軸長度呈高度相關(guān)(r=0.940,P=0.000)。 6.試驗(yàn)結(jié)束時(shí),B組實(shí)驗(yàn)眼的極限應(yīng)力和6%彈性模量分別為(7.988±3.677)MPa(P=0.002)和(19.938±4.871)MPa(P=0.001),較對(duì)側(cè)眼分別降低了34.21%和34.38%,極限應(yīng)變實(shí)驗(yàn)眼為(28.6±3.6)%(P=0.034),較對(duì)側(cè)眼增加了19.17%。甘油醛交聯(lián)后,C組實(shí)驗(yàn)眼的極限應(yīng)力和6%彈性模量分別為(9.244±0.806)MPa(P=0.001)和(26.180±4.388)MPa(P=0.031),較對(duì)側(cè)眼分別降低了23.13%和13.34%,極限應(yīng)變實(shí)驗(yàn)眼為(26.2±1.0)%(P=0.016),較對(duì)側(cè)眼增加了12.93%；D組實(shí)驗(yàn)眼的極限應(yīng)力為(12.476-2.507)MPa(P=0.580),較對(duì)側(cè)眼降低了5.50%,6%彈性模量為(30.446±3.410)MPa(P=0.314),較對(duì)側(cè)眼增加了6.53%,極限應(yīng)變?yōu)?23.8±1.8)%(P=0.253),較對(duì)側(cè)眼降低了4.42%。在D組(0.5mol/L甘油醛)攻膜交聯(lián)21天后與B組的實(shí)驗(yàn)眼相比,鞏膜的極限應(yīng)力增加了35.85%,6%彈性模量增加了52.70%,極限應(yīng)變降低了16.78%。不同濃度甘油醛交聯(lián)與對(duì)照組實(shí)驗(yàn)眼的應(yīng)力-應(yīng)變曲線有明顯的梯度變化,D組的應(yīng)力-應(yīng)變曲線接近正常對(duì)照組,高于其它3組。 7.組織學(xué)檢查：隨著豚鼠FDM的出現(xiàn),表現(xiàn)為眼外組織疏松,鞏膜厚度明顯降低,纖維排列整齊,脈絡(luò)膜厚度降低,可見血管腔,但未見大的竇腔,視網(wǎng)膜層次清晰。MMP-2在眼球表層結(jié)締組織、鞏膜表層結(jié)締組織、淺層鞏膜、脈絡(luò)膜實(shí)質(zhì)層以及視網(wǎng)膜外叢狀層陽性表達(dá)比正常對(duì)照眼明顯。甘油醛交聯(lián)后出現(xiàn)了眼球表面結(jié)締組織結(jié)構(gòu)變得相對(duì)致密,排列有序,與鞏膜連接緊密。鞏膜組織的厚度變薄,細(xì)胞密度增加。脈絡(luò)膜管腔擴(kuò)大,厚度變化不明顯。視網(wǎng)膜光感受器細(xì)胞核清晰,排列整齊,無炎性細(xì)胞浸潤。MMP-2在除眼表結(jié)締組織外的鞏膜表面結(jié)締組織,淺層鞏膜、脈絡(luò)膜實(shí)質(zhì)層以及視網(wǎng)膜外叢狀層的陽性表達(dá)明顯降低。 結(jié)論： 1.甘油醛是一種安全有效的交聯(lián)劑,能夠顯著增強(qiáng)鞏膜的生物力學(xué)強(qiáng)度。 2.頭套法形覺剝奪成功誘導(dǎo)出以豚鼠為對(duì)象的動(dòng)物近視模型,而且這種近視是以眼軸延長特別是玻璃體腔長度增加為主的軸性近視。 3.隨著眼軸的延長及近視度數(shù)的加深,鞏膜的生物力學(xué)強(qiáng)度明顯降低。 4.本實(shí)驗(yàn)進(jìn)一步證明了MMP-2與近視形成的相關(guān)性,而且發(fā)現(xiàn)甘油醛能夠降低MMP-2的陽性表達(dá)。 5.采用甘油醛交聯(lián)的方法能夠有效控制FDM動(dòng)物模型眼近視的發(fā)展。為防治PM提供新思路。
[Abstract]:Purpose of study

Objective : To study the effect of glyceraldehyde on the biomechanical properties of sclera and to investigate the effect of glyceraldehyde on the biomechanical properties of sclera and to investigate the inhibitory effect of glyceraldehyde on the biomechanical properties of sclera .

Research content and methods

1 . The cross - linking effect of glyceraldehyde on scleral strap in vitro : 15 three - week - old three - color guinea pigs were selected and divided into 3 groups according to the random number table method . The scleral bands of 2 mm 脳 6 mm were taken for the right eye . The ultimate stress ( 蟽max ( MPa ) , ultimate strain 蔚max ( % ) and 6 % elastic modulus E ( MPs ) of scleral strips were analyzed statistically .

2 . Establishment of a guinea pig FDM model : Using a 6 # milky white translucent latex balloon , the head cover is cut according to the shape of the head of the guinea pig to expose the left eye , the double ears and the mouth nose of the animal . The right eye shape deprivation model of the animal is made . The eye axis length , the length of the vitreous cavity and the power of the glass body are measured before the experiment is started , and the length and the power of the vitreous cavity are analyzed statistically .

3 . Posterior scleral cross - linking of glyceraldehyde : 35 three - week - old three - color guinea pigs were selected , the animals were divided into 5 groups according to the random number table method , 7 in each group , the right eye was the experimental eye , the left eye was the control eye . Group A : the right eye covered 7 days ;
Group B : The right eye was covered in 21 days , 0.9 % saline was injected at 1 , 8 , 15 days Tenon ' s capsule .
Group C : The right eye covered 21 days , 0.05 mol / L glyceraldehyde group 1 , 8 , 15 days Tenon ' s capsule was injected after balloon ;
Group D : The right eye was covered in 21 days , 0.5 mol / L glyceraldehyde group 1 , 8 , 15 days Tenon ' s capsule was injected after balloon ;
At the end of the experiment , the ultimate stress 蟽max ( MPa ) , the ultimate strain 蔚max ( % ) , the 6 % elastic modulus E ( MPa ) of the sclera in the five groups were measured and compared with the contralateral eye , and the effects of glyceraldehyde crosslinking on the biomechanical strength of the sclera of guinea pig were compared .

4 . The effects of posterior scleral cross - linking on the ultrastructure of FDM eyes in guinea pigs were observed : 15 guinea pigs were randomly selected from 18 guinea pigs with three - week - old three - color guinea pigs .

Results

1 . In vitro scleral experiment : The stress - strain curve of sclera was significantly changed in different concentration glyceraldehyde group and control group . The limit stress 蟽maax , ultimate strain smax , and 6 % elastic modulus E of the scleral were ( 7.198 鹵 0.981 ) MPa , ( 21.480 鹵 0.853 ) % and ( 22.808 鹵 2.159 ) MPa respectively . The limit stress 蟽max of the 0 . 05 mol / L glyceraldehyde group was ( 10.386 鹵 1.023 ) MPa ( P = 0.0012 ) and ( 14.851 鹵 1.602 ) MPa ( P = 0.000 ) , respectively , and the blank control group increased 44.30 % and 106.32 % respectively .
The limit strain smax of the 0.05 mol / L glyceraldehyde group was ( 19.320 鹵 0.672 ) % ( P = 0.002 ) and ( 14.140 鹵 1.001 ) % ( P = 0.000 ) , respectively , which were 10.06 % and 34.17 % respectively .

2 . After deprivation , the vitreous cavity length and the axial length of vitreous cavity and relative myopic degree increased in the experimental eyes . In group A , group B and C , the length of vitreous cavity , the length of the eye axis and the refractive power were significantly different from that of the contralateral eye ( P glass body cavity = 0 . 018 , 0.002 , 0.001 ;
P - eye axis = 0.019 , 0.002 , 0.000 ;
P refractive index = 0.000 , 0.005 , 0.001 ) . Compared with the contralateral eye , there was no significant difference in the length of vitreous cavity , the length of the vitreous cavity and the length of the vitreous cavity in group D and E ( P glass body cavity = 0.607 , 0.539 ;
P - eye axis = 0.607 , 0.539 , the difference was statistically significant ( P refractive index = 0.020 ) .

3 . At the end of the 21st day of the experiment , the refractive changes of the experimental eyes of B , C , D and E groups were ( 8.800 鹵 0.616 ) D , ( 7.236 鹵 2.198 ) D , ( 6.271 鹵 1.112 ) D and ( 0.934 鹵 0.158 ) D , respectively . The total difference was statistically significant ( F = 61.249 , P = 0.000 ) , and the difference was statistically significant ( PB = 0.000 , PC = 0.000 , PD = 0.000 ) ;
The changes of ocular axis in group B , C and D were higher than those in the control group , but the difference was not statistically significant ( PB = 0.430 , PC = 0.840 , PD = 0.386 ) .

4 . At the end of the 21st day of experiment , the increase of vitreous cavity length in group B and group E were ( 0.198 鹵 0.038 ) mm and ( 0.139 鹵 0.026 ) mm , respectively , and the difference was statistically significant ( t = - 3.689 , P = 0.002 ) .
The increase of ocular axial length in group B was ( 0.481 鹵 0.062 ) mm , E group was ( 0.438 鹵 0.068 ) mm , there was no significant difference ( t = - 1.295 , P = 0.216 ) .

In group B , the ocular axial length was negatively correlated with the refractive index ( r = - 0.832 , P = 0.000 ) .
In group B , the vitreous cavity length was negatively correlated with the refractive power ( r = - 0.804 , P = 0.000 ) .
There was a moderate negative correlation between the ocular axis length and the refractive power of the right eye ( r = - 0.604 , P = 0.000 ) .
The ocular axial length of both eyes was highly correlated ( r = 0.940 , P = 0.000 ) .

6 . At the end of experiment , the ultimate stress and 6 % elastic modulus of the experimental eyes of group B were ( 7.988 鹵 3.677 ) MPa ( P = 0.002 ) and ( 19.938 鹵 4.871 ) MPa ( P = 0.001 ) , respectively . The ultimate stress and 6 % elastic modulus of experimental eyes were ( 9.244 鹵 0.806 ) MPa ( P = 0.031 ) and ( 26.180 鹵 4.388 ) MPa ( P = 0.031 ) , respectively .
In group D , the ultimate stress was ( 12.476 - 2.507 ) MPa ( P = 0.580 ) , the side - eye was reduced by 5.50 % , the elastic modulus of 6 % was ( 30.446 鹵 3.410 ) MPa ( P = 0.314 ) , the limit strain was ( 23.8 鹵 1.8 ) % ( P = 0.253 ) .

7 . Histological examination : With the appearance of FDM in guinea pigs , the thickness of sclera decreased significantly , the fibrous arrangement was orderly , the thickness of the sclera was decreased , and the retinal layer was clear . The thickness of the scleral tissues became thinner and the density of the sclera was increased . The expression of MMP - 2 in the scleral surface connective tissue , scleral surface connective tissue , the superficial sclera , the choroidoidal substance and the outer retina of the sclera decreased significantly .

Conclusion :

1 . glyceraldehyde is a safe and effective crosslinking agent , which can significantly enhance the biomechanical strength of sclera .

2 . The myopia model of the guinea pig is induced successfully by the method of the method of headgear , and the myopia is the axial myopia which mainly increases the length of the vitreous cavity , especially the length of the vitreous cavity .

3 . With the extension of the ocular axis and the degree of myopia , the biomechanical strength of sclera decreased significantly .

4 . This experiment further proved the correlation between MMP - 2 and myopia , and found that glyceraldehyde could reduce the expression of MMP - 2 .

5 . The method of crosslinking glyceraldehyde can effectively control the development of ocular myopia in FDM animal model .

【學(xué)位授予單位】：天津醫(yī)科大學(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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