本文選題：豚鼠　切入點(diǎn)：離焦型近視　出處：《中南大學(xué)》2014年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：摘要 第一章RA和緊密連接蛋白ZO-1、Occludin在豚鼠離焦型近視眼視網(wǎng)膜色素上皮-脈絡(luò)膜復(fù)合體中的表達(dá) 目的：通過(guò)建立豚鼠離焦型近視模型,觀察視黃酸(RA)、緊密連接蛋白ZO-1和Occludin在視網(wǎng)膜色素上皮(RPE)-脈絡(luò)膜復(fù)合體中的動(dòng)態(tài)表達(dá)變化,探討豚鼠近視眼中RA、ZO-1、Occludin在近視眼中可能的調(diào)控作用。 方法：取3周齡三色豚鼠(體重約150-180g)30只(雌雄不限),隨機(jī)分為A組：正常組(n=15),豚鼠雙眼均不佩戴鏡片；B組：離焦組(n=15),豚鼠右眼均佩戴-6.00D的凹透鏡作為實(shí)驗(yàn)眼,左眼均不佩戴鏡片作為自身對(duì)照眼。分別于實(shí)驗(yàn)前、實(shí)驗(yàn)第3天、第7天、第14天均對(duì)各組豚鼠進(jìn)行屈光度、眼軸的檢測(cè),并在相應(yīng)時(shí)間點(diǎn)取視網(wǎng)膜色素上皮-脈絡(luò)膜復(fù)合體組織用高效液相色譜分析儀檢測(cè)其RA的含量,用Real-time PCR技術(shù)檢測(cè)緊密連接蛋白ZO-1、Occludin的mRNA含量,Western blot技術(shù)檢測(cè)緊密連接蛋白ZO-1、Occludin的蛋白含量,同時(shí)利用免疫熒光技術(shù)觀察其表達(dá)部位。 結(jié)果：1、屈光度和眼軸變化：隨著離焦時(shí)間的延長(zhǎng),A組豚鼠雙眼屈光度均呈正視化方向發(fā)展,眼軸呈正常生長(zhǎng)發(fā)育速度增長(zhǎng),各時(shí)間點(diǎn)雙眼屈光度、眼軸長(zhǎng)度比較差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。B組實(shí)驗(yàn)眼呈明顯近視漂移,離焦第14天時(shí)誘導(dǎo)出相對(duì)近視約4.70D,實(shí)驗(yàn)眼與自身對(duì)照眼屈光度比較差異均有統(tǒng)計(jì)學(xué)意義(P0.05)；實(shí)驗(yàn)眼相對(duì)自身對(duì)照眼眼軸增長(zhǎng)速度加快,離焦第14天時(shí)實(shí)驗(yàn)眼(8.18±0.09mm)與自身對(duì)照眼(7.68±0.06mm)眼軸長(zhǎng)度比較差異最大,有統(tǒng)計(jì)學(xué)意義(P0.05)。各時(shí)間點(diǎn)正常組與自身對(duì)照眼比較差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。 2、視黃酸表達(dá)變化：視黃酸在豚鼠實(shí)驗(yàn)眼視網(wǎng)膜色素上皮-脈絡(luò)膜復(fù)合體上的表達(dá)隨離焦時(shí)間延長(zhǎng)而上調(diào),實(shí)驗(yàn)眼在離焦第14天表達(dá)最高,表達(dá)量為137.85±1.02ng/100mg,其與自身對(duì)照眼(12.67±0.40ng/100mg)比較差異有統(tǒng)計(jì)學(xué)意義(P0.05)。各時(shí)間點(diǎn)正常組與自身對(duì)照眼比較差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。 3、緊密連接蛋白表達(dá)變化：緊密連接蛋白ZO-1、Occludin在豚鼠實(shí)驗(yàn)眼視網(wǎng)膜色素上皮-脈絡(luò)膜復(fù)合體中的mRNA和蛋白表達(dá)均隨離焦時(shí)間延長(zhǎng)而上調(diào),在離焦第14天表達(dá)最高,其與正常組比較差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。各時(shí)間點(diǎn)正常組與自身對(duì)照眼比較差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。免疫熒光檢測(cè)顯示ZO-1、Occludin主要表達(dá)于RPE-脈絡(luò)膜復(fù)合體組織的RPE層,其熒光表達(dá)結(jié)果變化趨勢(shì)與Real-time PCR和Western Blot檢測(cè)結(jié)果相符。 結(jié)論：通過(guò)給豚鼠佩戴-6.00D凹透鏡可以成功建立離焦型近視模型；視黃酸在豚鼠離焦型近視的發(fā)展過(guò)程中表達(dá)上調(diào)；緊密連接蛋白ZO-1、Occludin隨離焦時(shí)間延長(zhǎng)表達(dá)上調(diào),可能參與了近視的形成和發(fā)展。 第二章RA受體拮抗劑LE540對(duì)豚鼠離焦型近視眼視網(wǎng)膜色素上皮-脈絡(luò)膜復(fù)合體中視黃酸和緊密連接蛋白ZO-1、Occludin的影響 目的：觀察LE540對(duì)豚鼠離焦型近視眼視網(wǎng)膜色素上皮-脈絡(luò)膜復(fù)合體中視黃酸以及緊密連接蛋白ZO-1、Occludin表達(dá)的影響,探討視黃酸與緊密連接蛋白ZO-1、Occludin之間可能的調(diào)控關(guān)系。 方法：取3周齡三色豚鼠(體重約150-180g)90只(雌雄不限),隨機(jī)分為A組：正常組(n=15)：豚鼠雙眼均不佩戴鏡片,且不予任何藥物干預(yù)；B組：離焦組(n=15),豚鼠右眼均佩戴-6.00D凹透鏡,左眼均不佩戴鏡片,雙眼均不予任何藥物干預(yù)；C組：陰性對(duì)照組(n=15),豚鼠右眼均佩戴-6.00D凹透鏡,同時(shí)予玻璃體腔內(nèi)注射PBS5μL；D組：藥物干預(yù)組(n=45),豚鼠右眼均佩戴-6.00D凹透鏡,同時(shí)予玻璃體腔內(nèi)注射不同濃度RA受體拮抗劑LE540,根據(jù)LE540注射的不同濃度將D組分為D1組：10μM注射組(n=15)；D2組：501μM注射組(n=15)；D3組：100μM注射組(n=15)。以上各組左眼均為自身對(duì)照眼。在實(shí)驗(yàn)前、實(shí)驗(yàn)第3天、第7天、第14天對(duì)以上各組豚鼠進(jìn)行屈光度、眼軸的檢測(cè),并在相應(yīng)時(shí)間點(diǎn)取視網(wǎng)膜色素上皮-脈絡(luò)膜復(fù)合體用高效液相色譜分析儀檢測(cè)其RA的含量,在D1、D2、D3組中根據(jù)視網(wǎng)膜色素上皮復(fù)合體中RA含量的變化及對(duì)屈光度和眼軸長(zhǎng)度的影響,選擇藥物干預(yù)后對(duì)豚鼠離焦型近視影響最明顯的組別作為D組,運(yùn)用Real-time PCR技術(shù)檢測(cè)A、B、C、D組緊密連接蛋白ZO-1、Occludin的mRNA含量,Western blot技術(shù)檢測(cè)各組緊密連接蛋白ZO-1、Occludin的蛋白含量,同時(shí)利用免疫熒光技術(shù)觀察其表達(dá)部位及表達(dá)強(qiáng)度。 結(jié)果：1、屈光度和眼軸變化：A組豚鼠雙眼屈光度均呈正視化發(fā)展,雙眼眼軸呈正常生長(zhǎng)發(fā)育速度增長(zhǎng),各時(shí)間點(diǎn)雙眼屈光度、眼軸長(zhǎng)度比較差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。B組豚鼠右眼屈光度呈明顯近視漂移,右眼相對(duì)左眼眼軸增長(zhǎng)速度加快,實(shí)驗(yàn)第7天、第14天右眼分別與正常組以及左眼屈光度及眼軸比較均有統(tǒng)計(jì)學(xué)差異(P0.05)；C組雙眼屈光度及眼軸變化與B組一致,比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；D組隨離焦時(shí)間的延長(zhǎng),豚鼠各組右眼屈光度近視漂移、眼軸增長(zhǎng)趨勢(shì)隨LE540注射濃度的不同均被不同程度抑制,其中在D2(50μtM)組抑制趨勢(shì)最為明顯。而各時(shí)間點(diǎn)自身對(duì)照眼屈光度和眼軸與正常組比較差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。 2、LE540對(duì)視網(wǎng)膜色素上皮-脈絡(luò)膜復(fù)合體中RA表達(dá)的影響：A組豚鼠雙眼RA表達(dá)在各時(shí)間點(diǎn)均無(wú)明顯差異,比較無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。B組豚鼠右眼RA隨離焦時(shí)間延長(zhǎng)表達(dá)上調(diào)。C組豚鼠右眼RA表達(dá)變化與B組表達(dá)變化趨勢(shì)一致,比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。D組豚鼠右眼玻璃體腔注射不同濃度RA受體拮抗劑LE540后,各組RA表達(dá)上調(diào)趨勢(shì)隨LE540注射濃度的不同均受到不同程度抑制,其中D2組抑制作用最明顯。各時(shí)間點(diǎn)自身對(duì)照眼視黃酸表達(dá)與正常組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。 3、LE540對(duì)視網(wǎng)膜色素上皮-脈絡(luò)膜復(fù)合體中緊密連接蛋白的影響：A組豚鼠雙眼緊密連接蛋白ZO-1、Occludin隨離焦時(shí)間延長(zhǎng)無(wú)明顯變化。B組豚鼠右眼緊密連接蛋白ZO-1、Occludin的mRNA和蛋白表達(dá)隨離焦時(shí)間延長(zhǎng)而上調(diào),左眼自身對(duì)照眼表達(dá)均無(wú)明顯變化。C組豚鼠雙眼緊密連接蛋白ZO-1、Occludin表達(dá)變化與B組相近。D組豚鼠右眼玻璃體腔注射不同濃度RA受體拮抗劑LE540后,各組緊密連接蛋白ZO-1、Occludin的mRNA和蛋白表達(dá)上調(diào)趨勢(shì)受到明顯抑制。免疫熒光檢測(cè)顯示ZO-1、Occludin主要表達(dá)于RPE-脈絡(luò)膜復(fù)合體組織的RPE層,其熒光表達(dá)結(jié)果變化趨勢(shì)與Real-timePCR和Western Blot檢測(cè)結(jié)果相符。 結(jié)論：玻璃體腔內(nèi)注射LE540能有效的抑制豚鼠離焦型近視眼的發(fā)展；RA可能通過(guò)調(diào)控RPE-脈絡(luò)膜復(fù)合體中緊密連接蛋白ZO-1、Occludin的變化從而參與對(duì)近視的調(diào)控。圖9幅,表6個(gè),參考文獻(xiàn)66篇。
[Abstract]:abstract
The expression of RA and tightly connexin ZO-1, Occludin in the retinal pigment epithelium - choroid complex of the defocus myopia of guinea pigs
Objective: to establish the defocus myopia of guinea pig model, observation of retinoic acid (RA), tight junction proteins ZO-1 and Occludin in retinal pigment epithelium (RPE) - dynamic expression changes of choroid complex, to explore the guinea pig myopia RA, ZO-1, Occludin in the regulation of myopia may be.
Methods: 3 week old tricolor guinea pigs (weighing about 150-180g) 30 (male or female), were randomly divided into A groups: normal group (n=15), do not wear glasses in guinea pig eyes; group B: focus group (n=15), the right eye was wearing -6.00D guinea pig concave lens as the experimental eye and the left eye was don't wear lenses as their control. Respectively before experiment, experiment of third days, seventh days, fourteenth days for each group of guinea pigs were detected diopter, eye axis, and the content in the corresponding time points in complex retinal pigment epithelium choroid tissue by high performance liquid chromatography analyzer RA, detection Real-time PCR tight junction protein ZO-1, the content of mRNA Occludin, Western blot detection technology of tight junction protein ZO-1, protein Occludin, and observe the expression site using immunofluorescence technique.
Results: 1, refraction and axial changes with increasing defocus time, A group showed refraction in both eyes face direction, eye axis at a normal growth rate, refractive error at each time point, the axial length of the eyes showed no significant difference (P0.05) of.B group in experimental eyes significant myopic shift, fourteenth days of defocus induced myopia diopter were about 4.70D, there were statistically significant differences between experimental eyes and its (P0.05); experimental eyes relative self-control eyes axial growth speed, defocusing fourteenth days experimental eyes (8.18 + 0.09mm) and control group (7.68 + to itself 0.06mm) axial length difference, with statistical significance (P0.05). The normal group at each time point served as self-control showed no significant difference (P0.05).
2, retinoic acid expression: the expression of retinoic acid in guinea pig retinal pigment epithelium choroid complex eye with defocus time increased in experimental eyes defocus fourteenth days was the highest, the expression level was 137.85 + 1.02ng/100mg, which served as self-control (12.67 + 0.40ng/ 100mg) was statistically significant the difference at each time point (P0.05). The normal group served as self-control showed no significant difference (P0.05).
3, tight junction protein expression: the expression of tight junction protein ZO-1, Occludin and mRNA protein in retinal pigment epithelium of guinea pig experimental eyes - choroid complex increased with the defocus time increase in defocus for fourteenth days was the highest, compared with the normal group the differences were statistically significant (P0.05) at each time point. The normal group and the control eyes showed no significant difference (P0.05). Immunofluorescence showed ZO-1, RPE layer Occludin is mainly expressed in complex tissue RPE- choroid, the trend of change with Real-time PCR and Western Blot expression. The fluorescence detection results.
Conclusion: the guinea pig wearing -6.00D concave lens can successfully establish the defocus myopia model; the upregulation of the expression of retinoic acid in the process of the development of myopia in guinea pig usingdefocus type; tight junction protein ZO-1, Occludin with defocus prolonged expression may be involved in the formation and development of myopia.
The second chapter is about the effect of RA receptor antagonist LE540 on retinoic acid and tight junction protein ZO-1 and Occludin in retinal pigment epithelium choroidal complex of guinea pig.
Objective: To observe the effect of LE540 on the expression of retinoic acid and tight junction protein ZO-1 and Occludin in retinal pigment epithelium choroidal complex of guinea pig with defocused myopia, and to explore the possible regulatory relationship between retinoic acid and tight junction protein ZO-1 and Occludin.
鏂規(guī)硶錛氬彇3鍛ㄩ緞涓夎壊璞氶紶(浣撻噸綰，

本文編號(hào)：1615714