视黄酸对豚鼠近视眼视网膜色素上皮—脉络膜复合体作用的相关研究

发布时间：2018-03-15 10:51

  本文选题：豚鼠　切入点：离焦型近视　出处：《中南大学》2014年博士论文　论文类型：学位论文

【摘要】：摘要 第一章RA和紧密连接蛋白ZO-1、Occludin在豚鼠离焦型近视眼视网膜色素上皮-脉络膜复合体中的表达 目的：通过建立豚鼠离焦型近视模型,观察视黄酸(RA)、紧密连接蛋白ZO-1和Occludin在视网膜色素上皮(RPE)-脉络膜复合体中的动态表达变化,探讨豚鼠近视眼中RA、ZO-1、Occludin在近视眼中可能的调控作用。 方法：取3周龄三色豚鼠(体重约150-180g)30只(雌雄不限),随机分为A组：正常组(n=15),豚鼠双眼均不佩戴镜片；B组：离焦组(n=15),豚鼠右眼均佩戴-6.00D的凹透镜作为实验眼,左眼均不佩戴镜片作为自身对照眼。分别于实验前、实验第3天、第7天、第14天均对各组豚鼠进行屈光度、眼轴的检测,并在相应时间点取视网膜色素上皮-脉络膜复合体组织用高效液相色谱分析仪检测其RA的含量,用Real-time PCR技术检测紧密连接蛋白ZO-1、Occludin的mRNA含量,Western blot技术检测紧密连接蛋白ZO-1、Occludin的蛋白含量,同时利用免疫荧光技术观察其表达部位。 结果：1、屈光度和眼轴变化：随着离焦时间的延长,A组豚鼠双眼屈光度均呈正视化方向发展,眼轴呈正常生长发育速度增长,各时间点双眼屈光度、眼轴长度比较差异均无统计学意义(P0.05)。B组实验眼呈明显近视漂移,离焦第14天时诱导出相对近视约4.70D,实验眼与自身对照眼屈光度比较差异均有统计学意义(P0.05)；实验眼相对自身对照眼眼轴增长速度加快,离焦第14天时实验眼(8.18±0.09mm)与自身对照眼(7.68±0.06mm)眼轴长度比较差异最大,有统计学意义(P0.05)。各时间点正常组与自身对照眼比较差异均无统计学意义(P0.05)。 2、视黄酸表达变化：视黄酸在豚鼠实验眼视网膜色素上皮-脉络膜复合体上的表达随离焦时间延长而上调,实验眼在离焦第14天表达最高,表达量为137.85±1.02ng/100mg,其与自身对照眼(12.67±0.40ng/100mg)比较差异有统计学意义(P0.05)。各时间点正常组与自身对照眼比较差异均无统计学意义(P0.05)。 3、紧密连接蛋白表达变化：紧密连接蛋白ZO-1、Occludin在豚鼠实验眼视网膜色素上皮-脉络膜复合体中的mRNA和蛋白表达均随离焦时间延长而上调,在离焦第14天表达最高,其与正常组比较差异均有统计学意义(P0.05)。各时间点正常组与自身对照眼比较差异均无统计学意义(P0.05)。免疫荧光检测显示ZO-1、Occludin主要表达于RPE-脉络膜复合体组织的RPE层,其荧光表达结果变化趋势与Real-time PCR和Western Blot检测结果相符。 结论：通过给豚鼠佩戴-6.00D凹透镜可以成功建立离焦型近视模型；视黄酸在豚鼠离焦型近视的发展过程中表达上调；紧密连接蛋白ZO-1、Occludin随离焦时间延长表达上调,可能参与了近视的形成和发展。 第二章RA受体拮抗剂LE540对豚鼠离焦型近视眼视网膜色素上皮-脉络膜复合体中视黄酸和紧密连接蛋白ZO-1、Occludin的影响 目的：观察LE540对豚鼠离焦型近视眼视网膜色素上皮-脉络膜复合体中视黄酸以及紧密连接蛋白ZO-1、Occludin表达的影响,探讨视黄酸与紧密连接蛋白ZO-1、Occludin之间可能的调控关系。 方法：取3周龄三色豚鼠(体重约150-180g)90只(雌雄不限),随机分为A组：正常组(n=15)：豚鼠双眼均不佩戴镜片,且不予任何药物干预；B组：离焦组(n=15),豚鼠右眼均佩戴-6.00D凹透镜,左眼均不佩戴镜片,双眼均不予任何药物干预；C组：阴性对照组(n=15),豚鼠右眼均佩戴-6.00D凹透镜,同时予玻璃体腔内注射PBS5μL；D组：药物干预组(n=45),豚鼠右眼均佩戴-6.00D凹透镜,同时予玻璃体腔内注射不同浓度RA受体拮抗剂LE540,根据LE540注射的不同浓度将D组分为D1组：10μM注射组(n=15)；D2组：501μM注射组(n=15)；D3组：100μM注射组(n=15)。以上各组左眼均为自身对照眼。在实验前、实验第3天、第7天、第14天对以上各组豚鼠进行屈光度、眼轴的检测,并在相应时间点取视网膜色素上皮-脉络膜复合体用高效液相色谱分析仪检测其RA的含量,在D1、D2、D3组中根据视网膜色素上皮复合体中RA含量的变化及对屈光度和眼轴长度的影响,选择药物干预后对豚鼠离焦型近视影响最明显的组别作为D组,运用Real-time PCR技术检测A、B、C、D组紧密连接蛋白ZO-1、Occludin的mRNA含量,Western blot技术检测各组紧密连接蛋白ZO-1、Occludin的蛋白含量,同时利用免疫荧光技术观察其表达部位及表达强度。 结果：1、屈光度和眼轴变化：A组豚鼠双眼屈光度均呈正视化发展,双眼眼轴呈正常生长发育速度增长,各时间点双眼屈光度、眼轴长度比较差异均无统计学意义(P0.05)。B组豚鼠右眼屈光度呈明显近视漂移,右眼相对左眼眼轴增长速度加快,实验第7天、第14天右眼分别与正常组以及左眼屈光度及眼轴比较均有统计学差异(P0.05)；C组双眼屈光度及眼轴变化与B组一致,比较差异无统计学意义(P0.05)；D组随离焦时间的延长,豚鼠各组右眼屈光度近视漂移、眼轴增长趋势随LE540注射浓度的不同均被不同程度抑制,其中在D2(50μtM)组抑制趋势最为明显。而各时间点自身对照眼屈光度和眼轴与正常组比较差异均无统计学意义(P0.05)。 2、LE540对视网膜色素上皮-脉络膜复合体中RA表达的影响：A组豚鼠双眼RA表达在各时间点均无明显差异,比较无统计学意义(P0.05)。B组豚鼠右眼RA随离焦时间延长表达上调。C组豚鼠右眼RA表达变化与B组表达变化趋势一致,比较差异无统计学意义(P0.05)。D组豚鼠右眼玻璃体腔注射不同浓度RA受体拮抗剂LE540后,各组RA表达上调趋势随LE540注射浓度的不同均受到不同程度抑制,其中D2组抑制作用最明显。各时间点自身对照眼视黄酸表达与正常组比较差异无统计学意义(P0.05)。 3、LE540对视网膜色素上皮-脉络膜复合体中紧密连接蛋白的影响：A组豚鼠双眼紧密连接蛋白ZO-1、Occludin随离焦时间延长无明显变化。B组豚鼠右眼紧密连接蛋白ZO-1、Occludin的mRNA和蛋白表达随离焦时间延长而上调,左眼自身对照眼表达均无明显变化。C组豚鼠双眼紧密连接蛋白ZO-1、Occludin表达变化与B组相近。D组豚鼠右眼玻璃体腔注射不同浓度RA受体拮抗剂LE540后,各组紧密连接蛋白ZO-1、Occludin的mRNA和蛋白表达上调趋势受到明显抑制。免疫荧光检测显示ZO-1、Occludin主要表达于RPE-脉络膜复合体组织的RPE层,其荧光表达结果变化趋势与Real-timePCR和Western Blot检测结果相符。 结论：玻璃体腔内注射LE540能有效的抑制豚鼠离焦型近视眼的发展；RA可能通过调控RPE-脉络膜复合体中紧密连接蛋白ZO-1、Occludin的变化从而参与对近视的调控。图9幅,表6个,参考文献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.
鏂规硶锛氬彇3鍛ㄩ緞涓夎壊璞氶紶(浣撻噸绾，

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