视网膜变性大鼠视网膜CSPGs表达及CSPGs降解对光感受器凋亡的影响

发布时间：2018-08-09 16:13

【摘要】：目的： 1.建立碘酸钠诱导视网膜变性大鼠模型，探讨硫酸软骨素蛋白多糖(chondroitin sulfate proteoglycans，CSPGs)在碘酸钠诱导视网膜变性大鼠视网膜中的表达特点。 2.证实硫酸软骨素酶（chondroitinaseABC, ChABC）对视网膜变性大鼠中硫酸软骨素蛋白多糖的降解作用，及缓解视网膜光感受器细胞的凋亡，并初步探讨其缓解凋亡的可能机制。方法： 1.经腹腔注射新鲜配制的3%NaIO3（100mg·kg-1）建立视网膜变性动物模型。实验大鼠随机分成：正常对照组、造模14d组、造模28d组。取眼球做病理检查，苏木素-伊红（HE）染色及细胞凋亡检测，验证视网膜变性大鼠的建立；免疫荧光法观察视网膜上CSPGs表达的时空规律；逆转录-聚合酶链反应(RT-PCR)法检测CSPGs多功能蛋白聚糖（Versican) mRNA的表达情况。 2.造模后7d，6只视网膜变性大鼠玻璃体腔注射2μl ChABC酶作为治疗组，另6只视网膜变性大鼠玻璃体腔注射等量磷酸盐缓冲液(Phosphat bufferliquid,PBS)作为PBS组，6只视网膜变性大鼠和6只正常SD大鼠不行玻璃体腔注射作为对照组。玻璃体腔注射后3w，采用免疫荧光法观察各组大鼠视网膜CSPGs表达；RT-PCR法检测CSPGs多功能蛋白聚糖（Versican) mRNA表达，并检测光感受器凋亡情况。结果： 1.注射NaIO3后，大鼠视网膜出现变性改变，且光感受器发生渐进性凋亡，造模14d、28d凋亡率分别为(32.33±2.34)%、(41.67±2.58)%，各组间差异显著（F=409.81，P＜0.01）。造模14d组，CS-56在视锥视杆层、外核层、内核层、节细胞层表达；造模28d组，CSPGs继续扩增到外丛状层。随造模时间延长，各层荧光表达逐渐增强。造模组Versican mRNA表达(1.02±0.06）显著高于正常对照组(0.23±0.02)（F=487.23，P＜0.01）。 2.(1）偶见SD对照组大鼠CSPGs于脉络膜、内核层、节细胞层弱阳性表达，注射碘酸钠4w后大鼠CSPGs扩增到外核层、外丛状层，各层荧光明显增强，而同期ChABC酶治疗组大鼠CSPGs在视网膜各层表达明显减弱；VersicanmRNA表达与此一致，正常SD对照组、视网膜变性对照组、PBS组、治疗组Versican mRNA的灰度比分别是(0.18±0.02)、(0.92±0.03)、(0.98±0.06)和(0.30±0.01)，其中，视网膜变性对照组和PBS组灰度比无显著差异（P＞0.05），治疗组灰度比明显低于视网膜变性对照组（F=562.01，P＜0.01）。（2）正常SD对照组、视网膜变性对照组、PBS组、治疗组大鼠视网膜光感受器凋亡率分别为(7.33±1.03)%、(40.0±2.37)%、(41.83±2.32)、（35.2+1.94）%，视网膜变性对照组和PBS组凋亡率无显著差异（P＞0.05），治疗组凋亡率显著低于视网膜变性对照组（F=392.81，P＜0.01）。结论： 1.在碘酸钠诱导视网膜色素上皮细胞变性大鼠中，随时间延长CSPGs表达范围扩大，表达量增加。 2. ChABC酶能通过降解模型大鼠视网膜上异常沉积的CSPGs，减少光感受器细胞的凋亡，，从而促进模型大鼠视网膜损伤修复。
[Abstract]:Objective: 1. A rat model of retinal degeneration induced by sodium iodate was established to investigate the expression of chondroitin sulfate proteoglycansn (chondroitin sulfate) in the retina of rats with retinal degeneration induced by sodium iodate. 2. To confirm the degradation of chondroitin sulfate proteoglycan in retinal degeneration rats by chondroitin sulfate enzyme (chondroitinaseABC, ChABC) and to alleviate the apoptosis of retinal photoreceptor cells. Methods: 1. Retinal degeneration model was established by intraperitoneal injection of freshly prepared 3%NaIO3 (100mg kg-1). The experimental rats were randomly divided into normal control group, 14 d model making group and 28 d model making group. Histopathological examination, hematoxylin-eosin (HE) staining and apoptosis detection were used to verify the establishment of retinal degeneration rats, and the temporal and spatial regularity of CSPGs expression on retina were observed by immunofluorescence method. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of CSPGs multifunctional proteoglycan (Versican) mRNA. After 7 days, 6 rats with retinal degeneration were injected with 2 渭 l ChABC enzyme into vitreous body as treatment group. The other six rats with retinal degeneration were injected Phosphat buffer liquid PBS into vitreous cavity as control group. Six rats with retinal degeneration and six normal SD rats were injected intravitreous as control group. Three weeks after vitreous injection, the expression of CSPGs in the retina of rats in each group was observed by immunofluorescence method. The expression of CSPGs multifunctional proteoglycan (Versican) mRNA was detected by RT-PCR, and the apoptosis of photoreceptor was detected. Results: 1. The retinal degeneration and photoreceptor apoptosis were observed in rats after NaIO3 injection. The apoptotic rates were (32.33 卤2.34) and (41.67 卤2.58) at 14d and 28d, respectively (P < 0.01). The expression of CS-56 was observed in the optic cone rod layer, outer nuclear layer, nuclear layer and ganglion cell layer in 14 d group, and in 28 d group, the CSPGs continued to expand to the outer plexiform layer. With the prolongation of modeling time, the fluorescence expression in each layer increased gradually. The expression of Versican mRNA in the model group (1.02 卤0.06) was significantly higher than that in the control group (0.23 卤0.02) (P < 0.01). The expression of Versica mRNA in all layers of retina was significantly decreased in ChABC enzyme treatment group. The grayscale ratio of Versican mRNA in normal SD control group and retinal degeneration control group was (0.18 卤0.02), () 0.92 卤0.03), (0.98 卤0.06 and (0.30 卤0.01), respectively. There was no significant difference between the retinal degeneration control group and the PBS group (P > 0. 05). The grayscale ratio in the treatment group was significantly lower than that in the retinal degeneration control group (F 562 01) (P < 0. 01). (2), and the retinal degeneration control group was in the normal SD group (P < 0. 01). The apoptosis rate of retinal photoreceptor in the treatment group was (7.33 卤1.03), (40.0 卤2.37), (41.83 卤2.32), () respectively. There was no significant difference between the retinal degeneration control group and the PBS group (P > 0. 05). The apoptosis rate in the treatment group was significantly lower than that in the retinal degeneration control group (P < 0. 01). Conclusion: 1. In rats with retinal pigment epithelium denaturation induced by sodium iodate, the expression range of CSPGs was enlarged and the expression amount was increased. ChABC enzyme can reduce the apoptosis of photoreceptor cells by degrading the abnormal deposition of CSPGs on the retina of the model rats, thus promoting the repair of retinal injury in the model rats.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R774.13

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