【摘要】：目的： 1.建立碘酸鈉誘導視網(wǎng)膜變性大鼠模型，探討硫酸軟骨素蛋白多糖(chondroitin sulfate proteoglycans，CSPGs)在碘酸鈉誘導視網(wǎng)膜變性大鼠視網(wǎng)膜中的表達特點。 2.證實硫酸軟骨素酶（chondroitinaseABC, ChABC）對視網(wǎng)膜變性大鼠中硫酸軟骨素蛋白多糖的降解作用，及緩解視網(wǎng)膜光感受器細胞的凋亡，并初步探討其緩解凋亡的可能機制。 方法： 1.經(jīng)腹腔注射新鮮配制的3%NaIO3（100mg·kg-1）建立視網(wǎng)膜變性動物模型。實驗大鼠隨機分成：正常對照組、造模14d組、造模28d組。取眼球做病理檢查，蘇木素-伊紅（HE）染色及細胞凋亡檢測，驗證視網(wǎng)膜變性大鼠的建立；免疫熒光法觀察視網(wǎng)膜上CSPGs表達的時空規(guī)律；逆轉(zhuǎn)錄-聚合酶鏈反應(RT-PCR)法檢測CSPGs多功能蛋白聚糖（Versican) mRNA的表達情況。 2.造模后7d，6只視網(wǎng)膜變性大鼠玻璃體腔注射2μl ChABC酶作為治療組，另6只視網(wǎng)膜變性大鼠玻璃體腔注射等量磷酸鹽緩沖液(Phosphat bufferliquid,PBS)作為PBS組，6只視網(wǎng)膜變性大鼠和6只正常SD大鼠不行玻璃體腔注射作為對照組。玻璃體腔注射后3w，采用免疫熒光法觀察各組大鼠視網(wǎng)膜CSPGs表達；RT-PCR法檢測CSPGs多功能蛋白聚糖（Versican) mRNA表達，并檢測光感受器凋亡情況。 結(jié)果： 1.注射NaIO3后，大鼠視網(wǎng)膜出現(xiàn)變性改變，且光感受器發(fā)生漸進性凋亡，造模14d、28d凋亡率分別為(32.33±2.34)%、(41.67±2.58)%，各組間差異顯著（F=409.81，P＜0.01）。造模14d組，CS-56在視錐視桿層、外核層、內(nèi)核層、節(jié)細胞層表達；造模28d組，CSPGs繼續(xù)擴增到外叢狀層。隨造模時間延長，各層熒光表達逐漸增強。造模組Versican mRNA表達(1.02±0.06）顯著高于正常對照組(0.23±0.02)（F=487.23，P＜0.01）。 2.(1）偶見SD對照組大鼠CSPGs于脈絡膜、內(nèi)核層、節(jié)細胞層弱陽性表達，注射碘酸鈉4w后大鼠CSPGs擴增到外核層、外叢狀層，各層熒光明顯增強，而同期ChABC酶治療組大鼠CSPGs在視網(wǎng)膜各層表達明顯減弱；VersicanmRNA表達與此一致，正常SD對照組、視網(wǎng)膜變性對照組、PBS組、治療組Versican mRNA的灰度比分別是(0.18±0.02)、(0.92±0.03)、(0.98±0.06)和(0.30±0.01)，其中，視網(wǎng)膜變性對照組和PBS組灰度比無顯著差異（P＞0.05），治療組灰度比明顯低于視網(wǎng)膜變性對照組（F=562.01，P＜0.01）。（2）正常SD對照組、視網(wǎng)膜變性對照組、PBS組、治療組大鼠視網(wǎng)膜光感受器凋亡率分別為(7.33±1.03)%、(40.0±2.37)%、(41.83±2.32)、（35.2+1.94）%，視網(wǎng)膜變性對照組和PBS組凋亡率無顯著差異（P＞0.05），治療組凋亡率顯著低于視網(wǎng)膜變性對照組（F=392.81，P＜0.01）。 結(jié)論： 1.在碘酸鈉誘導視網(wǎng)膜色素上皮細胞變性大鼠中，隨時間延長CSPGs表達范圍擴大，表達量增加。 2. ChABC酶能通過降解模型大鼠視網(wǎng)膜上異常沉積的CSPGs，減少光感受器細胞的凋亡，，從而促進模型大鼠視網(wǎng)膜損傷修復。
[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 鹵2.34) and (41.67 鹵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 鹵0.06) was significantly higher than that in the control group (0.23 鹵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 鹵0.02), () 0.92 鹵0.03), (0.98 鹵0.06 and (0.30 鹵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 鹵1.03), (40.0 鹵2.37), (41.83 鹵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.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R774.13

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