【摘要】：目的：（1）建立小鼠脈絡(luò)膜新生血管（choroidal neovascularization，CNV）動物模型；（2）采用RT-PCR、Western blot和免疫熒光染色技術(shù)對NSA2（Nop seven-associated2）在C57BL/6J小鼠CNV動物模型眼組織中的表達情況進行研究，以期為探討濕性AMD的發(fā)病機制提供新的實驗依據(jù)和理論依據(jù)。 方法：（1）采用532nm氬激光誘導(dǎo)C57BL/6J小鼠CNV動物模型；（2）取光凝后14d組小鼠眼做脈絡(luò)膜鋪片，用CD31抗體標(biāo)記血管內(nèi)皮細(xì)胞的方法對CNV模型進行鑒定；（3）采用RT-PCR和Westernblotting方法檢測正常對照組和光凝后1d、3d、5d、7d和14d組小鼠CNV中NSA2mRNA和蛋白的表達變化情況進行研究，得出光凝后不同時間點NSA2在CNV模型鼠眼組織中表達的時間變化規(guī)律；（4）取光凝后7d組小鼠眼球做冰凍切片，用免疫熒光染色方法對NSA2蛋白在CNV模型鼠眼組織中的表達進行定位研究。 結(jié)果：（1）在本課題中，我們用532nm氬激光成功穩(wěn)定的誘導(dǎo)出了C57BL/6J小鼠CNV動物模型，能滿足該課題中所有實驗的需要。（2）PCR結(jié)果顯示NSA2mRNA在正常成年C57BL/6J小鼠視網(wǎng)膜-脈絡(luò)膜-鞏膜復(fù)合物中弱表達。視網(wǎng)膜光凝后，NSA2mRNA在CNV模型眼的視網(wǎng)膜-脈絡(luò)膜-鞏膜復(fù)合物中的表達有明顯的時間變化規(guī)律，，光凝后1d NSA2的表達開始上調(diào)，1~3d逐漸增強，3d達高峰，之后其表達逐漸減弱。其中，光凝后1d、3d、5d、7d組NSA2mRNA的表達明顯高于正常對照組，差異有統(tǒng)計學(xué)意義（P＜0.05）；光凝后14d組NSA2mRNA的表達與正常對照組相比無統(tǒng)計學(xué)意義（P＞0.05）；光凝后3d組NSA2mRNA的表達量明顯高于其余各組，差別有統(tǒng)計學(xué)意義（P＜0.05）。（3）Western blot結(jié)果顯示，NSA2蛋白在正常成年C57BL/6J小鼠視網(wǎng)膜-脈絡(luò)膜-鞏膜復(fù)合物中呈弱表達。光凝后1dNSA2蛋白的表達開始上調(diào)，3d達高峰，之后逐漸下降，14d組NSA2蛋白的表達與正常組相比仍有明顯差異（P＜0.05）；光凝后3d組和5d組NSA2蛋白的表達量明顯高于其余各組，差異明顯（P＜0.05），但3d組和5d組組間比較，未見明顯差異（P＞0.05）。（4）免疫熒光染色結(jié)果顯示NSA2蛋白在正常小鼠視網(wǎng)膜外叢狀層有較強表達；在光凝后7d組CNV模型鼠眼組織中，除了在視網(wǎng)膜外叢狀層中NSA2蛋白呈高表達外，在組織增生活躍的CNV形成區(qū)域也可檢測到較強的NSA2蛋白表達，差異有統(tǒng)計學(xué)意義（P＜0.05）。 結(jié)論：NSA2在CNV形成早期表達上調(diào)，具有明顯的時間變化規(guī)律，且在CNV區(qū)域有較強的表達，因此我們推斷NSA2可能在CNV形成這一病理過程中起著重要作用。
[Abstract]:Objective: (1) to establish the model of choroidal neovascularization (choroidal neovascularization,CNV) in mice; (2) the expression of NSA2 (Nop seven-associated2) in the eye tissue of C57BL/6J mice model of CNV was studied by RT-PCR,Western blot and immunofluorescence staining. In order to provide a new experimental and theoretical basis for the pathogenesis of wet AMD. Methods: (1) the animal model of CNV in C57BL/6J mice was induced by 532nm argon laser, (2) the eyes of 14 days after photocoagulation were taken for choroidal patch, and the CNV model was identified by using CD31 antibody labeled vascular endothelial cells (VECs). (3) RT-PCR and Westernblotting methods were used to detect the expression of NSA2mRNA and protein in CNV of normal control group and mice at day 1, day 3, day 5, day 7 and day 14 after photocoagulation. The temporal changes of NSA2 expression in the eye tissue of CNV model were obtained at different time points after photocoagulation. (4) 7 days after photocoagulation, the expression of NSA2 protein in the eye tissue of CNV model was studied by immunofluorescence staining. Results: (1) in this study, we successfully and stably induced the animal model of C57BL/6J mouse CNV with 532nm argon laser. The results of PCR showed that NSA2mRNA was weakly expressed in retina-choroid-sclera complex of normal adult C57BL/6J mice. After photocoagulation, the expression of NSA2mRNA in the retinal-choroid-scleral complex of CNV model eyes had obvious time-varying regularity. The expression of NSA2 began to upregulate on the 1st day after photocoagulation, gradually increased at the 1st day after photocoagulation, reached the peak at the 3rd day after photocoagulation, and then decreased gradually. The expression of NSA2mRNA in group 1, 3, 5, 7 days after photocoagulation was significantly higher than that in normal control group (P < 0.05), but the expression of NSA2mRNA in group 14 after photocoagulation had no statistical significance compared with that in normal control group (P > 0.05). The expression of NSA2mRNA in 3 days after photocoagulation was significantly higher than that in other groups (P < 0.05). (3) Western blot). The results showed that NSA2 protein was weakly expressed in the retinal choroid sclera complex of normal adult C57BL/6J mice. After photocoagulation, the expression of 1dNSA2 protein began to upregulate, reached the peak at 3 days, then decreased gradually. The expression of NSA2 protein in 14 days group was still significantly different from that in normal group (P < 0.05). The expression of NSA2 protein in 3 d and 5 d groups was significantly higher than that in other groups (P < 0.05), but there was a significant difference between 3 d group and 5 d group (P < 0.05). There was no significant difference (P > 0.05). (4). The results of immunofluorescence staining showed that NSA2 protein was strongly expressed in the outer plexiform layer of the normal mouse retina. In the eye tissue of CNV model group 7 days after photocoagulation, in addition to the high expression of NSA2 protein in the outer plexiform layer of retina, the strong expression of NSA2 protein could also be detected in the area of CNV formation where tissue proliferation was active. The difference was statistically significant (P < 0.05). Conclusion: the expression of NSA2 is up-regulated in the early stage of CNV formation, with obvious time-varying regularity and strong expression in CNV region. Therefore, we infer that NSA2 may play an important role in the formation of CNV.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R318.51

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