NSA2在激光诱导的小鼠脉络膜新生血管模型中表达的时相性和定位研究
发布时间：2019-03-27 13:22
【摘要】：目的：（1）建立小鼠脉络膜新生血管（choroidal neovascularization，CNV）动物模型；（2）采用RT-PCR、Western blot和免疫荧光染色技术对NSA2（Nop seven-associated2）在C57BL/6J小鼠CNV动物模型眼组织中的表达情况进行研究，以期为探讨湿性AMD的发病机制提供新的实验依据和理论依据。 方法：（1）采用532nm氩激光诱导C57BL/6J小鼠CNV动物模型；（2）取光凝后14d组小鼠眼做脉络膜铺片，用CD31抗体标记血管内皮细胞的方法对CNV模型进行鉴定；（3）采用RT-PCR和Westernblotting方法检测正常对照组和光凝后1d、3d、5d、7d和14d组小鼠CNV中NSA2mRNA和蛋白的表达变化情况进行研究，得出光凝后不同时间点NSA2在CNV模型鼠眼组织中表达的时间变化规律；（4）取光凝后7d组小鼠眼球做冰冻切片，用免疫荧光染色方法对NSA2蛋白在CNV模型鼠眼组织中的表达进行定位研究。 结果：（1）在本课题中，我们用532nm氩激光成功稳定的诱导出了C57BL/6J小鼠CNV动物模型，能满足该课题中所有实验的需要。（2）PCR结果显示NSA2mRNA在正常成年C57BL/6J小鼠视网膜-脉络膜-巩膜复合物中弱表达。视网膜光凝后，NSA2mRNA在CNV模型眼的视网膜-脉络膜-巩膜复合物中的表达有明显的时间变化规律，，光凝后1d NSA2的表达开始上调，1~3d逐渐增强，3d达高峰，之后其表达逐渐减弱。其中，光凝后1d、3d、5d、7d组NSA2mRNA的表达明显高于正常对照组，差异有统计学意义（P＜0.05）；光凝后14d组NSA2mRNA的表达与正常对照组相比无统计学意义（P＞0.05）；光凝后3d组NSA2mRNA的表达量明显高于其余各组，差别有统计学意义（P＜0.05）。（3）Western blot结果显示，NSA2蛋白在正常成年C57BL/6J小鼠视网膜-脉络膜-巩膜复合物中呈弱表达。光凝后1dNSA2蛋白的表达开始上调，3d达高峰，之后逐渐下降，14d组NSA2蛋白的表达与正常组相比仍有明显差异（P＜0.05）；光凝后3d组和5d组NSA2蛋白的表达量明显高于其余各组，差异明显（P＜0.05），但3d组和5d组组间比较，未见明显差异（P＞0.05）。（4）免疫荧光染色结果显示NSA2蛋白在正常小鼠视网膜外丛状层有较强表达；在光凝后7d组CNV模型鼠眼组织中，除了在视网膜外丛状层中NSA2蛋白呈高表达外，在组织增生活跃的CNV形成区域也可检测到较强的NSA2蛋白表达，差异有统计学意义（P＜0.05）。 结论：NSA2在CNV形成早期表达上调，具有明显的时间变化规律，且在CNV区域有较强的表达，因此我们推断NSA2可能在CNV形成这一病理过程中起着重要作用。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.51
本文编号：2448219
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.51
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本文编号：2448219
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