Caveolin-1在鼠视网膜新生血管形成中调控作用的研究
发布时间：2018-07-18 15:46
【摘要】：第一章Caveolin-1在鼠视网膜新生血管模型中的表达 目的建立氧诱导视网膜新生血管小鼠模型,观察小窝蛋白-1 (caveolin-1)在正常鼠和氧诱导视网膜新生血管模型鼠视网膜中的表达,探讨caveolin-1在视网膜新生血管形成过程中的作用。 方法取鼠龄7天(P7)的健康C57BL/6J小鼠(144只)随机分为模型组(72只)和正常对照组(72只),模型组置于75%±2%氧气浓度的密闭氧舱中5天,鼠龄12天时(P12)返回正常空气环境以建立氧诱导鼠视网膜新生血管模型；正常对照组置于正常空气环境中饲养不予任何处理。模型组和正常对照组均在鼠龄17天(P17)行FITC-Dextran左心室灌注视网膜铺片,荧光显微镜下观察视网膜血管分布和形态；组织切片HE染色观察突破视网膜内界膜的血管内皮细胞核数量。提取不同鼠龄(P13、P15、P17)模型组和正常对照组视网膜总RNA及蛋白质,采用RT-PCR及western blot技术检测视网膜组织中caveolin-1的表达；Western blot技术检测视网膜白蛋白(albumin)含量。 结果荧光造影结果显示模型组视网膜有大量新生血管形成及明显的荧光素渗漏；组织切片显示模型组有大量突破视网膜内界膜的新生血管内皮细胞核。模型组视网膜caveolin-1 mRNA和蛋白质表达水平随缺氧时间延长而上调,于P17明显上调；同时视网膜组织中白蛋白含量逐渐增加,与caveolin-1表达呈基本相同的上升趋势,两者在时间和表达趋势上是一致的。不同鼠龄正常对照组caveolin-1 mRNA和蛋白质表达无明显变化。 结论在氧诱导鼠视网膜新生血管形成过程中caveolin-1表达明显上调,其变化趋势与血-视网膜屏障破坏及视网膜新生血管形成具有时间上的一致性。caveolin-1可能参与调控视网膜新生血管形成。 第二章Caveolin-1 shRNA抑制鼠视网膜新生血管的实验研究 目的观察caveolin-1小发夹RNA (caveolin-1 shRNA)对血-视网膜屏障破坏和视网膜新生血管形成的抑制作用,探讨caveolin-1对视网膜新生血管的调控作用,为视网膜新生血管性疾病的治疗提供新的治疗靶点。 方法取鼠龄7天(P7)的健康C57BL/6J小鼠(144只)随机分为正常对照组(36只)、模型对照组(36只)、阴性对照组(36只)和caveolin-1 shRNA注射组(36只)。正常对照组置于正常空气环境中饲养不予任何处理。模型对照组、阴性对照组和caveolin-1 shRNA注射组置于75%±2%氧气浓度的密闭氧舱中5天,鼠龄12天时(P12)返回正常空气环境以建立氧诱导鼠视网膜新生血管模型；其中,模型对照组不做任何治疗；caveolin-1 shRNA注射组在P12时予玻璃体腔注射1ul脂质体-caveolin-1 shRNA重组质粒混合物；阴性对照组在P12时玻璃体腔注射1ul脂质体-阴性对照质粒混合物。以上各组在鼠龄17天(P17)分别采用RT-PCR和western blot技术检测视网膜caveolin-1 mRNA和蛋白质表达,western blot技术检测视网膜白蛋白表达。行FITC-Dextran造影视网膜铺片方法观察血管形态变化,组织切片HE染色观察并计数突破视网膜内界膜的血管内皮细胞核数目, 结果Caveolin-1 shRNA注射组视网膜caveolin-1 mRNA和蛋白质表达分别较模型对照组下调47.94%和54.76%,差异均有统计学意义(P0.05)；caveolin-1 shRNA注射组视网膜组织白蛋白含量较模型对照组下调56.32%,差异有统计学意义(P0.05)；荧光造影显示：caveolin-1 shRNA注射组较模型对照组视网膜新生血管明显减少,荧光渗漏明显减轻；组织切片显示caveolin-1 shRNA注射组突破视网膜内界膜的血管内皮细胞核数目较模型对照组减少51.3%,差异有统计学意义(P0.01)。 结论Caveolin-1 shRNA可以有效地抑制视网膜血管白蛋白渗漏,并抑制视网膜新生血管形成,进一步证明了caveolin-1在视网膜新生血管发生中的调控作用,为视网膜新生血管性疾病的治疗提供了新的靶点。
[Abstract]:Chapter 1 expression of Caveolin-1 in rat retinal neovascularization model
Objective to establish a mouse model of oxygen induced retinal neovascularization, and to observe the expression of fossa protein -1 (caveolin-1) in normal rat and oxygen induced retinal neovascularization model rat retina, and to explore the role of caveolin-1 in the formation of retinal neovascularization.
Methods the healthy C57BL/6J mice of 7 days of age (P7) were randomly divided into model group (72 rats) and normal control group (72 rats). The model group was placed in the closed oxygen chamber of 75% + 2% oxygen concentration for 5 days, and the rat age 12 days (P12) returned to the normal air environment to establish the oxygen induced retinal neovascularization model in the oxygen induced rat, and the normal control group was placed in the normal air environment. In the model group and the normal control group, the retinal vasculature was perfused in the left ventricle of FITC-Dextran at 17 days (P17), and the distribution and morphology of the retinal vessels were observed under the fluorescence microscope. The number of vascular endothelial cells that broke through the inner boundary membrane of the retina was observed by HE staining. The models of different age of rat (P13, P15, P17) were extracted. The total RNA and protein in the retina of the group and the normal control group were detected by RT-PCR and Western blot, and the content of retina albumin (albumin) was detected by Western blot.
The results showed that the retina of the model group had a large number of neovascularization and obvious fluorescein leakage, and the tissue section showed that the model group had a large number of neovascular endothelial nuclei breaking through the inner boundary membrane of the retina. The expression level of caveolin-1 mRNA and protein in the model group was up-regulated with the prolongation of the time of hypoxia and was obviously on the P17. At the same time, the content of albumin in retinal tissue increased gradually, and the expression of caveolin-1 showed the same upward trend, both in time and in the expression trend. The expression of caveolin-1 mRNA and protein in the normal control group had no obvious change.
Conclusion the expression of caveolin-1 is obviously up-regulated during the formation of retinal neovascularization in oxygen induced retina. The trend of the change is consistent with the disruption of the blood retinal barrier and the formation of retinal neovascularization in time..caveolin-1 may be involved in the regulation of the formation of retinal neovascularization.
Second chapter Caveolin-1 shRNA inhibits retinal neovascularization in rats
Objective To observe the inhibitory effect of caveolin-1 small hairpin RNA (caveolin-1 shRNA) on the destruction of blood retinal barrier and the formation of retinal neovascularization, and to explore the regulation of caveolin-1 on retinal neovascularization, and to provide new therapeutic targets for the treatment of retinal neovascularization.
Methods the healthy C57BL/6J mice of 7 days of age (P7) were randomly divided into normal control group (36 rats), model control group (36 rats), negative control group (36) and caveolin-1 shRNA injection group (36 rats). Normal control group was placed in normal air environment for no treatment. Model control group, negative control group and caveolin-1 shRNA injection group were set up. In the 75% + 2% oxygen concentration airtight oxygen tank 5 days, the rat age 12 days (P12) returned to the normal air environment to establish the oxygen induced rat retinal neovascularization model, and the model control group did not do any treatment; the caveolin-1 shRNA injection group injected the vitreous cavity with the 1ul liposome -caveolin-1 shRNA recombinant plasmid mixture at P12; negative pairs. The mixture of 1ul liposome negative control plasmid was injected into the vitreous cavity at P12. The expression of retina caveolin-1 mRNA and protein was detected by RT-PCR and Western blot in the 17 days of age (P17), and the expression of retinal albumin was detected by Western blot. Tissue sections were stained with HE to observe and count the number of endothelial cells that broke through the inner limiting membrane of the retina.
Results the expression of caveolin-1 mRNA and protein in the retina of the Caveolin-1 shRNA injection group was down 47.94% and 54.76%, respectively, and the difference was statistically significant (P0.05). The albumin content of retinal tissue in caveolin-1 shRNA injection group was 56.32% lower than that in the model control group, and the difference was statistically significant (P0.05); fluorescein angiography showed ca. Compared with the model control group, the retinal neovascularization in the veolin-1 shRNA injection group was significantly reduced and the fluorescence leakage was significantly reduced. The number of vascular endothelial nuclei in the caveolin-1 shRNA injection group was 51.3% less than that in the model control group, and the difference was statistically significant (P0.01).
Conclusion Caveolin-1 shRNA can effectively inhibit the leakage of retinal blood vessel albumin and inhibit the formation of retinal neovascularization, and further demonstrate the regulatory role of caveolin-1 in the development of retinal neovascularization, which provides a new target for the treatment of retinal neovascularization.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R774.1
[Abstract]:Chapter 1 expression of Caveolin-1 in rat retinal neovascularization model
Objective to establish a mouse model of oxygen induced retinal neovascularization, and to observe the expression of fossa protein -1 (caveolin-1) in normal rat and oxygen induced retinal neovascularization model rat retina, and to explore the role of caveolin-1 in the formation of retinal neovascularization.
Methods the healthy C57BL/6J mice of 7 days of age (P7) were randomly divided into model group (72 rats) and normal control group (72 rats). The model group was placed in the closed oxygen chamber of 75% + 2% oxygen concentration for 5 days, and the rat age 12 days (P12) returned to the normal air environment to establish the oxygen induced retinal neovascularization model in the oxygen induced rat, and the normal control group was placed in the normal air environment. In the model group and the normal control group, the retinal vasculature was perfused in the left ventricle of FITC-Dextran at 17 days (P17), and the distribution and morphology of the retinal vessels were observed under the fluorescence microscope. The number of vascular endothelial cells that broke through the inner boundary membrane of the retina was observed by HE staining. The models of different age of rat (P13, P15, P17) were extracted. The total RNA and protein in the retina of the group and the normal control group were detected by RT-PCR and Western blot, and the content of retina albumin (albumin) was detected by Western blot.
The results showed that the retina of the model group had a large number of neovascularization and obvious fluorescein leakage, and the tissue section showed that the model group had a large number of neovascular endothelial nuclei breaking through the inner boundary membrane of the retina. The expression level of caveolin-1 mRNA and protein in the model group was up-regulated with the prolongation of the time of hypoxia and was obviously on the P17. At the same time, the content of albumin in retinal tissue increased gradually, and the expression of caveolin-1 showed the same upward trend, both in time and in the expression trend. The expression of caveolin-1 mRNA and protein in the normal control group had no obvious change.
Conclusion the expression of caveolin-1 is obviously up-regulated during the formation of retinal neovascularization in oxygen induced retina. The trend of the change is consistent with the disruption of the blood retinal barrier and the formation of retinal neovascularization in time..caveolin-1 may be involved in the regulation of the formation of retinal neovascularization.
Second chapter Caveolin-1 shRNA inhibits retinal neovascularization in rats
Objective To observe the inhibitory effect of caveolin-1 small hairpin RNA (caveolin-1 shRNA) on the destruction of blood retinal barrier and the formation of retinal neovascularization, and to explore the regulation of caveolin-1 on retinal neovascularization, and to provide new therapeutic targets for the treatment of retinal neovascularization.
Methods the healthy C57BL/6J mice of 7 days of age (P7) were randomly divided into normal control group (36 rats), model control group (36 rats), negative control group (36) and caveolin-1 shRNA injection group (36 rats). Normal control group was placed in normal air environment for no treatment. Model control group, negative control group and caveolin-1 shRNA injection group were set up. In the 75% + 2% oxygen concentration airtight oxygen tank 5 days, the rat age 12 days (P12) returned to the normal air environment to establish the oxygen induced rat retinal neovascularization model, and the model control group did not do any treatment; the caveolin-1 shRNA injection group injected the vitreous cavity with the 1ul liposome -caveolin-1 shRNA recombinant plasmid mixture at P12; negative pairs. The mixture of 1ul liposome negative control plasmid was injected into the vitreous cavity at P12. The expression of retina caveolin-1 mRNA and protein was detected by RT-PCR and Western blot in the 17 days of age (P17), and the expression of retinal albumin was detected by Western blot. Tissue sections were stained with HE to observe and count the number of endothelial cells that broke through the inner limiting membrane of the retina.
Results the expression of caveolin-1 mRNA and protein in the retina of the Caveolin-1 shRNA injection group was down 47.94% and 54.76%, respectively, and the difference was statistically significant (P0.05). The albumin content of retinal tissue in caveolin-1 shRNA injection group was 56.32% lower than that in the model control group, and the difference was statistically significant (P0.05); fluorescein angiography showed ca. Compared with the model control group, the retinal neovascularization in the veolin-1 shRNA injection group was significantly reduced and the fluorescence leakage was significantly reduced. The number of vascular endothelial nuclei in the caveolin-1 shRNA injection group was 51.3% less than that in the model control group, and the difference was statistically significant (P0.01).
Conclusion Caveolin-1 shRNA can effectively inhibit the leakage of retinal blood vessel albumin and inhibit the formation of retinal neovascularization, and further demonstrate the regulatory role of caveolin-1 in the development of retinal neovascularization, which provides a new target for the treatment of retinal neovascularization.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R774.1
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