Unc5b-FC抑制小鼠视网膜新生血管生成的研究
发布时间：2018-08-18 17:01
【摘要】：第一章OIR模型鼠视网膜组织中netrin-1受体的表达 目的：研究已表明轴突生长因子-1(netrin-1)除诱导神经轴突生长外,还参与血管发育以及新生血管生成,我们的前期研究已经证明netrin-1shRNA可以抑制氧诱导视网膜病变(oxygen-induced retinopathy OIR)模型鼠的视网膜新生血管的形成。因此,本研究旨在探讨netrin-1的受体在OIR小鼠视网膜上的表达,以期明确何种受体介导netrin-1促进视网膜新生血管的形成。 方法：将出生后7天(P7)的C57BL/6J小鼠随机分为模型组及对照组,模型组24只,正常组21只。然后将出生后第7天(P7)模型组小鼠放入氧气含量为75%±2%的饲养箱中饲养,5天后(P12)返回到正常大气环境中饲养以诱导视网膜新生血管的形成。正常组则一直在空气中饲养。在出生后第17天(P17),分别取15只模型组小鼠和12只正常组小鼠,FITC-Dextran左心室灌注后行视网膜铺片,荧光显微镜下观察视网膜血管形态和分布(每组各3只)。组织学切片HE染色计算突破视网膜内界膜的新生血管内皮细胞核数量(每组各3只)。isolectinB4(血管内皮细胞标记物)染色标记血管内皮细胞,观察正常组和模型组血管形态的变化(每组各3只)。采用RT-PCR技术检测视网膜中netrin-1的7种受体unc5a,unc5b,unc5c,unc5d,DCC, neogenin,A2b的mRNA的表达差异(模型组3只)。采用免疫荧光法和isolectinB4染色检测netrin-1受体在视网膜上的表达定位(每组各3只)。提取视网膜蛋白质,采用Western blot技术检测P12、P17、P21模型组和正常组鼠视网膜组织中netrin-1的受体蛋白表达差异(每组各个时间点各3只,共18只)。 结果：FITC-Dextran荧光造影视网膜铺片结果显示,模型组小鼠在出生后第17天(P17)时视网膜有大量新生血管形成,视盘周围见大范围无灌注区。组织切片HE染色结果显示,模型组可见大量突破视网膜内界膜的新生血管内皮细胞核；isolectinB4染色结果显示,模型组突破视网膜内界膜的血管内皮细胞明显增多,且排列不规则。RT-PCR结果显示模型鼠视网膜组织中除unc5a外,netrin-1其余6种受体unc5b-d、DCC、A2b、neogenin的mRNA均在P17模型小鼠视网膜中表达。western-blot结果显示,仅unc5b在模型鼠视网膜新生血管形成活跃期(P17、P21天)表达上调,其余的5种受体unc5c、unc5d、DCC、 A2b、neogenin在模型组和正常组中表达水平无明显差异。免疫荧光染色结果显示,unc5b-d、DCC、A2b、neogenin6种受体均在视网膜的神经节细胞层、内网层和外网层表达。双标染色结果显示,仅unc5b和neogenin同时表达在模型组视网膜新生血管上。 结论：(1)受体unc5b在鼠视网膜新生血管形成过程中表达上调,且在视网膜新生血管内皮细胞上表达阳性,因此该受体可能参与了小鼠视网膜新生血管的形成。(2)受体neogenin在视网膜新生血管内皮细胞上表达阳性,故该因子也可能参与了小鼠视网膜血管新生。 第二章unc5b-FC抑制小鼠视网膜新生血管的形成 目的：观察unc5b-FC对鼠视网膜新生血管形成的抑制作用,进一步阐明unc5b对鼠视网膜的新生血管形成的调控作用。方法：将9只鼠龄为7天(P7)的C57BL/6J小鼠置于浓度为75%±2%高氧环境中,5天后(P12)返回正常氧环境中以诱导新生血管的形成。9只小鼠于出氧舱后当日(P12)一只眼玻璃体腔内注射1u1unc5b-FC (1ug/ul),对侧眼注射lu1PBS。5天后(P17),采用FITC-dextran左心室造影视网膜铺片观察视网膜血管形态的改变；组织学切片isolectinB4血管内皮细胞染色观察新生血管内皮细胞的形态,HE染色计算突破视网膜内界膜的新生血管内皮细胞核的数量。 结果：视网膜铺片结果显示,unc5b-FC注射眼视网膜荧光素渗漏现象较PBS注射眼明显减少。isolectinB4血管内皮细胞染色结果显示,unc5b-FC注射眼血管内皮细胞,尤其是突破内界膜的血管内皮细胞较PBS注射眼明显减少。组织学切片HE染色结果显示,unc5b-FC注射眼突破视网膜内界膜的血管内皮细胞核数较PBS注射眼明显减少(P0.05)。 结论：unc5b-FC能有效地抑制鼠视网膜新生血管的形成,unc5b可能参与了鼠视网膜新生血管的形成。
[Abstract]:Chapter 1 expression of netrin-1 receptor in OIR model rat retina
AIM: Studies have shown that axon growth factor-1 (netrin-1) is involved in angiogenesis and angiogenesis in addition to axon growth. Our previous studies have shown that netrin-1 shRNA can inhibit retinal neovascularization in oxygen-induced retinopathy (OIR) mice. The aim of this study was to investigate the expression of netrin-1 receptor in the retina of OIR mice and to determine which receptor mediates the formation of retinal neovascularization.
Methods: C57BL/6J mice were randomly divided into model group and control group at 7 days after birth (P7), 24 mice in model group and 21 mice in normal group. On the 17th day after birth (P17), 15 mice in the model group and 12 mice in the normal group were taken respectively. After left ventricular perfusion with FITC-Dextran, retinal slices were made. The morphology and distribution of retinal blood vessels were observed under fluorescence microscope (3 in each group). Histological slices of HE staining were used to calculate the neovascularization breaking through the retinal inner limiting membrane. Number of endothelial cell nuclei (3 in each group). Vascular endothelial cells were stained with Isolectin B4 (endothelial cell marker). Vascular morphological changes were observed in normal and model groups (3 in each group). The mRNA expression of netrin-1 receptors unc5a, unc5b, unc5c, unc5d, DCC, neogenin, A2b in retina was detected by RT-PCR. Immunofluorescence and Isolectin B4 staining were used to detect the expression and localization of netrin-1 receptor in the retina (3 in each group). Protein was extracted from the retina. The expression of netrin-1 receptor protein in the retina of P12, P17, P21 model group and normal group was detected by Western blot.
Results: The results of FITC-Dextran fluorescence imaging showed that a large number of neovascularization was found in the retina of the model group on the 17th day (P17) after birth, and a large area of non-perfusion was found around the optic disc. The results of RT-PCR showed that the expression of unc5b-d, DCC, A2b, and neogenin mRNA in the retina of P17 model mice except unc5a was detected in the retina of the model mice. The expression of unc5c, unc5d, DCC, A2b, and neogenin was up-regulated in the active phase of retinal neovascularization (P17, P21 days) in model group and normal control group. Immunofluorescence staining showed that unc5b-d, DCC, A2b, and neogenin receptors were all expressed in ganglion cell layer, inner and outer retinal layer of retina. Double labeled staining showed that only unc5b and neogenin were expressed in retinal neovascularization of model group.
Conclusion: (1) Receptor unc5b is up-regulated in retinal neovascularization and positive in retinal endothelial cells, so it may be involved in retinal neovascularization in mice. (2) Receptor neogenin is positive in retinal neovascular endothelial cells, so this factor may also be involved. Retinal angiogenesis in mice.
Second chapter unc5b-FC inhibits the formation of retinal neovascularization in mice.
AIM: To observe the inhibitory effect of unc5b-FC on retinal neovascularization in mice, and further elucidate the regulatory effect of unc5b on retinal neovascularization in mice. Methods: Nine C57BL/6J mice aged 7 days (P7) were exposed to 75% + 2% hyperoxia for 5 days (P12) to induce retinal neovascularization. Nine mice were injected with 1u1unc5b-FC (1ug/ul) into vitreous cavity in one eye on the same day (P12) after exposure to oxygen chamber, and lu1PBS was injected into the contralateral eye 5 days later (P17). The morphology of retinal vascular endothelial cells was observed by FITC-dextran left ventriculography. HE staining was used to calculate the number of endothelial cells that broke through the inner limiting membrane of the retina.
Result: The fluorescein leakage in the eyes injected with unc5b-FC was significantly less than that in the eyes injected with PBS. The results of Isolectin B4 staining showed that the number of endothelial cells injected with unc5b-FC, especially the endothelial cells breaking through the inner limiting membrane, was significantly lower than that in the eyes injected with PBS. The results showed that the number of endothelial cell nuclei in unc5b-FC injected eyes was significantly lower than that in PBS injected eyes (P 0.05).
Conclusion: unc5b-FC can effectively inhibit the formation of retinal neovascularization in rats, and unc5b may be involved in the formation of retinal neovascularization in rats.
【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
本文编号：2190112
[Abstract]:Chapter 1 expression of netrin-1 receptor in OIR model rat retina
AIM: Studies have shown that axon growth factor-1 (netrin-1) is involved in angiogenesis and angiogenesis in addition to axon growth. Our previous studies have shown that netrin-1 shRNA can inhibit retinal neovascularization in oxygen-induced retinopathy (OIR) mice. The aim of this study was to investigate the expression of netrin-1 receptor in the retina of OIR mice and to determine which receptor mediates the formation of retinal neovascularization.
Methods: C57BL/6J mice were randomly divided into model group and control group at 7 days after birth (P7), 24 mice in model group and 21 mice in normal group. On the 17th day after birth (P17), 15 mice in the model group and 12 mice in the normal group were taken respectively. After left ventricular perfusion with FITC-Dextran, retinal slices were made. The morphology and distribution of retinal blood vessels were observed under fluorescence microscope (3 in each group). Histological slices of HE staining were used to calculate the neovascularization breaking through the retinal inner limiting membrane. Number of endothelial cell nuclei (3 in each group). Vascular endothelial cells were stained with Isolectin B4 (endothelial cell marker). Vascular morphological changes were observed in normal and model groups (3 in each group). The mRNA expression of netrin-1 receptors unc5a, unc5b, unc5c, unc5d, DCC, neogenin, A2b in retina was detected by RT-PCR. Immunofluorescence and Isolectin B4 staining were used to detect the expression and localization of netrin-1 receptor in the retina (3 in each group). Protein was extracted from the retina. The expression of netrin-1 receptor protein in the retina of P12, P17, P21 model group and normal group was detected by Western blot.
Results: The results of FITC-Dextran fluorescence imaging showed that a large number of neovascularization was found in the retina of the model group on the 17th day (P17) after birth, and a large area of non-perfusion was found around the optic disc. The results of RT-PCR showed that the expression of unc5b-d, DCC, A2b, and neogenin mRNA in the retina of P17 model mice except unc5a was detected in the retina of the model mice. The expression of unc5c, unc5d, DCC, A2b, and neogenin was up-regulated in the active phase of retinal neovascularization (P17, P21 days) in model group and normal control group. Immunofluorescence staining showed that unc5b-d, DCC, A2b, and neogenin receptors were all expressed in ganglion cell layer, inner and outer retinal layer of retina. Double labeled staining showed that only unc5b and neogenin were expressed in retinal neovascularization of model group.
Conclusion: (1) Receptor unc5b is up-regulated in retinal neovascularization and positive in retinal endothelial cells, so it may be involved in retinal neovascularization in mice. (2) Receptor neogenin is positive in retinal neovascular endothelial cells, so this factor may also be involved. Retinal angiogenesis in mice.
Second chapter unc5b-FC inhibits the formation of retinal neovascularization in mice.
AIM: To observe the inhibitory effect of unc5b-FC on retinal neovascularization in mice, and further elucidate the regulatory effect of unc5b on retinal neovascularization in mice. Methods: Nine C57BL/6J mice aged 7 days (P7) were exposed to 75% + 2% hyperoxia for 5 days (P12) to induce retinal neovascularization. Nine mice were injected with 1u1unc5b-FC (1ug/ul) into vitreous cavity in one eye on the same day (P12) after exposure to oxygen chamber, and lu1PBS was injected into the contralateral eye 5 days later (P17). The morphology of retinal vascular endothelial cells was observed by FITC-dextran left ventriculography. HE staining was used to calculate the number of endothelial cells that broke through the inner limiting membrane of the retina.
Result: The fluorescein leakage in the eyes injected with unc5b-FC was significantly less than that in the eyes injected with PBS. The results of Isolectin B4 staining showed that the number of endothelial cells injected with unc5b-FC, especially the endothelial cells breaking through the inner limiting membrane, was significantly lower than that in the eyes injected with PBS. The results showed that the number of endothelial cell nuclei in unc5b-FC injected eyes was significantly lower than that in PBS injected eyes (P 0.05).
Conclusion: unc5b-FC can effectively inhibit the formation of retinal neovascularization in rats, and unc5b may be involved in the formation of retinal neovascularization in rats.
【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
【共引文献】
相关期刊论文 前2条
1 熊思齐;夏晓波;蒋剑;孙伟;;轴突导向因子-1 mRNA在氧诱导血管增生性视网膜病变中的表达[J];眼科研究;2009年02期
2 Xia Zhang;Jiaolian Liu;Siqi Xiong;Xiaobo Xia;Huizhuo Xu;;Expression of Netrin-1 in Diabetic Rat Retina[J];Eye Science;2013年03期
相关博士学位论文 前2条
1 谢涵;Netrin-1基因对胎盘血管生成作用的体内、外研究[D];华中科技大学;2011年
2 刘矫连;慢病毒介导的Netrin-1小发夹状RNA在视网膜新生血管形成中的调控作用[D];中南大学;2011年
相关硕士学位论文 前2条
1 林海华;脂筏—依赖性受体DCC信号在Netrin-1调节肝癌细胞极性中的作用研究[D];华中科技大学;2011年
2 赵淼;轴突诱向因子Netrin-1蛋白在早期糖尿病视网膜病变中的表达[D];首都医科大学;2013年
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