Unc5b-FC抑制小鼠視網(wǎng)膜新生血管生成的研究
發(fā)布時間：2018-08-18 17:01
【摘要】：第一章OIR模型鼠視網(wǎng)膜組織中netrin-1受體的表達 目的：研究已表明軸突生長因子-1(netrin-1)除誘導神經(jīng)軸突生長外,還參與血管發(fā)育以及新生血管生成,我們的前期研究已經(jīng)證明netrin-1shRNA可以抑制氧誘導視網(wǎng)膜病變(oxygen-induced retinopathy OIR)模型鼠的視網(wǎng)膜新生血管的形成。因此,本研究旨在探討netrin-1的受體在OIR小鼠視網(wǎng)膜上的表達,以期明確何種受體介導netrin-1促進視網(wǎng)膜新生血管的形成。 方法：將出生后7天(P7)的C57BL/6J小鼠隨機分為模型組及對照組,模型組24只,正常組21只。然后將出生后第7天(P7)模型組小鼠放入氧氣含量為75%±2%的飼養(yǎng)箱中飼養(yǎng),5天后(P12)返回到正常大氣環(huán)境中飼養(yǎng)以誘導視網(wǎng)膜新生血管的形成。正常組則一直在空氣中飼養(yǎng)。在出生后第17天(P17),分別取15只模型組小鼠和12只正常組小鼠,FITC-Dextran左心室灌注后行視網(wǎng)膜鋪片,熒光顯微鏡下觀察視網(wǎng)膜血管形態(tài)和分布(每組各3只)。組織學切片HE染色計算突破視網(wǎng)膜內界膜的新生血管內皮細胞核數(shù)量(每組各3只)。isolectinB4(血管內皮細胞標記物)染色標記血管內皮細胞,觀察正常組和模型組血管形態(tài)的變化(每組各3只)。采用RT-PCR技術檢測視網(wǎng)膜中netrin-1的7種受體unc5a,unc5b,unc5c,unc5d,DCC, neogenin,A2b的mRNA的表達差異(模型組3只)。采用免疫熒光法和isolectinB4染色檢測netrin-1受體在視網(wǎng)膜上的表達定位(每組各3只)。提取視網(wǎng)膜蛋白質,采用Western blot技術檢測P12、P17、P21模型組和正常組鼠視網(wǎng)膜組織中netrin-1的受體蛋白表達差異(每組各個時間點各3只,共18只)。 結果：FITC-Dextran熒光造影視網(wǎng)膜鋪片結果顯示,模型組小鼠在出生后第17天(P17)時視網(wǎng)膜有大量新生血管形成,視盤周圍見大范圍無灌注區(qū)。組織切片HE染色結果顯示,模型組可見大量突破視網(wǎng)膜內界膜的新生血管內皮細胞核；isolectinB4染色結果顯示,模型組突破視網(wǎng)膜內界膜的血管內皮細胞明顯增多,且排列不規(guī)則。RT-PCR結果顯示模型鼠視網(wǎng)膜組織中除unc5a外,netrin-1其余6種受體unc5b-d、DCC、A2b、neogenin的mRNA均在P17模型小鼠視網(wǎng)膜中表達。western-blot結果顯示,僅unc5b在模型鼠視網(wǎng)膜新生血管形成活躍期(P17、P21天)表達上調,其余的5種受體unc5c、unc5d、DCC、 A2b、neogenin在模型組和正常組中表達水平無明顯差異。免疫熒光染色結果顯示,unc5b-d、DCC、A2b、neogenin6種受體均在視網(wǎng)膜的神經(jīng)節(jié)細胞層、內網(wǎng)層和外網(wǎng)層表達。雙標染色結果顯示,僅unc5b和neogenin同時表達在模型組視網(wǎng)膜新生血管上。 結論：(1)受體unc5b在鼠視網(wǎng)膜新生血管形成過程中表達上調,且在視網(wǎng)膜新生血管內皮細胞上表達陽性,因此該受體可能參與了小鼠視網(wǎng)膜新生血管的形成。(2)受體neogenin在視網(wǎng)膜新生血管內皮細胞上表達陽性,故該因子也可能參與了小鼠視網(wǎng)膜血管新生。 第二章unc5b-FC抑制小鼠視網(wǎng)膜新生血管的形成 目的：觀察unc5b-FC對鼠視網(wǎng)膜新生血管形成的抑制作用,進一步闡明unc5b對鼠視網(wǎng)膜的新生血管形成的調控作用。方法：將9只鼠齡為7天(P7)的C57BL/6J小鼠置于濃度為75%±2%高氧環(huán)境中,5天后(P12)返回正常氧環(huán)境中以誘導新生血管的形成。9只小鼠于出氧艙后當日(P12)一只眼玻璃體腔內注射1u1unc5b-FC (1ug/ul),對側眼注射lu1PBS。5天后(P17),采用FITC-dextran左心室造影視網(wǎng)膜鋪片觀察視網(wǎng)膜血管形態(tài)的改變；組織學切片isolectinB4血管內皮細胞染色觀察新生血管內皮細胞的形態(tài),HE染色計算突破視網(wǎng)膜內界膜的新生血管內皮細胞核的數(shù)量。 結果：視網(wǎng)膜鋪片結果顯示,unc5b-FC注射眼視網(wǎng)膜熒光素滲漏現(xiàn)象較PBS注射眼明顯減少。isolectinB4血管內皮細胞染色結果顯示,unc5b-FC注射眼血管內皮細胞,尤其是突破內界膜的血管內皮細胞較PBS注射眼明顯減少。組織學切片HE染色結果顯示,unc5b-FC注射眼突破視網(wǎng)膜內界膜的血管內皮細胞核數(shù)較PBS注射眼明顯減少(P0.05)。 結論：unc5b-FC能有效地抑制鼠視網(wǎng)膜新生血管的形成,unc5b可能參與了鼠視網(wǎng)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.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R774.1
本文編號：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.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R774.1
【共引文獻】
相關期刊論文 前2條
1 熊思齊;夏曉波;蔣劍;孫偉;;軸突導向因子-1 mRNA在氧誘導血管增生性視網(wǎng)膜病變中的表達[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
相關博士學位論文 前2條
1 謝涵;Netrin-1基因對胎盤血管生成作用的體內、外研究[D];華中科技大學;2011年
2 劉矯連;慢病毒介導的Netrin-1小發(fā)夾狀RNA在視網(wǎng)膜新生血管形成中的調控作用[D];中南大學;2011年
相關碩士學位論文 前2條
1 林海華;脂筏—依賴性受體DCC信號在Netrin-1調節(jié)肝癌細胞極性中的作用研究[D];華中科技大學;2011年
2 趙淼;軸突誘向因子Netrin-1蛋白在早期糖尿病視網(wǎng)膜病變中的表達[D];首都醫(yī)科大學;2013年
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