Notch-Dll4在激光誘導(dǎo)的大鼠脈絡(luò)膜新生血管中的作用
發(fā)布時(shí)間：2018-10-21 16:27
【摘要】： 【研究背景】脈絡(luò)膜新生血管(CNV)是許多視網(wǎng)膜和脈絡(luò)膜疾病的共同病理表現(xiàn),常見CNV疾病包括年齡相關(guān)性黃斑變性(AMD)、特發(fā)性脈絡(luò)膜新生血管、眼組織胞漿菌病綜合征(POHS),以及眼底血管樣條紋(AS)、病理性近視和外傷性脈絡(luò)膜破裂等。新生血管從脈絡(luò)膜長(zhǎng)出后突破Bruch膜后長(zhǎng)入視網(wǎng)膜色素上皮下間隙或神經(jīng)上皮下,由于新生血管是不成熟的,易發(fā)生出血和滲漏等改變,繼而形成瘢痕,造成局部損傷,這些損傷好發(fā)于黃斑區(qū),對(duì)中心視力影響較大,因此CNV是造成視力喪失的重要原因之一。CNV的發(fā)生和發(fā)展是復(fù)雜的過(guò)程,受多種因素和因子精細(xì)而敏感的調(diào)控。血管內(nèi)皮生長(zhǎng)因子(VEGF)是目前已知的最強(qiáng)促血管生成因子,能夠誘導(dǎo)新生血管生成并使血管通透性增高�？筕EGF治療也是目前主流的CNV治療方式。本研究組已證實(shí)Notch信號(hào)通路同樣在CNV的生成中起重要作用,Notch信號(hào)通路中的Delta樣配體4(Dll4)基因是除VEGF外目前發(fā)現(xiàn)的另一可因半劑量等位基因缺失而使動(dòng)物在胚胎時(shí)期死亡的基因,Dll4在血管的發(fā)生發(fā)展中起重要作用。探討Dll4在CNV的發(fā)生中可能的作用及其機(jī)制,可以為臨床防治CNV相關(guān)疾病提供新的思路。 【目的】探討Notch信號(hào)通路中Dll4在激光誘導(dǎo)的大鼠脈絡(luò)膜新生血管生成中的作用機(jī)制。 【方法】將120只BN大鼠隨機(jī)分為正常組(18只)、光凝組(54只)、光凝后實(shí)驗(yàn)組(24只)和光凝后對(duì)照組(24只)。532nm激光誘導(dǎo)建立雙眼CNV模型。實(shí)驗(yàn)組在光凝后立刻玻璃體內(nèi)注射25mg.mL-1的VEGF抗體Avastin 10 L,對(duì)照組在光凝后注射0.01mmol.L-1 PBS10 L。采用免疫熒光染色法檢測(cè)7d時(shí)Dll4和VEGF在正常組(6只)和光凝組(6只)CNV中的表達(dá)。采用Western-blotting和RT-PCR方法檢測(cè)正常組(12只)和光凝后1d(12只)、3 d(12只)、7 d(12只)和14 d(12只)組CNV中Dll4和VEGF的蛋白和mRNA表達(dá)趨勢(shì)。7 d時(shí)采用Westernblotting和RT-PCR檢測(cè)實(shí)驗(yàn)組(12只)和對(duì)照組(12只)CNV中Dll4和VEGF蛋白及mRNA表達(dá)含量的差異,14 d時(shí)通過(guò)組織切片HE染色和脈絡(luò)膜鋪片法檢測(cè)實(shí)驗(yàn)組(12只)和對(duì)照組(12只)CNV生成的厚度和面積。 【結(jié)果】Dll4和VEGF在CNV區(qū)域有共表達(dá)。Dll4和VEGF蛋白和mRNA的表達(dá)趨勢(shì)一致,均在第一天表達(dá)開始上升,第7天時(shí)達(dá)到高峰,第14天時(shí)開始下降。玻璃體內(nèi)注射Avastin后,VEGF和Dll4表達(dá)均下降(P0.05),實(shí)驗(yàn)組CNV面積(24202±2608 m2,n=8)較對(duì)照組(37226±3208 m2,n=10)明顯減小(P0.01),實(shí)驗(yàn)組CNV厚度(53.68±5.89 m,n=9)較對(duì)照組(82.86±7.46 m,n=11)明顯降低(P0.01)。 【結(jié)論】Dll4在CNV生成中具有一定作用,可能參與了VEGF調(diào)控CNV生成的過(guò)程。
[Abstract]:[background] choroidal neovascularization (CNV) is a common pathological feature of many retinal and choroidal diseases. Common CNV diseases include age-related macular degeneration (AMD),) idiopathic choroidal neovascularization. Ocular histiocytoplasmosis syndrome (POHS), fundus vascular stripe (AS), (AS), myopia and traumatic choroidal rupture. After the neovascularization breaks through the Bruch membrane from the choroid, it grows into the subcutaneous space of retinal pigment or nerve. Because the neovascularization is immature, it is prone to haemorrhage and leakage, and then it forms scar and causes local injury. CNV is one of the important causes of visual loss. The occurrence and development of CNV is a complicated process, which is controlled by many factors and factors. Vascular endothelial growth factor (VEGF) is the strongest known angiogenic factor, which can induce angiogenesis and increase vascular permeability. Anti-VEGF therapy is also the mainstream of CNV treatment. Our team has confirmed that the Notch signaling pathway also plays an important role in the production of CNV. The Delta like ligand 4 (Dll4) gene in the Notch signaling pathway is another currently discovered gene in addition to VEGF that causes the loss of half dose alleles in animals at embryonic stage. The death gene, Dll4, plays an important role in the development of blood vessels. To explore the possible role and mechanism of Dll4 in the pathogenesis of CNV. [objective] to explore the role of Dll4 in laser-induced choroidal neovascularization in rats. [methods] 120 BN rats were randomly divided into normal group (n = 18), photocoagulation group (n = 54), experimental group (n = 24) and control group (n = 24). Bilateral CNV model was induced by 532nm laser. The VEGF antibody Avastin 10 L of 25mg.mL-1 was injected into the vitreous of the experimental group immediately after photocoagulation, and that of the control group was injected with 0.01mmol.L-1 PBS10 L after photocoagulation. Immunofluorescence staining was used to detect the expression of Dll4 and VEGF in normal group (6 rats) and photocoagulation group (6 rats) at day 7. Western-blotting and RT-PCR were used to detect the expression trend of Dll4 and VEGF protein and mRNA in CNV of normal group (12 rats), 1 day (12 rats), 3 days (12 rats), 7 days (12 rats) and 14 days (12 rats). Westernblotting and RT-PCR were used to detect the expression trend of Dll4 and VEGF protein and mRNA in CNV at 7 days. Westernblotting and RT-PCR were used to detect the expression of Dll4 and VEGF in the experimental group (12 rats) and the control group (12 rats) at 7 days. The difference of Dll4 and VEGF protein and mRNA expression in CNV. The thickness and area of CNV production in experimental group (12 rats) and control group (12 rats) were detected by HE staining and choroidal laying method at day 14. [results] Dll4 and VEGF coexpressed in CNV region. The expression trend of Dll4 and VEGF protein and mRNA were the same. The expression began to rise on the first day, peaked on the 7th day, and began to decrease on the 14th day. After intravitreous injection of Avastin, the expression of VEGF and Dll4 decreased (P0.05). The area of CNV in the experimental group (24202 鹵2608 m2) was significantly lower than that in the control group (37226 鹵3208 m2) (P0.01), and the thickness of CNV in the experimental group (53.68 鹵5.89 mNNM) was significantly lower than that in the control group (82.86 鹵7.46 mnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn Has a certain role in generation, It may be involved in the process of VEGF regulating CNV generation.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R773.4
本文編號(hào)：2285689
[Abstract]:[background] choroidal neovascularization (CNV) is a common pathological feature of many retinal and choroidal diseases. Common CNV diseases include age-related macular degeneration (AMD),) idiopathic choroidal neovascularization. Ocular histiocytoplasmosis syndrome (POHS), fundus vascular stripe (AS), (AS), myopia and traumatic choroidal rupture. After the neovascularization breaks through the Bruch membrane from the choroid, it grows into the subcutaneous space of retinal pigment or nerve. Because the neovascularization is immature, it is prone to haemorrhage and leakage, and then it forms scar and causes local injury. CNV is one of the important causes of visual loss. The occurrence and development of CNV is a complicated process, which is controlled by many factors and factors. Vascular endothelial growth factor (VEGF) is the strongest known angiogenic factor, which can induce angiogenesis and increase vascular permeability. Anti-VEGF therapy is also the mainstream of CNV treatment. Our team has confirmed that the Notch signaling pathway also plays an important role in the production of CNV. The Delta like ligand 4 (Dll4) gene in the Notch signaling pathway is another currently discovered gene in addition to VEGF that causes the loss of half dose alleles in animals at embryonic stage. The death gene, Dll4, plays an important role in the development of blood vessels. To explore the possible role and mechanism of Dll4 in the pathogenesis of CNV. [objective] to explore the role of Dll4 in laser-induced choroidal neovascularization in rats. [methods] 120 BN rats were randomly divided into normal group (n = 18), photocoagulation group (n = 54), experimental group (n = 24) and control group (n = 24). Bilateral CNV model was induced by 532nm laser. The VEGF antibody Avastin 10 L of 25mg.mL-1 was injected into the vitreous of the experimental group immediately after photocoagulation, and that of the control group was injected with 0.01mmol.L-1 PBS10 L after photocoagulation. Immunofluorescence staining was used to detect the expression of Dll4 and VEGF in normal group (6 rats) and photocoagulation group (6 rats) at day 7. Western-blotting and RT-PCR were used to detect the expression trend of Dll4 and VEGF protein and mRNA in CNV of normal group (12 rats), 1 day (12 rats), 3 days (12 rats), 7 days (12 rats) and 14 days (12 rats). Westernblotting and RT-PCR were used to detect the expression trend of Dll4 and VEGF protein and mRNA in CNV at 7 days. Westernblotting and RT-PCR were used to detect the expression of Dll4 and VEGF in the experimental group (12 rats) and the control group (12 rats) at 7 days. The difference of Dll4 and VEGF protein and mRNA expression in CNV. The thickness and area of CNV production in experimental group (12 rats) and control group (12 rats) were detected by HE staining and choroidal laying method at day 14. [results] Dll4 and VEGF coexpressed in CNV region. The expression trend of Dll4 and VEGF protein and mRNA were the same. The expression began to rise on the first day, peaked on the 7th day, and began to decrease on the 14th day. After intravitreous injection of Avastin, the expression of VEGF and Dll4 decreased (P0.05). The area of CNV in the experimental group (24202 鹵2608 m2) was significantly lower than that in the control group (37226 鹵3208 m2) (P0.01), and the thickness of CNV in the experimental group (53.68 鹵5.89 mNNM) was significantly lower than that in the control group (82.86 鹵7.46 mnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn Has a certain role in generation, It may be involved in the process of VEGF regulating CNV generation.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R773.4
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