Notch-Dll4在激光诱导的大鼠脉络膜新生血管中的作用
发布时间：2018-10-21 16:27
【摘要】： 【研究背景】脉络膜新生血管(CNV)是许多视网膜和脉络膜疾病的共同病理表现,常见CNV疾病包括年龄相关性黄斑变性(AMD)、特发性脉络膜新生血管、眼组织胞浆菌病综合征(POHS),以及眼底血管样条纹(AS)、病理性近视和外伤性脉络膜破裂等。新生血管从脉络膜长出后突破Bruch膜后长入视网膜色素上皮下间隙或神经上皮下,由于新生血管是不成熟的,易发生出血和渗漏等改变,继而形成瘢痕,造成局部损伤,这些损伤好发于黄斑区,对中心视力影响较大,因此CNV是造成视力丧失的重要原因之一。CNV的发生和发展是复杂的过程,受多种因素和因子精细而敏感的调控。血管内皮生长因子(VEGF)是目前已知的最强促血管生成因子,能够诱导新生血管生成并使血管通透性增高。抗VEGF治疗也是目前主流的CNV治疗方式。本研究组已证实Notch信号通路同样在CNV的生成中起重要作用,Notch信号通路中的Delta样配体4(Dll4)基因是除VEGF外目前发现的另一可因半剂量等位基因缺失而使动物在胚胎时期死亡的基因,Dll4在血管的发生发展中起重要作用。探讨Dll4在CNV的发生中可能的作用及其机制,可以为临床防治CNV相关疾病提供新的思路。 【目的】探讨Notch信号通路中Dll4在激光诱导的大鼠脉络膜新生血管生成中的作用机制。 【方法】将120只BN大鼠随机分为正常组(18只)、光凝组(54只)、光凝后实验组(24只)和光凝后对照组(24只)。532nm激光诱导建立双眼CNV模型。实验组在光凝后立刻玻璃体内注射25mg.mL-1的VEGF抗体Avastin 10 L,对照组在光凝后注射0.01mmol.L-1 PBS10 L。采用免疫荧光染色法检测7d时Dll4和VEGF在正常组(6只)和光凝组(6只)CNV中的表达。采用Western-blotting和RT-PCR方法检测正常组(12只)和光凝后1d(12只)、3 d(12只)、7 d(12只)和14 d(12只)组CNV中Dll4和VEGF的蛋白和mRNA表达趋势。7 d时采用Westernblotting和RT-PCR检测实验组(12只)和对照组(12只)CNV中Dll4和VEGF蛋白及mRNA表达含量的差异,14 d时通过组织切片HE染色和脉络膜铺片法检测实验组(12只)和对照组(12只)CNV生成的厚度和面积。 【结果】Dll4和VEGF在CNV区域有共表达。Dll4和VEGF蛋白和mRNA的表达趋势一致,均在第一天表达开始上升,第7天时达到高峰,第14天时开始下降。玻璃体内注射Avastin后,VEGF和Dll4表达均下降(P0.05),实验组CNV面积(24202±2608 m2,n=8)较对照组(37226±3208 m2,n=10)明显减小(P0.01),实验组CNV厚度(53.68±5.89 m,n=9)较对照组(82.86±7.46 m,n=11)明显降低(P0.01)。 【结论】Dll4在CNV生成中具有一定作用,可能参与了VEGF调控CNV生成的过程。
[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 卤2608 m2) was significantly lower than that in the control group (37226 卤3208 m2) (P0.01), and the thickness of CNV in the experimental group (53.68 卤5.89 mNNM) was significantly lower than that in the control group (82.86 卤7.46 mnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn Has a certain role in generation, It may be involved in the process of VEGF regulating CNV generation.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R773.4
本文编号：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 卤2608 m2) was significantly lower than that in the control group (37226 卤3208 m2) (P0.01), and the thickness of CNV in the experimental group (53.68 卤5.89 mNNM) was significantly lower than that in the control group (82.86 卤7.46 mnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn Has a certain role in generation, It may be involved in the process of VEGF regulating CNV generation.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R773.4
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