超声爆破微泡联合Avastin治疗兔眼脉络膜新生血管的实验研究

发布时间：2018-02-26 13:06

本文关键词： 超声微泡造影剂脉络膜新生血管血管内皮生长因子抗血管内皮生长因子单克隆抗体　出处：《重庆医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 脉络膜新生血管(CNV)与许多眼底疾病有关,是致盲的常见原因[1]。而目前的治疗方法存在许多缺点,如:方法复杂、昂贵、疗效欠稳定等。由于在CNV形成和发展过程中血管内皮生长因子(VEGF)发挥着中心调控作用,阻断VEGF的作用已成为目前国内外CNV治疗方案中研究的热点和重点。因此近年有学者将被美国FDA批准的通过抑制血管生成发挥抗癌作用的新药Avastin(即bevacizumab,血管内皮生长因子单克隆抗体)用于眼部新生血管性疾病,特别是年龄相关性黄斑变性的治疗,并取得了较好的治疗效果。而超声微泡造影剂作为一种新型声学造影剂,爆破过程中可有效增加局部组织通透性,提高局部药物浓度,以达到靶向治疗作用。本研究首先利用氩绿激光构建了兔眼脉络膜新生血管模型,其次将超声爆破微泡与抗血管内皮生长因子单克隆抗体Avastin (bevacizumab)联合,用于兔CNV的治疗,观察VEGF蛋白的表达和对CNV的抑制情况,为临床治疗CNV提供了一种新的思路。方法:动物模型的建造:用氩绿激光(波长514.5nm)距视乳头2~3个视盘直径的颞侧髓线上下视盘网膜密集处照射20个点,激光光斑间隔300μm,激光光斑直径50μm,激光功率0.7 W,曝光时间0.1 s,建造实验性色素兔脉络膜新生血管模型。在造模后第21d进行组织病理学和FFA观察,确定CNV的形成。造模成功后再进行以下研究:将CNV兔分为对照组:不做任何处理; Avastin组(A):玻璃体内注射Avastin;超声微泡+Avastin组(U+MB+A):玻璃体腔内注射Avastin及超声微泡,再用频率1MHZ,声强分别为0.5W/cm2超声辐照眼球60s,工作时间20%超,每周两次,处理后7d、14d、28d用免疫荧光及Western-blot检测VEGF蛋白表达,FFA观察CNV的抑制情况。结果:在分组处理后7d、14d、28d超声微泡+Avastin组免疫荧光及Western-blot检测VEGF蛋白表达均明显低于Avastin组(p0.05),FFA结果显示超声微泡+Avastin组荧光渗漏吸光度(A值)明显低于Avastin组(p0.05),且与对照组差异均有统计学意义(p0.05)。以28天作为疗效判定时间点,FFA检查结果显示Avastin组荧光渗漏平均强度为66.96±4.41 ,与对照组(119.60±6.57 )相比,差异有统计学意义(t=16.2952;p=0.0000);超声微泡+Avastin组为54.75±4.41,与Avastin组相比,差异有统计学意义(t=4.7955;p=0.0000)。且各组荧光渗漏强度随时间变化均呈下降趋势。分组处理后28d,组织免疫荧光及Western-blot检测VEGF蛋白表达结果显示,Avastin组为23.9825±3.3180与0.5666±0.0179,与对照组相比,差异有统计学意义(t=7.0327,p=0.0000;t=9.2596,P=0.0000);超声微泡+Avastin组为19.5636±1.5006与0.3214±0.030 ,与Avastin组相比,差异有统计学意义(t=2.9724,p=0.0140;t=17.1 937,p=0.0000),且各组VEGF蛋白表达随时间变化呈下降趋势。结论: 1、采用一定波长、功率的氩绿激光进行视网膜光凝方法,可较好复制实验性色素兔脉络膜新生血管模型。 2、玻璃体腔注射Avastin,对脉络膜新生血管有一定疗效 3、超声微泡与Avastin联合使用时,可有效增加Avastin对脉络膜新生血管的治疗效果。
[Abstract]:Choroidal neovascularization (CNV) associated with many fundus diseases, [1]. is a common cause of blindness and the current treatment methods have many disadvantages, such as complex, expensive, less effective and stable. The formation and development of vascular endothelial growth factor in the process of CNV (VEGF) plays a key role in regulating, blocking VEGF the effect has become the hotspot and focus of research at home and abroad CNV therapy. So in recent years scholars will be approved by the FDA to play a role in cancer drug Avastin through inhibition of angiogenesis (bevacizumab, vascular endothelial growth factor monoclonal antibody) for ocular neovascular diseases, especially in the treatment of age-related macular degeneration. And get a better therapeutic effect. Ultrasound microbubble contrast agent as a new contrast agent, the blasting process can effectively increase the local tissue permeability, improve local drug In order to achieve the concentration, the effect of targeted therapy. In this study, the use of argon green laser was constructed rabbit choroidal neovascularization model, followed by ultrasonic microbubble and anti VEGF monoclonal antibody Avastin (bevacizumab), for the treatment of rabbit CNV, to observe the expression of VEGF protein and inhibition of CNV. Provides a new way for the treatment of CNV.
Methods: to build animal model with argon green laser (wavelength 514.5nm) from the optic disc diameter 2~3 temporal pith line of optic disc intensive irradiation omentum 20 points, laser spot spacing 300 m, laser spot diameter of 50 m and 0.7 W laser power, exposure time of 0.1 s, the construction of experimental pigmented choroidal neovascularization in rabbits model. After modeling the 21d pathology and FFA observation, determine the formation of CNV.
The following research again after the success of the model: the CNV rabbits were divided into control group: no treatment; group Avastin (A): Intravitreal injection of Avastin; ultrasound microbubble group +Avastin (U+MB+A): Intravitreal injection of Avastin and ultrasound, then the frequency 1MHZ, sound intensity were 0.5W/cm2 ultrasound irradiation of eyeball 60s, working time of 20%, two times a week, after the treatment of 7D, 14d, 28d, VEGF protein was detected by immunofluorescence and the expression of Western-blot, inhibition of FFA observation of CNV.
Results: in group 7d after treatment, 14d, 28d ultrasound microbubble group +Avastin immunofluorescence detection of VEGF protein and Western-blot expression were significantly lower than that of Avastin group (P0.05), FFA results showed that ultrasound microbubble group +Avastin fluorescence leakage absorbance (A value) was significantly lower than that of Avastin group (P0.05) and the control group, and the differences were statistically significant (P0.05) for 28 days. As the effect of the judgment time, FFA examination showed that Avastin group average fluorescence leakage intensity was 66.96 + 4.41, and the control group (119.60 + 6.57) compared to the difference was statistically significant (t=16.2952; p=0.0000); ultrasoundmicrobubble +Avastin group is 54.75 + 4.41, compared with the Avastin group, a statistically significant differences (t=4.7955; p=0.0000). And each group of fluorescence leakage intensity decreased. After 28d, the expression of VEGF by immunofluorescence and the expression of Western-blot showed that the Avastin group is 23.9825 + 3.3180 With 0.5666 + 0.0179, compared with the control group, the difference was statistically significant (t=7.0327, p=0.0000; t=9.2596, P=0.0000); ultrasoundmicrobubble +Avastin group is 19.5636 + 1.5006 and 0.3214 + 0.030, compared with the Avastin group, the difference was statistically significant (t=2.9724, p=0.0140; t=17.1 937, p=0.0000), and each group of VEGF expression downward trend changes with time.
Conclusion:
1, the retinal photocoagulation method with a certain wavelength and power argon green laser can be used to reproduce the experimental pigmented rabbit choroidal neovascularization model.
2, the intravitreal injection of Avastin has a certain effect on the choroidal neovascularization.
3, the combined use of ultrasound microbubbles and Avastin can effectively increase the therapeutic effect of Avastin on choroidal neovascularization.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R774.1

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