超声爆破微泡联合抗血管内皮生长因子单克隆抗体bevacizumab治疗激光诱导的兔眼脉络膜新生血管

目的　观察超声爆破微泡联合抗血管内皮生长因子（VEGF）单克隆抗体bevacizumab（商品名Avastin）对兔脉络膜新生血管（CNV）的治疗效果。方法 30只有色家兔60只眼通过氩绿激光视网膜激光光凝的方法建立CNV模型。激光光凝后21 d行荧光素眼底血管造影（FFA），并于造影6~8 h后任意选择3只兔处死，摘取眼球，行苏木精-伊红（HE）染色组织学检查，判断CNV建模是否成功。于激光光凝后21 d,将27只CNV模型兔随机分成空白对照组、bevacizumb组及超声微泡+bevacizumb组，每组各9只兔。空白对照组不做任何处理，bevacizumab组玻璃体腔注射bevacizumab，超声微泡+bevacizumab组玻璃体腔注射超声微泡+bevacizumab。分别于处理后7、14、28 d对各组兔行FFA检查，观察CNV抑制情况并每组各处死3只兔取双眼行免疫荧光及蛋白质免疫印迹（Western blot）法检测视网膜、脉络膜VEGF蛋白的表达。以注射bevacizumab后28 d为疗效判断时间点，以FFA及组织蛋白学检测对比结果为疗效判断标准。结果　激光光凝后21 d组织病理学和FFA检查结果显示，CNV向视网膜内层增生，激光光凝区荧光渗漏明显。分组处理后28 d,FFA检查结果显示空白对照组有明显荧光渗漏，bevacizumab组荧光渗漏平均强度与空白对照组比较，差异有统计学意义（t=16.2952，P＜0.05）；超声微泡+bevacizumab组与bevacizumab组相比，差异有统计学意义（t=4.7955，P＜0.05）。各组荧光渗漏强度随时间变化均呈下降趋势。组织免疫荧光及Western blot检测结果显示，bevacizumab组VEGF蛋白表达与空白组和对照组相比，差异有统计学意义（t=7.0327，9.2596；P＜0.05）；超声微泡+bevacizumab组VEGF蛋白表达与bevacizumab组相比，差异有统计学意义（t=2.9724,17.1937；P＜0.05）。结论　超声微泡造影剂联合bevacizumab注射能够通过抑制VEGF的表达来增强CNV的治疗效果。

Ultrasonic microbubble combined with bevacizumab injection for choroidal neovascularization induced by phtocoagulation in rabbits

Objective To observe the therapeutic effect of ultrasonic microbubble combined with bevacizumab (Avastin) on choroidal neovascularization induced by photocoagulation in rabbits.Methods CNV was induced by photocoagulation with argon laser in 30 rabbits (60 eyes).All of the rabbits underwent fundus fluorecein angiography (FFA) 21 days after photocoagulation;6-8 hours later,3 rabbits were randomly chosen to be executed to having the immunohistochemical examination.Twenty-one days after photocoagulation,27 rabbits were divided randomly into 3 groups:bevacizumb,ultrasonic microbubble+bevacizumb,and control group;each group has 9 rabbits (18 eyes).The rabbits in control group had no interference treatment;while the rats in bevacizumb and ultrasonic microbubble+bevacizumb group underwent injection with bevacizumb or ultrasonic microbubble+bevacizumb respectively.FFA was performed on all of the rabbits 7,14,and 28 days after photocoagulation to observe the inhibition of CNV;immunofluorecence and Western blot were used to detect the expression of VEGF in retina and choroid.Twenty-eight days is the time point to determine the therapeutic efficacy.The expression of VEGF and the results of FFA were the sdandards of the judgement of therapeutic efficacy.Results Proliferaion of CNV to the retinal inner layer and the obvious leakage of fluoresein in the photocoagulation area indicated that the model of CNV was set up successfully.Twenty-eight days after injection,obvious fluorescent leakage was found in the control group,and the average fluorescent leakage in bevacizumab group differed much from the control group(t=16.2952,P<0.05);while the difference between ultrasonic microbubble+bevacizumb group and bevacizumab group was also significant (t=4.7955,P<0.05).At the same time point,the expression of VEGF in bevacizumab group detected by immunofluorecent assay and Western blot differed much from the control group (t=7.0327,9.2596;P<0.05),and the difference of VEGF between ultrasonic microbubble+bevacizumb group and bevacizumab group was significant (t=2.9724,17.1937;P<0.05).this experiment show that ultrasound combined bevacizumab intravitreal injection of the therapeutic effect of CNV superior to other groups(P<0.01).Conclusion Ultrasound microbubble combined with bevacizumab injection may improve the therapeutic effect on CNV by inhibiting the expression of VEGF.