VEGF siRNA联合凉血通络方治疗脉络膜新生血管的实验研究

Experimental study of VEGF siRNA combined Liangxue Tongluo decoction for choroidal neovascularization

目的研究VEGF siRNA联合凉血通络方对脉络膜新生血管(CNV)的治疗作用。方法 90只棕色挪威(BN)大鼠,右眼通过激光光凝方式建立CNV模型,随机分为空白对照组、VEGF siRNA组、联合用药组(VEGF siRNA+中药),每组30只。3组分别在光凝后第14 d行荧光素眼底血管造影检查(FFA)、眼球标本行组织病理切片光镜观察、蛋白印记检测、实时荧光定量PCR检测。结果 FFA:VEGF siRNA组及联合用药组在光凝后14 d,荧光素渗漏面积减少,与空白对照组相比差异均具有统计学意义(VEGF siRNA组t=2.627,P=0.014;联合用药组t=5.361,P=0.000),且联合用药组荧光渗漏面积小于VEGF siRNA组,差异具有统计学意义(t=2.733,P=0.011);HE染色:CNV最大中央厚度与FFA检查趋势一致。蛋白印记检测:光凝后14 d,VEGF siRNA组、联合用药组VEGF蛋白表达程度均较空白对照组表达减弱,差异有统计学意义(VEGF siRNA组t=2.495,P=0.019;联合用药组t=5.242, P=0.000),联合用药组VEGF蛋白表达低于VEGF siRNA组,差异有统计学意义(t=2.747,P=0.011)。光凝后14 d,VEGF siRNA组PEDF蛋白表达程度与对空白照组比较无明显差异(t=-1.847,P=0.076),联合用药组PEDF蛋白表达程度较空白对照组增强,差异存在统计学差异(t=-11.11,P=0.000)。实时荧光定量PCR检测与蛋白印记检测趋势一致。结论 VEGF siRNA联合凉血通络方能有效抑制脉络膜新生血管生长,可为CNV的治疗提供了新思路。

OBJECTIVE To investigate the effect of VEGF siRNA combined with Liangxue Tongluo decoction in the treatment of choroidal neovascularization(CNV) in rats.METHODS Ninety Brown Norway(BN) rats, the CNV model of the right eye established by laser photocoagulation were randomly divided into blank control group, VEGF siRNA group and combined treatment group, with 30 rats each. On day 14 th, fluorescein angiography(FFA), HE staining and real time PCR was performed in all right eyes. RESULTS Compared with blank control group,VEGF siRNA group and combined treatment group, the leakage area of fluorescein decreased after fourteen days of photocoagulation, and the difference was statistically significant(VEGF siRNA group t =2.627,P =0.014,combined treatment group t=5.361, P=0.000). The area of fluorescence leakage of the combined treatment group was smaller than VEGF siRNA group, and the difference was statistically significant(t = 2.733, P = 0.011).CNV maximum central thickness by HE stain ing was consistent with the trend of FFA examination. After fourteen days photocoagulation, Western blot examination showed that the expression of VEGF protein of the VEGF siRNA group and the combined treatment group was weaker than that of the blank control group, and the difference was statistically significant(VEGF siRNA group, t = 2.495, combined treatment group,P= 0.019;t = 5.242, P= 0.000), and the expression of VEGF protein of combined treatment group was lower than VEGF siRNA group, and the difference was statistically significant(t=2.747,P=0.011). After fourteen days photocoagulation, the expression of PEDF protein in the VEGF siRNA group was not significantly different from that of the blank control group(t=-1.847, P= 0.076). The expression of PEDF protein in the combined treatment group was stronger than that of the blank control group,and the difference was statistically significant(t=-11.11, P=0.000).VEGF mRNA and PEDF mRNA examination by real time PCR was consistent with the trend of FFA western blot. CONCLUSIONS VEGF siRNA combined with Liangxue Tongluo decoction could inhibit the growth of choroidal neovascularization effectively, which could provide new ideas for the treatment of CNV.