摘要背景 脉络膜新生血管(CNV)是眼内新生血管的重要表现形式.研究发现补体旁路途径的激活在激光诱导的CNV发展中起关键作用,旁路途径中的补体因子B(CFB)可以作为一个重要的靶点来阻断补体活化的旁路途径. 目的 探讨不同浓度重组CFB-siRNA(CFB-siRNA)抑制激光诱导大鼠CNV的效果及其作用机制.方法 取棕色挪威(BN)大鼠60只120只眼,采用随机数字表法将其分为空白对照组、实验对照组和25、50、75 μg CFB-siRNA组,每组12只鼠24只眼.实验对照组和CFB-siRNA组大鼠用激光光凝法建立CNV模型,不同剂量CFB-siRNA组于激光光凝后第1、3、5天分别经鼠尾静脉注射相应剂量的CFB-siRNA;实验对照组大鼠以同样方法注射生理盐水,空白对照组大鼠不给予任何干预措施.各组大鼠分别于光凝后第3、7、14、21、28天进行荧光素眼底血管造影( FFA),根据荧光素渗漏程度对各光凝斑评分并检测CNV的生长情况；采用免疫组织化学法检测各组大鼠脉络膜内血管内皮生长因子(VEGF)、Ⅷ因子的表达情况.分别于第7、14、21、28天用免疫组织化学法检测脉络膜中CFB、VEGF、转化生长因子p2(TGF-β2)蛋白的表达,用逆转录聚合酶链反应(RT-PCR)法观察CFB与VEGFmRNA、TGF-β2mRNA表达的关系. 结果 不同剂量CFB-siRNA组的CNV发生率明显低于实验对照组,差异均有统计学意义(P＜0.05)；75 μg CFB-siRNA治疗组CNV发生率明显低于25 μg CFB-siRNA组(P＜0 05).免疫组织化学检测结果显示,不同剂量CFB-siRNA组VEGF、Ⅷ因子在大鼠脉络膜中的表达较2个对照组均减弱,且75 μg CFB-siRNA组减弱程度高于25 μg CFB-siRNA组.光凝后14 ～ 21 d,各剂量CFB-siRNA组VEGF蛋白、Ⅷ因子和TGF-β2在脉络膜中的表达均低于空白对照组,各组总体比较差异有统计学意义(P＜0.05).光凝后7～21 d,不同剂量CFB-siRNA组的CFB在脉络膜中的表达明显低于空白对照组,各组总体比较差异均有统计学意义(P＜0.05).RT-PCR检测结果显示,实验对照组CFB表达持续增加,VEGF、TGF-β2呈曲线变化,21 d时为表达高峰；不同剂量CFB-siRNA组中CFB、VEGF、TGF-β2表达显著减少,差异均有统计学意义(P＜0.05). 结论 尾静脉注射CFB-siRNA能够有效抑制激光诱导的大鼠CNV生成,其抑制效果随着注射剂量的增加而增强.补体激活旁路途径在CNV生成中扮演着重要角色,抑制CFB可减少CNV生成中VEGF和TGF-β2的表达.

abstractsBackground Choriodal neovascularization is an important ocular manifestation of angiogenesis in eyes,which derives from the choroid capillaries.Recent studies have found that complement activation is playing a key role in the laser-induced CNV.Because of the key position of CFB in the alternative pathway,bytargeting CFB and blocking the alternative pathway may provide an approach to observe the role of this alternative pathway in the generation of CNV. Objective This study was to investigate the inhibitory effect of reconstructed complement factor B (CFB)-small interfering ribonucleic acid (siRNA) on choroidal neovascularization (CNV)and its mechanism. Methods Experimental CNV was induced by laser photocoagulation in 96 eyes of 48 clean Brown Norway rats.The rats were randomly divided into 4 groups.25,50 and 75 μg B factor siRNA were injected via caudal vein on 1 day,3,5 days after photocoagulation in different dose groups,and normal saline solution was injected at the same way in experimental control group.Other 12 normal rats were used as blank control group.Fundus fluorescein angiography(FFA) was performed on 3,7,14,21,28 days after injection of CFB-siRNA and CNV was scored.The expressions of vascular endothelial growth factor(VEGF) and factor Ⅷ in choroid were detected by immunochemistry.The expressions of CFB-siRNA,VEGF,transforming growth factor β2( TGF-β2 )proteins in choroid were determined using immunochemistry in 7,14,21,28 days,and the expressions of mRNA of CFB-siRNA,VEGF,TGF-β2 were examined by reverse transcription polymerase chain reaction(RT-PCR). Results FFA revealed that the CNV rates in various doses of CFB-siRNA groups were significant lower than those of experimental control group in various time points(P＜0.05),and those in 75 μg B factor siRNA were decreased in comparison with 25 μg B factor siRNA (P＜0.05).Immunochemistry showed that the intensities of the VEGF and factor Ⅶ expression in various doses of CFB-siRNA groups were weaker than the blank control group ( P ＜ 0.05 ).Compared with the control group,the expression of CFB reduced in 7 days,and then approached to the level near the control group.Fourteen to twenty-one days after injection of CFB-siRNA,VEGF and TGF-β2 depressions in different doses of CFB-siRNA groups were lower than blank control group( P＜0.05 ).CFB expression in choroid showed the lower levels in CFB-siRNA injection group compared with blank control group in from 7 through 21 days (P＜0.05).RT-PCR displayed the gradual increase of CFB mRNA and curve-like changes of VEGF and TGF-β2 with time prolong. Conclusions Recombinated CFB-siRNA can effectively inhibit laser-induced CNV by down-regulating the expression of VEGF and factor Ⅷ.Alternative pathway of complement plays an important role in the production of CNV.