中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2012年
4期
320-325
,共6页
马诚%尚庆丽%马景学%高建%修贺明%姚会娟%杨爱琴
馬誠%尚慶麗%馬景學%高建%脩賀明%姚會娟%楊愛琴
마성%상경려%마경학%고건%수하명%요회연%양애금
脉络膜新生血管/治疗%基因疗法%补体因子B%小干扰核糖核酸
脈絡膜新生血管/治療%基因療法%補體因子B%小榦擾覈糖覈痠
맥락막신생혈관/치료%기인요법%보체인자B%소간우핵당핵산
Choroidal neovascularization/management%Gene therapy%Complement factor B%Small interfering RNA
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.
揹景 脈絡膜新生血管(CNV)是眼內新生血管的重要錶現形式.研究髮現補體徬路途徑的激活在激光誘導的CNV髮展中起關鍵作用,徬路途徑中的補體因子B(CFB)可以作為一箇重要的靶點來阻斷補體活化的徬路途徑. 目的 探討不同濃度重組CFB-siRNA(CFB-siRNA)抑製激光誘導大鼠CNV的效果及其作用機製.方法 取棕色挪威(BN)大鼠60隻120隻眼,採用隨機數字錶法將其分為空白對照組、實驗對照組和25、50、75 μg CFB-siRNA組,每組12隻鼠24隻眼.實驗對照組和CFB-siRNA組大鼠用激光光凝法建立CNV模型,不同劑量CFB-siRNA組于激光光凝後第1、3、5天分彆經鼠尾靜脈註射相應劑量的CFB-siRNA;實驗對照組大鼠以同樣方法註射生理鹽水,空白對照組大鼠不給予任何榦預措施.各組大鼠分彆于光凝後第3、7、14、21、28天進行熒光素眼底血管造影( FFA),根據熒光素滲漏程度對各光凝斑評分併檢測CNV的生長情況；採用免疫組織化學法檢測各組大鼠脈絡膜內血管內皮生長因子(VEGF)、Ⅷ因子的錶達情況.分彆于第7、14、21、28天用免疫組織化學法檢測脈絡膜中CFB、VEGF、轉化生長因子p2(TGF-β2)蛋白的錶達,用逆轉錄聚閤酶鏈反應(RT-PCR)法觀察CFB與VEGFmRNA、TGF-β2mRNA錶達的關繫. 結果 不同劑量CFB-siRNA組的CNV髮生率明顯低于實驗對照組,差異均有統計學意義(P＜0.05)；75 μg CFB-siRNA治療組CNV髮生率明顯低于25 μg CFB-siRNA組(P＜0 05).免疫組織化學檢測結果顯示,不同劑量CFB-siRNA組VEGF、Ⅷ因子在大鼠脈絡膜中的錶達較2箇對照組均減弱,且75 μg CFB-siRNA組減弱程度高于25 μg CFB-siRNA組.光凝後14 ～ 21 d,各劑量CFB-siRNA組VEGF蛋白、Ⅷ因子和TGF-β2在脈絡膜中的錶達均低于空白對照組,各組總體比較差異有統計學意義(P＜0.05).光凝後7～21 d,不同劑量CFB-siRNA組的CFB在脈絡膜中的錶達明顯低于空白對照組,各組總體比較差異均有統計學意義(P＜0.05).RT-PCR檢測結果顯示,實驗對照組CFB錶達持續增加,VEGF、TGF-β2呈麯線變化,21 d時為錶達高峰；不同劑量CFB-siRNA組中CFB、VEGF、TGF-β2錶達顯著減少,差異均有統計學意義(P＜0.05). 結論 尾靜脈註射CFB-siRNA能夠有效抑製激光誘導的大鼠CNV生成,其抑製效果隨著註射劑量的增加而增彊.補體激活徬路途徑在CNV生成中扮縯著重要角色,抑製CFB可減少CNV生成中VEGF和TGF-β2的錶達.
배경 맥락막신생혈관(CNV)시안내신생혈관적중요표현형식.연구발현보체방로도경적격활재격광유도적CNV발전중기관건작용,방로도경중적보체인자B(CFB)가이작위일개중요적파점래조단보체활화적방로도경. 목적 탐토불동농도중조CFB-siRNA(CFB-siRNA)억제격광유도대서CNV적효과급기작용궤제.방법 취종색나위(BN)대서60지120지안,채용수궤수자표법장기분위공백대조조、실험대조조화25、50、75 μg CFB-siRNA조,매조12지서24지안.실험대조조화CFB-siRNA조대서용격광광응법건립CNV모형,불동제량CFB-siRNA조우격광광응후제1、3、5천분별경서미정맥주사상응제량적CFB-siRNA;실험대조조대서이동양방법주사생리염수,공백대조조대서불급여임하간예조시.각조대서분별우광응후제3、7、14、21、28천진행형광소안저혈관조영( FFA),근거형광소삼루정도대각광응반평분병검측CNV적생장정황；채용면역조직화학법검측각조대서맥락막내혈관내피생장인자(VEGF)、Ⅷ인자적표체정황.분별우제7、14、21、28천용면역조직화학법검측맥락막중CFB、VEGF、전화생장인자p2(TGF-β2)단백적표체,용역전록취합매련반응(RT-PCR)법관찰CFB여VEGFmRNA、TGF-β2mRNA표체적관계. 결과 불동제량CFB-siRNA조적CNV발생솔명현저우실험대조조,차이균유통계학의의(P＜0.05)；75 μg CFB-siRNA치료조CNV발생솔명현저우25 μg CFB-siRNA조(P＜0 05).면역조직화학검측결과현시,불동제량CFB-siRNA조VEGF、Ⅷ인자재대서맥락막중적표체교2개대조조균감약,차75 μg CFB-siRNA조감약정도고우25 μg CFB-siRNA조.광응후14 ～ 21 d,각제량CFB-siRNA조VEGF단백、Ⅷ인자화TGF-β2재맥락막중적표체균저우공백대조조,각조총체비교차이유통계학의의(P＜0.05).광응후7～21 d,불동제량CFB-siRNA조적CFB재맥락막중적표체명현저우공백대조조,각조총체비교차이균유통계학의의(P＜0.05).RT-PCR검측결과현시,실험대조조CFB표체지속증가,VEGF、TGF-β2정곡선변화,21 d시위표체고봉；불동제량CFB-siRNA조중CFB、VEGF、TGF-β2표체현저감소,차이균유통계학의의(P＜0.05). 결론 미정맥주사CFB-siRNA능구유효억제격광유도적대서CNV생성,기억제효과수착주사제량적증가이증강.보체격활방로도경재CNV생성중분연착중요각색,억제CFB가감소CNV생성중VEGF화TGF-β2적표체.
Background 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.
