重組CFB-siRNA在脈絡(luò)膜新生血管中抑制作用的實(shí)驗(yàn)研究

李 凡，尚慶麗，郝玉華，馬景學(xué)，葉存喜，王 鑫

(河北醫(yī)科大學(xué)第二醫(yī)院眼科，河北 石家莊 050000)

·論著·

重組CFB-siRNA在脈絡(luò)膜新生血管中抑制作用的實(shí)驗(yàn)研究

李 凡，尚慶麗*，郝玉華，馬景學(xué)，葉存喜，王 鑫

(河北醫(yī)科大學(xué)第二醫(yī)院眼科，河北 石家莊 050000)

目的補(bǔ)體旁路途徑在脈絡(luò)膜新生血管(choroidal neovascularization,CNV)的發(fā)生發(fā)展中起重要作用，補(bǔ)體因子B(complement factor B,CFB)作為補(bǔ)體旁路途徑的重要因子可成為阻斷補(bǔ)體活化的靶點(diǎn)。本研究探討重組CFB-siRNA在實(shí)驗(yàn)性CNV中的抑制作用與機(jī)制。方法取Brown Norway大鼠45只隨機(jī)分為空白對(duì)照組、實(shí)驗(yàn)對(duì)照組、實(shí)驗(yàn)組各15只(30只眼)。空白對(duì)照組不給予任何干預(yù)措施，實(shí)驗(yàn)對(duì)照組與實(shí)驗(yàn)組均激光光凝建立大鼠CNV模型。實(shí)驗(yàn)對(duì)照組在CFB表達(dá)高峰前日尾靜脈注射生理鹽水0.5 μL，實(shí)驗(yàn)組同一時(shí)間尾靜脈注射CFB-SiRNA(0.5 μL/75 μg)，均隔日注射1次，共注射3次。各組分別于光凝后3、7、14、21、28 d進(jìn)行熒光素眼底血管造影(fluorescein fundusangiography,FFA)，根據(jù)熒光素滲漏程度對(duì)各光凝斑評(píng)分并檢測(cè)CNV的生長(zhǎng)情況；采用免疫組織化學(xué)法檢測(cè)各組視網(wǎng)膜脈絡(luò)膜組織中CFB、血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor,VEGF)、堿性成纖維細(xì)胞因子(basic fibroblast growth factor,BFGF)的表達(dá)情況，并測(cè)定其灰度值。結(jié)果FFA顯示：實(shí)驗(yàn)組與實(shí)驗(yàn)對(duì)照組比較，在光凝后7、14、21、28 d CNV發(fā)生率差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。免疫組織化學(xué)檢測(cè)結(jié)果顯示：空白對(duì)照組正常大鼠CFB、VEGF和BFGF在視網(wǎng)膜和脈絡(luò)膜中的表達(dá)非常弱。CFB表達(dá)情況：光凝后3 d實(shí)驗(yàn)組與實(shí)驗(yàn)對(duì)照組CFB表達(dá)達(dá)最高峰，隨后表達(dá)減少，光凝后7 d仍有少量表達(dá)，光凝后14～28 d表達(dá)趨于穩(wěn)定。實(shí)驗(yàn)對(duì)照組VEGF、BFGF在光凝后7～14 d表達(dá)明顯增多，光凝后21 d達(dá)高峰；實(shí)驗(yàn)組VEGF、BFGF在光凝后14、21、28 d表達(dá)減少。3組CFB、VEGF和BFGF灰度值在組間、時(shí)點(diǎn)間、組間·時(shí)點(diǎn)間交互作用差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)論尾靜脈注射CFB-siRNA可抑制實(shí)驗(yàn)性CNV的發(fā)展，通過抑制CFB，阻斷補(bǔ)體旁路途徑，減少CNV形成過程中VEGF、BFGF的表達(dá)。

脈絡(luò)膜新生血管化；補(bǔ)體因子B；血管內(nèi)皮生長(zhǎng)因子

10.3969/j.issn.1007-3205.2017.11.015

脈絡(luò)膜新生血管(choroidal neovascularization,CNV)受多種因素調(diào)控[1-6]。關(guān)于CNV的確切發(fā)病機(jī)制至今尚未明確，也缺少理想有效的治療手段。近期研究發(fā)現(xiàn)補(bǔ)體旁路途徑的激活在實(shí)驗(yàn)性CNV的發(fā)展中起關(guān)鍵作用[7]，旁路途徑中補(bǔ)體因子B(complement factor B, CFB)可以作為一個(gè)重要的靶點(diǎn)來阻斷補(bǔ)體活化的旁路途徑。本研究采用RNAi技術(shù)，用CFB-siRNA將CFB沉默，從而阻斷旁路途徑，觀察CFB-siRNA對(duì)CFB、血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor,VEGF)、堿性成纖維細(xì)胞因子(basic fibroblast growth factor,BFGF)的抑制作用，旨在為CNV的治療開辟新的思路。

1 材 料 與 方 法

1.1 CNV動(dòng)物模型的制備 健康8～10周雄性Brown Norway(BN)大鼠45只(購(gòu)于北京維通利華實(shí)驗(yàn)動(dòng)物技術(shù)有限公司)，體質(zhì)量180～200 g，常規(guī)喂養(yǎng)，實(shí)驗(yàn)前經(jīng)裂隙燈、眼底鏡檢查雙眼均正常。10%水合氯醛(0.35 mL/100 g體質(zhì)量)腹腔注射麻醉，鹽酸丙美卡因滴眼液眼表麻醉，1.5 cm×3.0 cm載玻片作為角膜接觸鏡，應(yīng)用Coherent Novus-Omni氪離子激光治療機(jī)(美國(guó)Coherent公司)進(jìn)行激光光凝，光凝參數(shù)為：波長(zhǎng)647 nm，光斑直徑50 μm，功率260 mW，曝光時(shí)間0.05 s，距視盤2～3個(gè)視盤直徑均勻光凝9～10個(gè)點(diǎn)。激光光凝時(shí)產(chǎn)生氣泡者為擊穿Bruch膜的標(biāo)志。

1.2 方法

1.2.1 動(dòng)物分組 45只大鼠隨機(jī)分為3組，每組15只、30只眼，分別為空白對(duì)照組、實(shí)驗(yàn)對(duì)照組、實(shí)驗(yàn)組?？瞻讓?duì)照組不給予任何處理，實(shí)驗(yàn)對(duì)照組及實(shí)驗(yàn)組大鼠均氪激光光凝建立CNV模型。實(shí)驗(yàn)對(duì)照組在CFB表達(dá)高峰前尾靜脈注射生理鹽水0.5 μL；實(shí)驗(yàn)組在CFB表達(dá)高峰前日尾靜脈注射CFB-SiRNA(0.5 μL/75 μg)。注射時(shí)間：隔日注射1次，共注射3次。

1.2.2 熒光素眼底血管造影檢查 于光凝后3、7、14、21、28 d，隨機(jī)選取各組大鼠3只，腹腔注射10%熒光素鈉注射液(0.5 mL/kg)，進(jìn)行熒光素眼底血管造影(fluorescein fundusangiography,FFA)，觀察熒光素滲漏范圍及程度，將各時(shí)間點(diǎn)出現(xiàn)的CNV光凝斑數(shù)目除以該時(shí)間點(diǎn)的光凝斑總數(shù)(百分?jǐn)?shù))，記為該時(shí)段CNV的發(fā)生率。

1.2.3 免疫組織化學(xué)法檢測(cè)相關(guān)因子在視網(wǎng)膜脈絡(luò)膜中的表達(dá) 各組在上述時(shí)間點(diǎn)完成FFA檢查后6 h，待大鼠體內(nèi)熒光素染料排凈，使用過量的水合氯醛腹腔注射處死大鼠，隨即摘取眼球，固定于FAA固定液中。乙醇梯度脫水，全層石蠟包埋，修整，4～5 μm連續(xù)切片。按照試劑盒說明書行CFB、VEGF、BFGF免疫組織化學(xué)檢測(cè)。一抗分別為：兔多克隆抗B因子抗體，工作濃度0.5 g/L(美國(guó)Santa Cruz Biotechnology公司)；兔抗鼠單克隆抗VEGF抗體，工作濃度1 g/L(北京中杉金橋生物技術(shù)有限公司)；兔多克隆抗BFGF抗體，工作濃度1 g/L(北京中杉金橋生物技術(shù)有限公司)。0.01 mol/L檸檬酸鹽緩沖液高溫高壓修復(fù)法修復(fù)抗原，采用SABC法染色，AEC顯色，經(jīng)蘇木素復(fù)染后封片。于光學(xué)顯微鏡下進(jìn)行100倍和400倍顯微照相，采集陽(yáng)性片。采用HPIAS-1000型高清晰度彩色病理圖文分析系統(tǒng)觀察染色結(jié)果，測(cè)定灰度值。

1.3 統(tǒng)計(jì)學(xué)方法 應(yīng)用SPSS 16.0軟件分析數(shù)據(jù)。計(jì)量資料比較采用重復(fù)測(cè)量的方差分析；計(jì)數(shù)資料比較采用χ2檢驗(yàn)。P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié) 果

2.1 FFA檢查結(jié)果 光凝后3 d，實(shí)驗(yàn)組及實(shí)驗(yàn)對(duì)照組的激光斑部位早期均呈弱熒光，晚期熒光增強(qiáng)，熒光素滲漏不明顯，CNV的發(fā)生率差異無統(tǒng)計(jì)學(xué)意義(P>0.05)；光凝后7 d，2組激光光斑部位均有輕度熒光滲漏，實(shí)驗(yàn)組CNV發(fā)生率低于實(shí)驗(yàn)對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；光凝后14 d，2組激光光斑均呈強(qiáng)熒光，后期熒光素滲漏明顯，CNV發(fā)生率差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；光凝后21 d，實(shí)驗(yàn)對(duì)照組與實(shí)驗(yàn)組激光光斑部位出現(xiàn)清晰的不規(guī)則顆粒狀、環(huán)狀及條狀熒光(圖1，2)，晚期2組均形成邊界略模糊的高熒光區(qū)(圖3，4)，形如花瓣?duì)畹?組CNV發(fā)生率均達(dá)高峰，但實(shí)驗(yàn)組CNV發(fā)生率顯著低于實(shí)驗(yàn)對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；光凝后28 d，2組激光光斑仍有熒光滲漏，實(shí)驗(yàn)組CNV發(fā)生率仍低于實(shí)驗(yàn)組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。見表1。

表1 激光誘導(dǎo)CNV在不同時(shí)間的發(fā)生率Table 1 The incidence rate of laser-induced CNV in different time (例數(shù)，%)

2.2 CFB、VEGF和BFGF免疫組織化學(xué)結(jié)果 實(shí)驗(yàn)對(duì)照組CFB免疫組織化學(xué)檢測(cè)：光凝后CFB陽(yáng)性染色信號(hào)見于光凝損傷區(qū)的視網(wǎng)膜全層，損傷區(qū)光斑內(nèi)CFB表達(dá)不均勻，光斑近脈絡(luò)膜部位CFB陽(yáng)性信號(hào)的強(qiáng)度明顯高于光斑內(nèi)部(圖5)；光凝后3 d，光斑區(qū)可見大量紅色的團(tuán)狀CFB陽(yáng)性反應(yīng)物，陽(yáng)性表達(dá)達(dá)到最高峰(圖6)；光凝后7 d，光斑區(qū)內(nèi)CFB陽(yáng)性反應(yīng)物減少(圖7)；2～4周時(shí)陽(yáng)性表達(dá)趨于穩(wěn)定(圖8)。CFB表達(dá)高峰為光凝后3 d。3組CFB、VEGF和BFGF灰度值在組間、時(shí)點(diǎn)間、組間·時(shí)點(diǎn)間交互作用差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。見表2～4。

組別3d7d14d21d28d空白對(duì)照組 1．32×106±67862．521．33×106±38186．181．32×106±53474．921．31×106±37417．021．31×106±34647．26實(shí)驗(yàn)對(duì)照組 4．87×106±95672．653．58×106±84264．461．34×106±45242．631．24×106±59263．441．21×106±15463．23實(shí)驗(yàn)組 3．70×106±45711．622．25×106±86723．541．24×106±95471．661．21×106±34521．221．19×106±144．37組間 F=204．407 P=0．000時(shí)點(diǎn)間 F=139．624 P=0．000組間·時(shí)點(diǎn)間F=7．717 P=0．016

組別3d7d14d21d28d空白對(duì)照組 1．36×106±26459．891．34×106±41927．211．32×106±50150．471．33×106±48839．931．34×106±38702．89實(shí)驗(yàn)對(duì)照組 1．84×106±42854．373．57×106±24465．277．45×106±19473．849．65×106±39372．819．03×106±67859．33實(shí)驗(yàn)組 1．30×106±12243．571．21×106±15463．598．05×105±21897．147．23×105±36232．967．18×105±43408．38組間 F=249．368 P=0．000時(shí)點(diǎn)間 F=152．817 P=0．000組間·時(shí)點(diǎn)間F=7．945 P=0．008

組別3d7d14d21d28d空白對(duì)照組 1．31×106±50426．681．29×106±52464．381．29×106±44561．331．30×106±50921．191．30×106±53851．65實(shí)驗(yàn)對(duì)照組 1．26×106±45679．442．78×106±21495．936．74×106±27933．748．37×106±37650．248．12×106±37492．57實(shí)驗(yàn)組 1．20×106±29606．201．08×106±27574．109．86×105±63160．708．17×105±65660．788．09×105±25549．50組間 F=215．694 P=0．000時(shí)點(diǎn)間 F=156．314 P=0．000組間·時(shí)點(diǎn)間F=8．015 P=0．011

3 討 論

CNV可見于多種眼科疾病，進(jìn)一步探索其發(fā)病機(jī)制及臨床治療手段一直是眼科學(xué)者的關(guān)注重點(diǎn)。目前應(yīng)用于臨床上的治療方法主要是對(duì)于已經(jīng)生成的CNV具有一定的作用，并且需要反復(fù)多次注射，并不能從根源上防止CNV的發(fā)生、發(fā)展和復(fù)發(fā)。研究表明補(bǔ)體旁路途徑異常激活是黃斑區(qū)發(fā)生病變的誘因，可以引起黃斑萎縮、變性和新生血管形成[8-10]。CFB是補(bǔ)體旁路途徑的重要因子，本研究采用前期研究[11]成功構(gòu)建的CFB-siRNA將CFB沉默，阻斷補(bǔ)體旁路途徑，通過觀察VEGF、BFGF因子的變化，探討CFB-siRNA對(duì)實(shí)驗(yàn)性CNV的抑制作用，為從根本上治愈CNV提供可能性。

隨著對(duì)年齡相關(guān)性黃斑變性的不斷探索，研究者們發(fā)現(xiàn)補(bǔ)體旁路途徑及經(jīng)典途徑的多個(gè)重要因子均與CNV密切相關(guān)，如CFB、CFH、C2和C3[12-15]。其中C3作為補(bǔ)體旁路途徑激活的中心環(huán)節(jié)，是補(bǔ)體系統(tǒng)中最重要、含量最豐富的一個(gè)因子。CFB是C3激活劑前體，C3形成的C3b與CFB結(jié)合，旁路途徑就會(huì)被激活，隨后經(jīng)一系列瀑布式反應(yīng)，形成膜攻擊復(fù)合物(membrane attack complex,MAC)而導(dǎo)致細(xì)胞裂解[16]。那么，構(gòu)建CFB-siRNA將CFB沉默后，即可阻止C3b與CFB的相互作用，從而抑制旁路途徑C3的形成，同時(shí)減少旁路激活途徑中活化的C3轉(zhuǎn)化酶中的Bb片段，使之失去酶活性，加速C3轉(zhuǎn)化酶衰變。本研究空白對(duì)照組CFB在視網(wǎng)膜脈絡(luò)膜組織中的表達(dá)非常弱，且隨時(shí)間的改變CFB無明顯變化；而實(shí)驗(yàn)對(duì)照組光凝后損傷部位出現(xiàn)了大量CFB表達(dá)，在光凝后3 d達(dá)高峰，隨后表達(dá)逐漸減少，在光凝后7 d仍有少量表達(dá)。CFB作為補(bǔ)體旁路途徑的重要因子，光凝后3 d實(shí)驗(yàn)對(duì)照組中CFB表達(dá)達(dá)最高峰，說明了實(shí)驗(yàn)性CNV形成過程中伴隨著補(bǔ)體旁路途徑的激活。提示可通過CFB作為靶點(diǎn)阻斷補(bǔ)體旁路激活途徑，從而影響CNV的形成與發(fā)展。

免疫組織化學(xué)檢測(cè)結(jié)果顯示，空白對(duì)照組大鼠VEGF、BFGF在視網(wǎng)膜脈絡(luò)膜組織中的表達(dá)非常弱，且各時(shí)間點(diǎn)均無明顯變化。實(shí)驗(yàn)對(duì)照組光凝后7 d，兩因子開始出現(xiàn)表達(dá)并逐漸增多，光凝后21 d達(dá)高峰。而實(shí)驗(yàn)組尾靜脈注射CFB-siRNA后，在光凝后21 d兩因子的表達(dá)水平較實(shí)驗(yàn)對(duì)照組顯著減少，并持續(xù)至光凝后28 d。因此，筆者推測(cè)CFB與CNV的發(fā)病機(jī)制高度相關(guān)。通過氪激光破壞Bruch膜，導(dǎo)致視網(wǎng)膜的炎癥反應(yīng)，從而激活了補(bǔ)體旁路途徑，增加了CFB的表達(dá)，同時(shí)誘導(dǎo)了VEGF、BFGF的表達(dá)上調(diào)，脈絡(luò)膜毛細(xì)血管內(nèi)皮細(xì)胞必然隨之分化增殖，然后穿過破裂的Bruch膜進(jìn)入視網(wǎng)膜色素上皮層下或視網(wǎng)膜下腔形成CNV。也就是說，CFB在旁路途徑中扮演重要角色，可以對(duì)CFB的表達(dá)情況進(jìn)行調(diào)控，從而干預(yù)新生血管的形成過程，最終抑制CNV的形成發(fā)展。此外，VEGF和BFGF兩者關(guān)系密切，在新生血管形成的各個(gè)環(huán)節(jié)共同作用，兩者表達(dá)的增多從一定意義上促使和加速了CNV發(fā)生，推動(dòng)了CNV整個(gè)復(fù)雜發(fā)病機(jī)制的發(fā)展。

近年來有研究認(rèn)為補(bǔ)體異常激活造成MAC的生成和沉積增加，是參與CNV發(fā)生發(fā)展的重要因素之一[17-18]。補(bǔ)體系統(tǒng)的激活途徑有3種，包括經(jīng)典途徑、旁路途徑和凝集素途徑。補(bǔ)體激活后，一系列的生物學(xué)效應(yīng)隨之出現(xiàn)。C3自然水解開啟了旁路途徑的變化反應(yīng)，經(jīng)過C3、C5到C9、CFH、CFB等多個(gè)因子共同參與反應(yīng)，最終形成MAC。MAC也稱為C5b-9，是補(bǔ)體系統(tǒng)3條途徑激活后形成的共同末端效應(yīng)產(chǎn)物，旁路途徑被激活，形成C5轉(zhuǎn)化酶，C5轉(zhuǎn)化酶裂解產(chǎn)生C5a及C5b，C5b于細(xì)胞表面結(jié)合，與C6、C7、C8、C9形成末端補(bǔ)體復(fù)合物，其中結(jié)合在細(xì)胞膜上的稱為MAC。也就是說，MAC的不斷沉積，使生長(zhǎng)因子釋放增多，膜通透性必然隨之發(fā)生改變，脈絡(luò)膜血管內(nèi)皮細(xì)胞發(fā)生異常增生，即導(dǎo)致了CNV的產(chǎn)生[19]。但關(guān)于CNV中MAC主要依賴于哪條補(bǔ)體途徑生成尚不明確。本研究通過沉默CFB，對(duì)實(shí)驗(yàn)性CNV產(chǎn)生抑制作用，這就意味著旁路途徑是激光誘導(dǎo)CNV發(fā)生發(fā)展的主要途徑。補(bǔ)體旁路途徑中CFB的缺乏，促進(jìn)了C3b的裂解，進(jìn)而抑制C5轉(zhuǎn)化酶的生成，使得MAC的沉積減少。所以，MAC的沉積可能依賴于補(bǔ)體旁路途徑的異常激活發(fā)生改變。

在未來的研究中，需要進(jìn)一步檢測(cè)CFB-siRNA在體內(nèi)轉(zhuǎn)染的靶向性。并且本實(shí)驗(yàn)僅僅從蛋白水平檢測(cè)相關(guān)因子的表達(dá)，尚未從核酸水平進(jìn)行定量定性檢測(cè)，同時(shí)在給藥劑量上存在研究空間，在今后的實(shí)驗(yàn)中有待進(jìn)一步的研究。(本文圖見封三)

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InhibitionefficacyofCFB-siRNAonlaser-inducedchoroidalneovascularizationinrat

LI Fan, SHANG Qing-li*, HAO Yu-hua, MA Jing-xue, YE Cun-xi, WANG Xin

(DepartmentofOphthalmology,theSecondHospitalofHebeiMedicalUniversity,Shijiazhuang050000,China)

ObjectiveAlternative complement pathway plays important roles in the pathogenesis and development of choroidal neovascularization(CNV). Complement factor B(CFB), an essential factor for the alternative complement pathway, has been considered as a target to block the activity of complement. In this study, we aim to investigate the inhibitive effects and the potential mechanism of recombinant CFB-siRNA in the CNV.MethodsForty-five Brown Norway rats were randomly divided into: blank control(n=15), subject to no treatment; control group(n=15), which was subject to CNV induction by laser photocoagulation, followed by administration of normal saline(0.5 μL) via caudal vein injection one day before the expression peak of CFB, and test group, which was subject to laser photocoagulation to induce CNV, followed by administration of CFB-siRNA(0.5 μL/75 μg) via caudal vein injection one day before the expression peak of CFB. The injection of normal saline and CFB-siRNA was performed every two days for thrice. Fundus fluorescein angiography(FFA) was performed on day 3, 7, 14, 21, and 28 after laser photocoagulation. Photocoagulation score was determined according to the leakage of the fluorescein, based on which to detect the growth of CNV. Expression of CFB, VEGF, and BFGF in the retina and the choroid was measured using immunohistochemical method.ResultsFFA showed that the difference of CNV incidence between the experimental group and the experimental group was statistically significant at 7, 14, 21 and 28 days after photocoagulation(P<0.05). In the blank control, the expression of CFB, VEGF, and BFGF in the retina and choroid was comparatively lower as revealed by immunohistochemical method. For the expression of CFB, peak value was obtained 3 days after photocoagulation in the experimental and control group, followed by down-regulation of CFB. Low content of CFB was still detected 7 days after photocoagulation, while the expression was stable from day 14 to day 28. In the control group, significant up-regulation was observed in the BEGF and BFGF 7-14 days after photocoagulation, and reached the peak value on day 21 after photocoagulation. In the experimental group, down-regulation of BFGF and BFGF was noticed on day 14, 21, and 28 after photocoagulation.ConclusionAdministration of CFB-siRNA via caudal vein inhibits the progression of CNV through prohibiting CFB expression, blocking the alternative complement pathway, and down-regulating the expression of VEGF and BFGF in the pathogenesis of CNV.

choroidal neovascularization； complement factor B； vascular endothelial growth factors

2016-12-26；

2017-06-02

河北省應(yīng)用基礎(chǔ)研究計(jì)劃重點(diǎn)基礎(chǔ)研究項(xiàng)目(09966111D)

李凡(1982-)，女，河北大名人，河北省石家莊市第一醫(yī)院主治醫(yī)師，醫(yī)學(xué)碩士，從事眼科疾病診治研究。

*通訊作者。E-mail：qinglishang2013@sina.cn

R773.4

A

1007-3205(2017)11-1305-06

(本文編輯：劉斯靜)