金 惠，步雪峰，王穆彬，蘇春香，嚴(yán)玉蘭

(江蘇大學(xué) 1.附屬人民醫(yī)院 呼吸科 江蘇 鎮(zhèn)江 212002； 2.臨床醫(yī)學(xué)院，江蘇 鎮(zhèn)江 212001；3.附屬人民醫(yī)院 普外科， 江蘇 鎮(zhèn)江 212002)

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研究論文

重組新城疫病毒rl-RVG抑制人肺腺癌A549系細(xì)胞遷移及相關(guān)機(jī)制

金 惠1, 2，步雪峰3，王穆彬2，蘇春香2，嚴(yán)玉蘭1*

(江蘇大學(xué) 1.附屬人民醫(yī)院 呼吸科 江蘇 鎮(zhèn)江 212002； 2.臨床醫(yī)學(xué)院，江蘇 鎮(zhèn)江 212001；3.附屬人民醫(yī)院 普外科， 江蘇 鎮(zhèn)江 212002)

目的觀察表達(dá)狂犬病毒糖蛋白的重組新城疫病毒(rl-RVG)對(duì)人肺癌細(xì)胞系A(chǔ)549的遷移影響，初步探討其可能的機(jī)制。方法重組新城疫病毒rl-RVG直接感染A549細(xì)胞為rl-RVG實(shí)驗(yàn)組，新城疫病毒LaSota株處理的A549細(xì)胞組及未感染病毒的A549細(xì)胞組作為對(duì)照組。Western blot法檢測(cè)NDV-HN蛋白及狂犬病毒糖蛋白(RVG)，細(xì)胞增殖實(shí)驗(yàn)測(cè)定新城疫病毒使用的最佳作用濃度；劃痕實(shí)驗(yàn)及Transwell法測(cè)A549遷移；Western blot法及免疫熒光法檢測(cè)E-cadherin,MMP2蛋白表達(dá)。結(jié)果空白對(duì)照組無(wú)NDV、rl-RVG表達(dá)，rl-RVG僅在感染rl-RVG細(xì)胞組有表達(dá)，NDV在感染rl-RVG細(xì)胞組及感染NDV細(xì)胞組皆有表達(dá)；與空白對(duì)照組相比，rl-RVG及NDV稀釋濃度大于5×10-5對(duì)細(xì)胞的增殖有抑制作用(P<0.05)；rl-RVG組遷移的距離及細(xì)胞數(shù)明顯減少(P<0.05)。與空白對(duì)照組及NDV感染組相比，rl-RVG組E-cadherin蛋白表達(dá)水平上調(diào)(P<0.05)，MMP2蛋白表達(dá)水平減弱(P<0.05)。結(jié)論重組新城疫病毒rl-RVG能抑制人肺癌細(xì)胞系A(chǔ)549的遷移，并可能通過(guò)影響肺腺癌A549上皮細(xì)胞-間質(zhì)轉(zhuǎn)化(EMT)過(guò)程中的調(diào)控因子E-cadherin,MMP2蛋白而對(duì)細(xì)胞遷移而作用。

重組新城疫病毒RVG；肺癌；遷移；上皮細(xì)胞-間質(zhì)轉(zhuǎn)化

重組新城疫病毒(recombinant avirulent NDV LaSota strain expressing the rabies virus glycoproteinrl-RVG)作為基因治療腫瘤的載體，插入外源基因不影響NDV的結(jié)構(gòu)，也不影響病毒對(duì)腫瘤特異性抗腫瘤作用，對(duì)人正常細(xì)胞無(wú)不良反應(yīng)[1]。前期的研究中發(fā)現(xiàn)表達(dá)狂犬病毒糖蛋白的重組新城疫病毒(recombinant avirulent NDV LaSota strain expressing the rabies virus glycoprotein，rl-RVG)[2]對(duì)于人類肺腺癌A549細(xì)胞具有較強(qiáng)的生長(zhǎng)抑制作用，rl-RVG[2]對(duì)人肺腺癌A549細(xì)胞系有抑制增殖并促進(jìn)其凋亡作用[3]，提示rl-RVG可能會(huì)引起人肺腺癌的遷移及轉(zhuǎn)移活性減弱。但是rl-RVG對(duì)人肺腺癌A549細(xì)胞遷移的影響及可能機(jī)制無(wú)相關(guān)報(bào)道。所以本實(shí)驗(yàn)將表達(dá)狂犬病毒糖蛋白的重組新城疫病毒疫苗感染A549肺癌細(xì)胞，通過(guò)觀察A549細(xì)胞的遷移能力，進(jìn)一步探索 rl-RVG對(duì)肺腺癌細(xì)胞的作用。

1 材料與方法

1.1 材料

新城疫病毒LaSota系、rl-RVG和雞抗NDV血清一抗(中國(guó)農(nóng)業(yè)科學(xué)院哈爾濱研究所饋贈(zèng))；小鼠抗狂犬病毒ERA系G蛋白一抗、羊抗兔抗體β-actin(Santa Cruz公司)。MTT試劑盒(Sigma公司)； 兔抗E-cadherin,兔抗MMP2抗體(博士德公司)； HRP標(biāo)記的山羊抗兔IgG二抗(康為世紀(jì)公司)。人肺腺癌A549 細(xì)胞為江蘇大學(xué)基礎(chǔ)醫(yī)學(xué)研究所保存;DMEM及胎牛血清(維森特公司)。

1.2 細(xì)胞增殖實(shí)驗(yàn)(MTT)檢測(cè)A549細(xì)胞增殖

LaSota系NDV病毒液及rl-RVG病毒液的滴度都在雞胚半數(shù)感染量(50% egg infective doses，EID50)為109.8EID50/mL 左右[4]。用無(wú)血清DMEM將病毒原液稀釋至以下濃度: 1×10-3、1×10-4、5×10-5和1×10-6。對(duì)數(shù)增殖期A549細(xì)胞接種于96孔板上(5×104個(gè)/mL)，每孔100 μL過(guò)夜培養(yǎng)后換含20 mL/L胎牛血清的DMEM維持液，在NDV LaSota組和rl-RVG組加入病毒液，陰性空白對(duì)照組為PBS;每組設(shè)5個(gè)平行孔，DMEM完全培養(yǎng)基為調(diào)零孔。每孔過(guò)夜培養(yǎng)后加入20 μL MTT再培養(yǎng)4 h，后加入150 μL二甲基亞砜溶解，酶標(biāo)儀490 nm上測(cè)定吸光度(A)值，實(shí)驗(yàn)重復(fù)3 次，并用以下公式計(jì)算細(xì)胞活力值。細(xì)胞活力值=(實(shí)驗(yàn)組平均A值/空白對(duì)照組平均A值)×100%。

1.3 Western blot法檢測(cè)RVG、NDV HN和E-cadherin、MMP2蛋白的表達(dá)

對(duì)數(shù)增殖期A549細(xì)胞種植于6孔板上(5×104個(gè)/mL)，NDV-LaSota組和rl-RVG組加入10-6稀釋度病毒液，陰性對(duì)照為PBS組。培養(yǎng)24 h提取細(xì)胞蛋白經(jīng)120 g/L SDS-PAGE后轉(zhuǎn)膜; 50 g/L脫脂奶粉封閉1 h,分別用小鼠抗RVG抗體、雞抗NDV 抗體(1∶300)、羊抗兔抗體β-actin(1∶10 000)，4 ℃孵育過(guò)夜; 緩沖液(TBST)洗3 次，加入HRP標(biāo)記的山羊抗小鼠二抗和HRP標(biāo)記的兔抗雞二抗、室溫孵育2 h; TBST洗3次，ECL發(fā)光劑在Typhoon9400掃描儀上掃描。E-cadherin、MMP2檢測(cè)方法同上，一抗分別為兔抗E-cadherin抗體(1∶300)，兔抗MMP2抗體(1∶300)，二抗為山羊抗兔IgG(1∶10 000)。內(nèi)參為羊抗兔抗體β-actin，EC顯色后掃描，Image J圖片軟件處理數(shù)據(jù)。

1.4 細(xì)胞劃痕遷移實(shí)驗(yàn)

接種對(duì)數(shù)增殖期A549細(xì)胞于24孔板中(1×105個(gè)/孔)，用含10%胎牛血清的高糖培養(yǎng)基(DMEM)培養(yǎng)液培養(yǎng)24 h，用無(wú)菌200 μL槍頭在細(xì)胞層中縱向劃線，形成寬度均勻一致的無(wú)細(xì)胞傷口模型；加入空白對(duì)照組(DMEM培養(yǎng)基)和NDV、rl-RVG(10-6稀釋度)分別感染24 h;光鏡下觀察劃痕的寬度。

1.5 Transwell實(shí)驗(yàn)

用無(wú)血清DMEM調(diào)整對(duì)數(shù)增殖期A549細(xì)胞濃度為1.0×105個(gè)/mL，按100 μL/孔細(xì)胞量接種于放入24孔培養(yǎng)板中Transwell上室，下室加入含LaSota系NDV(10-6稀釋度)和rl-RVG(10-6稀釋度) 的DMEM 600 μL(對(duì)照組加入DMEM 600 μL)，37 ℃培養(yǎng)24 h。用棉簽擦去Transwell上室聚碳酸酯膜表面的細(xì)胞，PBS洗2遍后，將上室置于4%多聚甲醛中固定15 min。PBS洗2遍后，結(jié)晶紫染色20 min，再用PBS洗2遍后，在倒置熒光顯微鏡下對(duì)5個(gè)不同視野的穿過(guò)膜細(xì)胞計(jì)數(shù)(×200)，求平均值。

1.6 免疫熒光法檢測(cè)蛋白表達(dá)

取24孔板中NDV,rl-RVG處理24 h后的A549細(xì)胞,棄培養(yǎng)液,甲醛固定6 h,TBS洗滌,加triton X- 100、牛血清白蛋白400 μL, 37 ℃保持1 h,后加入E-cadherin，MMP2一抗, 4 ℃孵育24 h, TBS洗滌3遍,加二抗，室溫下孵育45 min,TBS洗滌3 遍。加核熒光抗體,照相。Image J圖片軟件處理數(shù)據(jù)。

1.7 統(tǒng)計(jì)學(xué)分析

2 結(jié)果

2.1 Western blot法檢測(cè)RVG及NDV-HN蛋白表達(dá)

感染A549細(xì)胞24 h后，發(fā)現(xiàn)RVG蛋白在NDV LaSota組和PBS組沒(méi)有表達(dá)，RVG蛋白可以在感染rl-RVG的A549細(xì)胞中穩(wěn)定表達(dá),并且能促進(jìn)NDV-HN蛋白在A549細(xì)胞中的表達(dá)(P<0.05)(圖1)。

2.2 rl-RVG病毒對(duì)A549細(xì)胞生長(zhǎng)的影響

細(xì)胞增殖實(shí)驗(yàn)(MTT)檢測(cè)A549細(xì)胞增殖:不同濃度NDV和rl-RVG作用A549細(xì)胞24 h后，細(xì)胞活力值隨溶度增加而升高，與空白對(duì)照組相比，rl-RVG及NDV稀釋濃度大于5×10-5對(duì)細(xì)胞的增殖有抑制作用(P<0.05) (圖2)，故選擇稀釋溶度為106倍的rl-RVG及NDV，作用于A549細(xì)胞24 h，對(duì)細(xì)胞增殖無(wú)明顯影響。

A.Westren blot; B.grayscale ratio; *P<0.05 compared with NDV-LaSota group圖1 rl-RVG感染A549細(xì)胞24 h后RVG蛋白和NDV蛋白表達(dá)Fig 1 The expression of RVG protein and NDV protein in A549 cells after infected by rl-RVG(n=5)

*P<0.05 compared with PBS control group圖2 不同溶度rl-RVG,NDV對(duì)肺癌A549細(xì)胞生長(zhǎng)的影響Fig 2 The cell growth of lung adenocarcinoma A549 was influenced after infected by different solubilities of rl-RVG and NDV

2.3 rl-RVG對(duì)肺腺癌A549 細(xì)胞遷移能力的影響

2.3.1 細(xì)胞劃痕遷移實(shí)驗(yàn):A549細(xì)胞劃痕后經(jīng)rl-RVG、NDV處理24 h，由劃痕邊緣向中央遷移后兩側(cè)距離分別為(465±39.9)μm，(778.6±6.7)μm,與對(duì)照組劃痕邊緣向中央遷移后兩側(cè)距離為(1 405±12.6)μm相比，rl-RVG感染后的A549細(xì)胞由劃痕邊緣向中央遷移的兩側(cè)距離較大(P<0.05) (圖3)。

2.3.2 Transwell實(shí)驗(yàn):Transwell 實(shí)驗(yàn)檢測(cè)，與對(duì)照組相比，rl-RVG處理組細(xì)胞穿過(guò)Transwell小室的細(xì)胞數(shù)明顯減少(P<0.05) (圖4)。

2.4 Western blot法及免疫熒光法檢測(cè)rl-RVG感染A549細(xì)胞后E-cadherin、MMP2的表達(dá)量

rl-RVG感染A549細(xì)胞后E-cadherin表達(dá)量增加，3組吸光度比值有統(tǒng)計(jì)學(xué)意義(P<0.05)，MMP2 66 ku/72 ku表達(dá)減弱,3組吸光度比值有統(tǒng)計(jì)學(xué)意義 (P<0.05)。免疫熒光法示rl-RVG組細(xì)胞膜上及細(xì)胞質(zhì)中的E-cadherin熒光強(qiáng)度升高，3組細(xì)胞熒光強(qiáng)度有統(tǒng)計(jì)學(xué)意義(P<0.05)；rl-RVG組細(xì)胞膜上及細(xì)胞質(zhì)中的MMP2熒光強(qiáng)度減弱(P<0.05)；NDV感染A549細(xì)胞，E-cadherin表達(dá)量及熒光強(qiáng)度也增加(P<0.05)，但弱于rl-RVG組，MMP2表達(dá)量及熒光強(qiáng)度也減弱(P<0.05),但強(qiáng)于rl-RVG組(圖5)。

3 討論

國(guó)內(nèi)外大量研究表明NDV具有抗腫瘤作用[5-7]，主要通過(guò)對(duì)腫瘤細(xì)胞的特異性溶瘤的直接細(xì)胞毒作用[5,8- 10]，引起腫瘤細(xì)胞增殖抑制并促進(jìn)其凋亡[3]。本研究中，劃痕實(shí)驗(yàn)及Transwell實(shí)驗(yàn)表明rl-RVG感染后的A549細(xì)胞組遷移明顯抑制。而Western blot法檢測(cè)結(jié)果顯示rl-RVG組NDV HN蛋白表達(dá)量較優(yōu)于NDV組，證明了RVG 能夠促進(jìn)NDV在細(xì)胞間的傳播,與報(bào)道實(shí)驗(yàn)結(jié)果一致。因此，rl-RVG抗腫瘤作用除NDV特異性溶瘤特性外，還可以通過(guò)抑制腫瘤細(xì)胞遷移得以實(shí)現(xiàn)。

而腫瘤細(xì)胞的遷移和對(duì)周圍組織和血管的侵襲是腫瘤細(xì)胞轉(zhuǎn)移的關(guān)鍵步驟[11]，上皮細(xì)胞-間質(zhì)轉(zhuǎn)化(epithelial mesenchymalt ransition,EMT)與腫瘤細(xì)胞的原位侵襲和遠(yuǎn)處轉(zhuǎn)移有著密切的關(guān)系。EMT是多細(xì)胞生物胚胎發(fā)生中的基礎(chǔ)過(guò)程,也存在于多種慢性疾病以及腫瘤的發(fā)生發(fā)展過(guò)程中, 它以上皮細(xì)胞極性喪失為主要特征。而參與EMT的重要調(diào)控因子有E-cadherin和MMP2[12- 13]。E-cadherin 是一類主要介導(dǎo)細(xì)胞間相互黏附的單鏈I型鈣依賴性跨膜蛋白，激活鈣黏蛋白, 促進(jìn)橋粒連接形成,維持正常上皮細(xì)胞的完整性及分化[14]，故而E-cadherin的異常表達(dá)被認(rèn)為與EMT的產(chǎn)生直接相關(guān)，在癌癥侵襲中起著重要作用。MMP2是具有Zn2+依賴性的最主要的降解型膠原酶蛋白水解酶，參與腫瘤新生血管形成、腫瘤細(xì)胞的侵襲和轉(zhuǎn)移灶的形成，與腫瘤EMT的發(fā)生密切相關(guān), MMP2在易發(fā)生早期轉(zhuǎn)移的小細(xì)胞肺癌中表達(dá)增高[15]。

圖3 通過(guò)劃痕實(shí)驗(yàn)檢測(cè)rl-RVG及NDV感染對(duì)A549細(xì)胞遷移能力影響

Arrow pointed to A549 cell; *P<0.05 compared with PBS group

A:Western blot; B:the grayscale ratio of MMP2, E-cadherin；C:immunofluorescence of MMP2 and E-cadherin, ①～③.MMP2 of PBS, NDV and rl-RVG group,respectively; ④～⑥.E-cadherin of PBS, NDV and rl-RVG group,respectively; D:single cell fluorsence intensity of MMP2,E-cadherin;*P<0.05 compared with PBS group;#P<0.05 compared with PBS and NDV group

圖5 rl-RVG及NDV 感染A549 細(xì)胞24 h 后的MMP2、E-cadherin蛋白Western blot及免疫熒光表達(dá)

Fig 5 Western blot and Immunofluorescence analysis of MMP2, E-cadherin protein expression in A549 cells after infected by rl-RVG and NDV on 24 hours

本研究Western blot結(jié)果表明，感染rl-RVG后E-cadherin表達(dá)增多、MMP2降低，提示rl-RVG抑制A549細(xì)胞遷移能力較強(qiáng)，rl-RVG對(duì)人肺癌細(xì)胞A549中E-cadherin、MMP2的合成和分布起一定的調(diào)節(jié)作用。rl-RVG可能通過(guò)增加A549細(xì)胞中的E-cadherin的合成, 減少M(fèi)MP2的合成增加抑制肺癌細(xì)胞的遷移；這一推測(cè)與劃痕愈合實(shí)驗(yàn)，Transwell實(shí)驗(yàn)的結(jié)果一致。

綜上所述，rl-RVG 感染A549后能抑制細(xì)胞生長(zhǎng)及遷移，并可能通過(guò)影響E-cadherin,MMP2的表達(dá)實(shí)施EMT而起作用，但關(guān)于rl-RVG對(duì)肺腺癌抗遷移的影響與肺腺癌EMT的相關(guān)性，還需體內(nèi)實(shí)驗(yàn)的進(jìn)一步證實(shí)。通過(guò)這次實(shí)驗(yàn)，為rl-RVG在臨床上用于肺癌患者術(shù)后的復(fù)發(fā)和轉(zhuǎn)移的治療提供了一定的實(shí)驗(yàn)依據(jù)。

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Recombinant avirulent newcastle disease virus rl-RVG inhibits migration of lung adenocarcinoma A549 and related mechanism

JIN Hui1,2, BU Xue-feng3, WANG Mu-bin2, SU Chun-xiang2, YAN Yu-lan1*

(1.Dept. of Respiratory Medicine，the Affiliated People’ s Hospital of Jiangsu University, Zhenjiang 212002；2.Clinical Medicine College of Jiangsu University, Zhenjiang 212001；3.Dept. of General Surgery，the Affiliated People’ s Hospital of Jiangsu University，Zhenjiang 212002,China)

Objective Observe the effects of rl-RVG on migration of lung adenocarcinoma A549cells, preliminarily explore potential mechanism.Methods The group infected with the rl-RVG was experimental group, The group infected with NDV and the group not infected with virous were control groups.The experimental detected for the expressions of RVG and NDV-HN proteins by Western blot．Cell growth experiment determined the best active concentrations of newcastle disease virus and rl-RVG；Observe the effects of rl-RVG and NDV on migration of lung adenocarcinoma A549 cells by scratch assay and Transwell method.The expression of E-cadherin and MMP2 was observed by Western blot and immunofluorescence.The LaSota strain of NDV was used as control group and PBS was the blank control. Results Neither RVG nor NDV proteins didn’t expressed in blank control group．RVG protein expressed in rl-RVG group and NDV protein expressed in both rl-RVG group and NDV group. Cell proliferation was inhibited in rl-RVG group and NDV group more significantly as compared with the blank control group(P<0.05). After infected with rl-RVG, migration distance and number of cells significantly reduced(P<0.05). After A549 cells were infected with rl-RVG, the expression of E-cadherin was enhanced(P<0.05)and the expression of MMP2 was decreased as compared with the blank control group and NDV group(P<0.05).Conclusions Recombinant avirulent newcastle disease virus can inhibit the migration of A549 cellsinvitro, which may be attributed to regulatory factors of E-cadherin and MMP2 in the procession of epithelial mesenchymal transition EMT.

rl-RVG; lung cancer; migration; epithelial mesenchymal transition

2014- 10- 09

2015- 01- 04

鎮(zhèn)江市社會(huì)發(fā)展基金(2013041)

1001-6325(2015)04-0508-06

R734.2

A

*通信作者(corresponding author)：ylyan2005@163.com