王祈璋 吳禎祥 詹宏量 錢藝芳 劉 英 張秋玉

(福建醫(yī)科大學(xué)免疫治療研究所，臨床醫(yī)學(xué)部，福州 350108)

mB7H4-Ig融合蛋白對(duì)咪喹莫特誘導(dǎo)小鼠銀屑病樣皮損的干預(yù)作用①

王祈璋 吳禎祥 詹宏量 錢藝芳 劉 英 張秋玉

(福建醫(yī)科大學(xué)免疫治療研究所，臨床醫(yī)學(xué)部，福州 350108)

目的探討mB7H4-Ig融合蛋白對(duì)咪喹莫特誘導(dǎo)小鼠銀屑病的干預(yù)作用。方法ELISA法測定高壓注射重組質(zhì)粒在體內(nèi)的表達(dá)水平；用咪喹莫特誘導(dǎo)小鼠銀屑病模型，治療組高壓注射mB7H4-Ig質(zhì)粒而對(duì)照組注射Flag-Ig質(zhì)粒，HE染色檢測皮損改變，流式細(xì)胞術(shù)測定外周血免疫細(xì)胞亞群比例。結(jié)果高壓注射后小鼠體內(nèi)可表達(dá)mB7H4-Ig融合蛋白；與對(duì)照組小鼠相比，治療組小鼠皮膚的銀屑病皮損炎癥癥狀及病理改變相對(duì)較輕，PASI 分?jǐn)?shù)降低，外周血中的中性粒細(xì)胞比例明顯降低而CD4+T細(xì)胞比例上調(diào)。結(jié)論mB7H4-Ig融合蛋白通過抑制炎癥反應(yīng)因而改善咪喹莫特誘導(dǎo)的小鼠銀屑病皮損病變，有望成為治療銀屑病新策略。

B7H4；銀屑??；咪喹莫特；中性粒細(xì)胞；T細(xì)胞

銀屑病是常見的四大頑固性皮膚病之一，近年的研究資料表明，該疾病是一種主要由T細(xì)胞及中性粒細(xì)胞等炎癥細(xì)胞所介導(dǎo)的自身免疫性疾病[1,2]。B7同源體4(B7 homology 4，B7-H4)是2003年由Sica等[3]先后發(fā)現(xiàn)的B7家族的新成員，又稱B7S1、VTCN1(V set domain-containing T cell activation inhibitor)，屬于免疫負(fù)調(diào)控分子[4]。類風(fēng)濕性關(guān)節(jié)炎等多種自身免疫性疾病的血清中可檢測到高水平的可溶性B7H4蛋白，膜型mB7H4-Ig蛋白具有抑制中性粒細(xì)胞發(fā)育及抑制T細(xì)胞增殖的作用而發(fā)揮免疫負(fù)調(diào)控作用[5,6]。有關(guān)B7H4與銀屑病相關(guān)的研究尚未見報(bào)道。鑒于銀屑病亦是自身免疫性炎癥性疾病，本項(xiàng)目擬觀察mB7H4-Ig融合蛋白對(duì)銀屑病模型的干預(yù)作用及作用機(jī)制，旨在為臨床治療銀屑病提供的新思路。

1 材料與方法

1.1材料

1.1.1動(dòng)物 8周齡雌性 BALB/c小鼠，體重20～22 g，購自上海斯萊克實(shí)驗(yàn)動(dòng)物有限公司[生產(chǎn)許可證號(hào)為SCXK(滬)2012-0002]，飼養(yǎng)于SPF級(jí)動(dòng)物室，實(shí)驗(yàn)動(dòng)物倫理審查號(hào)為2017-031。

1.1.2主要試劑 咪喹莫特乳膏(含5% IMQ)購自珠海聯(lián)邦制藥股份有限公司；凡士林乳膏為Unilever公司產(chǎn)品；PEI轉(zhuǎn)染試劑、DMEM及細(xì)胞培養(yǎng)基添加劑均購自Sigma公司；去內(nèi)毒素質(zhì)粒大抽提試劑盒購自QIAGEN公司；純化融合蛋白的Protein A柱購自GE公司；SDS-PAGE相關(guān)試劑購自Bio-Rad公司；HE染色試劑購自碧云天生物有限公司；流式抗體、HRP標(biāo)記的鏈霉親和素及TMB購自ebiosience公司；HRP-anti mouse IgG抗體及ECL顯影劑均購自CST公司；293T細(xì)胞為本室保存；mB7H4-Ig表達(dá)質(zhì)粒及對(duì)照質(zhì)粒Flag-Ig、mB7H4-hIg重組蛋白、小鼠B7H4單克隆抗體(6H3、4M5)均由耶魯大學(xué)癌癥研究中心陳列平教授贈(zèng)送。

1.2方法

1.2.1免疫印跡法鑒定mB7H4-Ig融合蛋白的表達(dá)及純度 mB7H4-Ig及Flag-Ig重組質(zhì)粒經(jīng)轉(zhuǎn)化、陽性菌擴(kuò)增培養(yǎng)后用去內(nèi)毒素質(zhì)粒大抽提試劑盒抽提;參照PEI轉(zhuǎn)染試劑的說明，將含有mB7H4胞外段和mIg2a-Fc基因融合拼接的重組質(zhì)粒(mB7H4-Ig)轉(zhuǎn)染293T細(xì)胞，5 d后收集10 ml培養(yǎng)上清經(jīng)Protein A柱純化，獲取mB7H4-Ig融合蛋白；純化蛋白加入含有或不含DTT的上樣緩沖液煮沸裂解，10% SDS-PAGE凝膠電泳及轉(zhuǎn)膜后，用抗B7H4的6H3單克隆抗體(1∶2 500)孵育過夜，次日加入HRP-anti mouse IgG二抗(1∶2 000)，ECL顯影并拍照。

1.2.2ELISA法分析高壓注射重組質(zhì)粒后血清中mB7H4-Ig水平 BALB/c 小鼠10只分成2組，通過尾靜脈分別高壓注射2種質(zhì)粒[30 μg/(2 ml·只)]，注射后第3、6、9、12天眼眶后靜脈叢取血，ELISA法分析血清中B7H4-mIg融合蛋白的表達(dá)水平：用抗mB7H4的6H3單克隆抗體包被ELISA板(2 μg/ml)4℃過夜，次日洗滌后加入1%BSA室溫封閉1 h，洗滌后加入 mB7H4-hIg重組蛋白(標(biāo)準(zhǔn)品，1 000、500、250、125、62.5、31.3、15.7、0 ng/ml)及待測血清37℃孵育2 h，洗滌后加入生物素標(biāo)記的抗mB7H4的4M5單克隆抗體(檢測抗體)37℃孵育1 h，洗滌后加入HRP標(biāo)記的鏈霉親和素室溫孵育1 h，加入TMB顯色，2 mol/L H2SO4終止反應(yīng),酶標(biāo)儀450 nm處讀取OD值。

1.2.3小鼠銀屑病模型的構(gòu)建及質(zhì)粒高壓注射 BALB/c小鼠30只隨機(jī)分3組，對(duì)照組、模型組及治療組各10只。將小鼠麻醉后，刮除其背部毛發(fā)，形成約2 cm ×3 cm大小的暴露區(qū)域，在剃毛區(qū)域涂抹咪喹莫特乳膏(含IMQ 5%)，劑量為62.5 mg/(只·d)；空白組使用等量凡士林乳膏；連續(xù)涂抹12 d。在建模前一天模型組和對(duì)照組進(jìn)行mB7H4-Ig及Flag-Ig重組質(zhì)粒的高壓注射，建模第5天每組取4只小鼠做皮損區(qū)HE染色分析，同時(shí)取外周血做流式染色分析；其余小鼠繼續(xù)觀察皮損進(jìn)展情況。

1.2.4小鼠的PSAI評(píng)分 參照文獻(xiàn)[7]，觀察上述各組皮損的變化情況，進(jìn)行小鼠的銀屑病樣皮損面積和疾病嚴(yán)重程度(Psoriasis area and severity index,PASI)的評(píng)分，依據(jù)PASI 評(píng)分標(biāo)準(zhǔn)對(duì)各組小鼠皮損的紅斑、鱗屑及浸潤增厚程度進(jìn)行積分估計(jì)(0～4分)，將三者積分相加得到總積分。

1.2.5小鼠涂藥皮膚的病理切片HE染色分析 小鼠脫頸處死后，按照九格法各組剪取涂抹區(qū)皮膚，經(jīng)10%甲醛固定、脫水、石蠟包埋及切片，HE常規(guī)染色，光鏡下觀察并拍照。

1.2.6流式分析法檢測外周血免疫細(xì)胞比例 各組小鼠外周血經(jīng)紅細(xì)胞裂解及洗滌后，加入熒光標(biāo)記抗體(FITC anti-CD11b、PE anti-CD4、APC anti-Gr-1、V450 anti-CD8)4℃孵育30 min，在BD Verse流式細(xì)胞儀進(jìn)行檢測分析。

2 結(jié)果

2.1mB7H4-Ig重組質(zhì)粒轉(zhuǎn)染、表達(dá)及純化 將含有mB7H4胞外段和mIg2a-Fc基因融合拼接的重組質(zhì)粒(mB7H4-Ig)轉(zhuǎn)染293T細(xì)胞，收集培養(yǎng)上清經(jīng)Protein A柱純化后獲取mB7H4-Ig融合蛋白。免疫印跡結(jié)果顯示(見圖1)，未經(jīng)DTT處理的目的蛋白分子量在50～150 kD之間，比融合蛋白理論值(52.2 kD)稍大，推測是由于該蛋白形成二聚體、多聚體所致；經(jīng)DTT處理去除二硫鍵后，目的蛋白分子量約50 kD，與預(yù)期的分子量相符。經(jīng)估算細(xì)胞培養(yǎng)上清中目的蛋白分泌量約在0.01 mg/ml左右。

2.2mB7H4-Ig重組質(zhì)粒高壓注射后血清中mB7H4水平 如圖2所示：A圖為以mB7H4-hIg純化蛋白作為標(biāo)準(zhǔn)品，利用結(jié)合不同位點(diǎn)的兩株抗mB7H4單克隆抗體6H3及4M5(生物素標(biāo)記)分別作為包被和檢測抗體，通過雙抗體夾心ELISA法繪制的標(biāo)準(zhǔn)曲線，擬合曲線方程為Y=489.4X-86.04(r=0.959 1)；B圖為高壓注射mB7H4-Ig及 Flag-Ig的重組質(zhì)粒不同時(shí)間后血清中mB7H4-Ig融合蛋白的表達(dá)水平，可見注射后的第1天即開始表達(dá)并達(dá)到最高水平，隨著時(shí)間增加，表達(dá)水平逐漸降低，第12天幾乎檢測不到蛋白的表達(dá)；而高壓對(duì)照組(Flag-Ig)未檢測到mB7H4蛋白的表達(dá)。結(jié)果表明，mB7H4-Ig重組質(zhì)粒高壓注射可在小鼠體內(nèi)獲得表達(dá)并釋放到血清中。

2.3mB7H4-Ig質(zhì)粒高壓注射對(duì)銀屑病小鼠皮損癥狀的影響 在建模前一天進(jìn)行高壓注射mB7H4-Ig融合蛋白及無關(guān)蛋白Flag-Ig的重組表達(dá)質(zhì)粒，隨后觀察涂藥區(qū)域皮損變化。如圖3所示，空白組(Vaseline)涂藥區(qū)域皮膚無明顯變化，而咪喹莫特(IMQ)涂藥區(qū)域皮膚在第3天皮膚出現(xiàn)紅斑、鱗屑、增厚等典型的銀屑病樣皮損，第5天癥狀最嚴(yán)重，第8天皮損改變開始自行消退。與對(duì)照組(IMQ-Flag-Ig)相比，治療組(IMQ-mB7H4-Ig)小鼠的皮膚受損癥狀較輕且消退較早，PASI評(píng)分尤其是浸潤增厚程度評(píng)分顯著降低(見圖4C)，提示mB7H4-Ig融合蛋白具有抑制咪喹莫特誘導(dǎo)小鼠銀屑病皮損的作用。

圖1 免疫印跡法鑒定mB7H4-Ig融合蛋白Fig.1 Analysis of purified mB7H4-Ig fusion protein by Western blotNote:M.Standard protein marker；1.Denatured fusion protein；2.Undenatured fusion protein.

圖2 重組質(zhì)粒高壓注射后小鼠血清中mB7H4水平Fig.2 Level of mB7H4 in mouse sera after hydrodynamic injection of recombinant plasmidsNote:A.Sensitive sandwich enzyme-linked immunosorbent assay (ELISA) was established to estimate mB7H4 fusion protein concentrations (left);B.The level of mB7H4 in mouse sera was examined on day 3,6,9,12 after tail vein hydrodynamic injection of recombinant mB7H4-Ig or Flag-Ig plasmids (right).

2.4各組小鼠涂藥區(qū)皮膚HE染色結(jié)果 在建模第5天處死小鼠，取涂藥區(qū)皮膚進(jìn)行HE染色，結(jié)果如圖5所示：空白組小鼠的皮膚表皮較薄，僅 2～3層，細(xì)胞形態(tài)正常；而IMQ處理的小鼠涂藥區(qū)皮膚可見表皮層明顯增厚、角化不全、新生血管增多、顆粒層減少和棘層肥厚、炎性細(xì)胞浸潤增多等銀屑病皮膚病理特征；與對(duì)照組相比，mB7H4-Ig高壓注射的治療組表皮層厚度降低、角化不全減少、炎癥細(xì)胞浸潤及血管增生擴(kuò)張等均有所減輕。由此可見，mB7H4-Ig融合蛋白具有緩解咪喹莫特誘導(dǎo)銀屑病樣皮損癥狀的作用。

2.5mB7H4-Ig高壓注射對(duì)銀屑病小鼠外周血免疫細(xì)胞亞群比例的影響 在建模第5天取各組小鼠外周血進(jìn)行流式染色分析。結(jié)果顯示：與空白組(Vaseline)相比，咪喹莫特(IMQ)涂藥組小鼠外周血細(xì)胞中的中性粒細(xì)胞(CD11b+Gr-1+)比例明顯增高(P=0.000 8)，CD4+T細(xì)胞比例明顯減少(P=0.001 2)，而單核細(xì)胞(CD11b+ly6C+)及CD8+T的比例差異不顯著(見表1)。有意義的是，與對(duì)照組(IMQ-Flag-Ig)相比，治療組(IMQ-mB7H4-Ig)小鼠外周血中的中性粒細(xì)胞比例升高幅度降低 (P=0.009)且CD4+T細(xì)胞比例略有上升(P=0.013 2)(表1，圖6)。這一結(jié)果說明，mB7H4-Ig融合蛋白可通過降低咪喹莫特誘導(dǎo)的炎癥細(xì)胞尤其是中性粒細(xì)胞的浸潤而減輕皮損的癥狀。

圖3 重組質(zhì)粒高壓注射后建模小鼠皮損癥狀變化Fig.3 Morphology of mouse skin after hydrodynamic injection of recombinant plasmidsNote:The gross observation of mouse lesional skin in different groups on day 3,day 5 and day 8. Vaseline:BALB/c mice were treated with vaseline as control; IMQ-mB7H4-Ig:BALB/c mice were treated with IMQ after hydrodynamic injection of mB7H4-Ig plasmids; IMQ-Flag-Ig:BALB/c mice were treated with IMQ after hydrodynamic injection of Flag-Ig plasmids.

圖4 重組質(zhì)粒高壓注射后各組小鼠的皮損PASI評(píng)分Fig.4 PASI scores of mouse skin lesion induced by IMQ after hydrodynamic injection of recombinant plasmidsNote:Scores of mouse skin lesions induced by IMQ. Erythema (A),scaling (B) and thickness (C) were scored daily on a scale from 0 to 4,then the cumulative scores (D) were calculated. The scores were expressed as the Mean±SEM,n=6.

圖5 重組質(zhì)粒高壓注射后各組小鼠涂藥區(qū)HE染色變化Fig.5 Hematoxylin eosin staining of mouse skin after hydrodynamic injection of recombinant plasmidsNote:HE staining of IMQ induced mouse skin lesions in different groups on day 5. Psoriasis-like lesions were characterized by increased vascularization (circles),inflammatory cell infiltration (arrows) and epidermal hyperplasia (double arrows). Vaseline:BALB/c mice were treated with vaseline as control; IMQ-mB7H4-Ig:BALB/c mice were treated with IMQ after hydrodynamic injection of mB7H4-Ig plasmids; IMQ-Flag-Ig:BALB/c mice were treated with IMQ after hydrodynamic injection of Flag-Ig plasmids.,×200 magnification (left),×400 magnification (right).

表1 重組質(zhì)粒高壓注射后各組小鼠外周血免疫細(xì)胞亞群比例變化

Note：Vaseline vs IMQ-Flag-Ig，1)P<0.000 1,2)P<0.001；IMQ-Flag-Ig vs IMQ-mB7H4-Ig，3)P<0.001,4)P<0.05. The percentages were expressed as the Mean±SEM,n=4.

圖6 重組質(zhì)粒高壓注射后各組小鼠外周血流式細(xì)胞術(shù)分析結(jié)果Fig.6 Flow cytometric analysis of murine peripheral blood after hydrodynamic injection of recombinant plasmidsNote:The proportion of CD11b+ Gr-1+ neutrophils in peripheral blood derived from different groups mice was analyzed by flow cytometry and analysis gates set on the lymphocytes (left);The proportion of CD4+ T,CD8+ T cells in peripheral blood was analyzed by flow cytometry and analysis gates set on CD45+ cells (right).

3 討論

銀屑病是一種常見的以表皮增殖和炎癥為特征的紅斑鱗屑性慢性炎癥性皮膚病。隨著對(duì)其發(fā)病的免疫機(jī)制研究的不斷深入,多數(shù)研究者認(rèn)為T細(xì)胞介導(dǎo)的免疫應(yīng)答在銀屑病皮損的發(fā)生及持續(xù)存在中起到了重要的作用。在銀屑病皮損局部可測到大量活化T細(xì)胞的浸潤，活化CD4+Th1細(xì)胞亞群通過釋放IFN-γ、TNF-α等的細(xì)胞因子加重局部的炎癥作用，后者通過促進(jìn)角質(zhì)形成細(xì)胞的增殖及抑制其發(fā)生凋亡而促進(jìn)銀屑病表皮細(xì)胞增生[8]。近年來，CD4+Th17及其釋放的細(xì)胞因子IL-17被視為銀屑病發(fā)病的關(guān)鍵性細(xì)胞亞群和細(xì)胞因子[9-11]。由于銀屑病存在著多種免疫學(xué)異常表現(xiàn)，抗TNF-α、IL-17A抗體等靶向活化T細(xì)胞的藥物在銀屑病臨床試驗(yàn)中顯現(xiàn)一定的療效[12，13]。

Chen課題組最早提出B7H4分子是一個(gè)重要的免疫負(fù)調(diào)控因子，具有抑制T細(xì)胞增殖的作用[3]；隨后，在李斯特菌感染的小鼠模型中發(fā)現(xiàn)，mB7H4-Ig融合蛋白可顯著抑制骨髓中中性粒細(xì)胞前體細(xì)胞的生成、增殖和吞噬能力[6]；在膠原誘導(dǎo)的小鼠關(guān)節(jié)炎模型中發(fā)現(xiàn)，高壓注射帶有mB7H4胞外段和mIg2a-Fc基因融合拼接的重組質(zhì)粒(mB7H4-Ig)具有抑制小鼠關(guān)節(jié)炎癥進(jìn)展的作用。鑒于銀屑病是一種中性粒細(xì)胞及T細(xì)胞浸潤的自身免疫性炎癥性疾病，我們推測B7H4-Ig融合蛋白可能具有緩解銀屑病臨床病變的作用。本研究通過構(gòu)建咪喹莫特誘導(dǎo)小鼠銀屑病樣皮損的模型，探討外源性mB7H4-Ig融合蛋白對(duì)銀屑病的干預(yù)作用及其可能的作用機(jī)制。

首先，我們將含有mB7H4胞外段和mIg2a-Fc基因融合拼接的重組質(zhì)粒(mB7H4-Ig)轉(zhuǎn)染293T細(xì)胞，Protein A柱純化獲得融合蛋白。但該融合蛋白在293T細(xì)胞中表達(dá)量有限，無法滿足體內(nèi)實(shí)驗(yàn)的需求。隨后，我們通過尾靜脈高壓注射mB7H4-Ig重組質(zhì)粒取代融合蛋白的直接注射。結(jié)果顯示，高壓注射法可誘導(dǎo)體內(nèi)表達(dá)mB7H4-Ig融合蛋白(見圖2)。選擇咪喹莫特作為構(gòu)建小鼠銀屑病模型的誘導(dǎo)劑，涂藥3 d后即可在涂藥區(qū)域觀察到紅斑、鱗屑、皮膚增厚等典型銀屑病改變，表明模型成功建立(見圖3)。在涂藥前一天高壓注射mB7H4-Ig重組質(zhì)粒進(jìn)行治療，治療后小鼠銀屑病樣皮損癥狀略緩解。PASI評(píng)分結(jié)果顯示，mB7H4-Ig重組質(zhì)粒干預(yù)組小鼠皮損的紅斑、鱗屑、皮膚增厚等評(píng)分均低于對(duì)照組。HE染色顯示，治療組皮損局部中性粒細(xì)胞等炎癥細(xì)胞浸潤、血管增生、表皮增厚等銀屑病的病理改變均較對(duì)照組顯著減輕(見圖5)。上述結(jié)果表明，mB7H4-Ig融合蛋白對(duì)銀屑病皮損有一定的干預(yù)作用。流式細(xì)胞分析顯示，治療組外周血中性粒細(xì)胞的比例顯著低于對(duì)照組。新近有研究表明，中性粒細(xì)胞可以分泌產(chǎn)生胞外誘捕網(wǎng)(Neutrophil extracellular traps，NET)促進(jìn)炎癥反應(yīng)，參與多種疾病自身免疫性損傷[14]。由此，我們推測，mB7H4-Ig融合蛋白可通過抑制中性粒細(xì)胞的生成或抑制其向皮損局部遷移或活化等機(jī)制而減輕銀屑病小鼠皮損癥狀及病理表型的改變，該結(jié)果與Zhu等報(bào)道m(xù)B7H4-Ig融合蛋白抑制中性粒細(xì)胞活化的結(jié)果一致[6]。

有趣的是，我們還觀察到咪喹莫特誘導(dǎo)的小鼠外周血淋巴細(xì)胞比例尤其是CD4+T細(xì)胞的比例顯著低于凡士林誘導(dǎo)組，mB7H4-Ig重組質(zhì)粒高壓后外周血CD4+T細(xì)胞的比例有所提高。已知，咪喹莫特是Toll樣受體7激動(dòng)劑(TLR7 agonist)。有的研究報(bào)道，咪喹莫特可結(jié)合皮膚Langerin(neg)常規(guī)型樹突狀細(xì)胞(Conventional dendritic cells)上TLR7受體，繼而促進(jìn)DC的活化并釋放大量IL-23，IL-23可促進(jìn)Th17細(xì)胞的分化及釋放IL-17，IL-23/IL-17軸是咪喹莫特的誘導(dǎo)小鼠皮膚發(fā)生銀屑病樣炎癥級(jí)聯(lián)反應(yīng)的重要因素[15]。2015年，Ramirez-Valle等[16]報(bào)道，咪喹莫特誘導(dǎo)的銀屑病模型皮膚局部存在一群Vγ4+γδT細(xì)胞，這群細(xì)胞具有記憶性T細(xì)胞的特征，IMQ再次誘導(dǎo)后可迅速活化并大量釋放IL-17A、IL-1β等炎癥因子，是介導(dǎo)銀屑病樣炎癥的主要細(xì)胞。我們的研究發(fā)現(xiàn)，伴隨著皮損炎癥的減輕，mB7H4-Ig治療后第5天及第8天小鼠外周血CD4+T細(xì)胞比例增高并接近未造模小鼠，而皮損局部浸潤的細(xì)胞比例亦顯著降低。因此，我們推測，造模后小鼠外周血CD4+T細(xì)胞的減少可能是CD4+效應(yīng)T細(xì)胞向皮損局部遷移所致，mB7H4-Ig減少效應(yīng)T細(xì)胞向皮損局部浸潤導(dǎo)致外周血CD4+T細(xì)胞比例略回升。后期研究將進(jìn)一步明確mB7H4-Ig作用的T細(xì)胞亞群及對(duì)炎癥性細(xì)胞因子表達(dá)水平的影響。

總之，本研究率先嘗試將mB7H4-Ig融合蛋白用于治療小鼠銀屑病皮損。結(jié)果表明，高壓注射B7H4-Ig融合蛋白可通過抑制中性粒細(xì)胞及CD4+T細(xì)胞的浸潤而達(dá)到減輕銀屑病皮損局部的炎癥病變。該研究結(jié)果為尋找臨床治療銀屑病的新靶點(diǎn)和新策略提供一定的理論基礎(chǔ)。

[1] Martin G,Guerard S,Fortin MM，etal.Pathological crosstalk in vitro between T lymphocytes and lesional keratinocytes in psoriasis:necessity of direct cell-to-cell contact [J].Lab Invest,2012,92(7):1058-1070.

[2] Guerard S,Allaeys I,Martin G，etal.Psoriatic keratinocytes prime neutrophils for an overproduction of superoxide anions [J].Arch Dermatol Res,2013,305(10):879-889.

[3] Sica GL,Choi IH,Zhu G，etal.B7-H4,a molecule of the B7 family,negatively regulates T cell immunity [J].Immunity,2003,18(6):849-861.

[4] Zang X,Loke P,Kim J，etal.B7x:a widely expressed B7 family member that inhibits T cell activation [J].Proc Natl Acad Sci USA,2003,100(18):10388-10392.

[5] Azuma T,Zhu G,Xu H，etal.Potential role of decoy B7-H4 in the pathogenesis of rheumatoid arthritis:a mouse model informed by clinical data [J].PLoS Med,2009,6(10):e1000166.

[6] Zhu G,Augustine MM,Azuma T，etal.B7-H4-deficient mice display augmented neutrophil-mediated innate immunity [J].Blood,2009,113(8):1759-1767.

[7] van de Kerkhof PC.The Psoriasis area and severity index and alternative approaches for the assessment of severity:persisting areas of confusion [J].Br J Dermatol,1997,137(4):661-662.

[8] Lowes MA,Suarez-Farinas M,Krueger JG.Immunology of psoriasis [J].Annu Rev Immunol,2014,32：227-255.

[9] Lowes MA,Russell CB,Martin DA，etal.The IL-23/T17 pathogenic axis in psoriasis is amplified by keratinocyte responses [J].Trends Immunol,2013,34(4):174-181.

[10] Kagami S,Rizzo HL,Lee JJ，etal.Circulating Th17,Th22,and Th1 cells are increased in psoriasis [J].J Invest Dermatol,2010,130(5):1373-1383.

[11] Girolomoni G,Mrowietz U,Paul C.Psoriasis:rationale for targeting interleukin-17 [J].Br J Dermatol,2012,167(4):717-724.

[12] Lee E,Zarei M,LaSenna C，etal.Psoriasis Targeted Therapy:Characterization of Interleukin 17A Expression in Subtypes of Psoriasis [J].J Drugs Dermatol,2015,14(10):1133-1136.

[13] Benoit S,Toksoy A,Brocker EB，etal.Treatment of recalcitrant pustular psoriasis with infliximab:effective reduction of chemokine expression [J].Br J Dermatol,2004,150(5):1009-1012.

[14] Pinegin B,Vorobjeva N,Pinegin V.Neutrophil extracellular traps and their role in the development of chronic inflammation and autoimmunity [J].Autoimmun Rev,2015,14(7):633-640.

[15] Wohn C,Ober-Blobaum JL,Haak S，etal.Langerin(neg)conventional dendritic cells produce IL-23 to drive psoriatic plaque formation in mice [J].Proc Natl Acad Sci U S A,2013,110(26):10723-10728.

[16] Ramirez-Valle F,Gray EE,Cyster JG.Inflammation induces dermal Vgamma4+ gammadeltaT17 memory-like cells that travel to distant skin and accelerate secondary IL-17-driven responses [J].Proc Natl Acad Sci U S A,2015,112(26):8046-8051.

[收稿2017-04-28 修回2017-06-20]

(編輯 倪 鵬)

EffectofmB7H4-Igfusionproteinonmousepsoriasis-likelesionsinducedbyimiquimod

WANGQi-Zhang,WUZhen-Xiang,ZHANHong-Liang，QIANYi-Fang，LIUYing,ZHANGQiu-Yu.
InstituteofImmunotherapy,SchoolofClinicalMedicine，F(xiàn)ujianMedicalUniversity,Fuzhou350108，China

Objective:To explore the effects of mB7H4-Ig fusion protein on mouse psoriasis-like lesions induced by imiquimod(IMQ).MethodsThe level of mB7H4-Ig fusion protein was detected by ELISA after hydrodynamic injection of recombinant plasmids.The therapeutic groups were injected with mB7H4-Ig plasmids and control groups were injected with Flag-Ig plasmids following IMQ treatment.5 days after treatment,the skin lesions were determined by hematoxylin-eosin staining and the percentage of immune cells in murine peripheral blood cells was measured by flow cytometric analysis.ResultsThe expression of mB7H4-Ig fusion protein was detected in mouse sera after hydrodynamic injection.Compared with control groups,mB7H4-Ig groups were alleviated,with decreased epidermal inflammation and lower PASI scores.The percentage of neutrophils in peripheral blood was reduced significantly in mB7H4-Ig groups,but the percentage of CD4+T cell was increased.ConclusionmB7H4-Ig fusion protein improved imiquimod induced psoriasis-like lesions by inhibiting the inflammatory response,indicating the potential therapeutic strategy of B7H4-Ig fusion in psoriasis.

B7 homology 4；Psoriasis;Imiquimod;Neutrophil;T cell

①本文為福建省自然科學(xué)基金(2017J01525)和福建省大學(xué)生創(chuàng)新創(chuàng)業(yè)訓(xùn)練計(jì)劃項(xiàng)目(201610392058)。

R392.12

A

1000-484X(2017)10-1509-06

10.3969/j.issn.1000-484X.2017.10.014

王祈璋(1995年-)，男，主要從事臨床醫(yī)學(xué)方面研究，E-mail：837490042@163.com。

及指導(dǎo)教師：張秋玉(1974年-)，女，醫(yī)學(xué)博士，副教授，碩士生導(dǎo)師，主要從事自身免疫、腫瘤免疫方面研究，E-mail：qiuyu.zhang@fjmu.edu.cn。