費(fèi)先艷張 新于成功,2▲
南京大學(xué)醫(yī)學(xué)院附屬鼓樓醫(yī)院消化科1(210008) 南京醫(yī)科大學(xué)鼓樓臨床學(xué)院2
?
淋巴細(xì)胞功能相關(guān)抗原-1調(diào)節(jié)Treg細(xì)胞對(duì)炎癥性腸病的療效觀察*
費(fèi)先艷1#張新1于成功1,2▲
南京大學(xué)醫(yī)學(xué)院附屬鼓樓醫(yī)院消化科1(210008)南京醫(yī)科大學(xué)鼓樓臨床學(xué)院2
背景:Treg細(xì)胞對(duì)炎癥性腸病(IBD)具有免疫保護(hù)作用,淋巴細(xì)胞功能相關(guān)抗原-1(LFA-1)參與了IBD時(shí)Treg 細(xì)胞的分化和遷移。目的:探討LFA-1調(diào)節(jié)Treg細(xì)胞對(duì)IBD療效的影響。方法:將LFA-1基因缺失小鼠和相同遺傳背景野生型小鼠各20只分別隨機(jī)分為炎癥組和治療組。小鼠飲用2.5% DSS溶液誘導(dǎo)結(jié)腸炎模型,治療組小鼠通過(guò)尾靜脈回輸體外誘導(dǎo)的Treg細(xì)胞。觀察小鼠一般情況和結(jié)腸組織學(xué)表現(xiàn),流式細(xì)胞術(shù)檢測(cè)外周血、脾臟、腸系膜淋巴結(jié)中Treg細(xì)胞比例,ELISA法檢測(cè)血清TGF-β1、IL-10、IL-17A、IFN-γ水平,實(shí)時(shí)PCR檢測(cè)結(jié)腸組織TGF-β1、IL-10 mRNA表達(dá)。結(jié)果:LFA-1缺失治療組結(jié)腸組織學(xué)評(píng)分與野生型治療組無(wú)明顯差異。LFA-1缺失治療組腸系膜淋巴結(jié)Treg細(xì)胞比例顯著高于相應(yīng)炎癥組(P<0.05);LFA-1缺失治療組外周血、脾臟、腸系膜淋巴結(jié)Treg細(xì)胞比例顯著低于野生型治療組(P<0.05)。與相應(yīng)炎癥組相比,野生型和LFA-1缺失治療組血清TGF-β1、IL-10以及結(jié)腸組織TGF-β1 mRNA表達(dá)顯著升高(P<0.05),IL-17A、IFN-γ以及IL-10 mRNA表達(dá)顯著降低(P<0.05)。野生型治療組血清TGF-β1、IL-10以及TGF-β1、IL-10 mRNA表達(dá)顯著高于LFA-1缺失治療組(P<0.05),IL-17A、IFN-γ顯著降低(P<0.05)。結(jié)論:LFA-1參與了Treg細(xì)胞功能的調(diào)控,能促進(jìn)Treg細(xì)胞分泌抗炎因子TGF-β1、IL-10等。Treg細(xì)胞治療LFA-1缺失結(jié)腸炎小鼠的療效低于野生型小鼠,可能與Treg細(xì)胞功能和細(xì)胞因子的分泌受到抑制相關(guān)。
淋巴細(xì)胞功能相關(guān)抗原1;T淋巴細(xì)胞,調(diào)節(jié)性;炎癥性腸??;細(xì)胞因子類
Correspondence to:YU Chenggong,Email:chenggong_yu@nju.edu.cn.
Background:Treg cells play an immunoprotective role in inflammatory bowel disease (IBD).Lymphocyte function-associated antigen-1 (LFA-1) is involved in the differentiation and migration of Treg cells in IBD.Aims:To investigate the efficacy of LFA-1 on IBD by regulating Treg cells.Methods:Twenty LFA-1 gene knockout (LFA-1-/-) mice and 20 wild type mice with same genetic background were randomly divided into colitis group and treatment group,respectively.Colitis model was induced by drinking 2.5% DSS solution.Mice in treatment group were injected with Treg cells induced in vitro through the tail vein.General condition and colon histology were examined.Percentages of Treg cells in peripheral blood,spleen and mesenteric lymph node were detected by flow cytometry.Serum levels of TGF-β1,IL-10,IL-17A and IFN-γ were measured by ELISA.mRNA expressions of TGF-β1 and IL-10 in colon tissue were detected by real time PCR.Results:No significant difference in colon histological score was found between LFA-1-/-treatment group and wild type treatment group.Percentage of Treg cells in mesenteric lymph node in LFA-1-/-treatment group was significantly higher than that in LFA-1-/-colitis group (P<0.05).Percentages of Treg cells in peripheral blood,spleen and mesenteric lymph node in LFA-1-/-treatment group were significantly lower than those in wild type treatment group (P<0.05).Compared with corresponding colitis group,serum levels of TGF-β1,IL-10 and colon tissue TGF-β1 mRNA expression in wild type treatment group and LFA-1-/-treatment group were significantly increased (P<0.05),levels of IL-17A,IFN-γ and expression of IL-10 mRNA were significantly decreased (P<0.05).Serum levels of TGF-β1,IL-10 and expressions of TGF-β1,IL-10 mRNA in wild type treatment group were significantly increased when compared with LFA-1-/-treatment group (P<0.05),levels of IL-17A and IFN-γ were significantly decreased (P<0.05).Conclusions:LFA-1 is involved in the regulation of Treg cells function,and can induce the secretion of anti-inflammatory cytokines TGF-β1 and IL-10.The efficacy of Treg cells in LFA-1-/-colitis mice is inferior to that in wild-type colitis mice,which may be related to the inhibiting of LFA-1 function and secretion of cytokines.
炎癥性腸病(IBD)包括潰瘍性結(jié)腸炎(UC)和克羅恩病(CD),主要表現(xiàn)為慢性非特異性腸道炎癥,其病因和發(fā)病機(jī)制不明。目前認(rèn)為環(huán)境、微生物、免疫、遺傳等因素參與了IBD的發(fā)病過(guò)程。異常的免疫應(yīng)答能引起腸道對(duì)微生物免疫耐受的失調(diào),進(jìn)而導(dǎo)致腸道組織損傷、炎癥的發(fā)生發(fā)展[1]。CD4+CD25+Foxp3+調(diào)節(jié)性T細(xì)胞(Treg細(xì)胞)在維持腸道免疫耐受和控制腸道炎癥中起有重要作用,動(dòng)物模型和臨床研究均已證實(shí)Treg細(xì)胞的缺失或功能異常與IBD病因?qū)W相關(guān)[2]。研究發(fā)現(xiàn),外源性輸注Treg細(xì)胞能通過(guò)抑制免疫應(yīng)答而控制IBD病情進(jìn)展[3]。淋巴細(xì)胞功能相關(guān)抗原-1(lymphocyte function-associated antigen-1,LFA-1)為淋巴細(xì)胞活化的重要協(xié)同刺激受體,可影響淋巴細(xì)胞激活、增殖、分化[4-5]。近年研究表明,LFA-1參與了Treg細(xì)胞的分化與功能調(diào)節(jié),其表達(dá)是Treg細(xì)胞發(fā)揮抑制功能的必要條件[6-8]。本研究的前期研究[9]發(fā)現(xiàn),LFA-1參與了IBD時(shí)Treg細(xì)胞的分化和遷移。本研究采用葡聚糖硫酸鈉(DSS)誘導(dǎo)結(jié)腸炎小鼠模型[10-11],觀察回輸體外誘導(dǎo)生成的Treg細(xì)胞對(duì)LFA-1基因缺失結(jié)腸炎的療效,旨在明確LFA-1對(duì)Treg細(xì)胞功能的影響。
一、實(shí)驗(yàn)動(dòng)物和主要試劑
SPF級(jí)LFA-1基因缺失C57BL/6J小鼠和相同遺傳背景野生型C57BL/6J小鼠(美國(guó)杰克遜實(shí)驗(yàn)室)各20只,雌雄各半,8~10周齡,體質(zhì)量18~23 g,飼養(yǎng)于南京鼓樓醫(yī)院動(dòng)物實(shí)驗(yàn)中心。
DSS(MP Biomedicals,LL.C),重組小鼠細(xì)胞因子IL-2、重組人細(xì)胞因子TGF-β1(RD),CD3、CD28單克隆抗體、Foxp3破膜固定套盒、FITC標(biāo)記抗鼠CD4抗體、APC標(biāo)記抗鼠CD25抗體、PE標(biāo)記抗鼠CD62L、Foxp3抗體(eBioscience,Inc.),CD4+CD62L+na?ve T磁珠分選試劑盒、CD4+CD25+Regulatory T磁珠分選試劑盒(Miltenyi Biotech),小鼠TGF-β1、IL-10、IL-17A、IFN-γ ELISA試劑盒(上海韻涵生物科技有限公司),TRizol裂解液、逆轉(zhuǎn)錄和熒光定量PCR試劑盒(TAKARA BIO INC.)。
二、研究方法
1.磁珠分選CD4+CD62L+na?ve T細(xì)胞和純度檢測(cè):無(wú)菌取2~3只小鼠脾臟并獲取單個(gè)核細(xì)胞懸液,取1×108個(gè)細(xì)胞用于分選和純度檢測(cè)。
2.na?ve T細(xì)胞體外誘導(dǎo)Treg細(xì)胞:分選前一天用無(wú)菌PBS包被96孔板,包被液中分別加入CD28單抗1 μg/mL和CD3單抗0.5 μg/mL,封板,4 ℃過(guò)夜備用。分選得到的na?ve T細(xì)胞加入AIM-V培養(yǎng)基將密度調(diào)整至1×106/mL,將細(xì)胞懸液以每孔200 μL加入96孔板,37 ℃ 5% CO2細(xì)胞培養(yǎng)箱培養(yǎng)4~5 d。取部分細(xì)胞用于檢測(cè)Foxp3誘導(dǎo)比例,剩余細(xì)胞計(jì)數(shù)后以CD4+CD25+Treg細(xì)胞磁珠分選后用于治療組小鼠的回輸。
3.流式細(xì)胞術(shù)檢測(cè)Foxp3誘導(dǎo)比例:取體外培養(yǎng)的細(xì)胞0.3×106~1×106個(gè)至不同流式管,同時(shí)單標(biāo)管均取一管未染色細(xì)胞作為同型對(duì)照,具體檢測(cè)步驟按說(shuō)明書(shū)操作。
4.磁珠分選CD4+CD25+Treg細(xì)胞和純度檢測(cè):將培養(yǎng)后的細(xì)胞用Treg試劑盒進(jìn)行分選,按流式抗體標(biāo)記說(shuō)明書(shū)行Treg細(xì)胞的純度檢測(cè)。
5.動(dòng)物分組、模型制備和標(biāo)本獲?。簩FA-1基因缺失小鼠和野生型小鼠分別隨機(jī)分為炎癥組和治療組,每組10只。四組小鼠均飲用2.5% DSS溶液5 d誘導(dǎo)結(jié)腸炎模型,第6天開(kāi)始治療組小鼠通過(guò)尾靜脈注射0.5×106個(gè)Treg細(xì)胞后正常飲水,炎癥組小鼠通過(guò)尾靜脈注射等體積PBS溶液后正常飲水。實(shí)驗(yàn)過(guò)程中觀察小鼠精神、活動(dòng)、飲食、排便情況,隔日記錄體質(zhì)量,第8天眼眶取血,記錄末次體質(zhì)量后處死小鼠。
學(xué)術(shù)性數(shù)據(jù)庫(kù)在“研究性教學(xué)”中的意義——新時(shí)代語(yǔ)境下本科生“學(xué)術(shù)性學(xué)習(xí)”研究系列之一 ………………… 簡(jiǎn)圣宇(2/62)
6.組織學(xué)評(píng)分:取回盲部至肛門口段全結(jié)腸,參照Dutra等[12]的標(biāo)準(zhǔn)行組織學(xué)評(píng)分。黏膜正常,無(wú)炎癥,0分;黏膜稍不規(guī)則,極少量白細(xì)胞浸潤(rùn),1分;黏膜不規(guī)則,少量白細(xì)胞浸潤(rùn),2分;黏膜紊亂,大量白細(xì)胞浸潤(rùn),血管密度高,結(jié)腸壁增厚,3分; 黏膜紊亂,白細(xì)胞透壁浸潤(rùn),杯狀細(xì)胞減少,血管密度高,結(jié)腸壁增厚,4分。
7.外周血、脾臟、腸系膜淋巴結(jié)中Treg細(xì)胞的檢測(cè):具體檢測(cè)方法按試劑盒說(shuō)明書(shū)進(jìn)行。
8.實(shí)時(shí)PCR法檢測(cè)結(jié)腸組織TGF-β1、IL-10 mRNA表達(dá):抽提結(jié)腸組織總RNA,逆轉(zhuǎn)錄成cDNA,行實(shí)時(shí)PCR。TGF-β1、IL-10和內(nèi)參GAPDH引物由Sangon公司合成。TGF-β1:F 5’-ATT CCT GGC GTT ACC TTG G-3’,R 5’-AGC CCT GTA TTC CGT CTC CT-3’;IL-10:F 5’-GCC TTA TCG GAA ATG ATC CA-3’,R 5’-AGG GTC TTC AGC TTC TCA CC-3’;GAPDH:F 5’-CAT GGC CTT CCG TGT TCC TA-3’,R 5’-TGT CAT CAT ACT TGG CAG GTT TCT-3’。具體步驟按熒光定量試劑盒說(shuō)明書(shū)操作。以2-ΔΔCt法計(jì)算目的基因mRNA相對(duì)表達(dá)量。
9.ELISA法檢測(cè)血清TGF-β1、IL-10、IL-17A、IFN-γ水平:具體步驟參照試劑盒說(shuō)明書(shū)進(jìn)行。
一、體外Treg細(xì)胞的誘導(dǎo)和檢測(cè)
CD4+CD62L+na?ve T細(xì)胞純度>95%(圖1A)。分選出的na?ve T細(xì)胞在IL-2和TGF-β1作用下誘導(dǎo)為iTreg細(xì)胞,其特異性轉(zhuǎn)錄因子Foxp3誘導(dǎo)的比例約70%(圖1B),Treg細(xì)胞純度>99%(圖1C)。
二、一般情況以及結(jié)腸大體和組織學(xué)表現(xiàn)
造模第3天,四組小鼠開(kāi)始出現(xiàn)精神萎靡、飲食減少、體質(zhì)量下降,第4天時(shí)四組均有部分小鼠出現(xiàn)肉眼血便。改為正常飲水后,炎癥組小鼠精神飲食稍有改善,但體質(zhì)量仍呈下降趨勢(shì);治療組小鼠精神飲食較炎癥組改善明顯,體質(zhì)量下降不明顯且最后呈上升趨勢(shì)。
野生型炎癥組結(jié)腸組織學(xué)評(píng)分顯著高于相應(yīng)治療組(P=0.031 3),而LFA-1缺失炎癥組和治療組相比差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.063 3)。兩組治療組結(jié)腸組織學(xué)評(píng)分差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.148 9)(表1、圖2)。野生型和LFA-1缺失炎癥組小鼠體質(zhì)量和終末結(jié)腸長(zhǎng)度較相應(yīng)治療組明顯降低(P=0.000 1,P=0.000 3);兩組治療組體質(zhì)量下降差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.739 6),終末結(jié)腸長(zhǎng)度差異有統(tǒng)計(jì)學(xué)意義(P=0.024 7)(表1)。
三、外周血、脾臟、腸系膜淋巴結(jié)中Treg細(xì)胞比例
LFA-1缺失治療組外周血、脾臟、腸系膜淋巴結(jié)Treg細(xì)胞比例顯著低于野生型治療組(P=0.000 5,P=0.000 1,P=0.000 2);野生型治療組外周血、脾臟、腸系膜淋巴結(jié)Treg細(xì)胞比例顯著高于相應(yīng)炎癥組(P=0.000 1,P=0.000 1,P=0.000 2);LFA-1缺失治療組外周血、脾臟Treg細(xì)胞比例與相應(yīng)炎癥組差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.180 5,P=0.175 0),但腸系膜淋巴結(jié)Treg細(xì)胞比例顯著高于相應(yīng)炎癥組(P=0.000 5)(表2)。
四、血清TGF-β1、IL-10、IL-17A、IFN-γ水平
LFA-1缺失治療組和野生型治療組血清TGF-β1、IL-10水平顯著高于相應(yīng)炎癥組(P<0.05);IL-17A、IFN-γ水平顯著低于相應(yīng)炎癥組(P<0.05)。LFA-1缺失治療組血清TGF-β1、IL-10水平顯著低于野生型治療組(P<0.05),IL-17A、IFN-γ水平顯著高于野生型治療組(P<0.05)(圖3)。
組別例數(shù)組織學(xué)評(píng)分體質(zhì)量下降值(g)終末結(jié)腸長(zhǎng)度(cm)野生型炎癥組103.00±0.274.99±0.345.24±0.11野生型治療組102.25±0.16*1.68±0.30**6.25±0.05**LFA-1缺失炎癥組102.25±0.166.01±0.275.09±0.15LFA-1缺失治療組102.63±0.181.81±0.23**6.06±0.07**#
與相應(yīng)炎癥組比較,*P<0.05,**P<0.01;#與野生型治療組比較,P<0.05
組別例數(shù)外周血脾臟腸系膜淋巴結(jié)野生型炎癥組100.64±0.061.41±0.072.69±0.13野生型治療組101.02±0.09*2.20±0.13*3.93±0.24*LFA-1缺失炎癥組100.35±0.110.97±0.091.62±0.14LFA-1缺失治療組100.50±0.42#1.12±0.07#2.43±0.10*#
*與相應(yīng)炎癥組比較,P<0.01;#與野生型治療組比較,P<0.01
五、結(jié)腸組織TGF-β1、IL-10 mRNA表達(dá)
LFA-1缺失治療組和野生型治療組結(jié)腸組織TGF-β1 mRNA表達(dá)顯著高于相應(yīng)炎癥組(P<0.05);IL-10 mRNA表達(dá)顯著低于相應(yīng)炎癥組(P<0.05)。LFA-1缺失治療組TGF-β1、IL-10 mRNA表達(dá)顯著低于野生型治療組(P<0.05)(圖4)。
A:CD4+ CD62L+ na?ve T細(xì)胞純度;B:Foxp3比例;C:Treg細(xì)胞純度
A:野生型炎癥組;B:野生型治療組;C:LFA-1缺失炎癥組;D:LFA-1缺失治療組
圖3 各組小鼠血清TGF-β1、IL-10、IL-17A、IFN-γ水平比較
圖4 各組小鼠結(jié)腸組織TGF-β1、IL-10 mRNA表達(dá)比較
近年IBD的發(fā)病率呈上升趨勢(shì),目前臨床治療僅能維持緩解和防止復(fù)發(fā),并不能完全治愈,甚至還造成一定的不良反應(yīng)。近年有研究提出基因和細(xì)胞療法在IBD治療中更有優(yōu)勢(shì),不良反應(yīng)較少見(jiàn)[13],其中Treg細(xì)胞已成為目前研究的熱點(diǎn)。許多藥物的治療旨在恢復(fù)Treg細(xì)胞數(shù)目和改善其功能。如研究發(fā)現(xiàn)英夫利西單抗能調(diào)控IBD血液和腸黏膜組織Foxp3的表達(dá)[14],而Foxp3是Treg細(xì)胞的特異性轉(zhuǎn)錄因子,為Treg細(xì)胞分化和發(fā)揮功能所必需[15-16],提示該藥物可能通過(guò)促進(jìn)Treg細(xì)胞分化從而發(fā)揮治療作用。由此可見(jiàn),調(diào)控Treg細(xì)胞分化和功能,能影響IBD的療效。
LFA-1是β2家族成員,與配體細(xì)胞間黏附分子-1(ICAM-1)的相互作用在淋巴細(xì)胞和免疫系統(tǒng)中起有重要作用,主要參與淋巴細(xì)胞增殖、活化、遷移和歸巢等。研究發(fā)現(xiàn)抑制LFA-1與ICAM-1的結(jié)合可治療自身免疫病,能影響T細(xì)胞活化、增殖和遷移,從而抑制免疫應(yīng)答[17]。此外,LFA-1還可影響Treg細(xì)胞的分化和功能發(fā)揮。研究[7]證實(shí)敲除LFA-1基因可導(dǎo)致外周Treg細(xì)胞數(shù)量和功能的降低。
目前關(guān)于敲除LFA-1對(duì)IBD中Treg細(xì)胞的影響未見(jiàn)報(bào)道。本實(shí)驗(yàn)中LFA-1缺失治療組小鼠體質(zhì)量下降和結(jié)腸組織學(xué)評(píng)分與野生型治療組無(wú)明顯差異,可能與回輸Treg細(xì)胞后觀察天數(shù)較短有關(guān)。但LFA-1缺失治療組終末結(jié)腸長(zhǎng)度較野生型治療組明顯縮短,外周血、脾臟、腸系膜淋巴結(jié)Treg細(xì)胞比例均顯著低于野生型治療組。提示LFA-1通過(guò)影響Treg細(xì)胞的分化和遷移,進(jìn)而影響了IBD的療效。與Wohler等[7]的研究結(jié)果一致。但LFA-1缺失治療組外周血、脾臟Treg細(xì)胞比例與相應(yīng)炎癥組無(wú)明顯差異,而腸系膜淋巴結(jié)Treg細(xì)胞比例顯著升高;提示Treg細(xì)胞在遷移至腸道淋巴結(jié)組織的過(guò)程中可能存在其他黏附分子的參與。
Treg細(xì)胞能通過(guò)多種機(jī)制抑制免疫應(yīng)答。Treg細(xì)胞治療IBD的機(jī)制主要為兩種:一是通過(guò)與靶細(xì)胞接觸,二是通過(guò)分泌TGF-β1、IL-10等抗炎細(xì)胞因子[18-19]。本研究?jī)山M治療組血清TGF-β1和IL-10水平均顯著高于相應(yīng)炎癥組,但LFA-1缺失治療組血清TGF-β1和IL-10水平顯著低于野生型治療組。說(shuō)明LFA-1能影響Treg細(xì)胞分泌抗炎因子TGF-β1、IL-10,從而影響IBD的療效。Wohler等[7]亦發(fā)現(xiàn)Treg細(xì)胞治療LFA-1缺失結(jié)腸炎的療效低于野生型。但LFA-1影響Treg細(xì)胞功能調(diào)控的機(jī)制需進(jìn)一步探究,可能為與靶細(xì)胞直接接觸起作用,或調(diào)控免疫抑制細(xì)胞因子的分泌。本研究還發(fā)現(xiàn),兩組治療組血清IL-17A和IFN-γ水平均顯著低于相應(yīng)炎癥組,且LFA-1缺失治療組血清IL-17A和IFN-γ水平高于野生型治療組,說(shuō)明Treg細(xì)胞發(fā)揮免疫效應(yīng)控制IBD的病理進(jìn)程受LFA-1的影響。為進(jìn)一步說(shuō)明LFA-1影響Treg細(xì)胞治療IBD的療效,本研究對(duì)腸道組織中TGF-β1、IL-10 mRNA表達(dá)進(jìn)行檢測(cè)。結(jié)果發(fā)現(xiàn)兩組治療組TGF-β1 mRNA表達(dá)顯著高于相應(yīng)炎癥組,IL-10 mRNA表達(dá)顯著降低;且LFA-1缺失治療組TGF-β1和IL-10 mRNA表達(dá)顯著低于野生型治療組。結(jié)合小鼠腸系膜淋巴結(jié)Treg細(xì)胞比例結(jié)果,進(jìn)一步說(shuō)明LFA-1參與了Treg細(xì)胞治療IBD時(shí)的分化、遷移和功能調(diào)控。其中IL-10是Treg細(xì)胞發(fā)揮免疫效應(yīng)的抗炎因子,但治療組結(jié)腸組織中IL-10 mRNA表達(dá)低于相應(yīng)炎癥組,可能與本實(shí)驗(yàn)回輸?shù)腡reg細(xì)胞為體外誘導(dǎo)有關(guān)。Karlsson等[20]發(fā)現(xiàn)體外誘導(dǎo)的Treg細(xì)胞表面表達(dá)的腸道歸巢分子CCR9和α4β7下降明顯。提示體外誘導(dǎo)的Treg細(xì)胞治療IBD時(shí)的遷移腸道組織受到影響。
雖然既往研究證實(shí)抗LFA-1可用于治療一些自身免疫病,但不能忽視其對(duì)Treg細(xì)胞分化和功能發(fā)揮的負(fù)向調(diào)控作用。本課題組的前期研究[9]和腦脊髓炎的動(dòng)物模型[21]均證實(shí)LFA-1基因缺失能減少體內(nèi)Treg細(xì)胞的數(shù)目。本實(shí)驗(yàn)證實(shí)LFA-1參與了Treg細(xì)胞功能的調(diào)控,LFA-1基因缺失能減少Treg細(xì)胞分泌細(xì)胞因子。LFA-1基因缺失小鼠回輸體外誘導(dǎo)的Treg細(xì)胞治療結(jié)腸炎的療效低于野生型小鼠,可能與Treg細(xì)胞的功能發(fā)揮和細(xì)胞因子的分泌受到抑制相關(guān)。說(shuō)明增加Treg細(xì)胞表面LFA-1表達(dá)可能為Treg細(xì)胞用于臨床治療提供新的思路,但相關(guān)結(jié)論仍需進(jìn)一步研究證實(shí)。
1Kaser A,Zeissig S,Blumberg RS.Inflammatory bowel disease[J].Annu Rev Immunol,2010,28:573-621.
2Izcue A,Coombes JL,Powrie F.Regulatory lymphocytes and intestinal inflammation[J].Annu Rev Immunol,2009,27:313-338.
3Mottet C,Uhlig HH,Powrie F.Cutting edge:cure of colitis by CD4+CD25+ regulatory T cells[J].J Immunol,2003,170 (8):3939-3943.
4Varga G,Nippe N,Balkow S,et al.LFA-1 contributes to signal Ⅰ of T-cell activation and to the production of T(h)1 cytokines[J].J Invest Dermatol,2010,130 (4):1005-1012.
5Smith A,Stanley P,Jones K,et al.The role of the integrin LFA-1 in T-lymphocyte migration[J].Immunol Rev,2007,218:135-146.
6Marski M,Kandula S,Turner JR,et al.CD18 is required for optimal development and function of CD4+CD25+ T regulatory cells[J].J Immunol,2005,175 (12):7889-7897.
7Wohler J,Bullard D,Schoeb T,et al.LFA-1 is critical for regulatory T cell homeostasis and function[J].Mol Immunol,2009,46 (11-12):2424-2428.
8Li L,Kim Js,Boussiotis VA.Rap1A regulates generation of T regulatory cells via LFA-1-dependent and LFA-1-independent mechanisms[J].Cell Immunol,2010,266 (1):7-13.
9姚仁玲,鄒曉平,于成功.淋巴細(xì)胞功能相關(guān)抗原-1調(diào)節(jié)Treg細(xì)胞對(duì)炎癥性腸病的影響[J].胃腸病學(xué),2013,18 (6):346-351.
10Wirtz S,Neufert C,Weigmann B,et al.Chemically induced mouse models of intestinal inflammation[J].Nat Protoc,2007,2 (3):541-546.
11Dieleman LA,Ridwan BU,Tennyson GS,et al.Dextran sulfate sodium-induced colitis occurs in severe combined immunodeficient mice[J].Gastroenterology,1994,107 (6):1643-1652.
12Dutra RC,Cola M,Leite DF,et al.Inhibitor of PI3Kγ ameliorates TNBS-induced colitis in mice by affecting the functional activity of CD4+CD25+FoxP3+ regulatory T cells[J].Br J Pharmacol,2011,163 (2):358-374.
13van der Marel S,Majowicz A,van Deventer S,et al.Gene and cell therapy based treatment strategies for inflammatory bowel diseases[J].World J Gastrointest Pathophysiol,2011,2 (6):114-122.
14Li Z,Arijs I,De Hertogh G,et al.Reciprocal changes of Foxp3 expression in blood and intestinal mucosa in IBD patients responding to infliximab[J].Inflamm Bowel Dis,2010,16 (8):1299-1310.
15Beissert S,Schwarz A,Schwarz T.Regulatory T cells[J].J Invest Dermatol,2006,126 (1):15-24.
16Hori S,Nomura T,Sakaguchi S.Control of regulatory T cell development by the transcription factor Foxp3[J].Science,2003,299 (5609):1057-1061.
17Kavanaugh AF,Davis LS,Jain RI,et al.A phase Ⅰ/Ⅱ open label study of the safety and efficacy of an anti-ICAM-1 (intercellular adhesion molecule-1; CD54) monoclonal antibody in early rheumatoid arthritis[J].J Rheumatol,1996,23 (8):1338-1344.
18Boden EK,Snapper SB.Regulatory T cells in inflammatory bowel disease[J].Curr Opin Gastroenterol,2008,24 (6):733-741.
19Mayne CG,Williams CB.Induced and natural regulatory T cells in the development of inflammatory bowel disease[J].Inflamm Bowel Dis,2013,19 (8):1772-1788.
20Karlsson F,Martinez NE,Gray L,et al.Therapeutic evaluation of ex vivo-generated versus natural regulatory T-cells in a mouse model of chronic gut inflammation[J].Inflamm Bowel Dis,2013,19 (11):2282-2294.
21Gültner S,Kuhlmann T,Hesse A,et al.Reduced Treg frequency in LFA-1-deficient mice allows enhanced T effector differentiation and pathology in EAE[J].Eur J Immunol,2010,40 (12):3403-3412.
(2015-11-03收稿;2015-12-11修回)
Efficacy of Lymphocyte Function-associated Antigen-1 on Inflammatory Bowel Disease by Regulating Treg Cells
FEI Xianyan1,ZHANG Xin1,YU Chenggong1,2.
1Department of Gastroenterology,the Affiliated Drum Tower Hospital of Nanjing University Medical School,Nanjing (210008);2Drum Tower School of Clinical Medicine,Nanjing Medical University,Nanjing
Lymphocyte Function-Associated Antigen-1;T-Lymphocytes,Regulatory;Inflammatory Bowel Disease;Cytokines
DOI:10.3969/j.issn.1008-7125.2016.07.003
*本課題為國(guó)家自然科學(xué)基金面上項(xiàng)目(81170359)
#Email:xianyan0228@163.com
▲本文通信作者,Email:chenggong_yu@nju.edu.cn.