張 敏 李媛媛 張東波 于成功,#
南京中醫(yī)藥大學(xué)中西醫(yī)結(jié)合鼓樓臨床學(xué)院1(210008) 南京大學(xué)醫(yī)學(xué)院附屬鼓樓醫(yī)院消化內(nèi)科2 南京大學(xué)醫(yī)學(xué)院消化科3
Faecalibacteriumprausnitzii對(duì)LFA-1基因敲除的結(jié)腸炎小鼠中Treg細(xì)胞和細(xì)胞因子的影響*
張 敏1李媛媛2張東波3于成功1,2#
南京中醫(yī)藥大學(xué)中西醫(yī)結(jié)合鼓樓臨床學(xué)院1(210008) 南京大學(xué)醫(yī)學(xué)院附屬鼓樓醫(yī)院消化內(nèi)科2南京大學(xué)醫(yī)學(xué)院消化科3
背景:Faecalibacteriumprausnitzii(Fp)可促進(jìn)Treg細(xì)胞的分化,淋巴細(xì)胞功能相關(guān)抗原-1(LFA-1)亦參與Treg細(xì)胞的分化調(diào)節(jié)。目的:探討Fp對(duì)LFA-1基因敲除(LFA-1-/-)的結(jié)腸炎小鼠中Treg細(xì)胞和細(xì)胞因子的影響。方法:將同樣遺傳背景下野生型小鼠和LFA-1-/-小鼠隨機(jī)分為野生型對(duì)照組、野生型治療組、LFA-1-/-對(duì)照組、LFA-1-/-治療組。小鼠飲用DSS溶液誘導(dǎo)結(jié)腸炎模型,治療組小鼠予Fp灌胃。觀察各組小鼠一般狀況和組織病理學(xué)評(píng)分,以流式細(xì)胞術(shù)檢測(cè)脾臟和腸系膜淋巴結(jié)中Treg細(xì)胞比例,ELISA法檢測(cè)外周血清IL-10、TGF-β1含量,實(shí)時(shí)PCR法檢測(cè)結(jié)腸組織IL-10、TGF-β1 mRNA表達(dá)。結(jié)果:與相應(yīng)對(duì)照組相比,野生型治療組結(jié)腸組織學(xué)評(píng)分顯著下降(P<0.05);野生型治療組和LFA-1-/-治療組脾臟和腸系膜淋巴結(jié)中Treg細(xì)胞比例顯著升高(P<0.05),血清IL-10、TGF-β1含量顯著升高(P<0.01),TGF-β1 mRNA表達(dá)顯著升高(P<0.05);LFA-1-/-治療組IL-10 mRNA表達(dá)顯著降低(P<0.01)。與野生型治療組相比,LFA-1-/-治療組血清TGF-β1含量顯著降低(P<0.05),IL-10、TGF-β1 mRNA表達(dá)顯著降低(P<0.05)。結(jié)論:Fp在LFA-1-/-小鼠中可上調(diào)Treg細(xì)胞比例,促進(jìn)Treg細(xì)胞分泌抗炎因子IL-10、TGF-β1。Fp治療野生型結(jié)腸炎小鼠的療效優(yōu)于LFA-1-/-小鼠,可能與LFA-1缺失對(duì)Treg細(xì)胞功能的發(fā)揮和細(xì)胞因子分泌受限有關(guān)。
Faecalibacteriumprausnitzii; 淋巴細(xì)胞功能相關(guān)抗原1; T淋巴細(xì)胞, 調(diào)節(jié)性; 結(jié)腸炎; 白細(xì)胞介素10; 轉(zhuǎn)化生長(zhǎng)因子β1
炎癥性腸病(IBD)為腸道慢性、復(fù)發(fā)性炎癥性疾病,主要包括潰瘍性結(jié)腸炎(UC)和克羅恩病(CD)。目前認(rèn)為腸道菌群參與腸黏膜免疫系統(tǒng)的異常反應(yīng)。Treg細(xì)胞能減輕炎癥反應(yīng),提高免疫耐受。Faecalibacteriumprausnitzii(Fp)是一種具有抗炎和免疫調(diào)節(jié)作用的腸道共生菌。有研究[1]發(fā)現(xiàn),F(xiàn)p及其產(chǎn)物在結(jié)腸炎動(dòng)物模型中具有促進(jìn)Treg細(xì)胞分化和抗炎因子分泌的作用。本研究的前期研究[2]已表明,淋巴細(xì)胞功能相關(guān)抗原(LFA)-1對(duì)Treg細(xì)胞的分化和功能有影響,但Fp對(duì)LFA-1基因敲除(LFA-1-/-)時(shí)影響Treg細(xì)胞分化調(diào)控及其作用機(jī)制目前尚不清楚。本研究通過DSS誘導(dǎo)小鼠結(jié)腸炎模型,旨在探討Fp對(duì)LFA-1-/-結(jié)腸炎小鼠中Treg細(xì)胞和細(xì)胞因子的影響。
一、實(shí)驗(yàn)動(dòng)物和主要試劑
1. 實(shí)驗(yàn)動(dòng)物:LFA-1-/-C57BL/6J小鼠和同樣遺傳背景的野生型C57BL/6J小鼠(美國(guó)Jackson Laboratory)各20只,飼養(yǎng)于南京鼓樓醫(yī)院無特定病原體級(jí)動(dòng)物實(shí)驗(yàn)中心,7~8周齡,雌雄各半,體質(zhì)量20~23 g。
2. 主要試劑:DSS(MP Biomedicals公司),F(xiàn)p(ATCC 27766,美國(guó)模式菌種保藏中心),F(xiàn)ITC標(biāo)記的抗鼠CD4抗體、APC標(biāo)記的抗鼠CD25抗體、PE標(biāo)記的抗鼠Foxp3抗體以及小鼠白細(xì)胞介素-10(IL-10)、轉(zhuǎn)化生長(zhǎng)因子-β1(TGF-β1) ELISA試劑盒(eBioscience, Inc.),紅細(xì)胞裂解液(MILTENYI BIOTEC),引物、反轉(zhuǎn)錄、PCR試劑盒和Trizol裂解液(TAKARA BIO INC.)。
二、研究方法
1. Fp菌培養(yǎng):將Fp接種至LYHBHI培養(yǎng)基于37 ℃厭氧箱中培養(yǎng)48 h。根據(jù)600 nm波長(zhǎng)處吸光度值結(jié)合平板菌落計(jì)數(shù)計(jì)算細(xì)菌濃度,以PBS重選并調(diào)整濃度至1×109CFU/mL。
2. 動(dòng)物模型制備和分組:將40只野生型小鼠和LFA-1-/-小鼠隨機(jī)分為野生型對(duì)照組、野生型治療組、LFA-1-/-對(duì)照組和LFA-1-/-治療組,每組各10只。小鼠飲用3.5% DSS溶液誘導(dǎo)結(jié)腸炎模型,治療組小鼠給予0.2 mL/10 g Fp每天灌胃1次,對(duì)照組以等量PBS灌胃,每天1次,連續(xù)7 d。造模期間觀察小鼠的狀況、活動(dòng)和排便情況,每日稱重。造模第8天,頸椎脫臼法處死小鼠,眼眶取血,無菌取脾臟、腸系膜淋巴結(jié)和結(jié)腸,測(cè)量小鼠回盲部至肛門的結(jié)腸長(zhǎng)度。
3. 組織學(xué)評(píng)分:取結(jié)腸炎病變顯著部位,以中性甲醛固定,行HE染色。參照Dutra等[3]的標(biāo)準(zhǔn)行組織學(xué)評(píng)分。0分,黏膜結(jié)構(gòu)正常,無炎癥;1分,黏膜隱窩損壞1/3伴較少炎性細(xì)胞浸潤(rùn);2分,黏膜隱窩損壞2/3伴少量炎性細(xì)胞浸潤(rùn);3分,黏膜結(jié)構(gòu)損壞嚴(yán)重涉及固有層伴大量炎性細(xì)胞浸潤(rùn);4分,黏膜結(jié)構(gòu)紊亂,炎性細(xì)胞大量浸潤(rùn),甚至有透壁反應(yīng),結(jié)腸壁僵硬增厚。
4. 流式細(xì)胞術(shù)檢測(cè)脾臟和腸系膜淋巴結(jié)中Treg細(xì)胞比例:無菌取小鼠新鮮脾臟和腸系膜淋巴結(jié),充分研磨后脾臟細(xì)胞加入紅細(xì)胞裂解液。予PBS重懸后計(jì)數(shù),取1×106個(gè)細(xì)胞加入表面抗體APC-CD25 0.8 μL、FITC-CD4 1.0 μL,振蕩、避光20 min;加入固定破膜劑800 μL,避光1~2 h;加入PE-Foxp3 2.5 μL,避光孵育45 min,PBS重懸至100 μL 后上流式細(xì)胞儀檢測(cè)。
5. ELISA法檢測(cè)外周血IL-10、TGF-β1含量:具體檢測(cè)步驟按照試劑盒說明書進(jìn)行操作。
6. 實(shí)時(shí)PCR法檢測(cè)結(jié)腸組織IL-10、TGF-β1 mRNA表達(dá):各組隨機(jī)抽取4只小鼠,提取結(jié)腸總RNA,逆轉(zhuǎn)錄合成cDNA,行PCR反應(yīng)。IL-10引物序列:F:5’-GCC TTA TCG GAA ATG ATC CA-3’,R:5’-AGG GTC TTC AGC TTC TCA CC-3’。TGF-β1引物序列:F:5’-ATT CCT GGC GTT ACC TTG G-3’,R:5’-AGC CCT GTA TTC CGT CTC CT-3’。內(nèi)參GAPDH引物序列:F:5’-CAT GGC CTT CCG TGT TCC TA-3’,R:5’-TGT CAT CAT ACT TGG CAG GTT TCT-3’。反應(yīng)條件:95 ℃ 30 min;95 ℃ 30 s,95 ℃ 5 s,60 ℃ 34 s,共40個(gè)循環(huán);60 ℃ 1 min,95 ℃ 15 s。以2-△△Ct法計(jì)算目的基因的表達(dá)。
三、統(tǒng)計(jì)學(xué)分析
一、小鼠的一般狀況、結(jié)腸長(zhǎng)度和病理變化
造模后第3天,兩組對(duì)照組小鼠開始出現(xiàn)進(jìn)食減少,活動(dòng)量少、精神萎靡、便質(zhì)稀,體質(zhì)量下降,第5天對(duì)照組小鼠開始出現(xiàn)部分血便,癥狀隨時(shí)間延長(zhǎng)加重,鏡下可見黏膜正常結(jié)構(gòu)破壞,大量炎性細(xì)胞浸潤(rùn),杯狀細(xì)胞減少;兩組治療組癥狀均較相應(yīng)對(duì)照組減輕,鏡下炎性細(xì)胞浸潤(rùn)程度減輕,同時(shí)體質(zhì)量下降減少(圖1)。
野生型對(duì)照組和LFA-1-/-對(duì)照組結(jié)腸長(zhǎng)度較相應(yīng)治療組明顯縮短(P<0.05),且野生型治療組較LFA-1-/-治療組明顯增加(P<0.05)。野生型對(duì)照組結(jié)腸組織病理學(xué)評(píng)分較相應(yīng)治療組顯著升高(P<0.05),而LFA-1-/-對(duì)照組與LFA-1-/-治療組之間無明顯差異(P>0.05)。兩組治療組結(jié)腸組織病理學(xué)評(píng)分無明顯差異(P>0.05)(表1)。
表1 各組結(jié)腸長(zhǎng)度和組織病理學(xué)評(píng)分(±s)
*與相應(yīng)對(duì)照組比較,P<0.05;▲與野生型治療組比較,P<0.05
二、脾臟和腸系膜淋巴結(jié)中Treg細(xì)胞比例
野生型治療組和LFA-1-/-治療組小鼠脾臟和腸系膜淋巴結(jié)中Treg細(xì)胞比例較相應(yīng)對(duì)照組均顯著升高(P<0.05);野生型治療組脾臟中Treg細(xì)胞比例顯著高于LFA-1-/-治療組(P<0.01),而兩組治療組腸系膜淋巴結(jié)中Treg細(xì)胞比例無明顯差異(P>0.05)(圖2)。
三、血清IL-10、TGF-β1含量
野生型治療組和LFA-1-/-治療組血清IL-10、TGF-β1含量較相應(yīng)對(duì)照組顯著升高(P<0.01),且野生型治療組血清TGF-β1含量顯著高于LFA-1-/-治療組(P<0.05),而兩組治療組血清IL-10含量無明顯差異(P>0.05)(表2)。
A:野生型對(duì)照組;B:野生型治療組;C:LFA-1-/-對(duì)照組;D:LFA-1-/-治療組
與相應(yīng)對(duì)照組比較,*P<0.05,**P<0.01
組 別IL?10(pg/mL)TGF?β1(pg/mL)IL?10mRNA表達(dá)TGF?β1mRNA表達(dá)野生型對(duì)照組83.51±6.53134.70±10.311.27±0.041.06±0.03野生型治療組121.00±11.08??174.10±9.91??1.07±0.141.19±0.07?LFA?1-/-對(duì)照組71.54±6.36114.60±8.251.03±0.040.47±0.13LFA?1-/-治療組105.10±7.24??140.20±8.16??▲0.61±0.09??▲0.70±0.06?▲
與相應(yīng)對(duì)照組比較,*P<0.05,**P<0.01;▲與野生型治療組比較,P<0.05
四、結(jié)腸組織IL-10、TGF-β1 mRNA表達(dá)
與相應(yīng)對(duì)照組相比,野生型治療組和LFA-1-/-治療組結(jié)腸組織TGF-β1 mRNA表達(dá)顯著升高(P<0.05),野生型治療組IL-10 mRNA表達(dá)無明顯差異(P>0.05),LFA-1-/-治療組IL-10 mRNA表達(dá)顯著降低(P<0.01)。野生型治療組結(jié)腸組織IL-10、TGF-β1 mRNA表達(dá)均顯著高于LFA-1-/-治療組(P<0.05)(表2)。
Fp對(duì)維護(hù)腸道菌群的穩(wěn)態(tài)起重要作用,可分泌丁酸鹽和未知產(chǎn)物。有研究發(fā)現(xiàn),F(xiàn)p具有免疫調(diào)節(jié)和抗炎的作用[4]。本課題組的前期動(dòng)物實(shí)驗(yàn)[5]發(fā)現(xiàn),對(duì)于TNBS誘導(dǎo)的實(shí)驗(yàn)性結(jié)腸炎大鼠,F(xiàn)p及其上清液通過增加脾臟中Treg細(xì)胞比例而發(fā)揮治療作用,但具體機(jī)制不明。說明益生菌對(duì)IBD的治療具有舉足輕重的作用。Treg細(xì)胞可降低炎性反應(yīng),抑制炎性進(jìn)展,在維持免疫穩(wěn)態(tài)、抗炎等過程中扮演重要角色。研究證明,Treg細(xì)胞數(shù)量減少或功能下降與IBD的發(fā)展相關(guān)[6]。動(dòng)物實(shí)驗(yàn)[7]顯示,通過體內(nèi)輸注Treg細(xì)胞可減輕重癥聯(lián)合免疫缺陷小鼠的結(jié)腸炎癥狀。臨床研究[8]表明,與正常人相比,IBD患者外周血Treg細(xì)胞比例顯著下降。因此,通過提高Treg細(xì)胞比例治療IBD已成為目前研究的熱點(diǎn)。Treg細(xì)胞分為nTreg細(xì)胞和iTreg細(xì)胞,兩者具有相似的抑制功能[9]。在人體內(nèi),生理情況下nTreg細(xì)胞分布于脾臟和淋巴結(jié)中,而iTreg細(xì)胞主要位于腸道相關(guān)淋巴結(jié)中[10-11]。本研究流式細(xì)胞術(shù)結(jié)果顯示野生型結(jié)腸炎小鼠給予Fp治療后,脾臟和腸系膜淋巴結(jié)Treg細(xì)胞比例顯著升高,同時(shí)癥狀減輕,結(jié)腸長(zhǎng)度增加,組織病理學(xué)評(píng)分降低,說明Fp可有效治療結(jié)腸炎小鼠;LFA-1-/-結(jié)腸炎小鼠經(jīng)Fp治療后,組織病理學(xué)評(píng)分與相應(yīng)對(duì)照組無明顯差異,考慮LFA-1對(duì)Treg細(xì)胞功能有調(diào)控作用,進(jìn)而影響了Fp治療結(jié)腸炎的療效。
LFA-1為β2家族成員,可與細(xì)胞間黏附分子-1(ICAM-1)結(jié)合,對(duì)淋巴細(xì)胞的遷移、活化、歸巢等有顯著作用[12]。動(dòng)物實(shí)驗(yàn)[13-14]表明,敲除LFA-1基因可導(dǎo)致外周Treg細(xì)胞比例和功能的降低。本研究中,F(xiàn)p治療后可提高LFA-1-/-小鼠脾臟和腸系膜淋巴結(jié)Treg細(xì)胞比例,提示Fp通過多種途徑調(diào)控Treg細(xì)胞的分化。本實(shí)驗(yàn)中,野生型治療組脾臟中Treg細(xì)胞比例高于LFA-1-/-治療組,提示Fp與LFA-1可能協(xié)同促進(jìn)Treg細(xì)胞分化,但具體機(jī)制不明;而兩組腸系膜淋巴結(jié)中Treg細(xì)胞比例無明顯差異,考慮到細(xì)胞活性、溫度、抗體結(jié)合度等條件可影響流式細(xì)胞術(shù),實(shí)驗(yàn)結(jié)果可能會(huì)有一定誤差。
Treg細(xì)胞通過分泌IL-10和TGF-β而在IBD中發(fā)揮抗炎作用。有研究表明,TGF-β mRNA表達(dá)可證實(shí)Treg細(xì)胞的存在[15]。Schramm等[16]發(fā)現(xiàn),外周Treg細(xì)胞比例升高是由于轉(zhuǎn)基因小鼠高表達(dá)TGF-β。TGF-β具有抑制炎癥的作用,在自身免疫中發(fā)揮重要作用,TGF-β1缺陷小鼠可引起全身多器官炎癥,并可發(fā)生早期死亡[17]。IL-10主要由巨噬細(xì)胞和單核細(xì)胞產(chǎn)生,具有抗炎作用。IL-10缺失可導(dǎo)致與CD相似的結(jié)腸炎,且CD的復(fù)發(fā)與低含量IL-10相關(guān)[18]。本研究?jī)山M治療組血清IL-10和TGF-β1含量均高于相應(yīng)對(duì)照組,說明Fp抗炎的機(jī)制可能與促進(jìn)抗炎因子的分泌有關(guān)。LFA-1-/-治療組中血清TGF-β1含量顯著低于野生型治療組,而IL-10含量無明顯差異。Wohler等[14]的研究亦發(fā)現(xiàn),Treg細(xì)胞治療LFA-1-/-結(jié)腸炎小鼠的療效不如野生型小鼠。
本研究還發(fā)現(xiàn),野生型和LFA-1-/-結(jié)腸炎小鼠經(jīng)Fp治療后,TGF-β1 mRNA表達(dá)均顯著升高,但野生型治療組IL-10 mRNA表達(dá)與相應(yīng)對(duì)照組無明顯差異,這一結(jié)果與野生型治療組外周血IL-10含量較相應(yīng)對(duì)照組升高不一致,考慮到體內(nèi)促炎因子如IL-17A等可影響IL-10的分泌,前期研究[19]已表明Fp可下調(diào)促炎因子IL-17A。考慮到IL-17A對(duì)IL-10的分泌有負(fù)反饋?zhàn)饔?,有利于維持腸道內(nèi)促炎因子/抗炎因子的平衡,維持腸道內(nèi)穩(wěn)態(tài)。有臨床研究[20]表明,UC患者T細(xì)胞中IL-10 mRNA表達(dá)明顯升高,結(jié)腸中IL-10陽(yáng)性細(xì)胞含量亦明顯升高。本研究中LFA-1-/-治療組IL-10 mRNA表達(dá)較相應(yīng)對(duì)照組顯著降低,考慮LFA-1缺失影響了外周Treg細(xì)胞向腸道組織的遷移。LFA-1-/-小鼠經(jīng)Fp治療后IL-10、TGF-β1 mRNA表達(dá)顯著低于野生型治療組,與LFA-1缺失影響Treg細(xì)胞的分化遷移有關(guān)。而Fp在LFA-1缺失的情況下,起調(diào)控Treg細(xì)胞相關(guān)炎性因子表達(dá)的作用。
綜上所述,雖然LFA-1能調(diào)控Treg細(xì)胞的分化和遷移以及相關(guān)細(xì)胞因子的基因表達(dá),但不能忽視Fp對(duì)Treg細(xì)胞的作用。本實(shí)驗(yàn)發(fā)現(xiàn),F(xiàn)p在LFA-1缺失的情況下能上調(diào)Treg細(xì)胞比例,促進(jìn)抗炎因子TGF-β1 mRNA表達(dá)。Fp治療LFA-1-/-結(jié)腸炎小鼠的療效低于野生型小鼠,可能與LFA-1缺失對(duì)Treg細(xì)胞功能的發(fā)揮和細(xì)胞因子表達(dá)受限有關(guān)。進(jìn)一步探究Fp與LFA-1協(xié)同作用的機(jī)制,可為今后治療IBD提供新思路。
1 Qiu X, Zhang M, Yang X, et al.Faecalibacteriumprausnitziiupregulates regulatory T cells and anti-inflammatory cytokines in treating TNBS-induced colitis[J]. J Crohns Colitis, 2013, 7 (11): e558-e568.
2 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.
3 Dutra 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.
4 Sokol H, Seksik P, Furet JP, et al. Low counts ofFaecalibacteriumprausnitziiin colitis microbiota[J]. Inflamm Bowel Dis, 2009, 15 (8): 1183-1189.
5 楊曉彤, 張明明, 洪娜, 等.Faecalibacteriumprausnitzii對(duì)實(shí)驗(yàn)性結(jié)腸炎大鼠Foxp3+Treg細(xì)胞的影響[J]. 胃腸病學(xué), 2012, 17 (3): 141-145.
6 Boden EK, Snapper SB. Regulatory T cells in inflammatory bowel disease[J]. Curr Opin Gastroenterol, 2008, 24 (6): 733-741.
7 Ogino H, Nakamura K, Ihara E, et al. CD4+CD25+regulatory T cells suppress Th17-responses in an experimental colitis model[J]. Dig Dis Sci, 2011, 56 (2): 376-386.
8 王軼, 劉雪平, 趙治彬, 等. 外周血CD4+CD25+FOXP3+調(diào)節(jié)性T細(xì)胞與炎癥性腸病疾病活動(dòng)度的關(guān)系[J]. 胃腸病學(xué), 2010, 15 (4): 218-221.
9 Horwitz DA, Zheng SG, Gray JD. Natural and TGF-beta-induced Foxp3(+)CD4(+) CD25(+) regulatory T cells are not mirror images of each other[J]. Trends Immunol, 2008, 29 (9): 429-435.
10 Mayne 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.
11 Barnes MJ, Powrie F. Regulatory T cells reinforce intestinal homeostasis[J]. Immunity, 2009, 31 (3): 401-411.
12 李猛, 高春記. LFA-1與配體ICAM-1黏附分子功能的研究進(jìn)展[J]. 中國(guó)實(shí)驗(yàn)血液學(xué)雜志, 2008, 16 (1): 213-216.
13 Li 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.
14 Wohler 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.
15 Becker C, Fantini MC, Neurath MF. TGF-beta as a T cell regulator in colitis and colon cancer[J]. Cytokine Growth Factor Rev, 2006, 17 (1-2): 97-106.
16 Schramm C, Huber S, Protschka M, et al. Blessing TGFbeta regulates the CD4+CD25+T-cell pool and the expression of Foxp3invivo[J]. Int Immunol, 2004, 16 (9): 1241-1249.
17 Annes JP, Munger JS, Rifkin DB. Making sense of latent TGFbeta activation[J]. J Cell Sci, 2003, 116 (Pt 2): 217-224.
18 Kühn R, L?hler J, Rennick D, et al. Interleukin-10-deficient mice develop chronic enterocolitis[J]. Cell, 1993, 75 (2): 263-274.
19 張明明.Faecalibacteriumprausnitzii抑制白介素17表達(dá)和對(duì)大鼠結(jié)腸炎的預(yù)防作用[D]. 南京大學(xué), 2012.
20 Melgar S, Yeung MM, Bas A, et al. Over-expression of interleukin 10 in mucosal T cells of patients with active ulcerative colitis[J]. Clin Exp Immunol, 2003, 134 (1): 127-137.
(2016-08-01收稿;2016-09-06修回)
Effect ofFaecalibacteriumprausnitziion Treg Cells and Cytokines in Colitis Mice with LFA-1 Knockout
ZHANGMin1,LIYuanyuan2,ZHANGDongbo3,YUChenggong1,2.
1ClinicalCollegeofChineseandWesternIntegratedMedicine,NanjingUniversityofChineseMedicine/DrumTowerHospital,Nanjing(210008);2DepartmentofGastroenterology,theAffiliatedDrumTowerHospitalofNanjingUniversityMedicalSchool,Nanjing;3DepartmentofGastroenterology,NanjingUniversityMedicalSchool,Nanjing
YU Chenggong, Email: chenggong_yu@nju.edu.cn.
Faecalibacteriumprausnitzii; Lymphocyte Function-Associated Antigen-1; T-Lymphocytes, Regulatory; Colitis; Interleukin-10; Transforming Growth Factor beta1
10.3969/j.issn.1008-7125.2017.03.006
國(guó)家自然科學(xué)基金項(xiàng)目(81470819)
#本文通信作者,Email: chenggong_yu@nju.edu.cn
Background: It has been widely accepted thatFaecalibacteriumprausnitzii(Fp) induces the differentiation of Treg cells. Lymphocyte function-associated antigen-1 (LFA-1) is also involved in the differentiation of Treg cells. Aims: To investigate the effect of Fp on Treg cells and cytokines in colitis mice with LFA-1 knockout (LFA-1-/-). Methods: Twenty wild type mice and twenty LFA-1-/-mice with same genetic background were randomly divided into wild type control group, wild type treatment group, LFA-1-/-control group and LFA-1-/-treatment group. Colitis model was induced by drinking DSS solution. Mice in the two treatment groups were intragastrically administrated with Fp. General status and histopathological score were assessed. Percentages of Treg cells in spleen and mesenteric lymph nodes were measured by flow cytometry. Serum levels of IL-10 and TGF-β1 were measured by ELISA. mRNA expressions of IL-10 and TGF-β1 in colonic tissue were detected by real time PCR. Results: Compared with corresponding control groups, histopathological score was significantly decreased in wild type treatment group (P<0.05); percentages of Treg cells in spleen and mesenteric lymph nodes were significantly increased (P<0.05), serum levels of IL-10 and TGF-β1 were significantly increased (P<0.01), expression of TGF-β1 mRNA was significantly increased in wild type treatment group and LFA-1-/-treatment group (P<0.05); expression of IL-10 mRNA was significantly decreased in LFA-1-/-treatment group (P<0.01). Compared with wild type treatment group, serum level of TGF-β1 was significantly decreased (P<0.05), and mRNA expressions of IL-10 and TGF-β1 were significantly decreased in LFA-1-/-treatment group (P<0.05). Conclusions: Fp can up-regulate the percentages of Treg cells and enhance the secretion of anti-inflammatory cytokines IL-10 and TGF-β1 in LFA-1-/-mice. The therapeutic efficacy for colitis in wild type mice is superior to that in LFA-1-/-mice, which may be related to the inhibition of function of Treg cells and secretion of cytokines due to LFA-1 knockout.