孫 琳，安 卓，李常虹，劉 蕊，趙金霞，劉湘源

(北京大學(xué)第三醫(yī)院風(fēng)濕免疫科， 北京 100191)

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·論著·

半乳糖凝集素1在臍帶來(lái)源間充質(zhì)干細(xì)胞調(diào)控類風(fēng)濕關(guān)節(jié)炎患者T細(xì)胞功能中的作用

孫 琳，安 卓，李常虹，劉 蕊，趙金霞，劉湘源△

(北京大學(xué)第三醫(yī)院風(fēng)濕免疫科， 北京 100191)

目的：明確半乳糖凝集素1(galectin-1)在臍帶間充質(zhì)干細(xì)胞(umbilical cord mesenchymal stem cells, UC-MSCs)調(diào)控類風(fēng)濕關(guān)節(jié)炎(rheumatoid arthritis, RA)患者T細(xì)胞功能中的作用。方法： 應(yīng)用慢病毒構(gòu)建galectin-1低表達(dá)的UC-MSCs，即UC-MSCs(Gal-1-)，采用直接接觸培養(yǎng)方式分別將UC-MSCs、UC-MSCs(Gal-1-)與RA患者的CD4+T細(xì)胞共培養(yǎng)。設(shè)立陰性對(duì)照組(CD4+T)、陽(yáng)性對(duì)照組[CD4+T培養(yǎng)過(guò)程中加入植物血凝素(phytohae-magg lutinin，PHA)2 mg/L]、UC-MSCs-CD4+T共培養(yǎng)組、UC-MSCs(對(duì)照shRNA)-CD4+T共培養(yǎng)組及UC-MSCs(Gal-1-)-CD4+T共培養(yǎng)組。MTS法檢測(cè)CD4+T細(xì)胞增殖，ELISA方法檢測(cè)共培養(yǎng)上清液中腫瘤壞死因子α(tumor necrosis factors α, TNF-α)水平，流式細(xì)胞術(shù)檢測(cè)Th1/Th2/Th17細(xì)胞亞型。結(jié)果： UC-MSCs和UC-MSCs(對(duì)照shRNA)與CD4+T細(xì)胞共培養(yǎng)組可顯著抑制PHA誘導(dǎo)的CD4+T細(xì)胞增殖及TNF-α表達(dá)，而UC-MSCs(Gal-1-)組對(duì)此無(wú)明顯抑制作用。與陰性對(duì)照組(8.51%±2.04%)相比，UC-MSCs及UC-MSCs(對(duì)照shRNA)組可顯著抑制Th1細(xì)胞分化(分別為4.83%±1.37%和5.13%±0.87%，P值分別為0.012和0.018)，而UC-MSCs(Gal-1-)組對(duì)此無(wú)明顯抑制作用(6.41%±0.96%，P=0.101)。與陰性對(duì)照組相比，UC-MSCs及UC-MSCs(對(duì)照shRNA)組可上調(diào)Th2并下調(diào)Th17比例，但差異并無(wú)統(tǒng)計(jì)學(xué)意義，UC-MSCs(Gal-1-)組的這一作用不明顯。結(jié)論： UC-MSCs可抑制RA患者CD4+T細(xì)胞的增殖、活化并可降低Th1細(xì)胞比例，但敲低galectin-1分子的表達(dá)后該作用減弱，提示galectin-1參與了UC-MSCs抑制RA患者CD4+T細(xì)胞功能的過(guò)程。

關(guān)節(jié)炎，類風(fēng)濕；間質(zhì)干細(xì)胞；半乳糖凝集素類

間充質(zhì)干細(xì)胞(mesenchymal stem cells, MSCs)是一種具有多向分化潛能的干細(xì)胞，可來(lái)源于骨髓、脂肪和臍帶組織，該細(xì)胞具有非特異性的免疫抑制和抗增殖作用，對(duì)多種自身免疫性疾病有潛在的治療作用[1-3]。在類風(fēng)濕關(guān)節(jié)炎(rheumatoid arthritis, RA)中，MSCs的治療潛能越來(lái)越受到關(guān)注。研究證實(shí)臍帶來(lái)源間充質(zhì)干細(xì)胞(Umbilical cord mesenchymal stem cells UC-MSCs)能有效抑制RA患者的T細(xì)胞及成纖維細(xì)胞樣滑膜細(xì)胞(fibroblast like synoviocyte, FLS)活化，UC-MSCs可減輕膠原誘導(dǎo)性關(guān)節(jié)炎(collagen induced arthritis, CIA)小鼠的滑膜炎癥和骨破壞[4]。國(guó)內(nèi)應(yīng)用UC-MSCs靜脈輸注治療RA也取得了良好效果[5]，這提示UC-MSCs有可能成為RA的新療法。T細(xì)胞活化是RA發(fā)病的中心環(huán)節(jié)，RA患者的滑膜及外周血中可見(jiàn)大量寡克隆增殖的T細(xì)胞，此外，患者還存在輔助性T細(xì)胞(helper T cell，Th)(Th1/Th2細(xì)胞)的分化異常，但目前關(guān)于UC-MSCs對(duì)RA患者CD4+T細(xì)胞的免疫調(diào)控機(jī)制尚不清楚，限制了此種新療法的應(yīng)用推廣。

半乳糖凝集素1(galectin-1)屬于galectins家族即β-半乳糖苷結(jié)合蛋白家族[6]。galectin-1被認(rèn)為是免疫反應(yīng)的負(fù)調(diào)節(jié)蛋白，可以抑制活化T細(xì)胞的增殖，促進(jìn)其凋亡，抑制Th1型和Th17型細(xì)胞因子的分泌且促進(jìn)Th2型細(xì)胞因子的分泌[7-8]。近年來(lái)有研究顯示，galectin-1在骨髓間充質(zhì)干細(xì)胞的免疫調(diào)節(jié)中發(fā)揮重要作用[9-11]。我們前期的研究也證實(shí)UC-MSCs高表達(dá)galectin-1[12]，因此推測(cè)galectin-1參與UC-MSCs調(diào)節(jié)RA患者CD4+T細(xì)胞功能的過(guò)程。本研究應(yīng)用慢病毒shRNA感染UC-MSCs，敲低galectin-1分子的表達(dá)，以明確galectin-1在UC-MSCs調(diào)控RA患者CD4+T細(xì)胞功能中的作用。

1 資料與方法

1.1 研究對(duì)象取材

新生兒臍帶取自北京大學(xué)第三醫(yī)院產(chǎn)科足月剖宮產(chǎn)的健康產(chǎn)婦，留取RA患者外周血，磁珠法分選CD4+T細(xì)胞。本研究獲得北京大學(xué)第三醫(yī)院醫(yī)學(xué)倫理委員會(huì)批準(zhǔn)(IRB00006761．2012011)，所有研究對(duì)象均簽署知情同意書。

1.2 主要試劑

FasGrow人間充質(zhì)干細(xì)胞條件培養(yǎng)基購(gòu)自北京百樂(lè)通生物技術(shù)有限公司，鼠抗人細(xì)胞表面標(biāo)記抗體購(gòu)自美國(guó)BD Biosciences公司，成骨、成脂分化誘導(dǎo)培養(yǎng)基購(gòu)自廣州賽業(yè)生物科技有限公司，慢病毒Ad/galectin-1 shRNA構(gòu)建于上海吉?jiǎng)P基因化學(xué)技術(shù)有限公司，MTS試劑購(gòu)自普洛麥格(北京)生物技術(shù)有限公司，腫瘤壞死因子α(tumor necrosis factors α, TNF-α)ELISA檢測(cè)試劑盒購(gòu)自Raybio公司，PerCP-Cy5-CD3、FITC-CD8、PE-IFN-γ、PE-IL-4及PE-IL-17流式抗體購(gòu)自eBioscience公司。

1.3 UC-MSCs的分離培養(yǎng)與鑒定

取臍帶放入高壓滅菌磷酸緩沖鹽溶液(phosphate buffer saline，PBS)中，反復(fù)沖洗后小心撕取動(dòng)、靜脈周圍的Wharton’s jelly組織，剪成小塊接種于培養(yǎng)瓶中，培養(yǎng)7～10 d后顯微鏡下觀察組織塊周圍呈集落生長(zhǎng)的成纖維狀細(xì)胞。取培養(yǎng)至P3代的UC-MSCs制成單細(xì)胞懸液，加入藻紅蛋白(P-phycoerythrin, PE)或異硫氰酸熒光素(fluorescein isothiocyanate，F(xiàn)ITC)標(biāo)記的單克隆抗體，流式細(xì)胞術(shù)檢測(cè)細(xì)胞表面分子CD29、CD73、CD90、CD105、CD14、CD34、CD45及人類白細(xì)胞抗原(human leukocyte antigen，HLA)-DR的陽(yáng)性率。將P3代UC-MSCs接種于6孔板中，將培養(yǎng)液改為成骨/成脂誘導(dǎo)分化培養(yǎng)液進(jìn)行誘導(dǎo)，最終固定細(xì)胞后分別用茜素紅和油紅O對(duì)誘導(dǎo)后的細(xì)胞進(jìn)行染色。

1.4 構(gòu)建galectin-1低表達(dá)的UC-MSCs

構(gòu)建Ad/galectin-1 shRNA及對(duì)照shRNA慢病毒，轉(zhuǎn)染UC-MSCs，構(gòu)建galectin-1低表達(dá)的UC-MSCs，即UC-MSCs(Gal-1-)。應(yīng)用PCR和Western blot法檢測(cè)galectin-1的表達(dá)水平，驗(yàn)證干擾表達(dá)的效果。

1.5 galectin-1對(duì)UC-MSCs調(diào)控RA患者CD4+T增殖及分泌TNF-α的影響

采用直接接觸方式將UC-MSCs和CD4+T共培養(yǎng)，共培養(yǎng)前UC-MSCs均經(jīng)鈷60照射。設(shè)立陰性對(duì)照組(CD4+T)、陽(yáng)性對(duì)照組[CD4+T培養(yǎng)過(guò)程中加入植物血凝素(phytohaemagg lutinin，PHA)2 mg/L]、UC-MSCs-CD4+T共培養(yǎng)組、UC-MSCs(對(duì)照shRNA)-CD4+T共培養(yǎng)組及UC-MSCs(Gal-1-)-CD4+T共培養(yǎng)組。培養(yǎng)時(shí)間為48 h，每組細(xì)胞設(shè)3個(gè)復(fù)孔，實(shí)驗(yàn)終止時(shí)采用MTS法檢測(cè)CD4+T細(xì)胞的增殖情況，另外，在相同條件下收集各組培養(yǎng)上清液，ELISA法檢測(cè)TNF-α水平。

1.6 galectin-1對(duì)UC-MSCs調(diào)控RA患者Th細(xì)胞分化的影響

各組細(xì)胞培養(yǎng)72 h，加入佛波酯、離子霉素和莫能霉素，培養(yǎng)5 h，收集懸浮的CD4+T細(xì)胞，每組又分為對(duì)照管和實(shí)驗(yàn)管。實(shí)驗(yàn)管分別加入PerCP-Cy5-抗CD3、FITC-抗CD8 mAb，4 ℃避光放置30 min對(duì)細(xì)胞表面抗原進(jìn)行染色，固定破膜后分別加入PE-抗IFN-γ/PE-抗IL-4/PE-抗IL-17 mAb，對(duì)照管加入同型對(duì)照抗體，4 ℃避光孵育30 min后采用流式細(xì)胞儀檢測(cè)分析。

1.7 統(tǒng)計(jì)學(xué)分析

所有實(shí)驗(yàn)數(shù)據(jù)均采用SPSS 16.0統(tǒng)計(jì)軟件進(jìn)行分析，實(shí)驗(yàn)結(jié)果均以均數(shù)±標(biāo)準(zhǔn)差表示，多組間計(jì)量資料采用單因素方差分析，P<0.05表示差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 UC-MSCs的鑒定

流式細(xì)胞儀檢測(cè)細(xì)胞表面分子標(biāo)志，結(jié)果顯示細(xì)胞高表達(dá)CD29(99.87%)、CD44(99.82%)、CD73(93.46%)、CD90(99.17%)和CD105(98.93%)，不表達(dá)CD14(0.12%)、CD34(0.48%)及CD45(0.22%)3種造血細(xì)胞表面標(biāo)記和免疫排斥相關(guān)分子HLA-DR(0.19%)。UC-MSCs在成骨和成脂誘導(dǎo)分化培養(yǎng)基作用后，誘導(dǎo)成功的成骨細(xì)胞胞質(zhì)內(nèi)可見(jiàn)被茜素紅染成桔紅色鈣結(jié)節(jié)，而誘導(dǎo)成功的脂肪細(xì)胞胞質(zhì)內(nèi)可見(jiàn)被油紅O著色的脂滴。免疫表型分析及成骨、成脂誘導(dǎo)分化結(jié)果均符合國(guó)際細(xì)胞治療協(xié)會(huì)提出的間充質(zhì)干細(xì)胞的鑒定標(biāo)準(zhǔn)[12]。

2.2 UC-MSCs(Gal-1-)細(xì)胞的建立及檢測(cè)

galectin-1-shRNA慢病毒siRNA序列為GCTGCCAGATGGATACGAA，采用GV248慢病毒載體。UC-MSCs中加入不同含量的galectin-1-shRNA慢病毒，培養(yǎng)后在熒光顯微鏡下觀察，結(jié)果顯示MOI(multiplicity of infection)為50時(shí)，轉(zhuǎn)染效率達(dá)到90%以上(圖1)。嘌呤霉素篩選細(xì)胞后，用PCR及Western blot檢測(cè)細(xì)胞中g(shù)alectin-1的表達(dá)，結(jié)果顯示轉(zhuǎn)染galectin-1-shRNA慢病毒后UC-MSCs(Gal-1-)中g(shù)alectin-1的表達(dá)明顯下調(diào)(圖2)。

A, light microscope; B, fluorescence microscopy. UC-MSCs, umbilical cord mesenchymal stem cells.

圖1 Galectin-1-shRNA慢病毒轉(zhuǎn)染UC-MSCs

Figure1 Galectin-1-shRNA lentivirus transfected to UC-MSCs

A, the mRNA level of galectin by RT-PCR; B, the protein level of galectin by Western blot. 1, UC-MSCs(Gal-1); 2, UC-MSCs (negative control shRNA); 3, UC-MSCs. UC-MSCs, umbilical cord mesenchymal stem cells.

圖2 慢病毒轉(zhuǎn)染后UC-MSCs中g(shù)alectin-1的表達(dá)

Figure2 The expression of galectin-1 on lentivirus shRNA transfected UC-MSCs

2.3 galectin-1可影響UC-MSCs對(duì)RA-CD4+T增殖及分泌TNF-α的抑制作用

MTS結(jié)果顯示，UC-MSCs-CD4+T共培養(yǎng)組可顯著抑制PHA誘導(dǎo)的CD4+T細(xì)胞的增殖(圖3)及TNF-α表達(dá)(圖4)，UC-MSCs(對(duì)照shRNA)-CD4+T共培養(yǎng)組同樣可以明顯抑制PHA對(duì)CD4+T的增殖作用及TNF-α表達(dá)，而UC-MSCs(Gal-1-)組無(wú)明顯抑制作用。

2.4 敲低galectin-1可影響UC-MSCs對(duì)RA患者Th亞型的調(diào)控作用

與對(duì)照組(8.51%±2.04%)相比，UC-MSCs及UC-MSCs(對(duì)照shRNA)組可顯著抑制Th1細(xì)胞分化(4.83%±1.37%和5.13%±0.87%，P值分別為0.012和0.018)，而UC-MSCs(Gal-1-)組對(duì)其無(wú)明顯抑制作用(6.41%±0.96%，P=0.101，圖5)。

1, negative control; 2, PHA 2 mg/L; 3, PHA+UC-MSCs; 4, PHA+UC-MSCs(negative control shRNA); 5, PHA+UC-MSCs(Gal-1). * P<0.05, compared with the other groups; # P<0.05, compared with the PHA group; & P<0.05, compared with the UC-MSCs group. PHA, phytohaemagg lutinin; UC-MSCs, umbilical cord mesenchymal stem cells.

圖3 Galectin-1表達(dá)影響UC-MSCs對(duì)CD4 T細(xì)胞增殖的抑制作用

Figure3 Galectin-1 affected the inhibition effect of UC-MSCs on CD4T cell

1, negtive control; 2, PHA 2 mg/L; 3, PHA+UC-MSCs; 4, PHA+UC-MSCs (negative control shRNA); 5, PHA+UC-MSCs(Gal-1). * P<0.05, compared with the other groups; # P<0.05, compared with the PHA group; & P<0.05, compared with the UC-MSCs group. Abbreviations as in Figure 3.

圖4 Galectin-1對(duì)UC-MSCs調(diào)控CD4 T細(xì)胞分泌TNF-α的影響

Figure4 Galectin-1 affected the TNF-α level of CD4 T cell regulated by UC-MSCs

與陰性對(duì)照組相比，UC-MSCs及UC-MSCs(對(duì)照shRNA)組可上調(diào)Th2并下調(diào)Th17比例，但差異無(wú)統(tǒng)計(jì)學(xué)意義，UC-MSCs(Gal-1-)組的這一作用不明顯，Th2分別為2.71%±1.15%、4.13%±0.74%、3.57%±0.76%、3.01%±0.34%，P值分別為0.060、0.221、0.644；Th17分別為1.67%±0.45%、0.89%±0.44%、0.83%±0.38%、1.41%±0.62%，P值分別為0.085、0.068、0.518。

3 討論

RA是一種以慢性關(guān)節(jié)炎癥和骨質(zhì)破壞為主要特征的全身自身免疫性疾病，如果不經(jīng)過(guò)正規(guī)治療，可發(fā)生關(guān)節(jié)畸形，嚴(yán)重影響患者的生活質(zhì)量。目前RA治療多采用甲氨蝶呤、來(lái)氟米特等慢作用抗風(fēng)濕藥物，雖然生物制劑(如TNF-α拮抗劑)的應(yīng)用使得更多患者受益，但RA的整體臨床緩解率并沒(méi)有顯著提高，臨床上仍有部分患者的病情難以控制，因此尋找新的治療方法至關(guān)重要。

MSCs是一種具有多向分化潛能的干細(xì)胞，可來(lái)源于骨髓、脂肪和臍帶組織，該細(xì)胞具有非特異性的免疫抑制和抗增殖作用，對(duì)多種自身免疫性疾病有潛在的治療作用。在RA中，MSCs的治療潛能越來(lái)越受到關(guān)注。MSCs在關(guān)節(jié)炎動(dòng)物模型及RA中作用的研究也越來(lái)越多，大部分研究表明，骨髓來(lái)源的MSCs(BM-MSCs)能夠阻止CIA鼠的關(guān)節(jié)炎進(jìn)展并減少骨及軟骨的破壞[13-15]，可治療難治性RA[16]，但因抽取骨髓為侵襲性操作，供者難于接受，且BM-MSCs存在高度病毒污染的可能，并隨年齡增長(zhǎng)，其細(xì)胞數(shù)量和擴(kuò)增、分化能力均出現(xiàn)明顯下降趨勢(shì)[17]。臍帶來(lái)源的MSCs因取材方便、易于體外培養(yǎng)擴(kuò)增等優(yōu)點(diǎn)，目前已逐漸成為MSCs的首選來(lái)源。研究證實(shí)，UC-MSCs能抑制RA患者T細(xì)胞及FLS活化，用其治療CIA模型動(dòng)物可減輕實(shí)驗(yàn)動(dòng)物的滑膜炎癥和骨破壞。國(guó)內(nèi)應(yīng)用UC-MSCs靜脈輸注及關(guān)節(jié)腔注射治療RA也取得良好效果，這提示UC-MSCs有可能成為RA的新療法。

galectin-1是一個(gè)相對(duì)分子質(zhì)量為14.5×103的蛋白質(zhì)，由定位在染色體22q13.1的單基因編碼，其結(jié)構(gòu)較為簡(jiǎn)單，含有單一的糖類識(shí)別結(jié)構(gòu)域。galectin-1是典型的細(xì)胞質(zhì)蛋白，但在細(xì)胞表面、細(xì)胞外基質(zhì)、胞質(zhì)和核內(nèi)均發(fā)現(xiàn)有它的表達(dá)，其與細(xì)胞表面復(fù)合糖上的β-半乳糖苷相結(jié)合，從而參與細(xì)胞黏附、生長(zhǎng)調(diào)節(jié)和免疫反應(yīng)等多種生物學(xué)功能。galectin-1被認(rèn)為是免疫反應(yīng)的負(fù)調(diào)節(jié)蛋白，可以抑制活化T細(xì)胞的增殖，促進(jìn)其凋亡，抑制Th1型和Th17型細(xì)胞因子的分泌且促進(jìn)Th2型細(xì)胞因子的分泌[7-8]。galectin-1與T細(xì)胞表面的CD7結(jié)合，激活A(yù)P-1活性并下調(diào)Bcl-2的表達(dá)可能是galectin-1誘導(dǎo)凋亡的分子機(jī)制[18]。有研究發(fā)現(xiàn)，galectin-1在RA發(fā)病中發(fā)揮負(fù)性調(diào)控的作用[19]。外源性給予galectin-1蛋白可減輕CIA的滑膜炎癥和骨破壞。敲除小鼠galectin-1基因后再用Ⅱ型膠原(CII)誘導(dǎo)關(guān)節(jié)炎模型發(fā)現(xiàn)，Gal-1-/-鼠更易出現(xiàn)關(guān)節(jié)炎且炎癥程度顯著高于野生型鼠，血清中無(wú)galectin-1的鼠中IgG1和IgG2型抗CII抗體水平同樣顯著高于野生型鼠[20]。我們前期的研究發(fā)現(xiàn)，UC-MSCs高表達(dá)galectin-1，因此本研究采用慢病毒構(gòu)建galectin-1低表達(dá)的UC-MSCs，觀察galectin-1對(duì)UC-MSCs調(diào)控RA患者CD4+T細(xì)胞功能的影響。

A, flow cytometric staining of intracellular cytokines; B, the proportion of CD4 IFN-γ T cell. Group 1, negative control; Group 2, UC-MSCs-CD4 T; Group 3, UC-MSCs (negative control shRNA)-CD4 T; Group 4, UC-MSCs(Gal-1)-CD4 T. * P<0.05, compared with the negative control group.

圖5 Galectin-1對(duì)UC-MSCs調(diào)控Th1細(xì)胞的影響

Figure5 Galectin-1 affected the inhibition effect of UC-MSCs on Th1 cell

本研究利用GV248慢病毒載體構(gòu)建galectin-1-shRNA慢病毒，轉(zhuǎn)染UC-MSCs，經(jīng)PCR及Western blot鑒定其galectin-1的表達(dá)水平明顯下調(diào)，成功建立了UC-MSC(Gal-1-)；再采用細(xì)胞直接接觸方式將UC-MSCs及UC-MSC(Gal-1-)和CD4+T細(xì)胞共培養(yǎng)，檢測(cè)galectin-1的表達(dá)不同對(duì)UC-MSCs抑制CD4+T細(xì)胞功能的影響。T細(xì)胞活化是RA發(fā)病的中心環(huán)節(jié)，MTS及ELISA結(jié)果顯示，UC-MSCs可顯著抑制PHA誘導(dǎo)的CD4+T細(xì)胞的增殖活化，而敲低galectin-1后此抑制作用顯著減弱。本研究這一結(jié)果與文獻(xiàn)報(bào)道一致，Najar等[9]的研究發(fā)現(xiàn)，下調(diào)BM-MSCs中g(shù)alectin-1分子的表達(dá)后，其對(duì)T細(xì)胞的免疫抑制作用減弱，且T細(xì)胞活化后分泌的促炎性細(xì)胞因子水平降低。RA患者還存在Th1/Th2/Th17細(xì)胞的分化異常，RA患者體內(nèi)存在Th1型細(xì)胞和細(xì)胞因子(如IFN-γ)表達(dá)的增加，提示Th1型CD4+T細(xì)胞在RA中發(fā)揮重要作用。本研究結(jié)果顯示，UC-MSCs可顯著下調(diào)RA患者Th1的比例，但敲低galectin-1后此抑制作用明顯減弱，此外，UC-MSCs可上調(diào)RA患者Th2細(xì)胞比例并下調(diào)Th17分化，但差異均無(wú)統(tǒng)計(jì)學(xué)意義，考慮可能納入例數(shù)較少且不同RA患者外周血中Th2及Th17的比例相差較大影響統(tǒng)計(jì)結(jié)果。

本研究證實(shí)，在體外，UC-MSCs可抑制RA患者CD4+T細(xì)胞的增殖活化，且顯著下調(diào)Th1細(xì)胞比例，但敲低galectin-1分子的表達(dá)后該作用減弱，提示galectin-1參與了UC-MSCs抑制RA患者CD4+T細(xì)胞功能的過(guò)程。

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(2016-08-05收稿)

(本文編輯：任英慧)

Role of galectin-1 in regulation of umbilical cord mesenchymal stem cells on T cells of rheumatoid arthritis

SUN Lin, AN Zhuo, LI Chang-hong, LIU Rui, ZHAO Jin-xia, LIU Xiang-yuan△

(Department of Rheumatology and Immunology, Peking University Third Hospital, Beijing 100191, China)

Objective： The therapeutic potential of umbilical cord mesenchymal stem cells (UC-MSCs) in rheumatoid arthritis (RA) has attracted more and more attention, because of it can suppress the various inflammatory effects of T cells. Galectin-1 is highly expressed in UC-MSCs, as the first lectin mediating the immunomodulatory effect of MSCs. Our study will investigate the effects of galectin-1 in regulation of UC-MSCs on rheumatoid arthritis T cells. Methods: Lentivirus transfected shRNA technique was used to knock down the expression of galectin-1 in UC-MSCs to construct UC-MSCs(Gal-1-). The effects of UC-MSCs and UC-MSCs(Gal-1-) on CD4+T cells in RA patients were investigated by contact system, including negative control group (CD4+T cells), positive control group [CD4+T-phytohemagg lutinin (PHA)], UC-MSCs-CD4+T cells co-culture group, UC-MSCs(control shRNA)-CD4+T cells co-culture group, and UC-MSCs(Gal-1-)-CD4+T cells co-culture group. The proliferation of CD4+T cells was detected by MTS assay. The level of tumor necrosis factors α (TNF-α) in cells supernatant was detected by enzyme linked immunosorbent assay (ELISA). The effect of UC-MSCs on helper T cell (Th) subset was detected by flow cytometry. Results:Invitro, UC-MSCs were capable of inhibiting PHA induced proliferation of CD4+T cells from RA patients, but UC-MSCs(Gal-1-) did not show the significant inhibitory effect. Galectin-1 affect the TNF-α level of CD4+T cells regulated by UC-MSCs. UC-MSCs and UC-MSCs(control shRNA) significantly inhibited the expression of TNF-α in PHA-induced CD4+T cells. However, UC-MSCs(Gal-1-) had no significant inhibitory effect. Furthermore, the Th1 cells were also significantly suppressed by UC-MSCs and UC-MSCs(control shRNA) (4.83%±1.37% and 5.13%±0.87%，P=0.012 andP=0.018). These was no significant difference in the proportion of the Th1 cells between the control group and UC-MSCs(Gal-1-) group (8.51%±2.04% and 6.41%±0.96%，P=0.101). The Th2 cells were protected after silence galectin-1 in UC-MSCs, whereas there was no significant difference. The proportion of Th17 was decreased by co-culture with UC-MSCs and UC-MSCs(control shRNA), but these was also no significant difference. Conclusion: UC-MSCs can inhibit the proliferation and differentiation of CD4+T cells from RA patients, but these effect declined after knocking down the expression of galectin-1. Galectin-1 maybe take part in the regulation of UC-MSCs on rheumatoid arthritis CD4+T cells.

Arthritis, rheumatoid; Mesenchymal stem cells; Galectins

國(guó)家自然科學(xué)基金(81273293)資助 Supported by the National Natural Science Foundation of China (81273293)

時(shí)間：2016-10-31 16：28：46

http://www.cnki.net/kcms/detail/11.4691.R.20161031.1628.016.html

R593.22

A

1671-167X(2016)06-0964-06

10.3969/j.issn.1671-167X.2016.06.008

△ Corresponding author’s e-mail, liu-xiangyuan@263.net