林曉濱，　陳　穎，　楊德壕，　謝甬淋，　畢　涌，　柯建明，　陳志博，　蘇中錢，　厲　向，　張　旭△

(溫州醫(yī)科大學(xué)附屬第一醫(yī)院 1超聲影像科，2神經(jīng)內(nèi)科，浙江 溫州 325000； 3武警浙江省總隊(duì)杭州醫(yī)院康復(fù)科，浙江 杭州 310000)

脂肪源性干細(xì)胞對(duì)多發(fā)性硬化患者Th17的免疫調(diào)控作用*

林曉濱1，陳穎2，楊德壕2，謝甬淋3，畢涌2，柯建明2，陳志博2，蘇中錢2，厲向2，張旭2△

(溫州醫(yī)科大學(xué)附屬第一醫(yī)院1超聲影像科，2神經(jīng)內(nèi)科，浙江 溫州 325000；3武警浙江省總隊(duì)杭州醫(yī)院康復(fù)科，浙江 杭州 310000)

[摘要]目的： 探討人脂肪源性干細(xì)胞(hASCs)對(duì)多發(fā)性硬化(MS)患者外周血輔助性T細(xì)胞17(Th17)的免疫調(diào)控作用機(jī)制。方法: 分離、純化脂肪組織中的hASCs。采用密度梯度離心法分離MS患者外周血單個(gè)核細(xì)胞(PBMCs)，磁珠分選CD4+T細(xì)胞，體外刺激細(xì)胞向Th17極化，并加入不同比例的hASCs(hASCs∶CD4+T為1∶4和1∶10)共培養(yǎng)4 d，設(shè)立添加anti-LIF抗體組；流式細(xì)胞術(shù)檢測(cè)共培養(yǎng)后Th17細(xì)胞占CD4+T細(xì)胞的比例，real-time PCR檢測(cè)白細(xì)胞介素6受體(IL-6R)、白細(xì)胞介素23受體(IL-23R)、白血病抑制因子受體(LIFR)、維甲酸相關(guān)孤兒受體γt(RORγt)及白血病抑制因子(LIF)的mRNA水平變化；ELISA法檢測(cè)共培養(yǎng)體系上清液中LIF的水平。結(jié)果: 分離的hASCs經(jīng)流式細(xì)胞術(shù)鑒定可基本判定為hASCs；PBMCs經(jīng)磁珠法分選后獲得90%以上純度的CD4+T細(xì)胞。共培養(yǎng)后，1∶4組和1∶10組中Th17細(xì)胞所占比例下降，且存在高濃度抑制效應(yīng)；共培養(yǎng)后RORγt、IL-6R和IL-23R的mRNA表達(dá)水平下降，LIFR和LIF的mRNA表達(dá)水平均升高；加入anti-LIF抗體后，Th17細(xì)胞比例回升至對(duì)照組水平；RORγt和IL-6R的mRNA表達(dá)水平回升；ELISA檢測(cè)各組LIF的水平，共培養(yǎng)組LIF分泌均較對(duì)照組明顯增多，加入anti-LIF抗體后明顯減少。結(jié)論: hASCs可抑制MS患者Th17細(xì)胞的分化，其作用可能與其分泌LIF、通過IL-6/LIF軸競(jìng)爭(zhēng)性抑制有關(guān)。

[關(guān)鍵詞]多發(fā)性硬化； 脂肪源性干細(xì)胞； Th17細(xì)胞； 白血病抑制因子； 白細(xì)胞介素6

多發(fā)性硬化(multiple sclerosis，MS)是一種中樞神經(jīng)系統(tǒng)(central nervous system，CNS)慢性脫髓鞘性的自身免疫性疾病。參與此自身免疫應(yīng)答過程的眾多效應(yīng)細(xì)胞中，輔助性T細(xì)胞17(T helper 17 cells，Th17)細(xì)胞起著最關(guān)鍵的決定性作用[1]。近年來，間充質(zhì)干細(xì)胞(mesenchymal stem cells，MSCs)移植為神經(jīng)系統(tǒng)疾病如MS、腦卒中等治療帶來了希望[2]。人脂肪源性干細(xì)胞(human adipose-derived stem cells，hASCs)是來源于脂肪的間充質(zhì)干細(xì)胞，具有免疫調(diào)節(jié)效應(yīng)，其調(diào)節(jié)能力涉及多種免疫細(xì)胞[3]，其機(jī)制包括細(xì)胞之間的接觸作用和分泌可溶性的免疫調(diào)節(jié)因子[4]。白血病抑制因子(leukemia inhibitory factor，LIF)是hASCs分泌的其中一種免疫調(diào)節(jié)因子，可通過LIF-LIF受體(LIF receptor,LIFR)機(jī)制抑制CD4+T細(xì)胞向Th17細(xì)胞分化，并且誘導(dǎo)T細(xì)胞無能而抑制免疫反應(yīng)的進(jìn)展[5]，而且在ASCs介導(dǎo)的抑制淋巴細(xì)胞增殖中起著關(guān)鍵作用[6]。MSCs對(duì)Th17細(xì)胞的免疫調(diào)節(jié)作用研究結(jié)論尚不統(tǒng)一，且hASCs對(duì)Th17細(xì)胞分化的作用及確切機(jī)制，目前相關(guān)報(bào)導(dǎo)較少。因此，本研究主要為體外利用 hASCs與MS患者外周血CD4+T細(xì)胞體外共培養(yǎng)，檢測(cè)Th17細(xì)胞細(xì)胞數(shù)量的變化，及細(xì)胞信號(hào)通路的變化，并進(jìn)一步探討hASCs調(diào)控Th17細(xì)胞分化的可能機(jī)制，是否與分泌LIF相關(guān)。

材料和方法

1標(biāo)本與試劑

人脂肪組織標(biāo)本取自我院整形外科抽脂手術(shù)患者。排除患有傳染病、自身免疫性疾病以及其它重大疾病的患者，捐贈(zèng)者簽署知情同意書。外周血來自我院住院且符合McDonald診斷標(biāo)準(zhǔn)的多發(fā)性硬化患者，經(jīng)患者知情同意及通過醫(yī)院倫理委員會(huì)批準(zhǔn)。高糖DMEM培養(yǎng)基(Hyclone)；胰蛋白酶(Anleresco)；I型膠原酶(博蘊(yùn)公司)；胎牛血清(Gibco)；hASCs表型鑒定流式抗體、細(xì)胞因子、LIF ELISA試劑盒(ebioscience)；淋巴細(xì)胞分離液(天津?yàn)笊?；CD4+T Cell Isolation Kit II(美天妮)；Th17表面流式抗體(BD)； anti-LIF(R&D)；逆轉(zhuǎn)錄試劑盒(Thermo)；SYRB Green染料(ABI)；刺激劑PMA/Ionomycin mixture和阻斷劑BFA/Monensin mixture(聯(lián)科生物)；固定破膜劑(BD)。

2方法

2.1hASCs的體外培養(yǎng)和生物學(xué)鑒定從抽脂手術(shù)患者中得到廢棄的脂肪組織，分離、純化hASCs，進(jìn)行原代與傳代培養(yǎng)后，并觀察其生物性狀。選取3～5代hASCs，進(jìn)行免疫細(xì)胞化學(xué)染色鑒定加入抗人CD44-FITC、CD105-APC、CD29-FITC、CD73-PE、CD31-PE、HLA-DR-APC、CD13-FITC、CD34-APC和CD49d-PE，對(duì)照組加入FITC-IgG1、APC-IgG1和PE-IgG1作為同型對(duì)照。

2.2人外周血提取外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cells，PBMCs)無菌采集患者靜脈血，采用淋巴細(xì)胞分離液進(jìn)行密度梯度離心法獲得單個(gè)核細(xì)胞，用紅細(xì)胞裂解液去除摻雜的紅細(xì)胞，用臺(tái)盼藍(lán)染液染色檢查PBMCs細(xì)胞存活率，鏡檢要求細(xì)胞存活率應(yīng)在95 %以上。

2.3磁珠分選(magnetic-activated cell sorting，MACS)CD4+T細(xì)胞分離得到的外周血單個(gè)核細(xì)胞反復(fù)洗滌去除血小板，采用免疫磁珠法分選CD4+T細(xì)胞陰選的試劑盒，按說明選擇合適的分離柱和分選器進(jìn)行分選，最后獲得的細(xì)胞加入CD4-PE-cy5.5抗體，用流式細(xì)胞術(shù)檢測(cè)分選前后CD4+T細(xì)胞純度。

2.4hASCs與CD4+T細(xì)胞體外共培養(yǎng)分離出MS患者的CD4+T細(xì)胞調(diào)整至4×108/L，接種于24孔培養(yǎng)板，用anti-CD3/CD28刺激增殖，添加促進(jìn)Th17細(xì)胞分化的細(xì)胞因子白細(xì)胞介素1β(interleukin-1β，IL-1β)、白細(xì)胞介素6(interleukin-6，IL-6)、轉(zhuǎn)化生長(zhǎng)因子β(transforming growth factor β，TGF-β)和白細(xì)胞介素23(interleukin-23，IL-23)，使細(xì)胞向Th17細(xì)胞極化，再與hASCs共培養(yǎng)4 d。實(shí)驗(yàn)組分別為hASCs∶CD4+T為1∶4、1∶10和1∶4加anti-LIF組；對(duì)照組為hASCs組和CD4+T組。取第3～5代hASCs按比例加入各孔，共培養(yǎng)4 d后，收集各個(gè)孔細(xì)胞用于流式檢測(cè)Th17細(xì)胞。

2.5Real-time PCRTRIzol法提取各組細(xì)胞的RNA，用分光光度計(jì)檢測(cè)其總RNA濃度和純度。取所提的RNA 1 μg參照逆轉(zhuǎn)錄試劑盒將其逆轉(zhuǎn)錄合成雙鏈cDNA，real-time PCR檢測(cè)，其體系為:SYBR Green 10 μL，上游引物(10 pmol/L)1 μL，下游引物(10 pmol/L)1 μL，cDNA模板1 μL，ddH2O 7 μL。引物序列見表1。應(yīng)用Applied Biosystems 7500 Real-Time PCR System對(duì)其進(jìn)行分析。分析維甲酸受體相關(guān)孤兒受體γt(retinoid-related orphan receptor，RORγt)、白細(xì)胞介素6受體(interleukin-6 receptor，IL-6R)、白細(xì)胞介素23受體(interleukin-23 receptor，IL-23R)、LIF和LIFR的mRNA表達(dá)情況。

2.6ELISA法檢測(cè)上清中LIF的表達(dá)收集各組培養(yǎng)的上清液，參照試劑盒說明進(jìn)行操作，設(shè)置3個(gè)復(fù)孔。在酶標(biāo)儀上檢測(cè)450 nm處測(cè)量A值，計(jì)算標(biāo)本濃度。

3統(tǒng)計(jì)學(xué)處理

采用SPSS 20.0軟件對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析，檢驗(yàn)實(shí)驗(yàn)結(jié)果正態(tài)性和方差齊性，計(jì)量資料均以均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示，各組間的均數(shù)比較采用單因素方差分析，各組均數(shù)兩兩比較采用SNK-q檢驗(yàn)，以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

表1　引物序列

結(jié)果

1hASCs的體外培養(yǎng)和生物學(xué)鑒定

分離的hASCs呈現(xiàn)類似纖維細(xì)胞樣的生長(zhǎng)狀態(tài)，細(xì)胞貼壁良好，倒置顯微鏡下呈長(zhǎng)梭形、編織狀，隨培養(yǎng)時(shí)間的延長(zhǎng)及數(shù)次傳代后其細(xì)胞形態(tài)逐漸變得均一，可以分離出純度較高的hASCs。本實(shí)驗(yàn)分離得的細(xì)胞表面高表達(dá)CD44、CD29、CD73、CD105和CD13，陽性率達(dá)95%以上，CD49d部分表達(dá)，幾乎不表達(dá)CD31、CD34和HLA-DR，基本可判定為hASCs，見圖1。

Figure 1.Morphological observation and surface marker detection of human adipose-derived stem cells (hASCs). A: hASCs at passage 3 under microscope (×100); B: the characteristics of the surface markers on hASCs detected by flow cytometry. The images showed that isolated hASCs positively expressed CD13, CD29, CD44, CD73 and CD105, but hardly expressed CD31, CD34 and HLA-DR.

圖1第3代hASCs細(xì)胞形態(tài)觀察及其表面標(biāo)志物表達(dá)情況

2hASCs對(duì)Th17細(xì)胞的免疫調(diào)控作用

2.1人外周血單個(gè)核細(xì)胞的分離離心20 min后，取出離心管觀察，離心管內(nèi)分為4層，自上而下分別為淡黃色的血漿層、乳白色云霧狀的淋巴細(xì)胞層、透明的淋巴細(xì)胞分離液層和紅細(xì)胞層。吸取淋巴細(xì)胞層，清洗后得到PBMCs。臺(tái)盼藍(lán)染色后顯微鏡下觀察細(xì)胞存活率達(dá)95%以上。

2.2流式細(xì)胞術(shù)檢測(cè)分選前后CD4+T細(xì)胞分選前，PBMCs經(jīng)流式細(xì)胞術(shù)檢測(cè)顯示有3群細(xì)胞，左下角為紅細(xì)胞群與細(xì)胞碎片，中間為淋巴細(xì)胞群，右上角為單核細(xì)胞群，選中淋巴細(xì)胞群，設(shè)門分析，結(jié)果顯示CD4+T細(xì)胞在PBMCs中所占比例為25%～35%。分選后，目的細(xì)胞經(jīng)過流式細(xì)胞術(shù)檢測(cè)，顯示2群細(xì)胞，左側(cè)為細(xì)胞碎片，右側(cè)為淋巴細(xì)胞群，選中淋巴細(xì)胞群，設(shè)門分析顯示CD4+T細(xì)胞所占比例為90%以上，這表明分選后所得的細(xì)胞大部分為CD4+T細(xì)胞，見圖2。

Figure 2.The percentage of CD4+T cells before (upper panel) and after (bottom panel) magnetic-activated cell sorting (MACS) detcted by flow cytometry, suggestting that the percentage of CD4+T cells in the PBMCs was 25%～35% before MACS and above 90% after MACS.

圖2流式細(xì)胞術(shù)檢測(cè)分選前后CD4+T細(xì)胞比例

2.3共培養(yǎng)體系細(xì)胞形態(tài)學(xué)特征CD4+T淋巴細(xì)胞呈懸浮狀態(tài)，不貼壁，為均勻透亮的圓形細(xì)胞；hASCs為長(zhǎng)梭形的貼壁細(xì)胞；共培養(yǎng)第2天開始觀察，淋巴細(xì)胞活化后形態(tài)發(fā)生變化，體積逐漸變大，數(shù)量增多；hASCs仍貼壁生長(zhǎng)，體積增大，見圖3。

Figure 3.The images of cultured hASCs, CD4+T cells as control groups and co-cultured CD4+T cells with hASCs in the ratio of 4∶1 or 10∶1.

圖3對(duì)照組與實(shí)驗(yàn)組細(xì)胞形態(tài)觀察

2.4hASCs對(duì)Th17細(xì)胞的抑制作用共培養(yǎng)4 d后，流式細(xì)胞術(shù)分別檢測(cè)實(shí)驗(yàn)組和對(duì)照組中Th17細(xì)胞(IL-17A+CD4+)占CD4+T細(xì)胞的比例。對(duì)照組Th17細(xì)胞的比例為(10.99±2.33)%，實(shí)驗(yàn)組中hASCs∶CD4+T為1∶4組Th17細(xì)胞的比例為(6.64±2.74)%，1∶10組Th17細(xì)胞的比例為(8.03±2.27)%。其中1∶4組、1∶10組與對(duì)照組相比，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)，實(shí)驗(yàn)組中1∶4組與1∶10組的Th17細(xì)胞比例差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)果表明hASCs抑制Th17細(xì)胞的分化，且hASCs所占比例越大，抑制作用越明顯，呈濃度依賴性，見圖4。

Figure 4.Flow cytometry was used to analyze the proportion of Th17 (IL-17A+CD4+) cells in each group. The numbers represents the percentage of double-positive cells. Mean±SD.n=4.**P<0.01vs0;△P<0.05vs1∶4.

圖4流式細(xì)胞術(shù)檢測(cè)實(shí)驗(yàn)組和對(duì)照組Th17細(xì)胞的比例

2.5Real-time PCR檢測(cè)IL-6R、RORγt、IL-23R、LIFR和LIF的mRNA表達(dá)與hASCs共培養(yǎng)后，1∶4組和1∶10組的CD4+T細(xì)胞中IL-6R、IL-23R和RORγt的mRNA表達(dá)量均較對(duì)照組降低；而LIFR的mRNA表達(dá)量較對(duì)照組升高(P<0.01)。共培養(yǎng)后的1∶4組和1∶10組的hASCs中LIF的mRNA表達(dá)量較不參與共培養(yǎng)的hASCs顯著升高(P<0.01)。IL-6R、RORγt、IL-23R、LIFR和LIF的mRNA表達(dá)在1∶4與1∶10實(shí)驗(yàn)組間差異無統(tǒng)計(jì)學(xué)意義(P>0.05)，見圖5。

Figure 5.Real-time PCR was used to detect the mRNA expression of RORγt, IL-6R, LIFR, IL-23R and LIF in each group.Mean±SD.n=4.**P<0.01vs0 or hASCs alone.

圖5Real-time PCR檢測(cè)LIFR、LIF、IL-6R、 RORγt和IL-23R的mRNA表達(dá)水平

3hASCs抑制Th17細(xì)胞分化的可能機(jī)制

3.1流式細(xì)胞術(shù)檢測(cè)各組Th17細(xì)胞選擇hASCs與CD4+T比例為1∶4的組進(jìn)行抗體中和實(shí)驗(yàn)。共培養(yǎng)4 d 后，取各組細(xì)胞用于流式檢測(cè)，對(duì)照組Th17細(xì)胞占CD4+T細(xì)胞的比例為(12.90±0.40)%，與hASCs共培養(yǎng)組Th17細(xì)胞的比例為(7.48±1.14)%，添加anti-LIF組Th17細(xì)胞的比例為(13.20±1.04)%；和hASCs共培養(yǎng)組與對(duì)照組相比，Th17細(xì)胞比例減低，而加入anti-LIF中和LIF后，Th17細(xì)胞上調(diào)(P<0.01)。結(jié)果說明，hASCs能抑制Th17細(xì)胞的分化，而anti-LIF則能解除hASCs的抑制作用，見圖6。

Figure 6.CD4+T cells were cultured with or without hASCs in the presence or absence of anti-LIF antibody， and flow cytometry was used to determine the proportion of Th17 (IL-17A+CD4+) cells. When the anti-LIF antibody was added into co-culture system, the ratio of Th17 cells increased and reached to the control level. Mean±SD.n=4.**P<0.01vsCD4+T;△△P<0.01vsCD4+T+hASCs.

圖6流式細(xì)胞術(shù)檢測(cè)Th17細(xì)胞占CD4+T細(xì)胞的比例情況

3.2ELISA檢測(cè)各組LIF的分泌水平共培養(yǎng)4 d 后，通過ELISA分別檢測(cè)對(duì)照組(CD4+T組、hASCs組)、與hASCs共培養(yǎng)組(hASCs∶CD4+T為1︰4)和添加anti-LIF組上清液中LIF的分泌，測(cè)得CD4+T組中LIF的濃度為(598.68±285.90)ng/L，hASCs組中LIF的濃度為(430.60±317.86)ng/L，與hASCs共培養(yǎng)組中LIF的濃度為(1 049.74±37.26)ng/L，添加anti-LIF組LIF的濃度為(103.76±67.85)ng/L；與hASCs共培養(yǎng)組分泌的LIF與對(duì)照組相比明顯增多，加anti-LIF中和LIF后，LIF分泌量較與hASCs共培養(yǎng)組明顯下降，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)，見圖7。

3.3Real-time PCR檢測(cè)RORγt和IL-6R的mRNA表達(dá)Real-time PCR結(jié)果顯示，與對(duì)照組相比，hASCs共培養(yǎng)組中 RORγt的mRNA的表達(dá)量下降，差異有統(tǒng)計(jì)學(xué)顯著性(P<0.01)；加anti-LIF后，RORγt的mRNA表達(dá)水平上調(diào)，與對(duì)照組相比，差異無統(tǒng)計(jì)學(xué)意義。與對(duì)照組相比，與hASCs共培養(yǎng)組中IL-6R mRNA的表達(dá)量減低(P<0.01)；加anti-LIF后，IL-6R的mRNA表達(dá)水平上調(diào)，與對(duì)照組相比，差異無統(tǒng)計(jì)學(xué)意義，見圖7。

Figure 7.The level of LIF in the supernatant and the mRNA expression of IL-6R and RORγt were tested by ELISA and real-time PCR, respectively. Mean±SD.n=4.**P<0.01vsCD4+T;△P<0.05,△△P<0.01vsCD4+T+hASCs.

圖7各組LIF的分泌水平及RORγt和IL-6R的mRNA表達(dá)情況

討論

近年來成體干細(xì)胞-間充質(zhì)干細(xì)胞的免疫調(diào)節(jié)功能被越來越多地認(rèn)識(shí)和研究，成為當(dāng)今熱門的學(xué)科。相對(duì)于骨髓MSCs來講，hASCs可以避免取材帶來的痛苦；hADSs來源相對(duì)豐富，且取材方便，體外分離和培養(yǎng)方法成熟；hASCs較骨髓來源的MSCs有更強(qiáng)大的體外克隆能力，因此脂肪間充質(zhì)干細(xì)胞作為理想種子細(xì)胞應(yīng)用于臨床治療具有良好醫(yī)學(xué)前景。Najar等[6]研究證明hASCs相較MSCs具有更強(qiáng)的免疫調(diào)節(jié)效應(yīng)，其調(diào)節(jié)能力涉及多種免疫細(xì)胞，包括抑制T淋巴細(xì)胞、B淋巴細(xì)胞、NK細(xì)胞的免疫效應(yīng)，并且誘導(dǎo)免疫耐受等[3]，目前hASCs被視為治療多種自身免疫疾病如MS、移植物抗宿主病、類風(fēng)濕性關(guān)節(jié)炎等較為理想的細(xì)胞[7]。Th17細(xì)胞亞群是MS免疫反應(yīng)和髓鞘脫失最主要的效應(yīng)細(xì)胞，IL-17A是Th17細(xì)胞分泌的主要炎癥因子，因此，抑制Th17細(xì)胞介導(dǎo)的炎癥反應(yīng)是治療MS的關(guān)鍵突破。黃雪瓊等[8]研究發(fā)現(xiàn)MSCs能抑制CD4+T細(xì)胞亞群向Th17方向分化，糾正Th17/Treg 失衡。本實(shí)驗(yàn)通過體外分離、純化hASCs，實(shí)現(xiàn)在體外擴(kuò)增，再通過體外實(shí)驗(yàn)證明hASCs能抑制CD4+T細(xì)胞向Th17細(xì)胞分化的免疫調(diào)節(jié)并探索其可能的機(jī)制。流式細(xì)胞技術(shù)檢測(cè)hASCs的表面分子，均高表達(dá)CD44、CD105、CD29和CD73，這些是基質(zhì)細(xì)胞基本的表面標(biāo)志分子；而內(nèi)皮祖細(xì)胞的表面標(biāo)記分子CD31表達(dá)很低，HLA-DR幾乎無表達(dá)[9]，可基本判斷為hASCs，這為后續(xù)實(shí)驗(yàn)的準(zhǔn)確和可靠性提供了保障。

Th17細(xì)胞亞群是初始CD4+T細(xì)胞在 TGF-β、IL-6和IL-21協(xié)同刺激下，激活STAT3磷酸化，進(jìn)而激活標(biāo)志性轉(zhuǎn)錄因子RORγt后分化產(chǎn)生， RORγt誘導(dǎo)IL-17A和IL-17E的表達(dá)，促進(jìn)Th17分化，且Th17細(xì)胞的擴(kuò)增需要IL-23維持[10]。而TGF-β和IL-6同時(shí)存在是可以大量誘導(dǎo)RORγt的表達(dá)，進(jìn)一步誘導(dǎo)IL-17A和IL-17E的產(chǎn)生，加重炎癥和自身免疫反應(yīng)。Th17細(xì)胞分泌獨(dú)特的促炎因子IL-17A、IL-17F、IL-21和IL-22，IL-17是Th17細(xì)胞分泌導(dǎo)致MS炎癥損傷最主要的炎癥介質(zhì)。因此，抑制Th17細(xì)胞介導(dǎo)的炎癥反應(yīng)是治療MS的關(guān)鍵突破。本研究通過體外實(shí)驗(yàn)證明hASCs能抑制CD4+T細(xì)胞向Th17細(xì)胞分化，這種抑制作用存在高濃度抑制效應(yīng)，即加入的hASCs比例越大，抑制作用越明顯。Najar等[6]在hASCs對(duì)T淋巴細(xì)胞增殖的抑制作用實(shí)驗(yàn)中認(rèn)為，hASCs∶T為1∶20時(shí)，無抑制作用，這將為hASCs用于體內(nèi)實(shí)驗(yàn)和移植治療的劑量選擇做一參考。

本實(shí)驗(yàn)研究發(fā)現(xiàn)加入hASCs后RORγt mRNA表達(dá)減少，這表明hASCs可減少Th17特異性轉(zhuǎn)錄因子RORγt表達(dá)，并且伴隨IL-6R和IL-23R的mRNA表達(dá)水平下調(diào)，但LIFR的mRNA表達(dá)水平升高；然而基因水平的改變與hASCs濃度并無明顯關(guān)系，這表明Th17抑制減低可能與RORγt、IL-6R、IL-23R和LIFR改變相關(guān)。本研究結(jié)果發(fā)現(xiàn)了hASCs使CD4+T細(xì)胞的IL-6R mRNA表達(dá)水平下調(diào)而LIFR mRNA表達(dá)水平上調(diào)的結(jié)果，據(jù)此我們進(jìn)一步探索hASCs可能的免疫調(diào)節(jié)機(jī)制。

LIF是hASCs分泌的可溶性細(xì)胞因子， hASCs可通過分泌LIF抑制淋巴細(xì)胞增殖[6]，但是hASCs抑制Th17細(xì)胞分化的機(jī)制是否與分泌LIF有關(guān)，尚缺乏相關(guān)報(bào)道。本研究顯示，hASCs與CD4+T細(xì)胞共培養(yǎng)組中，LIF的分泌量較對(duì)照組(CD4+T組、hASCs)明顯升高，加入可溶性中和抗體anti-LIF中和LIF后，LIF濃度下降，與此同時(shí)，hASCs對(duì)Th17細(xì)胞分化的抑制作用亦解除。我們通過real-time PCR進(jìn)一步從分子水平檢測(cè) RORγt和IL-6R的表達(dá)水平，發(fā)現(xiàn)人Th17細(xì)胞特異性的轉(zhuǎn)錄因子 RORγt及IL-6R的mRNA表達(dá)與Th17細(xì)胞比例水平一致，隨著LIF分泌的增多，IL-6R表達(dá)受抑制，當(dāng)中和LIF后，IL-6R的表達(dá)水平上升。LIF是一種多功能的細(xì)胞因子，屬于IL-6家族中的成員，gp130受體是IL-6家族成員信號(hào)轉(zhuǎn)導(dǎo)通路的基本亞單位，IL-6R是由gp130組成的二聚體，LIF受體則是由gp130/gp190組成的異物二聚體，在特定環(huán)境下，IL-6和LIF通過相互競(jìng)爭(zhēng)受體而拮抗對(duì)方的效應(yīng)[11]。研究表明，LIF是體內(nèi)一種重要的免疫負(fù)調(diào)因子，LIF基因敲除鼠的皮炎嚴(yán)重程度遠(yuǎn)遠(yuǎn)高于正常鼠，而慢病毒攜帶LIF轉(zhuǎn)染小鼠后，癥狀明顯減輕，這與早期的研究認(rèn)為L(zhǎng)IF治療可減少同種異體皮膚移植的免疫排斥反應(yīng)是相一致的[12]。體外研究認(rèn)為L(zhǎng)IF誘導(dǎo)初始CD4+T細(xì)胞向Foxp3+Treg細(xì)胞分化，發(fā)揮免疫耐受作用，而IL-6是重要的促炎因子，誘導(dǎo)初始CD4+T細(xì)胞向Th17細(xì)胞分化，是MS、類風(fēng)濕性關(guān)節(jié)炎等自身免疫性疾病的重要炎癥因子[13]。IL-6/LIF軸與Th17細(xì)胞和Treg細(xì)胞的分化密切相關(guān)，IL-6與IL-1β、IL-23共同參與促進(jìn)Th17細(xì)胞分化，LIF則單獨(dú)參與激活Treg細(xì)胞特異性的轉(zhuǎn)錄因子Foxp3，促進(jìn)Treg細(xì)胞的分化。利用IL-6/LIF軸在治療MS等自身免疫性疾病中取得了重大突破。本研究認(rèn)為hASCs通過分泌LIF，作用于CD4+T細(xì)胞表面的LIFR受體，與IL-6受體IL-6R相互拮抗，從而抑制CD4+T細(xì)胞向Th17細(xì)胞分化。但是，LIF是否通過增加Treg細(xì)胞的表達(dá)從而抑制Th17細(xì)胞，未來可進(jìn)一步深入研究。

本實(shí)驗(yàn)成功分離、純化及鑒定了hASCs，建立了免疫磁珠法分選外周血，成功獲得高純度CD4+T細(xì)胞作為后續(xù)研究。體外開展的hASCs和CD4+T細(xì)胞共培養(yǎng)證明了hASCs可以抑制MS患者Th17細(xì)胞分化，機(jī)制可能與其分泌LIF，通過IL-6/ LIF軸競(jìng)爭(zhēng)性抑制有關(guān)。利用hASCs的免疫調(diào)節(jié)作用可以降低MS的Th17細(xì)胞分化，改善免疫炎癥。已有研究發(fā)現(xiàn)骨髓MSCs在MS動(dòng)物模型和臨床試驗(yàn)中已初顯成效，對(duì)MS有一定治療作用[14]。運(yùn)用hASCs移植來治療MS患者將會(huì)有廣闊的臨床應(yīng)用前景。

[參考文獻(xiàn)]

[1]Axtell RC, de Jong BC, Boniface K, et al. T helper type 1 and 17 cells determine efficacy of interferon-β in multiple sclerosis and experimental encephalomyelitis[J]. Nat Med, 2010, 16(4):406-412.

[2]Uccelli A, Laroni A, Freedman MS. Mesenchymal stem cells for the treatment of multiple sclerosis and other neurological diseases[J]. Lancet Neurol, 2011, 10(7):649-656.

[3]Akiyama K, Chen C, Wang DD, et al. Mesenchymal-stem-cell-induced immunoregulation involves FAS-ligand-/FAS-mediated T cell apoptosis[J]. Cell Stem Cell, 2012, 10(5):544-555.

[4]English K. Mechanisms of mesenchymal stromal cell immunomodulation[J]. Immunol Cell Biol, 2013, 91(1):19-26.

[5]Gao W, Thompson L, Zhou Q, et al. Treg versus Th17 lymphocyte lineages are cross-regulated by LIF versus IL-6[J]. Cell Cycle, 2009, 8(9):1444-1450.

[6]Najar M, Raicevic G, Boufker HI, et al. Adipose-tissue-derived and Wharton′s Jelly-derived mesenchymal stromal cells suppress lymphocyte responses by secreting leukemia inhibitory factor[J]. Tissue Eng Part A, 2010, 16(11): 3537-3546.

[7]Gruber HE, Deepe R, Hoelscher GL, et al. Human adipose-derived mesenchymal stem cells: direction to a phenotype sharing similarities with the disc, gene expression profiling, and coculture with human annulus cells[J]. Tissue Eng Part A, 2010, 16(9):2843-2860.

[8]黃雪瓊，檀衛(wèi)平，吳葆菁，等. 骨髓間充質(zhì)干細(xì)胞對(duì)重癥哮喘患兒外周血 Th17/Treg的免疫調(diào)節(jié)作用[J]. 中國(guó)病理生理雜志, 2014, 30(9):1694-1697,1702.

[9]Mitchell JB,McIntosh K,Zvonic S,et al. Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers[J]. Stem Cells, 2006, 24(2):376-385.

[10]Zhu JF,Yamane H, Paul WE. Differentiation of effector CD4+T cell population[J]. Annu Rev Immunol, 2010, 28:445-489.

[11]Metcalfe SM. LIF in the regulation of T-cell fate and as a potential therapeutic[J]. Genes Immun, 2011, 12(3):157-168.

[12]Zhu M, Oishi K, Lee SC, et al. Studies using leukemia inhibitory factor (LIF) knockout mice and a LIF adenoviral vector demonstrate a keyanti-inflammatory role for this cytokine in cutaneous inflammation[J]. J Immunol, 2001, 166(3):2049-2054.

[13]Bettelli E, Carrier Y, Gao W, et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells[J]. Nature, 2006, 441(7090):235-238.

[14]郝飛，梁戰(zhàn)華，李愛萍，等. 骨髓間充質(zhì)干細(xì)胞應(yīng)用于多發(fā)性硬化的基礎(chǔ)及臨床研究進(jìn)展[J]. 中國(guó)病理生理雜志, 2014, 21(2):134-137.

(責(zé)任編輯： 林白霜， 羅森)

Adipose-derived stem cells mediate immunosuppression of Th17 in multiple sclerosis

LIN Xiao-bin1, CHEN Ying2, YANY De-hao2, XIE Yong-lin3, BI Yong2, KE Jian-ming2, CHEN Zhi-bo2, SU Zhong-qian2, LI Xiang2, ZHANG Xu2

(1DepartmentofUltrasound，2DepartmentofNeurology,TheFirstAffiliatedHospital,WenzhouMedicalUniversity,Wenzhou325000,China;3DepartmentofRehabilitation,ZhejiangProvincialHospitalofChineseArmedPoliceForce,Hangzhou310000,China.E-mail:drzhangxu@live.cn)

[ABSTRACT]AIM: To investigate how human adipose-derived stem cells (hASCs) regulates the differentiation of Th17 cells in multiple sclerosis. METHODS: hASCs were isolated from the adipose tissues. Magnetic-activated cell sorting (MACS) kit was used to isolate CD4+ T cells from peripheral blood mononuclear cells (PBMCs) which were isolated by density gradient centrifugation. The percentage of CD4+ T cells was detected by flow cytometry. The activated CD4+ T cells were co-cultured with hASCs for about 4 d at different ratios of hASCs to CD4+ T cells (1∶4 and 1∶10) in a Th17 polarised condition. Another group adding anti-leukemia inhibitory factor (LIF) antibody was set up. Th17 cell proportion of the CD4+ T cells was determined by flow cytometry. The level of LIF in the supernatant of co-cultured system was measured by ELISA. The mRNA expression of retinoid-related orphan receptor γt (RORγt), interleukin-6 receptor (IL-6R), interleukin-23 receptor (IL-23R), LIF and leukemia inhibitory factor receptor (LIFR) was detected by real-time PCR. RESULTS: The result of flow cytometry suggested there were mainly hASCs, and the percentage of CD4+ T cells in the PBMCs were above 90% after MACS. The Th17 cell proportion decreased in 1∶4 and 1∶10 co-cultured groups in a dose-dependent manner. The mRNA expression of IL-6R, IL-23R and RORγt was downregulated and the expression of LIFR and LIF was up-regulated. When the anti-LIF was added into the co-cultured system, the ratio of Th17 cells increased and reached to the control level. The protein level of LIF obviously increased after co-cultured. After anti-LIF added, the mRNA expression of RORγt and IL-6R was up-regulated. CONCLUSION: hASCs inhibits the differentiation of Th17 cells from multiple sclerosis patients through the competitive inhibition of LIF/IL-6 by secreting LIF.

[KEY WORDS]Multiple sclerosis; Adipose-derived stem cells; Th17 cells; Leukemia inhibitory factor; Interleukin-6

[文章編號(hào)]1000- 4718(2016)01- 0051- 07

[收稿日期]2015- 07- 13[修回日期] 2015- 10- 08

*[基金項(xiàng)目]浙江省自然科學(xué)基金資助項(xiàng)目(No. LY14H130002； No. LY13H090010)；溫州市科技計(jì)劃(No. Y20140278)

通訊作者△Tel: 0577-55579372; E-mail: drzhangxu@live.cn

[中圖分類號(hào)]R741.05

[文獻(xiàn)標(biāo)志碼]A

doi:10.3969/j.issn.1000- 4718.2016.01.009