人胰腺癌細(xì)胞培養(yǎng)上清對(duì)樹(shù)突狀細(xì)胞TIM-3表達(dá)及其功能的影響*

夏媛媛,　蔡長(zhǎng)青,　盧椏楠,　甘慧泉，　周泉波

(1廣東醫(yī)學(xué)院，廣東 湛江 524023； 2廣東省第二人民醫(yī)院腫瘤二科，廣東 廣州 510317； 3中山大學(xué)孫逸仙紀(jì)念醫(yī)院麻醉科，廣東 廣州 510120； 4廣東省人民醫(yī)院檢驗(yàn)科，廣東 廣州 510080；5中山大學(xué)孫逸仙紀(jì)念醫(yī)院肝膽胰外科，廣東 廣州 510120)

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人胰腺癌細(xì)胞培養(yǎng)上清對(duì)樹(shù)突狀細(xì)胞TIM-3表達(dá)及其功能的影響*

夏媛媛1,2,蔡長(zhǎng)青2△,盧椏楠3,甘慧泉4，周泉波5△

(1廣東醫(yī)學(xué)院，廣東 湛江 524023；2廣東省第二人民醫(yī)院腫瘤二科，廣東 廣州 510317；3中山大學(xué)孫逸仙紀(jì)念醫(yī)院麻醉科，廣東 廣州 510120；4廣東省人民醫(yī)院檢驗(yàn)科，廣東 廣州 510080；5中山大學(xué)孫逸仙紀(jì)念醫(yī)院肝膽胰外科，廣東 廣州 510120)

目的： 研究人胰腺癌微環(huán)境對(duì)樹(shù)突狀細(xì)胞(DCs)T細(xì)胞免疫球蛋白及黏蛋白-3(TIM-3)表達(dá)及其功能的影響，初步探討腫瘤微環(huán)境調(diào)節(jié)DCs上TIM-3表達(dá)的可能機(jī)制。方法：流式細(xì)胞術(shù)檢測(cè)腫瘤浸潤(rùn)樹(shù)突狀細(xì)胞(TIDC)以及癌旁組織、胰腺癌患者和健康人外周血誘導(dǎo)DCs上TIM-3的表達(dá)；觀察人胰腺癌細(xì)胞培養(yǎng)液上清對(duì)健康人外周血單個(gè)核細(xì)胞經(jīng)rhGM-CSF和rhIL-4誘導(dǎo)擴(kuò)增制備DCs上 TIM-3表達(dá)的影響；酶聯(lián)免疫吸附法(ELISA)檢測(cè)TIM-3+DCs組和對(duì)照組的DCs分別與凋亡胰腺癌細(xì)胞Capan-2共培養(yǎng)上清中細(xì)胞因子IFN-β和IL-12水平。結(jié)果：胰腺癌組織中TIDC上TIM-3的表達(dá)明顯高于癌旁組織及患者和健康人外周血的DCs(P<0.01)。人胰腺癌細(xì)胞株Canpan-2、SW1990和Panc-1的上清液較人皮膚成纖維細(xì)胞Hs27顯著上調(diào)DCs上TIM-3表達(dá)(P<0.05)，聯(lián)合阻斷VEGF、IL-10和PGE2可明顯降低Canpan-2細(xì)胞上清對(duì)DCs上TIM-3的上調(diào)作用(P<0.05)。TIM-3高表達(dá)DCs組較低表達(dá)組分泌的IL-12和IFN-β水平低，而阻斷TIM-3后，IFN-β和IL-12水平均升高(P<0.01)。而這種升高趨勢(shì)可在加入DNase和RNase后消失。結(jié)論：人胰腺癌TIDC上TIM-3表達(dá)升高導(dǎo)致其固有免疫功能受損；腫瘤細(xì)胞分泌的VEGF、IL-10和PGE2可能參與TIM-3的表達(dá)調(diào)控。

胰腺癌； 樹(shù)突狀細(xì)胞； 腫瘤微環(huán)境； T細(xì)胞免疫球蛋白及黏蛋白-3

胰腺癌是常見(jiàn)的消化系腫瘤之一，由于手術(shù)及放化療對(duì)治療效果的局限性[1]，腫瘤免疫治療已成為新的研究熱點(diǎn)，腫瘤微環(huán)境對(duì)腫瘤的發(fā)生、發(fā)展以及免疫逃逸的影響更是引起人們的廣泛關(guān)注。目前認(rèn)為，樹(shù)突狀細(xì)胞(dendritic cells，DCs)是功能最強(qiáng)大的專職抗原提呈細(xì)胞(antigen-presenting cells，APC)。腫瘤及其營(yíng)造的微環(huán)境能夠通過(guò)各種機(jī)制導(dǎo)致DCs的分化和成熟障礙，抑制DCs抗原提呈和激活T細(xì)胞的能力[2-3]。研究表明[4-5]：T細(xì)胞免疫球蛋白及黏蛋白-3(T-cell immunoglobulin and mucin-3, TIM-3)對(duì)DCs參與的腫瘤免疫起負(fù)性調(diào)節(jié)作用。

那么，在胰腺癌中DCs是否表達(dá)TIM-3？ TIM-3是否參與了胰腺癌DCs介導(dǎo)的固有免疫應(yīng)答？腫瘤微環(huán)境通過(guò)何種途徑調(diào)節(jié)其表達(dá)？本課題通過(guò)觀察人胰腺癌組織中腫瘤浸潤(rùn)樹(shù)突狀細(xì)胞(tumor-infiltrating dendritic cells,TIDC)上TIM-3的表達(dá)情況，分析胰腺癌細(xì)胞通過(guò)腫瘤微環(huán)境影響TIDC表型的可能因素，進(jìn)一步探討TIM-3對(duì)TIDC 固有免疫的影響。為研究胰腺癌細(xì)胞免疫逃逸機(jī)制及尋找新的胰腺癌的免疫治療靶點(diǎn)打下基礎(chǔ)。

材　料　和　方　法

1材料

1.1主要試劑與儀器重組人粒-巨噬細(xì)胞集落刺激因子(rhGM-CSF)、重組人白細(xì)胞介素4(rhIL-4)、RPMI-1640和DMEM培養(yǎng)基(HyClone);胎牛血清(FBS)、0.25%胰蛋白酶(Gibco);淋巴細(xì)胞分離液(TBD);抗人CD1α單克隆抗體(monoclonal antibo-dy, mAb)、抗人S-100 mAb和IL-10 mAb(Abcam);免疫組化試劑盒(博士德生物公司);抗人CD11c-FITC抗體及同型對(duì)照抗體(BD);抗人TIM-3-PECy7抗體(eBioscience);TIM-3 阻斷抗體(BioLegend);VEGFR2 mAb(R&D);PGE2受體EP2封閉肽(AH6809)(Cayman);人IL-12和IFN-β ELISA試劑盒(Elabscience);CCK-8(Dojindo)。順鉑(cis-diaminodichloroplatin,DDP)凍干粉(齊魯制藥);AnnexinⅤ/PI凋亡檢測(cè)試劑盒(凱基公司);DNase和RNase(TaKaRa)；流式細(xì)胞儀(FACSCalibur，BD);低速離心機(jī)和低溫高速離心機(jī)(Beckman)；細(xì)胞培養(yǎng)箱(Forma Series Ⅱ, Thermo)；超凈臺(tái)(VS-840K-U, 蘇凈);倒置普通光學(xué)顯微鏡和熒光顯微鏡(Nikon);酶標(biāo)儀(Denley Dragon Wellscan MK2)。

1.2標(biāo)本來(lái)源人胰腺癌患者手術(shù)標(biāo)本及外周血4例均來(lái)源中山大學(xué)孫逸仙紀(jì)念醫(yī)院肝膽外科，瘤體重量4.20～7.82 g，平均6.04 g。患者年齡48～70歲，平均60.25歲，男7例、女2例，均符合腫瘤手術(shù)切除指征，未發(fā)現(xiàn)遠(yuǎn)處轉(zhuǎn)移，術(shù)后病理診斷為胰腺癌；癌旁組織取自距腫瘤切緣2～3 cm的正常胰腺組織。健康人外周血濃縮白細(xì)胞來(lái)源于廣州市中心血站。

本研究符合醫(yī)學(xué)倫理學(xué)標(biāo)準(zhǔn)，經(jīng)醫(yī)院倫理委員會(huì)批準(zhǔn)，得到患者家屬的知情同意。

1.3細(xì)胞株來(lái)源人胰腺癌細(xì)胞株SW1990、Capan-2、Panc-1及人皮膚成纖維細(xì)胞(human skin fibroblast) Hs27由中山大學(xué)孫逸仙紀(jì)念醫(yī)院肝膽外科實(shí)驗(yàn)室提供。

2方法

2.1人胰腺癌組織免疫組化檢測(cè)將人胰腺癌組織及癌旁胰腺組織手術(shù)標(biāo)本進(jìn)行常規(guī)固定、包埋、切片，分別用鼠抗CD1α單抗及鼠抗S-100單抗作為Ⅰ抗，生物素化的羊抗鼠IgG作為Ⅱ抗，參照SABC免疫組化試劑盒書(shū)說(shuō)明書(shū)對(duì)胰腺癌組織及癌旁組織切片進(jìn)行免疫組織化學(xué)染色，DAB顯色后在光學(xué)顯微鏡下觀察染色結(jié)果。

2.2人胰腺癌組織及癌旁組織中浸潤(rùn)性DCs的制備手術(shù)新鮮腫瘤組織經(jīng)0.25%胰蛋白酶和0.04%Ⅳ型膠原酶37 ℃充分消化，組織碎塊研磨，200目孔徑鋼網(wǎng)濾過(guò)收集單細(xì)胞懸液，洗滌，調(diào)整細(xì)胞密度1×1010/L，淋巴細(xì)胞分離液分離細(xì)胞后調(diào)整細(xì)胞密度為1×109/L，37 ℃、5% CO2細(xì)胞培養(yǎng)箱中培養(yǎng)過(guò)夜，次日收集懸浮細(xì)胞，洗滌，用含2%FBS的PBS重懸細(xì)胞。

2.3外周血DCs的誘導(dǎo)及體外培養(yǎng)胰腺癌患者及健康人外周血經(jīng)淋巴細(xì)胞分離液分離單個(gè)核細(xì)胞(peripheral blood mononuclear cells，PBMC)，用含10% FBS的RPMI-1640培養(yǎng)液重懸細(xì)胞，調(diào)整為(2～3)×109/L，加入6孔板(每孔2 mL)，37 ℃、5% CO2細(xì)胞培養(yǎng)箱培養(yǎng)4 h，洗去懸浮的淋巴細(xì)胞，取貼壁單個(gè)核細(xì)胞加入含10% FBS的RPMI-1640培養(yǎng)液，補(bǔ)充終濃度為100 μg/L的 rhGM-CSF和50 μg/L 的rhIL-4，每3 d換液并補(bǔ)充相應(yīng)濃度的rhGM-CSF、rhIL-4，第6天收獲未成熟的樹(shù)突狀細(xì)胞(immature dendritic cells, imDC)，備用。臺(tái)盼蘭染色，檢測(cè)活細(xì)胞率。

2.4流式細(xì)胞術(shù)檢測(cè)DCs表面TIM-3的表達(dá)各組DCs用流式洗液(PBS加入2% FBS)洗2次后，重懸并分別加入抗人TIM-3-PECy7抗體和抗人CD11c-FITC抗體，4 ℃避光孵育30 min, 洗滌2次后上機(jī)檢測(cè)。

2.5流式細(xì)胞術(shù)分選CD11c+DCs將外周血誘導(dǎo)培養(yǎng)獲得的imDC 用CD11c-FITC抗體標(biāo)記，4 ℃避光孵育30 min, PBS再洗2次后上流式細(xì)胞儀進(jìn)行CD11c+DCs的無(wú)菌分選，鑒定純度。分選獲得CD11c+DCs離心濃縮(1 000 r/min,5 min)，用含10% FBS的RPMI-1640培養(yǎng)液重懸，備用。分選流式細(xì)胞儀嚴(yán)格執(zhí)行無(wú)菌維護(hù)流程[6]。

2.6細(xì)胞培養(yǎng)及胰腺癌細(xì)胞培養(yǎng)上清的制備人胰腺癌細(xì)胞株(Capan-2、SW1990、Panc-1)與人皮膚成纖維細(xì)胞株(HS27)在含10% FBS的RPMI-1640培養(yǎng)液中，37 ℃、5% CO2細(xì)胞培養(yǎng)箱培養(yǎng)，常規(guī)傳代。將胰腺癌細(xì)胞株與Hs27細(xì)胞經(jīng)胰酶消化后，以5×108/L密度接種于10 cm培養(yǎng)皿中(10 mL)，37 ℃、5% CO2培養(yǎng)48 h后收集培養(yǎng)上清，離心(1 500 r/min， 5 min)去除細(xì)胞碎片，微孔濾膜(0.22 μm)過(guò)濾后分裝，-80 ℃保存?zhèn)溆谩?/p>

2.7胰腺癌細(xì)胞培養(yǎng)液上清對(duì)DCs表達(dá)TIM-3的影響健康人外周血單核細(xì)胞體外誘導(dǎo)培養(yǎng)至第6天生成的imDC，分成2組，第1組分別加入含有25%的3種胰腺癌細(xì)胞上清的培養(yǎng)液，第2組加入含25% HS27細(xì)胞上清的培養(yǎng)液，培養(yǎng)48 h, 收集各組DCs，流式細(xì)胞術(shù)檢測(cè)TIM-3的表達(dá)情況。

2.8阻斷胰腺癌細(xì)胞培養(yǎng)液上清中VEGF、IL-10和PGE2對(duì)DCs表達(dá)TIM-3的影響收集體外誘導(dǎo)培養(yǎng)至第6天生成的imDC,分為4組，第1、2、3組在含25%Capan-2細(xì)胞上清的培養(yǎng)液中分別加入anti-VEGFR2抗體、anti-IL-10抗體和PGE2受體EP2封閉肽，第4組在含25%Capan-2細(xì)胞上清的培養(yǎng)液中聯(lián)合加入anti-VEGF-R2抗體、anti-IL-10抗體和PGE2受體EP2封閉肽，培養(yǎng)48 h，流式細(xì)胞術(shù)檢測(cè)TIM-3的表達(dá)情況。

2.9CCK-8法測(cè)藥物細(xì)胞毒性試驗(yàn)取對(duì)數(shù)生長(zhǎng)期的胰腺癌細(xì)胞，胰酶消化后1 000 r/min離心5 min后棄上清，用10% FBS的RPMI-1640重懸細(xì)胞，調(diào)整細(xì)胞密度為5×107/L，每孔取100 μL種96孔板，放置于37 ℃、5% CO2細(xì)胞培養(yǎng)箱培養(yǎng)24 h，待細(xì)胞貼壁后，加入10 μL含不同濃度藥物的培養(yǎng)基，每種濃度設(shè)5個(gè)復(fù)孔，培養(yǎng)24 h后吸棄含藥物的培養(yǎng)基，每孔加入100 μL新鮮含10% FBS的RPMI-1640培養(yǎng)液及10 μL CCK-8,孵育1 h后觀察顯色程度，酶標(biāo)儀測(cè)定在450 nm處的吸光度(A)值。GraphPad Prism 5軟件計(jì)算藥物不同濃度抑制率，得到IC50。

2.10DCs與凋亡的Capan-2細(xì)胞共培養(yǎng)的分組取對(duì)數(shù)生長(zhǎng)期的Capan-2細(xì)胞，分別按不同濃度加入DDP，作用時(shí)間分別為12、24、36、48 h，胰酶消化收集細(xì)胞，按AnnexinⅤ/PI凋亡檢測(cè)試劑盒說(shuō)明書(shū)操作，流式細(xì)胞術(shù)測(cè)細(xì)胞凋亡率，篩選出DDP作用Capan-2細(xì)胞的最佳作用濃度和時(shí)間。將健康人外周血單核細(xì)胞經(jīng)體外誘導(dǎo)培養(yǎng)、流式分選的CD11c+DCs分成4組，第1組加入含有25%Capan-2細(xì)胞上清的培養(yǎng)液培養(yǎng)48 h， 第2組加入含25%成纖維細(xì)胞上清的培養(yǎng)液培養(yǎng)48 h，第3組在第1組基礎(chǔ)上再加入TIM-3單克隆抗體。將前3組DCs與用順鉑處理后凋亡的Capan-2細(xì)胞按5∶1比例混勻共培養(yǎng) 24 h，第4組在第3組處理的DCs基礎(chǔ)上與DNase和RNase處理的凋亡Capan-2細(xì)胞共培養(yǎng)，收集各組培養(yǎng)液，4 ℃、1 500 r/min離心10 min，取上清液，待測(cè)。

2.11ELISA檢測(cè)各組DCs分泌的細(xì)胞因子IFN-β和IL-12的水平在酶標(biāo)包被板上設(shè)立標(biāo)準(zhǔn)孔，每組2個(gè)復(fù)孔，具體步驟按試劑說(shuō)明書(shū)進(jìn)行，運(yùn)用酶標(biāo)儀自動(dòng)分析檢測(cè)結(jié)果，讀出具體檢測(cè)值。實(shí)驗(yàn)重復(fù)3次，取平均值作為最終結(jié)果。

3統(tǒng)計(jì)學(xué)處理

采用SPSS 13.0軟件，對(duì)所有數(shù)據(jù)均進(jìn)行正態(tài)性檢驗(yàn), 正態(tài)分布的計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差(mean±SD) 表示，組間比較采用單因素方差分析(one-way ANOVA)，方差齊組間多重比較采用LSD分析，方差不齊組間多重比較采用Dunnett’s T3分析。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié)　　果

1胰腺癌組織及癌旁組織中DCs的觀察

免疫組化觀察9例癌旁胰腺組織和胰腺癌組織中均發(fā)現(xiàn)有浸潤(rùn)的CD1α+S-100+DCs,見(jiàn)圖1。

2胰腺癌患者TIDC、癌旁組織中DCs、外周血及健康人外周血DCs上TIM-3表達(dá)的比較

流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示胰腺癌組織中TIDC上TIM-3表達(dá)為(71.79±11.74)%,高于癌旁組織中、胰腺癌患者及健康人外周血DCs上TIM-3表達(dá)，數(shù)據(jù)分別為(11.46±6.68)%、(9.38±3.94)%和(4.10±2.24)%，差異顯著(P<0.01)。臺(tái)盼蘭染色檢測(cè)各組DCs存活率均大于93%，見(jiàn)圖2。

Figure 1.Immunohistochemistry detection of TIDC from pancreatic cancer tissues and adjacent tissues in human. A: CD1α+DCs in para-carcinoma tissue; B: S-100+DCs in para-carcinoma tissue; C: CD1α+DCs in pancreatic cancer tissue; D： S-100+DCs in pancreatic cancer tissue; E： CD1α+DCs in pancreatic cancer tissue; F： S-100+DCs in pancreatic cancer tissue (A～D:×100; E, F:×400).

圖1胰腺癌腫瘤組織及癌旁組織中分布的DCs

Figure 2.Expression of TIM-3 on surface of TIDC isolated from human pancreatic cancer and of myeloid dendritic cells (MDC) from peripheral blood. A: TIM-3 expression on DCs from pancreatic cancer tissues [TIDC(Pt)], para-carcinoma tissues[PcDC(Pt)] and peripheral blood of the same patients [MDC(Pt)]or healthy population [MDC(HP)], analyzed by flow cytometry. Numbers in top right quadrants indicate percentages of TIM-3+CD11c+cells among CD11c+cells. B: percentages of TIM-3+CD11c+cells among CD11c+cells in the 4 groups. Mean±SD.n=4.**P<0.01vsTIDC(Pt).

圖2分離自胰腺癌組織的TIDC及體外誘導(dǎo)的MDC表面TIM-3的表達(dá)

3胰腺癌細(xì)胞培養(yǎng)液上清對(duì)DCs表面TIM-3表達(dá)的影響

流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示:含25%Capan-2、SW1990和Panc-1胰腺癌細(xì)胞培養(yǎng)液上清的3組DCs上TIM-3的相對(duì)表達(dá)量分別為(44.52±8.62)%、(52.05±6.48)%和(47.90±10.38)%,均高于Hs27細(xì)胞組TIM-3相對(duì)表達(dá)量[(12.67±4.27)%](P<0.05)。而含胰腺癌細(xì)胞上清的3組數(shù)值比較無(wú)顯著差異,見(jiàn)圖3。

Figure 3.Expression of TIM-3 on surface of DCs treated for 48 h with supernatants of Capan-2, SW1990 and Panc-1 tumor cells or Hs27 cells (25% in total medium). Mean±SD.n=4.*P<0.05vsHs27.

圖3胰腺癌細(xì)胞及Hs27細(xì)胞上清對(duì)DCs上TIM-3表達(dá)的影響

4阻斷胰腺癌上清中VEGF、IL-10和PGE2對(duì)DCs表達(dá)TIM-3的影響

分別在含25% Capan-2細(xì)胞上清的培養(yǎng)液中加入anti-VEGF-R2抗體、anti-IL-10抗體和PGE2受體EP2封閉肽，培養(yǎng)imDC 48 h后，流式細(xì)胞術(shù)檢測(cè)DCs上 TIM-3相對(duì)表達(dá)量，3組表達(dá)率分別為(38.09±5.87)%、(43.12±5.08)%和(39.53±4.60)%,與未加抗體組[(44.52±8.62)%]比較，有降低趨勢(shì)，但差異無(wú)統(tǒng)計(jì)學(xué)意義;聯(lián)合阻斷3種細(xì)胞因子作用后，TIM-3的表達(dá)水平明顯下調(diào)[(27.44±5.55)%](P<0.05)，但并未完全抑制其表達(dá)，見(jiàn)圖4。

5DCs與凋亡胰腺癌細(xì)胞Capan-2共培養(yǎng)上清中細(xì)胞因子IFN-β、IL-12水平

CCK-8法測(cè)得DDP對(duì)Capan-2胰腺癌細(xì)胞24 h的IC50為13.6 mg/L。2倍IC50(27.2 mg/L)的DDP,作用于Capan-2細(xì)胞，12 h開(kāi)始凋亡增加，36 h凋亡逐漸達(dá)到高峰值，48 h后以晚期凋亡和壞死為主(圖5)，于是選擇27.2 mg/L作用36 h為DDP致Capan-2細(xì)胞凋亡的濃度和時(shí)間。凋亡Capan-2細(xì)胞與各組CD11c+DCs(流式分選CD11c+DCs純度>98%，圖6)共培養(yǎng)，TIM-3高表達(dá)DCs組(Capan-2上清組)、TIM-3低表達(dá)DCs組(Hs27上清組)、TIM-3高表達(dá)DCs+TIM-3 mAb組和TIM-3高表達(dá)DCs+DNase+RNase組上清中細(xì)胞因子IL-12和IFN-β水平比較結(jié)果見(jiàn)圖7。

Figure 4.Expression of TIM-3 on surface of DCs treated with VEGF, IL-10 and PGE2inhibitors.A: expression of TIM-3 on surface of DCs treated for 48 h with supernatants of Capan-2 cells untreated or treated with anti-VEGFR2 (vascular endothelial growth factor receptor 2), anti-IL-10 and EP2 blocker (EP2 receptor bloking peptide) alone, or a combination of all three inhibitors； B: percentages of TIM-3+CD11c+cells among CD11c+cells in the 5 groups. Mean±SD.n=4.*P<0.05vscombination.

圖4抑制胰腺癌細(xì)胞培養(yǎng)液上清中VEGF、IL-10和PGE2對(duì)DCs表達(dá)TIM-3的影響

Figure 5.The percentages of apoptotic Capan-2 cells induced by DDP (27.2 mg/L) at different time points. UT: untreated.

圖52倍IC50(27.2 mg/L)的DDP作用不同時(shí)間后Capan-2細(xì)胞的凋亡情況

討　　論

近期有學(xué)者研究報(bào)道，肺癌及結(jié)直腸腺癌荷瘤小鼠腫瘤組織浸潤(rùn)的DCs及晚期肺癌、胃腺癌等患者腫瘤相關(guān)性DCs (tumor-associated DCs,TADC) 均高表達(dá)TIM-3，而癌旁組織、脾和外周血中DCs表面TIM-3低表達(dá)或幾乎不表達(dá)[5]。TIM-3表達(dá)與核酸介導(dǎo)的固有免疫密切相關(guān)。

TIM-3是2002 年Monney 等[7]發(fā)現(xiàn)并鑒定的Th1細(xì)胞表面的含免疫球蛋白和黏蛋白結(jié)構(gòu)域的跨膜蛋白。TIM-3 可通過(guò)與其天然配體半乳糖凝集素-9結(jié)合，誘導(dǎo)Th1、Th17及Tc細(xì)胞凋亡或抑制其分化[8]。研究顯示TIM-3還表達(dá)于多種固有免疫細(xì)胞亞群包括人NK細(xì)胞、巨噬細(xì)胞、未成熟的DCs[9-10]。而目前人胰腺癌TADC上TIM-3表達(dá)情況未見(jiàn)有報(bào)道。

Figure 6.Purity of CD11c+DCs detected by flow cytometry after sorting. The left curve indicates the control antibody-labeled DCs, while the right M1 indicates percentage of CD11c+DCs labeled by fluoresence.

圖6流式細(xì)胞術(shù)分選CD11c+DC的純度檢測(cè)

Figure 7.TIM-3 suppressed IL-12 and IFN-β production in DCs. The levels of IL-12 and IFN-β produced by four groups of dendritic cells after co-culture with apopto-tic Capan-2 cells at the ratio of 5∶1 for 24 h, detected by ELISA. A: Capan-2 Sup+DCs; B: Hs27 Sup+DCs; C: Capan-2 Sup+DCs+TIM-3 mAb; D: Capan-2 Sup+DCs+TIM-3 mAb+DNase+RNase. Mean±SD.n=4.**P<0.01vsC;▲▲P<0.01vsB.

圖7DCs上TIM-3表達(dá)對(duì)IL-12和IFN-β分泌的影響

本研究通過(guò)免疫組化發(fā)現(xiàn)胰腺癌患者腫瘤組織中存在大量浸潤(rùn)性CD1α+S-100+DCs。我們進(jìn)一步研究發(fā)現(xiàn)：胰腺癌組織中TIDC上TIM-3的表達(dá)明顯高于癌旁組織中DCs及患者和健康人外周血DCs，提示胰腺癌局部微環(huán)境可能與DCs上TIM-3 表達(dá)增高有關(guān)。研究腫瘤微環(huán)境中TIDC表型及功能狀態(tài)變化的原因，有助于深入認(rèn)識(shí)腫瘤免疫逃逸機(jī)制，找到促進(jìn)機(jī)體抗腫瘤免疫應(yīng)答的治療方法。

許多研究表明TIDC的分化和成熟發(fā)生障礙與腫瘤復(fù)雜的微環(huán)境關(guān)系密切[2-3]。在胰腺癌組織中，癌細(xì)胞可以產(chǎn)生細(xì)胞因子(如TGF-β、IL-10和IL-6)，又可以表達(dá)表面分子(如VEGF、Fas-L、PD-L1和IDO)[11-13]，腫瘤微環(huán)境內(nèi)還存在許多免疫抑制細(xì)胞(如CAFs、tolerogenic DCs、MDSCs、immunosuppressive TAMs和Treg cells)[14]，這些免疫抑制細(xì)胞可通過(guò)消耗精氨酸、產(chǎn)生ROS和NO來(lái)抑制抗腫瘤免疫，與Treg細(xì)胞分泌的轉(zhuǎn)化生長(zhǎng)因子 β(transforming growth factor β, TGF-β)和白細(xì)胞介素10(interleukin-10,IL-10)等共同形成一個(gè)免疫抑制環(huán)境，影響DCs分子表型和功能。我們通過(guò)研究人胰腺癌細(xì)胞培養(yǎng)液上清對(duì)DCs上TIM-3表達(dá)的影響，結(jié)果發(fā)現(xiàn)含有Capan-2、SW1990和Panc-1 3種胰腺癌細(xì)胞株上清的培養(yǎng)液對(duì)DCs上TIM-3表達(dá)均有不同程度上調(diào)作用；當(dāng)在Capan-2細(xì)胞上清中聯(lián)合加入VEGFR2 mAb、IL-10 mAb和PGE2受體EP2阻斷劑時(shí)， DCs上TIM-3表達(dá)量有明顯下調(diào)，但并未完全抑制其表達(dá)，而分別單獨(dú)加入VEGFR2 mAb、IL-10 mAb和PGE2受體EP2阻斷劑后，TIM-3表達(dá)則并無(wú)明顯下調(diào)，提示胰腺癌微環(huán)境中參與DCs表達(dá)TIM-3調(diào)節(jié)的因素可能是多方面的，其中VEGF、IL-10及炎癥因子PGE2等可能共同參與了對(duì)DCs上TIM-3表達(dá)的正性調(diào)節(jié)。且已有研究表明在體內(nèi)腫瘤免疫抑制微環(huán)境中這些細(xì)胞因子相互影響，對(duì)DCs的調(diào)節(jié)作用更為廣泛。VEGF是一種重要的血管生成刺激因子，由腫瘤細(xì)胞釋放后，對(duì)腫瘤的血管生成起著關(guān)鍵作用，還可以抑制CD34+前體細(xì)胞向DCs的分化, 在抑制腫瘤微環(huán)境中TIDC 的成熟過(guò)程中發(fā)揮著重要作用，導(dǎo)致腫瘤逃避機(jī)體的免疫監(jiān)視[15]。而IL-10能抑制DCs 的分化成熟及抗原提呈能力、表達(dá)免疫抑制性受體[16]，同時(shí)IL-10 的分泌又受到TGF-β的影響，TGF-β可顯著促進(jìn)IL-10 的分泌[17]。另外免疫炎性反應(yīng)也是腫瘤微環(huán)境的重要特征之一，在人和小鼠腫瘤內(nèi)腫瘤相關(guān)巨噬細(xì)胞(tumor-assocciated macrophages，TAMs)，能夠產(chǎn)生大量的炎性細(xì)胞因子，腫瘤本身在 Toll樣受體(Toll-like recepters，TLRs)配體激活后也會(huì)產(chǎn)生大量炎性細(xì)胞因子[18]，PGE2在大腸癌、原發(fā)性胃癌、乳腺癌等多種腫瘤組織中均發(fā)現(xiàn)高表達(dá)[19-21]，是腫瘤微環(huán)境中重要的炎癥因子，參與了TIDC免疫抑制的發(fā)生[22]并且PGE2可通過(guò)EGFR-MAPK信號(hào)通路上調(diào)VEGF的表達(dá)[22-23]；復(fù)雜的腫瘤免疫抑制的微環(huán)境[24-25]，使得腫瘤局部免疫應(yīng)答逐漸趨于負(fù)性調(diào)節(jié)，形成免疫耐受是導(dǎo)致腫瘤細(xì)胞免疫逃逸的重要原因。

綜上所述，腫瘤微環(huán)境中負(fù)性免疫抑制因子及炎癥因子可能是造成TIDC上TIM-3表達(dá)上調(diào)的原因之一。

本研究還初步探討了TIM-3對(duì)TIDC功能的影響。IL-12是一種Th1型細(xì)胞因子，主要由成熟樹(shù)突狀細(xì)胞產(chǎn)生，是Th0細(xì)胞向Th1細(xì)胞分化的主要誘導(dǎo)因子，參與腫瘤細(xì)胞免疫，與NK細(xì)胞、T淋巴細(xì)胞等協(xié)同發(fā)揮抗腫瘤作用[26-29]。INF-β屬于Ⅰ型干擾素，能促進(jìn)CD4+T細(xì)胞分泌IFN-γ，可激活NK細(xì)胞，進(jìn)一步增強(qiáng)NK細(xì)胞非特異性殺傷靶細(xì)胞的作用。檢測(cè)IFN-β和IL-12水平，可以反映DCs固有免疫功能。細(xì)胞毒性藥物DDP是胰腺癌化療的一線用藥，能引起腫瘤細(xì)胞凋亡，同時(shí)會(huì)釋放內(nèi)源性核酸分子(DNA或RNA)導(dǎo)抗腫瘤的免疫應(yīng)答[30]。我們通過(guò)實(shí)驗(yàn)發(fā)現(xiàn)TIM-3+DCs與凋亡胰腺癌細(xì)胞Capan-2共培養(yǎng)后上清中細(xì)胞因子IL-12和IFN-β水平較對(duì)照組水平低，而阻斷TIM-3后，IFN-β和IL-12水平明顯升高，但加入DNase和RNase后，這種升高趨勢(shì)被消除，提示胰腺癌微環(huán)境中TIDC上表達(dá)的TIM-3可能參與了核酸介導(dǎo)的固有免疫應(yīng)答的負(fù)性調(diào)節(jié)，TIDC上TIM-3 的高表達(dá)可能抑制了化療誘導(dǎo)的腫瘤細(xì)胞死亡后釋放的內(nèi)源性核酸對(duì)DCs固有免疫免疫應(yīng)答的激活，這也可能是導(dǎo)致胰腺癌對(duì)化療產(chǎn)生耐藥原因之一。而阻斷TIM-3是否可以增強(qiáng)胰腺癌化療的效果、促進(jìn)胰腺癌核酸疫苗抗腫瘤免疫應(yīng)答有待于體內(nèi)實(shí)驗(yàn)的驗(yàn)證。

本研究結(jié)果表明：TIM-3是胰腺癌腫瘤微環(huán)境中DCs固有免疫激活的負(fù)性調(diào)節(jié)因子之一；腫瘤細(xì)胞分泌的VEGF、IL-10和PGE2可能參與了對(duì)TIM-3表達(dá)的調(diào)控。研究為胰腺癌以DCs為基礎(chǔ)的免疫治療提供新的思路。但是，引起TIM-3高表達(dá)的轉(zhuǎn)錄因子及其下游的信號(hào)通路如何？在不同組織環(huán)境TIM-3通過(guò)什么途徑對(duì)固有免疫、適應(yīng)性免疫進(jìn)行調(diào)節(jié)？如何改善免疫抑制性的微環(huán)境提高DCs抗腫瘤效應(yīng)？這一系列問(wèn)題都有待進(jìn)一步探索。

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(責(zé)任編輯： 林白霜， 羅森)

Effect of human pancreatic cancer cell supernatant on expression of TIM-3 and function of dendritic cells

XIA Yuan-yuan1,2, CAI Chang-qing2, LU Ya-nan3, GAN Hui-quan4,ZHOU Quan-bo5

(1GuangdongMedicalCollege,Zhanjiang524023,China;2DepartmentⅡofOncology,TheSecondPeople’sHospitalofGuangdongProvince,Guangzhou510317,China;3DepartmentofAnesthesiology,SunYat-senMemorialHospital,SunYat-senUniversity,Guangzhou510120,China;4DepartmentofClinicalLaboratory,GuangdongGeneralHospital,Guangzhou510080,China;5DepartmentofHepatopancreatobiliarySurgery,SunYat-senMemorialHospital,SunYat-senUniversity,Guangzhou510120,China.E-mail:cchangq@163.com;zhquanbo@126.com)

AIM: To investigate the influence and mechanisms of human pancreatic cancer tumor microenvironments on T-cell immunoglobulin mucin-3 (TIM-3) expression and function of dendritic cells (DCs). METHODS: Tumor-infiltrating dendritic cells (TIDC) and para-carcinoma tissue DCs were isolated by Ficoll-Hypaque density centrifugation from trypsinized pancreatic carcinoma tissues, and the peripheral blood mononuclear cells were isolated from pancreatic cancer patients or healthy people. The expression of TIM-3 on DCs was detected by flow cytometry. DCs isolated from healthy people peripheral blood mononuclear cells were induced by rhGM-CSF and IL-4. The expression of TIM-3 in the DCs treated with the culture supernatants of Capan-2, SW1990 and Panc-1 pancreatic cancer cells or human skin fibroblast (Hs27) cells for 48 h, and in the DCs treated with supernatants of Capan-2 cells, anti-VEGF-R2, anti-IL-10 and EP2 receptor blocking peptide were evaluated by flow cytometry. The releases of IFN-β and IL-12 in the culture supernatants of DCs pretreated with monoclonal antibody (mAb) to TIM-3 or DNase+RNase, followed by stimulation with apoptotic Capan-2 cells, were detected by ELISA. RESULTS: DCs in tumor microenvironments had higher expression of TIM-3 than the DCs from para-carcinoma tissues and pancreatic cancer patient or healthy people peripheral blood (P<0.01). TIM-3 expression in the DCs treated with the culture supernatants of Capan-2, SW1990 and Panc-1 pancreatic cancer cells for 48 h was much higher than that in Hs27 cells (P<0.05). Treatment with a combination of anti-VEGF-R2, anti-IL-10 and EP2 receptor blocking peptide largely diminished the upregulation of TIM-3 on the DCs mediated by Capan-2 cell supernatants (P<0.05). The concentrations of IFN-β and IL-12 in the DCs with high expression level of TIM-3 were lower than those in the DCs with low TIM-3 expression level. Treatment with mAb to TIM-3 resulted in much more IFN-β and IL-12 releases (P<0.01), but DNase+RNase made this effect disappear. CONCLUSION: TIM-3 serves as a negative regulator of DCs innate immune responses in the pancreatic cancer microenvironments. The secretion of soluble factors to tumor microenvironment by pancreatic cancer cells, including IL-10, VEGF and PGE2, may contribute to the regulation of TIM-3 expression.

Pancreatic cancer; Dendritic cells; Tumor microenvironment; T-cell immunoglobulin and mucin-3

1000- 4718(2016)04- 0628- 09

2015- 11- 02

2016- 01- 13

國(guó)家自然科學(xué)基金資助項(xiàng)目(No.81000917;No.81370059)

Tel: 020-89168114； E-mail: cchangq@163.com; zhquanbo@126.com

R730.23

A

10.3969/j.issn.1000- 4718.2016.04.009

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