IL-6在巨噬細(xì)胞向M2型極化過程中的誘導(dǎo)作用

劉苗 唐榮 姜毅

·論著·

IL-6在巨噬細(xì)胞向M2型極化過程中的誘導(dǎo)作用

劉苗 唐榮 姜毅

目的 將小鼠巨噬細(xì)胞系RAW264.7細(xì)胞和骨髓瘤細(xì)胞系KM3細(xì)胞共培養(yǎng)，探討IL-6在巨噬細(xì)胞向M2型極化的過程的作用。方法 取對(duì)數(shù)生長(zhǎng)期細(xì)胞，分為3組：A組(KM3細(xì)胞組)，B組(RAW264.7細(xì)胞組)，C組(RAW264.7細(xì)胞+KM3細(xì)胞組)。分別予以ACTA(IL-6特異性抑制劑激活素A)或rIL-6(重組人IL-6)處理后，檢測(cè)腫瘤相關(guān)M2型巨噬細(xì)胞表達(dá)標(biāo)志F4/80+ CD206+的比例。RT-PCR和Westernblot方法檢測(cè)各組細(xì)胞因子CCL22、IL-10、IL-12、TNF-αmRNA和蛋白表達(dá)量。ELISA法檢測(cè)各組細(xì)胞培養(yǎng)上清中IL-6的含量。結(jié)果 RAW264.7和KM3細(xì)胞共培養(yǎng)24 h后，與對(duì)照組相比，M2 型巨噬細(xì)胞比例顯著增加(P<0.05)；予以ACTA處理后M2型巨噬細(xì)胞表達(dá)明顯下降(P<0.05)；而予以rIL-6處理后M2型巨噬細(xì)胞表達(dá)明顯上調(diào)(P<0.05)。與B組(RAW264.7細(xì)胞組)相比，C組(RAW264.7細(xì)胞+KM3細(xì)胞)M2型巨噬細(xì)胞相關(guān)細(xì)胞因子CCL22，IL-10的mRNA和蛋白表達(dá)水平明顯上調(diào)(P<0.05)，而M1型巨噬細(xì)胞相關(guān)因子IL-12，TNF-α的mRNA和蛋白表達(dá)水平明顯下調(diào)(P<0.05)。與A組、B組相比，C組細(xì)胞上清液中IL-6含量在24 h、48 h、72 h時(shí)間點(diǎn)均明顯上調(diào)(P<0.05)。通過濃度梯度改變RAW264.7細(xì)胞或KM3細(xì)胞的數(shù)量，發(fā)現(xiàn)RAW264.7細(xì)胞數(shù)量的改變顯著影響了IL-6的表達(dá)水平。結(jié)論 將RAW264.7細(xì)胞和KM3細(xì)胞共培養(yǎng)后，后者能誘導(dǎo)RAW264.7細(xì)胞向腫瘤相關(guān)M2型巨噬細(xì)胞轉(zhuǎn)化， IL-6在上述轉(zhuǎn)化過程中發(fā)揮重要作用。

巨噬細(xì)胞；骨髓瘤；IL-6；極化

白細(xì)胞介素-6(IL-6)是一種多功能的細(xì)胞因子，能調(diào)節(jié)多種生理功能，包括基因激活、細(xì)胞增殖和分化[1,2]。近年來，大量的研究表明IL-6參與了多種腫瘤的進(jìn)展過程，促進(jìn)腫瘤細(xì)胞生長(zhǎng)，是惡性腫瘤預(yù)后不良的重要標(biāo)志[3,4]。M2型巨噬細(xì)胞是腫瘤組織中數(shù)量最多的抗原提呈細(xì)胞，其在腫瘤的發(fā)展、浸潤(rùn)、轉(zhuǎn)移及促進(jìn)腫瘤血管生成中發(fā)揮重要作用。有研究表明，IL-6與巨噬細(xì)胞極化相關(guān)[5,6]。本研究將小鼠巨噬細(xì)胞系RAW264.7細(xì)胞和骨髓瘤細(xì)胞系KM3細(xì)胞作為研究對(duì)象，探討IL-6是否參與了巨噬細(xì)胞向M2型極化的過程。

1 材料與方法

1.1 試劑 新生牛血清及IMEM培養(yǎng)基購(gòu)于Gibco公司，PI標(biāo)記的抗小鼠F4/80抗體和FITC標(biāo)記的抗小鼠CD206抗體購(gòu)自eBioscience公司，Trizol試劑盒購(gòu)于上海生工生物有限公司,RT-PCR試劑盒購(gòu)于Invitrogen公司，相關(guān)一抗體(CCL22、IL-10、IL-12、TNF-α多克隆抗體)購(gòu)自Cell signal公司，相應(yīng)二抗體購(gòu)自Pierce公司，IL-6特異性抑制劑激活素A及鼠源重組白細(xì)胞介素6(rIL-6)購(gòu)自輝瑞公司，蛋白電泳marker購(gòu)自Fermentas公司。ELISA試劑盒購(gòu)于美國(guó)BD公司。

1.2 方法

1.2.1 細(xì)胞培養(yǎng)：RAW264.7細(xì)胞 和KM3細(xì)胞購(gòu)自華中科技大學(xué)同濟(jì)醫(yī)學(xué)院基礎(chǔ)學(xué)院，RAW264.7細(xì)胞 和KM3細(xì)胞分別在IMEM培養(yǎng)基(含10%Gibco新生牛血清，100 U/ml青霉素，100 μg/ml鏈霉素)中常規(guī)培養(yǎng)。

1.2.2 實(shí)驗(yàn)分組：實(shí)驗(yàn)分為：A組：KM3細(xì)胞組，B組：RAW264.7細(xì)胞組，C組：RAW264.7細(xì)胞+KM3細(xì)胞組，RAW264.7細(xì)胞接種于6孔板的數(shù)目為1×105/孔， KM3細(xì)胞接種于6孔板的數(shù)目為5×105/孔。

1.2.3 流式細(xì)胞術(shù)檢測(cè)：各組細(xì)胞培養(yǎng)24 h后，檢測(cè)腫瘤相關(guān)M2型巨噬細(xì)胞表達(dá)標(biāo)志F4/80+ CD206+的比例。將C組細(xì)胞分為2組，D組：RAW264.7細(xì)胞+KM3細(xì)胞+ ACTA(IL-6特異性抑制劑激活素A)；E組：RAW264.7細(xì)胞+KM3細(xì)胞+ rIL-6(重組人IL-6)。通過流式細(xì)胞術(shù)檢測(cè)M2型巨噬細(xì)胞的比例。采用PI標(biāo)記F4/80抗體，F(xiàn)ITC標(biāo)記CD206抗體。

1.2.4 RT-PCR方法：檢測(cè)各組CCL22、IL-10、IL-12、TNF-α基因mRNA表達(dá)收集各組細(xì)胞，提取總RNA，檢測(cè)各組細(xì)胞因子CCL22、IL-10、IL-12、TNF-α的mRNA 表達(dá)。方法如下：①逆轉(zhuǎn)錄：根據(jù)Trizol一步法抽提總RNA，分光光度計(jì)測(cè)定RNA濃度。將抽提的總RNA為模板，以O(shè)ligo(dT)15為反轉(zhuǎn)錄引物，各樣品取相同量的RNA逆轉(zhuǎn)錄成cDNA。②PCR擴(kuò)增： 設(shè)計(jì)引物，取PCR產(chǎn)物8 μl,加5× Loading buffer 2 μl,2%瓊脂糖凝膠電泳。溴化乙錠染色，凝膠成像儀成象，并對(duì)電泳條帶進(jìn)行分析。

1.2.5 Westernblot方法：檢測(cè)各組CCL22、IL-10、IL-12、TNF-α蛋白表達(dá)收集各組細(xì)胞進(jìn)行以下實(shí)驗(yàn)。①蛋白抽提與定量:各組細(xì)胞加入200 μl蛋白裂解液，混勻，充分裂解后提取總蛋白。②免疫印跡檢測(cè)目的蛋白:配置SDS-PAGE濃縮膠與分離膠，本實(shí)驗(yàn)濃縮膠為3.9%, 分離膠為8%和10%,凝膠聚合1 h。在電泳緩沖液中進(jìn)行電泳，然后將蛋白質(zhì)轉(zhuǎn)移至硝酸纖維素膜上。在5%脫脂奶粉中進(jìn)行抗體封閉，然后加入相應(yīng)的一抗：CCL22(1∶2 000)，IL-10(1∶2 500)，IL-12(1∶2 000)，TNF-α(1∶1 000)4℃冰箱過夜后，將硝酸纖維素膜用TBST液反復(fù)沖洗，再加入相應(yīng)的二抗，搖床搖1 h后取出，TBST液反復(fù)沖洗，并以兔抗GAPDH(1∶1 000)作為對(duì)照。隨后進(jìn)行顯影及曝光，計(jì)算蛋白條帶的吸光度值(IA=平均吸光度×面積)。

1.2.6 ELISA方法檢測(cè):①取各組細(xì)胞培養(yǎng)上清液，按照ELISA試劑盒說明書進(jìn)行操作，測(cè)定細(xì)胞因子IL-6含量；②取RAW264.7細(xì)胞和KM3細(xì)胞，按照細(xì)胞混合比例不同，分為8組，C1：RAW264.7細(xì)胞數(shù)為 1×105/孔,KM3細(xì)胞數(shù)為5×105/孔； C2:RAW264.7細(xì)胞數(shù)為1×105/孔； KM3細(xì)胞數(shù)為2.5×105/孔; C3:RAW264.7細(xì)胞數(shù)為1×105/孔,KM3細(xì)胞數(shù)為1.25×105/孔; C4:RAW264.7細(xì)胞數(shù)為1×105/孔,KM3細(xì)胞數(shù)為0.625×105/孔; C5:KM3細(xì)胞數(shù)為5×105/孔，RAW264.7細(xì)胞數(shù)為1×105/孔; C6:KM3細(xì)胞數(shù)為5×105/孔，RAW264.7細(xì)胞數(shù)為0.5×105/孔; C7:KM3細(xì)胞數(shù)為5×105/孔，RAW264.7細(xì)胞數(shù)為0.25×105/孔; C8:KM3細(xì)胞數(shù)為5×105/孔，RAW264.7細(xì)胞數(shù)為0.125×105/孔。培養(yǎng)24 h后取上清液，測(cè)定IL-6的含量。

2 結(jié)果

2.1 流式細(xì)胞術(shù)檢測(cè)結(jié)果 培養(yǎng)24 h后，A組和B組僅可見少量(2.46%,2.37%)M2型巨噬細(xì)胞表達(dá)。與A組和B組相比，C組M2型巨噬細(xì)胞(10.35%)比例顯著增加，差異有統(tǒng)計(jì)學(xué)意義(t=4.379,P<0.05;t=3.726,P<0.05)。干預(yù)24 h后，D組M2型巨噬細(xì)胞表達(dá)的比例為1.07%，與C組相比，D組M2型巨噬細(xì)胞表達(dá)明顯下降(t=5.201,P<0.05); E組M2型巨噬細(xì)胞表達(dá)的比例為25.12%，與C組相比，E組M2型巨噬細(xì)胞表達(dá)明顯上調(diào)(t=3.592,P<0.05)。見圖1，表1。

2.2 RT-PCR檢測(cè)結(jié)果 與B組比較，C組的M2型巨噬細(xì)胞相關(guān)細(xì)胞因子CCL22，IL-10的mRNA表達(dá)水平明顯增高(t=2.993,P<0.05;t=4.162,P<0.05)，而M1型巨噬細(xì)胞相關(guān)因子IL-12，TNF-α的mRNA表達(dá)水平降低(t=3.386,P<0.05;t=4.359,P<0.05)。見圖2，表2。

A組B組C組D組E組

圖1 干預(yù)24 h后不同細(xì)胞組腫瘤相關(guān)M2型巨噬細(xì)胞表達(dá)的比例

注: 與KM3細(xì)胞組比較,*P<0.05；與RAW264.7細(xì)胞組比較,#P<0.05;與RAW264.7細(xì)胞+KM3細(xì)胞比較,△P<0.05

圖2 RT-PCR 電泳結(jié)果

M:marker：1：RAW264.7細(xì)胞組， 2：RAW264.7細(xì)胞+KM3細(xì)胞

指標(biāo)RAW264.7細(xì)胞組RAW264.7細(xì)胞+KM3細(xì)胞組F值P值CCL220.4236±0.01280.5367±0.0239*5228.689<0.05IL-10 0.7269±0.03230.8642±0.0156*4529.332<0.05IL-12 0.5717±0.01960.3673±0.0241*3274.126<0.05TNF-α0.6425±0.03340.4565±0.0183*5176.457<0.05

注: 與RAW264.7細(xì)胞組比較,*P<0.05

2.3Westernblot檢測(cè)結(jié)果 與B組比較，C組的M2型巨噬細(xì)胞相關(guān)細(xì)胞因子CCL22，IL-10的蛋白表達(dá)水平明顯增高(t=4.302,P<0.05;t=5.265,P<0.05)，而M1型巨噬細(xì)胞相關(guān)因子IL-12，TNF-α的蛋白表達(dá)水平降低(t=3.889,P<0.05;t=4.747,P<0.05)。見圖3，表3。

2.4 ELISA檢測(cè)結(jié)果 與A組、B組相比，C組細(xì)胞上清液中IL-6含量在24 h、48 h、72 h時(shí)間點(diǎn)均明顯上調(diào)(P<0.05),在72 h時(shí)，結(jié)果示C組IL-6的含量是A組2.61倍，B組2.35倍。見圖4，表4。

圖3 Western blot 電泳結(jié)果

注: 1：RAW264.7細(xì)胞組;2：RAW264.7細(xì)胞+KM3細(xì)胞

指標(biāo)RAW264.7細(xì)胞組RAW264.7細(xì)胞+KM3細(xì)胞組F值P值CCL220.3757±0.02210.4809±0.0178*3377.657<0.05IL-10 0.6524±0.01950.8715±0.0269*5903.332<0.05IL-12 0.6063±0.03460.5126±0.0332*4041.256<0.05TNF-α0.9356±0.02730.8177±0.0247*2832.747<0.05

注: 與RAW264.7細(xì)胞組比較,*P<0.05

圖4 3組細(xì)胞上清液中IL-6表達(dá)水平

注:A：KM3細(xì)胞組，B：RAW264.7細(xì)胞組，C：RAW264.7細(xì)胞+KM3細(xì)胞

干預(yù)時(shí)間KM3細(xì)胞組RAW264.7細(xì)胞組RAW264.7細(xì)胞+KM3細(xì)胞組24h11.5623±0.536615.3447±0.675820.1892±0.3139*#48h13.1145±0.678216.8361±0.559425.2123±0.2775*#72h17.5523±0.499519.4943±0.394645.8115±0.4138*#

注: 與KM3細(xì)胞組比較,*P<0.05;與RAW264.7細(xì)胞組比較,#P<0.05

C2組、C3組、C4 組IL-6的表達(dá)水平分別為C1 組的92.1%,91.2%,85.7%,僅C4組與C1組的差異有統(tǒng)計(jì)學(xué)意義(t=4.634,P<0.05)。C6組,C7組,C8組IL-6的表達(dá)水平分別為C5 組的66.7%,57.8%,46.5%,差異均有統(tǒng)計(jì)學(xué)意義(t=4.208,P<0.05;t=5.537,P<0.05;t=3.901,P<0.05)。見圖5，表5、6。

圖5 不同比例混合的共培養(yǎng)細(xì)胞上清液中IL-6表達(dá)水平

注: C1:RAW264.7細(xì)胞數(shù)為1×105/孔,KM3細(xì)胞數(shù)為5×105/孔; C2:RAW264.7細(xì)胞數(shù)為1×105/孔,KM3細(xì)胞數(shù)為2.5×105/孔;C3:RAW264.7細(xì)胞數(shù)為1×105/孔,KM3細(xì)胞數(shù)為1.25×105/孔;C4:RAW264.7細(xì)胞數(shù)為1×105/孔,KM3細(xì)胞數(shù)為0.625×105/孔;C5: KM3細(xì)胞數(shù)為5×105/孔，RAW264.7細(xì)胞數(shù)為1×105/孔;C6: KM3細(xì)胞數(shù)為5×105/孔，RAW264.7細(xì)胞數(shù)為0.5×105/孔;C7: KM3細(xì)胞數(shù)為5×105/孔，RAW264.7細(xì)胞數(shù)為0.25×105/孔;C8: KM3細(xì)胞數(shù)為5×105/孔，RAW264.7細(xì)胞數(shù)為0.125×105/孔

3 討論

細(xì)胞因子IL-6參與了機(jī)體多種功能，研究發(fā)現(xiàn)，IL-6與腫瘤的發(fā)生及發(fā)展密切相關(guān)，IL-6參與了惡性腫瘤的免疫逃逸，是惡性腫瘤預(yù)后不良的重要標(biāo)志[7,8]。研究表明，降低IL-6的活性能夠恢復(fù)骨髓瘤細(xì)胞對(duì)化療藥物的敏感性[9]。

巨噬細(xì)胞的激活狀態(tài)主要分為兩種類型：M1型和M2型。 M1型巨噬細(xì)胞主要由Th1型細(xì)胞因子激活，表現(xiàn)為高表達(dá)MHCII，產(chǎn)生大量TNF-α、IL-12和NO，可直接殺傷病原微生物和腫瘤細(xì)胞，M1型巨噬細(xì)胞參與促炎反應(yīng)，具有免疫監(jiān)視作用[10,11]。M2型巨噬細(xì)胞主要由Th2型細(xì)胞因子激活，其表達(dá)標(biāo)志主要為F4/80+ CD206+，通過分泌抑制性細(xì)胞因子IL-10、CCL22、TGF-β等下調(diào)機(jī)體的免疫應(yīng)答，M2型巨噬細(xì)胞與抗炎反應(yīng)及腫瘤疾病相關(guān)[12,13]。研究發(fā)現(xiàn)，M2型巨噬細(xì)胞在在腫瘤的發(fā)展、浸潤(rùn)、轉(zhuǎn)移及促進(jìn)腫瘤血管生成中發(fā)揮重要作用[14，15]。

炎癥微環(huán)境在腫瘤的進(jìn)展中發(fā)揮著重要作用，巨噬細(xì)胞是腫瘤微環(huán)境中浸潤(rùn)炎性細(xì)胞的主要成分。腫瘤細(xì)胞周圍基質(zhì)中分泌的細(xì)胞因子和酶構(gòu)成的局部微環(huán)境，決定了巨噬細(xì)胞的類型和功能。研究表明，腫瘤微環(huán)境中相關(guān)細(xì)胞因子IL-6能誘導(dǎo)巨噬細(xì)胞向M2型極化，后者廣泛參與了腫瘤的免疫逃逸[16-18]。

表5 不同比例混合的共培養(yǎng)細(xì)胞上清液中IL-6表達(dá)水平 ±s

注: 與C1組(RAW264.7細(xì)胞數(shù)為1×105/孔,KM3細(xì)胞數(shù)為5×105/孔)比較，*P<0.05

表6 不同比例混合的共培養(yǎng)細(xì)胞上清液中IL-6表達(dá)水平 ±s

注: 與C5組(KM3細(xì)胞數(shù)為5×105/孔，RAW264.7細(xì)胞數(shù)為1×105/孔)比較,*P<0.05

本研究顯示，KM3細(xì)胞與RAW264.7細(xì)胞共培養(yǎng)后，M2型巨噬細(xì)胞所占比例增加，CCL22、IL-10等M2型細(xì)胞因子表達(dá)水平上調(diào)，IL-12、TNF-α等M1型細(xì)胞因子表達(dá)水平下調(diào)，提示KM3細(xì)胞能誘導(dǎo)RAW264.7細(xì)胞向M2型巨噬細(xì)胞轉(zhuǎn)化。通過對(duì)RAW264.7向M2型巨噬細(xì)胞轉(zhuǎn)化的分子機(jī)制深入研究發(fā)現(xiàn)， 當(dāng)RAW264.7細(xì)胞和KM3細(xì)胞共培養(yǎng)后，培養(yǎng)上清液中IL-6高表達(dá)。研究發(fā)現(xiàn)，多種腫瘤細(xì)胞分泌IL-6，暴露于IL-6或者自分泌IL-6的癌細(xì)胞具有較強(qiáng)的侵襲力和抗藥性。

本研究通過濃度梯度改變RAW264.7細(xì)胞或KM3細(xì)胞的數(shù)量，觀察IL-6表達(dá)量的變化，旨在分辨IL-6主要是由何種細(xì)胞分泌。結(jié)果發(fā)現(xiàn)，C2、C3、C4組的濃度分別是C1 組的92.1%、91.2%、85.7%，僅C4組與C1組的差異有統(tǒng)計(jì)學(xué)意義(P<0.05)， C6、C7、C8組的濃度分別是C5組的66.7%、57.8%、46.5%, 差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)果表明IL-6的表達(dá)主要受RAW264.7細(xì)胞數(shù)量影響更為明顯，我們推測(cè)IL-6主要通過RAW264.7細(xì)胞分泌。進(jìn)一步研究發(fā)現(xiàn)，激活素A(IL-6特異性抑制劑)能抑制RAW264.7細(xì)胞向M2型巨噬細(xì)胞轉(zhuǎn)化，重組IL-6能促進(jìn)上述轉(zhuǎn)化過程。

總之，本研究初步證實(shí)骨髓瘤細(xì)胞能誘導(dǎo)巨噬細(xì)胞向M2型轉(zhuǎn)化，IL-6參與了上述過程，其具體機(jī)制仍需進(jìn)一步研究。

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The induction effect of IL-6 in polarization process of macrophages to M2 type in vitro

LIUMiao,TANGRong,JIANGYi.

DepartmentofPaediatrics,People’sHospitalAffiliatedtoWuhanUniversity,Wuhan430060,China

Objective To investigate the effects of IL-6 in polarization process of macrophages to M2 type,with macrophage cell line-RAW264.7 of mice being co-cultured with myeloma cell line KM3 in vitro.Methods The cells at exponential growth phase were divided into three groups: group A (KM3 cell),group B (RAW264.7 cell),group C (RAW264.7 cell +KM3 cell).After the cells were treated with ACTA (a specific inhibitor of IL-6) or rIL-6 (recombinant human IL-6),respectively,the proportion of F4/80+ CD206+ cells was detected. The expression levels of CCL22, IL-10, IL-12, TNF-α lpha were measured by RT-PCR and Western Blot, respectively. The content of IL-6 in culture supernatant was detected by ELISA.Results After RAW264.7 cells and KM3 cells were co-cultured for 24 hours, as compared with that in control group, the proportion of M2 type macrophages was significantly increased (P<0.05),moreoverafterthecellsweretreatedbyACTA,theexpressionofM2typemacrophageswasobviouslydecreased(P<0.05),however,afterthecellsweretreatedbyrIL-6,theproportionofM2macrophageswassignificantlyincreased(P<0.05).AscomparedwiththoseingroupB,theexpressionlevelsofM2macrophagerelatedcytokines-CCL22andIL-10mRNAandproteinweresignificantlyup-regulatedingroupC(P<0.05),buttheexpressionlevelsofM1macrophagerelatedcytokines-IL-12,TNF-αlphamRNAandproteinwereobviouslydown-regulated(P<0.05).AscomparedwiththoseingroupAandgroupB,thelevelsofIL-6inculturesupernatantweresignificantlyincreasedat24h, 48h, 72htimepointsingroupC(P<0.05).WhenthecellcountsofRAW264.7cellsorKM3cellswerechangedbydifferentconcentrationgradient,theexpressionlevelsofIL-6weremoreeasilyinfluencedbythechagesofRAW264.7cellnumber.Conclusion After RAW264.7 cells are co-cultured with KM3 cells in vitro, the latter can induce the transformation of RAW264.7 cell into tumor-associated M2 macrophages, moreover, IL-6 may play an important role in the transformation process.

macrophage；myeloma；IL-6；polarization

10.3969/j.issn.1002-7386.2017.08.001

項(xiàng)目來源：湖北省自然科學(xué)基金項(xiàng)目(編號(hào)：2014CFB395)

430060 湖北省武漢市,武漢大學(xué)人民醫(yī)院兒科

姜毅，430060 湖北省武漢市,武漢大學(xué)人民醫(yī)院兒科;

E-mail: jiangyiwd@126.com

R 730.2

A

1002-7386(2017)08-1125-05

2016-12-13)