腫瘤相關(guān)巨噬細(xì)胞與骨肉瘤

劉政選 王曉旭 符勇 方松清

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腫瘤相關(guān)巨噬細(xì)胞與骨肉瘤

劉政選 王曉旭 符勇 方松清

骨肉瘤是發(fā)病率最高的骨惡性腫瘤，在兒童及青少年中最為常見。研究發(fā)現(xiàn)，腫瘤微環(huán)境在骨肉瘤的發(fā)生發(fā)展中發(fā)揮重要功能，其中腫瘤相關(guān)巨噬細(xì)胞(TAM)廣泛參與骨肉瘤生長、血管新生、轉(zhuǎn)移及干細(xì)胞樣表型表達(dá)等。該文對(duì)TAM與骨肉瘤的研究現(xiàn)狀作一綜述。

骨肉瘤；腫瘤相關(guān)巨噬細(xì)胞；腫瘤微環(huán)境

骨肉瘤是最常見的骨惡性腫瘤，好發(fā)于股骨、脛骨和肱骨等長骨干骺端，以局部侵犯和早期肺部轉(zhuǎn)移為主要特點(diǎn)，患者預(yù)后極差[1]。近幾十年來，關(guān)于骨肉瘤患者總生存情況的研究一直處于瓶頸狀態(tài)[2]。隨著研究不斷深入，在發(fā)現(xiàn)針對(duì)骨肉瘤實(shí)質(zhì)細(xì)胞的治療不能對(duì)患者預(yù)后有明顯改善后，越來越多的研究轉(zhuǎn)向腫瘤微環(huán)境方面[3]。既往研究[4]顯示，在常見的惡性腫瘤中，腫瘤微環(huán)境與腫瘤惡性表型密切相關(guān)，且可促進(jìn)惡性腫瘤血管新生、局部浸潤及轉(zhuǎn)移，是惡性腫瘤進(jìn)展的危險(xiǎn)因素。巨噬細(xì)胞作為腫瘤微環(huán)境中含量最多的炎性細(xì)胞，與人類多種惡性腫瘤密切相關(guān)，為影響惡性腫瘤患者預(yù)后的獨(dú)立因素[5]。

1 腫瘤相關(guān)巨噬細(xì)胞來源及分型

腫瘤微環(huán)境是指由間質(zhì)細(xì)胞、細(xì)胞外間質(zhì)、細(xì)胞因子及趨化因子等構(gòu)成的局部病理環(huán)境，它是一個(gè)動(dòng)態(tài)變化的整體，可與腫瘤細(xì)胞相互作用：一方面，微環(huán)境影響腫瘤細(xì)胞，為腫瘤生長、血管生成及轉(zhuǎn)移提供條件；另一方面，腫瘤自身也影響著所處微環(huán)境的理化狀態(tài)。腫瘤微環(huán)境在骨肉瘤發(fā)生發(fā)展中起著重要作用，其細(xì)胞組成主要包括間充質(zhì)干細(xì)胞(MSC)、破骨細(xì)胞、成纖維細(xì)胞和炎性細(xì)胞，其中巨噬細(xì)胞是骨肉瘤微環(huán)境中含量最多的炎性細(xì)胞。

巨噬細(xì)胞來源于單核細(xì)胞，可遷移到不同組織中，分化為功能各異的細(xì)胞，在組織中起維持平衡、促進(jìn)炎癥反應(yīng)及調(diào)節(jié)免疫的作用。與正常組織中的巨噬細(xì)胞相同，腫瘤相關(guān)巨噬細(xì)胞(TAM)也來源于外周血循環(huán)的單核-巨噬細(xì)胞，在趨化因子CCL2、CCL5、血管內(nèi)皮生長因子(VEGF)、轉(zhuǎn)化生長因子(TGF)-β等作用下，單核細(xì)胞向腫瘤組織中浸潤，繼而在局部微環(huán)境影響下被誘導(dǎo)極化成M1型和M2 型TAM。

2 TAM對(duì)腫瘤發(fā)生發(fā)展的影響

M1型TAM(即經(jīng)典激活的巨噬細(xì)胞)由脂多糖(LPS)和干擾素(IFN)-γ等刺激極化而來，高表達(dá)白細(xì)胞介素(IL)-1、IL-12、 IL-23、活性氧簇(ROS)和一氧化氮(NO)，低表達(dá)IL-4 和IL-10，有較強(qiáng)的抗原提呈能力，主要功能為殺滅被吞噬的細(xì)菌、病毒及腫瘤細(xì)胞，從而抑制腫瘤的發(fā)生發(fā)展[6]。炎癥介質(zhì)IL-10 或IL-4 可促進(jìn)TAM向M2型極化，M2型TAM高表達(dá)IL-10、清道夫受體CD163及甘露糖受體CD206等，低表達(dá)IL-12和IL-23，抗原提呈能力較弱，在組織修復(fù)、免疫抑制和促進(jìn)腫瘤生長中扮演重要角色[6]。局部微環(huán)境發(fā)生變化時(shí)，M1型TAM與M2型TAM可相互轉(zhuǎn)化；激活或阻斷TAM的Notch信號(hào)轉(zhuǎn)導(dǎo)通路可使TAM向M1型或M2型極化[7]。阻斷TAM的核因子-κB(NF-κB)信號(hào)轉(zhuǎn)導(dǎo)通路可使TAM向M1型極化，表現(xiàn)為高表達(dá)IL-12，低表達(dá)IL-10，從而抑制腫瘤的發(fā)生發(fā)展[8]。目前研究認(rèn)為，眾多惡性腫瘤如肝細(xì)胞癌[9]、胰腺癌[10]、胃癌[11]等細(xì)胞間質(zhì)中高度浸潤的TAM主要表現(xiàn)為M2型，可促進(jìn)腫瘤發(fā)生發(fā)展，為影響患者預(yù)后的危險(xiǎn)因素。目前TAM在骨肉瘤中的表型尚未明確，其對(duì)骨肉瘤的生長、血管新生、轉(zhuǎn)移及干細(xì)胞樣表型的影響各有不同，甚至存在分歧。

2.1 對(duì)骨肉瘤生長的影響

多種信號(hào)轉(zhuǎn)導(dǎo)通路、分子與骨肉瘤的生長有關(guān)。Yang等[12]研究發(fā)現(xiàn)，通過轉(zhuǎn)染使骨肉瘤中的組蛋白修飾因子MacroH2A高表達(dá)能下調(diào)細(xì)胞周期素D及細(xì)胞周期素依賴性蛋白激酶(CDK)表達(dá)，從而抑制骨肉瘤細(xì)胞增殖。Ma等[13]通過體外實(shí)驗(yàn)發(fā)現(xiàn)，夾竹桃苷能抑制骨肉瘤中Wnt/β-catenin信號(hào)轉(zhuǎn)導(dǎo)通路，同時(shí)降低基質(zhì)金屬蛋白酶(MMP)-2和MMP-9的活性，導(dǎo)致骨肉瘤細(xì)胞凋亡。Li等[14]研究發(fā)現(xiàn)，微小RNA(miRNA)-143能抑制B細(xì)胞淋巴瘤基因(Blc)-2表達(dá)，激活含半胱氨酸的天冬氨酸蛋白水解酶-3，導(dǎo)致骨肉瘤細(xì)胞凋亡。

多數(shù)惡性腫瘤中TAM呈M2型，可通過分泌內(nèi)皮生長因子(EGF)等生長因子直接或間接促進(jìn)腫瘤細(xì)胞生長[15]。Hu等[16]研究發(fā)現(xiàn)，M2型TAM可上調(diào)細(xì)胞周期蛋白D1表達(dá)，促進(jìn)子宮內(nèi)膜癌細(xì)胞增殖。Yang等[17]研究發(fā)現(xiàn)，M2型TAM能促進(jìn)Bcl-2表達(dá)，抑制乳腺癌細(xì)胞凋亡。Xiao等[18]研究證實(shí)，骨肉瘤組織中大量M2型TAM浸潤,與骨肉瘤的增殖密切相關(guān)。體內(nèi)實(shí)驗(yàn)[19]發(fā)現(xiàn)，注射氯膦酸鹽可減少骨肉瘤小鼠模型中的巨噬細(xì)胞數(shù)量，使小鼠腫瘤體積顯著減?。幻庖呓M化檢測顯示，骨肉瘤的移植瘤多為F4/80(一種巨噬細(xì)胞標(biāo)志物)陽性，且移植瘤的TAM表現(xiàn)為M2型。骨肉瘤細(xì)胞高表達(dá)EGF,但骨肉瘤的生長僅受內(nèi)皮生長因子受體(EFGR)及TAM調(diào)控，與EGF無關(guān)。Pahl等[15]研究證實(shí)，脂質(zhì)胞壁三肽與IFN-γ共同誘導(dǎo)極化的M1型TAM能抑制骨肉瘤細(xì)胞生長；當(dāng)骨肉瘤細(xì)胞株與西妥昔單抗共同培養(yǎng)時(shí)，IL-10誘導(dǎo)極化的M2型TAM也能抑制骨肉瘤細(xì)胞的生長。因此，M2型TAM在某些因素作用下，也可能抑制骨肉瘤的生長。但TAM對(duì)骨肉瘤生長的影響機(jī)制尚待進(jìn)一步研究。

2.2 對(duì)骨肉瘤血管新生的影響

腫瘤血管新生是指在原有血管的基礎(chǔ)上，各種因素通過內(nèi)皮細(xì)胞增殖、細(xì)胞外基質(zhì)(ECM)降解及內(nèi)皮細(xì)胞遷移、重構(gòu)，以出芽的方式形成新的血管，進(jìn)入腫瘤組織內(nèi)，為腫瘤發(fā)生發(fā)展提供必要條件，是腫瘤十大特征之一。促進(jìn)腫瘤血管新生的因子包括VEGF 、EGF、成纖維細(xì)胞生長因子(FGF)及TGF-β等，其中以VEGF的促血管生成作用最為重要，它能與內(nèi)皮細(xì)胞的特異性受體flt和flt/KDR結(jié)合，促進(jìn)內(nèi)皮細(xì)胞增殖，刺激新生血管形成[20]。同時(shí)，MMP能破壞ECM，誘導(dǎo)內(nèi)皮細(xì)胞遷移，也能促進(jìn)血管新生[21]。骨肉瘤的發(fā)生發(fā)展過程也需要血管。骨肉瘤中的VEGF能激活磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信號(hào)轉(zhuǎn)導(dǎo)通路，使骨肉瘤微血管密度(MVD)增加，促進(jìn)血管生成[22]。大量研究發(fā)現(xiàn)，精子相關(guān)抗原-9[23]、骨膜蛋白[24]及IL-6[25]均能通過提高骨肉瘤中VEGF的表達(dá)促進(jìn)其血管新生。骨肉瘤組織中高表達(dá)的低氧誘導(dǎo)因子(HIF)-1α與MVD的增加呈正相關(guān)，是患者預(yù)后不良的重要因素[26]。

TAM能分泌各種促血管生成因子(如VEGF、FGF、MMP-9等)參與血管新生的每個(gè)過程。大量研究[27]證實(shí)，TAM易聚集在腫瘤組織的低氧部位，而腫瘤組織的低氧環(huán)境能促使TAM中HIF-1α活性增高，從而分泌大量VEGF及MMP等促血管生成因子，促進(jìn)血管新生。研究發(fā)現(xiàn)，在多種惡性腫瘤如膠質(zhì)母細(xì)胞瘤[28]、乳腺癌[29]及胃癌[30]等中，TAM能通過促進(jìn)腫瘤組織中VEGF的表達(dá)以促進(jìn)腫瘤血管新生，是導(dǎo)致患者預(yù)后不良的重要因素。Werno等[31]研究發(fā)現(xiàn)，在敲除HIF-1α基因的乳腺癌中，TAM向M2型極化，腫瘤組織血管再生能力減弱。上述研究結(jié)果與一般認(rèn)為M2型TAM促進(jìn)腫瘤血管新生的觀點(diǎn)相悖，有待進(jìn)一步論證。Segaliny等[32]研究發(fā)現(xiàn)，IL-34能使巨噬細(xì)胞在骨肉瘤組織中聚集，促進(jìn)骨肉瘤血管新生。因此，VEFG是骨肉瘤血管新生最重要的影響因素，骨肉瘤組織中浸潤的TAM可能通過促進(jìn)VEFG表達(dá)來誘導(dǎo)骨肉瘤新生血管生成，但具體機(jī)制尚需進(jìn)一步探索。

2.3 抑制骨肉瘤轉(zhuǎn)移

腫瘤轉(zhuǎn)移是指腫瘤從原發(fā)灶通過淋巴管、血管或直接蔓延等多種途徑到達(dá)其他部位繼續(xù)生長的過程。上皮間質(zhì)化(EMT)是指在多種因素影響下，上皮細(xì)胞黏附能力減弱，而運(yùn)動(dòng)能力增強(qiáng)，失去細(xì)胞極性，轉(zhuǎn)變?yōu)榫哂修D(zhuǎn)移能力的間充質(zhì)表型的上皮細(xì)胞的過程，是腫瘤發(fā)生轉(zhuǎn)移的起始階段。Notch信號(hào)轉(zhuǎn)導(dǎo)通路能激活骨肉瘤EMT，促進(jìn)骨肉瘤轉(zhuǎn)移[33]；Wnt信號(hào)轉(zhuǎn)導(dǎo)通路的配體及受體在骨肉瘤細(xì)胞株中高表達(dá)，能提高骨肉瘤轉(zhuǎn)移的能力[34]。骨肉瘤組織中的VEGF[35]、埃茲蛋白[36]及趨化因子受體CXCR3[37]等水平也與骨肉瘤的轉(zhuǎn)移呈正相關(guān)。Cheng等[38]研究發(fā)現(xiàn)，重樓皂苷Ⅶ能激活p38絲裂原活化蛋白激酶(p38 MAPK)信號(hào)轉(zhuǎn)導(dǎo)通路，下調(diào)MMP-2及MMP-9水平，減少ECM降解，抑制骨肉瘤轉(zhuǎn)移。

在腫瘤轉(zhuǎn)移過程中，TAM既能上調(diào)MMP水平使ECM降解[39]，又能通過激活多種信號(hào)轉(zhuǎn)導(dǎo)通路激活EMT[40]。Liu等[41]研究發(fā)現(xiàn)，M2型TAM可通過激活Toll樣受體(TLR)4/IL-10信號(hào)轉(zhuǎn)導(dǎo)通路促進(jìn)波形蛋白、上皮細(xì)胞鈣黏蛋白等間充質(zhì)細(xì)胞標(biāo)志物表達(dá)，提高M(jìn)MP-9、MMP-2的活性，激活EMT，介導(dǎo)胰腺癌轉(zhuǎn)移。Lee等[42]研究發(fā)現(xiàn)，M2型TAM可激活Gas6/Axl-NF-κB17信號(hào)轉(zhuǎn)導(dǎo)通路，促進(jìn)EMT，介導(dǎo)口腔癌轉(zhuǎn)移。Lin等[43]研究發(fā)現(xiàn)，M2型TAM能分泌趨化因子CCL18，下調(diào)miRNA-98 和miRNA-27b基因表達(dá)，激活EMT，介導(dǎo)乳腺癌轉(zhuǎn)移。

綜上所述，多數(shù)惡性腫瘤中高度浸潤的TAM主要表現(xiàn)為M2型，可促進(jìn)腫瘤轉(zhuǎn)移。Buddingh等[44]研究發(fā)現(xiàn)，在高度惡性的骨肉瘤中，TAM同時(shí)具備M1型及M2型TAM的特點(diǎn)，且TAM數(shù)量越多，骨肉瘤患者發(fā)生轉(zhuǎn)移的概率越低，生存期越長，提示在高度惡性骨肉瘤中，TAM可能主要表現(xiàn)為M1型TAM的特點(diǎn)，即抑制骨肉瘤轉(zhuǎn)移，但TAM如何抑制骨肉瘤轉(zhuǎn)移尚需進(jìn)一步研究。

2.4 上調(diào)骨肉瘤腫瘤干細(xì)胞樣表型

腫瘤干細(xì)胞(CSC)是惡性腫瘤中一小群具有自我更新能力、高度耐藥性及多種分化潛能且增殖迅速的細(xì)胞，與腫瘤的發(fā)生、復(fù)發(fā)、轉(zhuǎn)移及耐藥密切相關(guān)[45]。Lapidot等[46]首次通過實(shí)驗(yàn)發(fā)現(xiàn)人類急性髓性白血病中存在CSC，之后學(xué)者們陸續(xù)從多種實(shí)體腫瘤如胰腺癌、肝癌、肺癌及胃癌等中分離出CSC。Gibbs等[47]于2005年首次從骨肉瘤中分離出CSC，其特征為高表達(dá)MSC特有的表面標(biāo)志物如CD133、CD177 和Sro-1。隨著研究的深入，學(xué)者們[48]發(fā)現(xiàn)Oct3/4和Nanog蛋白等也可作為骨肉瘤CSC樣表型的標(biāo)志物。TGF-β1信號(hào)轉(zhuǎn)導(dǎo)通路[49]、Notch信號(hào)轉(zhuǎn)導(dǎo)通路[50]能上調(diào)骨肉瘤CSC樣表型，而Wnt信號(hào)轉(zhuǎn)導(dǎo)通路[51]能抑制骨肉瘤CSC樣表型上調(diào)。

研究[52]表明，TAM能促進(jìn)CSC樣表型上調(diào)。TAM能通過激活TGF-β1信號(hào)轉(zhuǎn)導(dǎo)通路導(dǎo)致EMT，促進(jìn)肝細(xì)胞癌CSC樣表型上調(diào)，但EMT介導(dǎo)CSC樣表型上調(diào)具體機(jī)制尚需進(jìn)一步研究[53]。此外，TAM也可通過調(diào)控EGFR/Stat3/Sox-2信號(hào)轉(zhuǎn)導(dǎo)通路上調(diào)Scal-1和ABCG2等CSC樣表型[54]。在大多數(shù)惡性腫瘤中，TAM通過不同信號(hào)轉(zhuǎn)導(dǎo)通路上調(diào)CSC樣表型。因此，骨肉瘤中的TAM也有可能通過TGF-β1信號(hào)轉(zhuǎn)導(dǎo)通路促進(jìn)EMT，上調(diào)CSC樣表型。而TAM上調(diào)骨肉瘤CSC樣表型的機(jī)制以及骨肉瘤中CSC對(duì)TAM極化的影響，有待進(jìn)一步研究與探索。

3 結(jié)語

TAM在腫瘤發(fā)生發(fā)展過程中扮演重要角色。促進(jìn)M2型TAM向M1型轉(zhuǎn)化，從而抑制腫瘤的發(fā)生發(fā)展是目前研究的熱點(diǎn)。研究[55]表明，丙酮酸激酶(PK)M2能促進(jìn)TAM向M2型極化。Liu等[56]研究發(fā)現(xiàn)，PKM2在骨肉瘤中高表達(dá)，其為導(dǎo)致骨肉瘤患者預(yù)后不良的重要因素，這或許能為骨肉瘤TAM的研究提供新的方向。TAM在骨肉瘤中的作用研究尚處于初始階段，存在諸多問題。大多數(shù)研究將CD163作為M2型TAM的表型標(biāo)志物，但CD163在樹突狀細(xì)胞等中也有表達(dá)[57]。TAM在骨肉瘤中主要表現(xiàn)為何種表型可能與骨肉瘤分期及分化程度有關(guān)。骨肉瘤微環(huán)境如何影響TAM表型變化及TAM與骨肉瘤相互作用機(jī)制，尚需進(jìn)一步探索。

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(收稿：2016-09-15；修回：2016-10-08)

(本文編輯：李圓圓)

湖南省教育廳資助項(xiàng)目(15C1215)

421000 湖南衡陽， 南華大學(xué)附屬第二醫(yī)院骨科(劉政選、王曉旭、符勇)； 421000 湖南衡陽， 南華大學(xué)臨床技能中心(方松清)

方松清 E-mail: fangsongqing123@163.com

10.3969/j.issn.1673-7083.2016.06.011