居頌文

(南京醫(yī)科大學(xué)附屬蘇州醫(yī)院北區(qū)，江蘇蘇州215008)

·論著·

結(jié)腸癌患者血漿外泌體表面膜結(jié)合型轉(zhuǎn)化生長(zhǎng)因子β1表達(dá)及其生物學(xué)作用

居頌文

(南京醫(yī)科大學(xué)附屬蘇州醫(yī)院北區(qū)，江蘇蘇州215008)

目的 觀察結(jié)腸癌患者血漿外泌體(exosomes)表面膜結(jié)合型轉(zhuǎn)化生長(zhǎng)因子β1(mTGF-β1)表達(dá)，探討其生物學(xué)作用。方法 選取結(jié)腸癌患者20例(觀察組)，同期體檢健康者10例(對(duì)照組)，提取兩組血漿exosomes，采用流式細(xì)胞儀分析exosomes表面mTGF-β1表達(dá)。從結(jié)腸癌患者肝素抗凝全血中分離外周血單個(gè)核細(xì)胞(PBMCs)，分選獲得純化的CD4+T細(xì)胞，采用CD3、CD28誘導(dǎo)細(xì)胞增殖，將細(xì)胞隨機(jī)分為三組，A組加入TGF-β1阻斷性單抗處理的結(jié)腸癌患者血漿exosomes(1 μg/mL)，B組加入IgG1同型對(duì)照抗體處理的結(jié)腸癌患者血漿exosomes(1 μg/mL)，C組加入健康人血漿exosomes(1 μg/mL)，培養(yǎng)3天時(shí)進(jìn)行臺(tái)盼藍(lán)染色計(jì)數(shù)活細(xì)胞數(shù)；采用ELISA法檢測(cè)細(xì)胞培養(yǎng)上清液中TNF-α和IFN-γ水平。結(jié)果 觀察組血漿exosomes表面mTGF-β1表達(dá)陽(yáng)性率為36.52%±4.26%，高于對(duì)照組的9.23%±3.77%(P<0.01)。與C組比較，B組CD4+T細(xì)胞數(shù)量及其分泌的TNF-α、IFN-γ水平均降低(P均<0.05)；與B組比較，A組CD4+T細(xì)胞數(shù)量及其分泌的TNF-α、IFN-γ水平均增加(P均<0.05)。結(jié)論 結(jié)腸癌患者血漿exosomes表面高表達(dá)mTGF-β1，mTGF-β1表達(dá)增加可能是構(gòu)成結(jié)腸癌免疫抑制環(huán)境的重要因素，可促進(jìn)結(jié)腸癌的發(fā)生、發(fā)展。

結(jié)腸癌；膜結(jié)合型轉(zhuǎn)化生長(zhǎng)因子β1；外泌體

外泌體(exosomes)是由多種動(dòng)物活細(xì)胞分泌的來(lái)源于多囊體的小囊泡，具有雙層膜結(jié)構(gòu)，是直徑30～100 nm的扁平或球形小體，廣泛分布于人體血漿、唾液、乳汁、尿液等體液中[1]。近年研究表明，exosomes不僅可促進(jìn)細(xì)胞新陳代謝過(guò)程中代謝產(chǎn)物的排出，也是細(xì)胞間進(jìn)行物質(zhì)、信息交流的重要媒介，其中腫瘤細(xì)胞來(lái)源的exosomes參與腫瘤侵襲、轉(zhuǎn)移及耐藥等病理過(guò)程[2,3]。轉(zhuǎn)化生長(zhǎng)因子β1(TGF-β1)是具有免疫抑制作用的細(xì)胞因子，可抑制機(jī)體的抗腫瘤免疫反應(yīng)[4]，而膜結(jié)合型轉(zhuǎn)化生長(zhǎng)因子β1(mTGF-β1)是調(diào)節(jié)性T細(xì)胞(Treg)及骨髓來(lái)源的抑制性細(xì)胞(MDSCs)發(fā)揮免疫抑制作用的重要效應(yīng)分子[5,6]。 本研究觀察結(jié)腸癌患者血漿exosomes表面TGF-β1表達(dá)，探討其生物學(xué)作用。

1 資料與方法

1.1 臨床資料 選取2014年1月～2015年6月南京醫(yī)科大學(xué)附屬蘇州醫(yī)院北區(qū)及蘇州大學(xué)附屬第二醫(yī)院收治的結(jié)腸癌患者20例(結(jié)腸癌組)，男13例、女7例，年齡31～75歲、平均56.8歲，TMN分期Ⅱ期1例、Ⅲ期13例、Ⅳ期6例，入組前均未進(jìn)行放化療。選取同期體檢健康者10例作為對(duì)照組，男5例、女5例，年齡28～60歲、平均39.3歲。兩組性別、年齡具有可比性。

1.2 血漿exosomes表面mTGF-β1表達(dá)檢測(cè) 觀察組入院時(shí)、對(duì)照組體檢時(shí)均留取外周靜脈血，離心取血漿，采用ExoQuick exosome precipitation solution試劑盒(SBI公司)提取血漿中的exosomes，具體操作參照試劑盒說(shuō)明書(shū)。采用微珠(Aldehyde/Sulfate Latex Beads，4% w/v，4 μm，Life technologies)包被exosomes，取30 μg exosomes與微珠混合，加入PBS調(diào)整體積至100 μL，室溫反應(yīng)2 h，加入100 mmol/L甘氨酸溶液終止反應(yīng)。包被exosomes的微珠標(biāo)記不同的熒光抗體和同型對(duì)照抗體，流式細(xì)胞儀檢測(cè)exosomes表面CD9、CD63表達(dá)，結(jié)果顯示結(jié)腸癌患者血漿中分離獲得的exosomes表達(dá)CD9、CD63，兩組表達(dá)量相近，提示血漿exosomes提取成功。采用流式細(xì)胞儀檢測(cè)兩組血漿exosomes表面mTGF-β1表達(dá)陽(yáng)性率。

1.3 外周血單個(gè)核細(xì)胞(PBMCs)CD4+T細(xì)胞增殖情況及TNF-α、IFN-γ分泌水平檢測(cè) 采用聚蔗糖-泛影葡胺密度梯度離心法從結(jié)腸癌患者肝素抗凝全血中分離PBMCs，采用流式細(xì)胞儀(BD FACSAria Ⅱ)分選獲得純化的CD4+T細(xì)胞。采用CD3單抗(1 μg/mL)包被96孔板，4 ℃過(guò)夜，棄包被液，備用。采用含10%胎牛血清的RPMI 1640培養(yǎng)基培養(yǎng)純化的CD4+T細(xì)胞，將細(xì)胞密度調(diào)整至3×105個(gè)/mL，接種至包被CD3單抗的96孔板，100 μL/孔，加入CD28單抗(1 μg/mL)。將細(xì)胞隨機(jī)分為三組，A組加入TGF-β1阻斷性單抗處理的結(jié)腸癌患者血漿exosomes(1 μg/mL)，B組加入IgG1同型對(duì)照抗體處理的結(jié)腸癌患者血漿exosomes(1 μg/mL)，C組加入健康人血漿exosomes(1 μg/mL)，培養(yǎng)3天時(shí)行臺(tái)盼藍(lán)染色計(jì)數(shù)活細(xì)胞數(shù)；收集細(xì)胞培養(yǎng)液上清，采用ELISA法檢測(cè)細(xì)胞因子TNF-α和IFN-γ水平。

TGF-β1阻斷性單抗處理的結(jié)腸癌患者血漿exosomes獲得方法：exosomes與TGF-β1阻斷性抗體(Biolegend公司)混合(質(zhì)量比1∶1)，4 ℃反應(yīng)15 min，加入PBS，超速離心(36 000 r/min，1.5 h)，棄上清，洗滌兩遍，去除未結(jié)合的抗體，收集阻斷TGF-β1的exosomes沉淀，加入PBS重新溶解，備用；TGF-β1同型對(duì)照處理的結(jié)腸癌患者血漿exosomes獲得方法：exosomes與IgG1同型對(duì)照抗體混合(質(zhì)量比1∶1)，其余操作同前。

2 結(jié)果

觀察組、對(duì)照組血漿exosomes表面mTGF-β1表達(dá)陽(yáng)性率分別為36.52%±4.26%、9.23%±3.77%，兩組比較P<0.01。與C組比較，B組CD4+T細(xì)胞數(shù)量及其分泌的TNF-α、IFN-γ水平均降低(P均<0.05)；與B組比較，A組CD4+T細(xì)胞數(shù)量及其分泌的TNF-α、IFN-γ水平均增加(P均<0.05)。見(jiàn)表1。

表1 三組CD4+T細(xì)胞數(shù)量及其分泌的TNF-α、IFN-γ水平比較

注：與B組比較，*P<0.05。

3 討論

Exosomes是由多種動(dòng)物活細(xì)胞分泌的小囊泡體，含有大量的蛋白質(zhì)、脂質(zhì)、RNA和microRNA等成分，并可將這些成分傳遞給附近或遠(yuǎn)處的受體細(xì)胞，進(jìn)而介導(dǎo)一系列的生物學(xué)功能[1～3]。研究發(fā)現(xiàn)，樹(shù)突狀細(xì)胞(DCs)來(lái)源的exosomes在體內(nèi)可刺激CD8+T細(xì)胞依賴的抗腫瘤效應(yīng)，導(dǎo)致腫瘤消退[7,8]；腫瘤細(xì)胞來(lái)源的exosomes在腫瘤細(xì)胞侵襲、轉(zhuǎn)移、耐藥及免疫調(diào)節(jié)過(guò)程中發(fā)揮重要作用，亦可通過(guò)抑制DCs、NK、CD4+和CD8+T細(xì)胞等的抗腫瘤免疫反應(yīng)以及誘導(dǎo)調(diào)節(jié)性T細(xì)胞(Treg)和骨髓來(lái)源的抑制性細(xì)胞(MDSCs)在腫瘤免疫逃逸中發(fā)揮作用[9～14]。乳腺癌細(xì)胞通過(guò)攜帶高水平miR-122的exosomes在轉(zhuǎn)移前微環(huán)境中抑制非腫瘤細(xì)胞的葡萄糖攝取，為即將轉(zhuǎn)移的腫瘤細(xì)胞提供一個(gè)高葡萄糖環(huán)境[11]。研究發(fā)現(xiàn)，exosomes表面的整合素種類決定著靶器官的特異性整合素移靶向性，例如與肺轉(zhuǎn)移有關(guān)的exosomes表面整合素為α6β4和α6β1，整合素αvβ5則與肝轉(zhuǎn)移有關(guān)[9]。在腎上皮細(xì)胞癌中，具有耐藥性的腫瘤細(xì)胞會(huì)通過(guò)exosomes傳遞與耐藥相關(guān)的長(zhǎng)鏈非編碼RNA(lncRNA)藥物敏感性受體結(jié)合，介導(dǎo)腫瘤細(xì)胞耐藥[10]。Exosomes能抑制單核細(xì)胞分化為DCs和抑制DCs成熟，亦能激活髓系來(lái)源的MDSCs的抑制活性[9,14]。腫瘤exosomes含有膜蛋白HSP72，通過(guò)Hsp72/TLR2途徑促進(jìn)MDSCs的免疫抑制功能[15]。Abusamra等[16]研究發(fā)現(xiàn)，前列腺癌細(xì)胞來(lái)源的exosomes表達(dá)FasL，與T細(xì)胞表面的Fas受體結(jié)合，誘導(dǎo)T細(xì)胞凋亡。

TGF-β1是具有免疫抑制作用的細(xì)胞因子，可抑制T細(xì)胞增殖，抑制IFN-γ、IL-12等細(xì)胞因子產(chǎn)生，通過(guò)抑制機(jī)體的抗腫瘤免疫反應(yīng)促進(jìn)腫瘤的發(fā)生、發(fā)展[17]。研究發(fā)現(xiàn)，TGF-β1上調(diào)殺傷抑制性受體NKG2A表達(dá)，抑制殺傷活化性受體NKG2D和NKp30在NK細(xì)胞上的表達(dá)，從而抑制NK細(xì)胞的抗腫瘤活性[18，19]。本研究結(jié)果顯示，結(jié)腸癌患者血漿中exosomes表面表達(dá)mTGF-β1顯著升高；結(jié)腸癌患者血漿exosomes能抑制CD4+T細(xì)胞增殖以及IFN-γ和TNF-α的產(chǎn)生，而采用TGF-β1阻斷性單抗可顯著抑制exosomes對(duì)CD4+T細(xì)胞增殖及IFN-γ、TNF-α產(chǎn)生的抑制作用；上述結(jié)果提示，結(jié)腸癌患者血漿exosomes表面高表達(dá)mTGF-β1，mTGF-β1表達(dá)升高可能是構(gòu)成結(jié)腸癌免疫抑制環(huán)境的重要因素，可促進(jìn)結(jié)腸癌的發(fā)生、發(fā)展。

[1] Thery C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function[J]. Nat Rev Immunol, 2002,2(8):569-579.

[2] Azmi AS, Bao B, Sarkar FH. Exosomes in cancer development, metastasis, and drug resistance: a comprehensive review[J]. Cancer Metastasis Rev, 2013,32(3-4):623-642.

[3] Beach A, Zhang HG, Ratajczak MZ, et al. Exosomes: an overview of biogenesis, composition and role in ovarian cancer[J]. J Ovarian Res, 2014(25):7-14.

[4] Yang L, Pang YL, Moses HL, et al. TGF-β and immune cells: an important regulatory axis in the tumormicroenvironment and progression[J]. Trends Immunol, 2010,31(6):220-227.

[5] Nakamura K, Kitani A, Strober W, et al. Cell contact-dependentimmunosuppression by CD4(+)CD25(+) regulatory T cells is mediated by cellsurface-bound transforming growth factor beta[J]. J Exp Med, 2001,194(4):629-644.

[6] Li HQ, Han,YM, Guo QL, et al. Cancer-expanded myeloid-derived suppressor cells induce anergyof NK cells through membrane-bound TGF-β1[J]. J Immunol, 2009,182(1):240-249.

[7] Pitt JM, Charrier M, Viaud S,et al. Dendritic cell-derived exosomes as immunotherapies in the fight against cancer[J]. J Immunol, 2014,193(3):1006-1011.

[8] 張?jiān)谠?李希德,劉葉,等.肺癌細(xì)胞裂解物負(fù)載對(duì)樹(shù)突狀細(xì)胞分泌的exosome誘導(dǎo)抗腫瘤作用的影響[J].山東醫(yī)藥,2011,51(31):4-6.

[9] Zhang HG, Grizzle WE. Exosomes and cancer: a newly described pathway of immune suppression[J]. Clin Cancer Res, 2011,17 (5):959-964.

[10] Taylor DD, Gercel-Taylor C. Exosomes/microvesicles: mediators of cancer-associated immunosuppressive microenvironments[J]. Semin Immunopathol, 2011,33(5):441-454.

[11] Fong MY, Zhou WY, Liu L, et al. Breast-cancer-secreted miR-122 reprograms glucose metabolism in premetastatic niche to promote metastasis[J]. Nature Cell Biology, 2015,17(2):183-194.

[12] Hoshino A, Costa-Silva B, Shen TL, et al. Tumorexosome integrins determine organotropic metastasis[J]. Nature, 2015,527(7578):329-335.

[13] Qu L, Ding J, Chen C, et al. Exosome-transmitted lncARSR promotes sunitinib resistance in renal cancer by acting as a competing endogenous RNA[J]. Cancer Cell, 2016,29(5):653-668.

[14] Ashiru O, Boutet P, Fernández-Messina L, et al. Natural killer cell cytotoxicity is suppressed by exposure to the human NKG2D ligand MICA*008 that is shed by tumor cells in exosomes[J]. Cancer Res, 2010,70(2):481-489.

[15] Chalmin F, Sylvain Ladoire S, Mignot G, et al. Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells[J]. J Clin Invest, 2010,120(2):457-471.

[16] Abusamra AJ, Zhong Z, Zheng X, et al.Tumor exosomes expressing Fas ligand mediate CD8+T-cell apoptosis[J]. Blood Cells Mol Dis, 2005,35(2):169-173.

[17] Oh SA, Li MO. TGF-β: guardian of T cell function[J]. J Immunol, 2013,191(8):3973-3979.

[18] Witham TF, Villa L, Yang T, et al. Expression of a soluble transforming growth factor-beta (TGFbeta) receptor reduces tumorigenicity by regulating natural killer (NK) cell activity against 9L gliosarcoma in vivo[J]. J Neurooncol, 2003,64(1-2):63-69.

[19] Zhang HG, Grizzle WE. Exosomesand cancer: a newly described pathway of immune suppression[J]. Clin Cancer Res, 2011,17(5):959-964.

Expression of mTGF-β1on the surface of exosomes in plasma from patients with colorectal cancer and its biological function

JUSongwen

(NorthDistrictofSuzhouHospitalAffiliatedtoNanjingMedicalUniversity,Suzhou215008,China)

Objective To explore the membrane-bound transforming growth factor-β1(mTGF-β1) expression on the surface of exosomes in the plasma from patients with colorectal cancer and its biological function. Methods The expression of mTGF-β1on the surface of exosomes from 20 samples of patients with colorectal cancer (observation group) and 10 healthy donors (control group) were analyzed by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated from heparin anticoagulated whole blood of colon cancer patients, and CD4+T cells were purified and obtained by sorting. Meanwhile, CD3 and CD28 were used to induce cell proliferation, and the cells were randomly divided into three groups: groups A, B and C, group A was added with 1 μg/ml exosomes from plasma of patients treated with anti-TGF-β1monoclonal antibody (mAb), group B was added with 1 μg/ml exosomes from plasma of patients treated with control IgG1mAb and group C was added with 1 μg/mL exosomes from plasma of healthy donors. The cell count was calculated by Trypan blue stainin, and the levels of TNF-α and IFN-γ was detected by ELISA on day 3 after culture. Results The positive rate of mTGF-β1expression in the observation group was 36.52%±4.26%, which was higher than that (9.23%±3.77%) in the control group (P<0.01). Compared with group C, the number of CD4+T cells and the levels of TNF-α and IFN-γ secreted in the group B were all lower than those in the group C (allP<0.05). Compared with group B, the number of CD4+T cells and the levels of TNF-α and IFN-γ secreted in the group A were higher (allP<0.05). Conclusion The mTGF-β1expression is highly expressed on the surface of exosomes in the plasma from patients with colorectal cancer, and the increased expression of mTGF-β1may play an important role in the colon cancer immunosuppressive environment, which promotes the occurrence and development of colon cancer.

colorectal carcinoma; membrane-bound transforming growth factor-β1; exosomes

國(guó)家自然科學(xué)基金資助項(xiàng)目(81373149)；江蘇省自然科學(xué)基金資助項(xiàng)目(BK20151195)；蘇州市科技計(jì)劃項(xiàng)目(SYS201363)。

居頌文(1977-)，男，副研究員，研究方向?yàn)槟[瘤免疫學(xué)。E-mail: szjusw@sina.com

10.3969/j.issn.1002-266X.2016.44.001

R735.3

A

1002-266X(2016)44-0001-03

2016-01-12)