結腸癌患者血漿外泌體表面膜結合型轉化生長因子β1表達及其生物學作用

居頌文

(南京醫科大學附屬蘇州醫院北區，江蘇蘇州215008)

·論著·

結腸癌患者血漿外泌體表面膜結合型轉化生長因子β1表達及其生物學作用

居頌文

(南京醫科大學附屬蘇州醫院北區，江蘇蘇州215008)

目的 觀察結腸癌患者血漿外泌體(exosomes)表面膜結合型轉化生長因子β1(mTGF-β1)表達，探討其生物學作用。方法 選取結腸癌患者20例(觀察組)，同期體檢健康者10例(對照組)，提取兩組血漿exosomes，采用流式細胞儀分析exosomes表面mTGF-β1表達。從結腸癌患者肝素抗凝全血中分離外周血單個核細胞(PBMCs)，分選獲得純化的CD4+T細胞，采用CD3、CD28誘導細胞增殖，將細胞隨機分為三組，A組加入TGF-β1阻斷性單抗處理的結腸癌患者血漿exosomes(1 μg/mL)，B組加入IgG1同型對照抗體處理的結腸癌患者血漿exosomes(1 μg/mL)，C組加入健康人血漿exosomes(1 μg/mL)，培養3天時進行臺盼藍染色計數活細胞數；采用ELISA法檢測細胞培養上清液中TNF-α和IFN-γ水平。結果 觀察組血漿exosomes表面mTGF-β1表達陽性率為36.52%±4.26%，高于對照組的9.23%±3.77%(P<0.01)。與C組比較，B組CD4+T細胞數量及其分泌的TNF-α、IFN-γ水平均降低(P均<0.05)；與B組比較，A組CD4+T細胞數量及其分泌的TNF-α、IFN-γ水平均增加(P均<0.05)。結論 結腸癌患者血漿exosomes表面高表達mTGF-β1，mTGF-β1表達增加可能是構成結腸癌免疫抑制環境的重要因素，可促進結腸癌的發生、發展。

結腸癌；膜結合型轉化生長因子β1；外泌體

外泌體(exosomes)是由多種動物活細胞分泌的來源于多囊體的小囊泡，具有雙層膜結構，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不僅可促進細胞新陳代謝過程中代謝產物的排出，也是細胞間進行物質、信息交流的重要媒介，其中腫瘤細胞來源的exosomes參與腫瘤侵襲、轉移及耐藥等病理過程[2,3]。轉化生長因子β1(TGF-β1)是具有免疫抑制作用的細胞因子，可抑制機體的抗腫瘤免疫反應[4]，而膜結合型轉化生長因子β1(mTGF-β1)是調節性T細胞(Treg)及骨髓來源的抑制性細胞(MDSCs)發揮免疫抑制作用的重要效應分子[5,6]。 本研究觀察結腸癌患者血漿exosomes表面TGF-β1表達，探討其生物學作用。

1 資料與方法

1.1 臨床資料 選取2014年1月～2015年6月南京醫科大學附屬蘇州醫院北區及蘇州大學附屬第二醫院收治的結腸癌患者20例(結腸癌組)，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，入組前均未進行放化療。選取同期體檢健康者10例作為對照組，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表達檢測 觀察組入院時、對照組體檢時均留取外周靜脈血，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明書。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調整體積至100 μL，室溫反應2 h，加入100 mmol/L甘氨酸溶液終止反應。包被exosomes的微珠標記不同的熒光抗體和同型對照抗體，流式細胞儀檢測exosomes表面CD9、CD63表達，結果顯示結腸癌患者血漿中分離獲得的exosomes表達CD9、CD63，兩組表達量相近，提示血漿exosomes提取成功。采用流式細胞儀檢測兩組血漿exosomes表面mTGF-β1表達陽性率。

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

TGF-β1阻斷性單抗處理的結腸癌患者血漿exosomes獲得方法：exosomes與TGF-β1阻斷性抗體(Biolegend公司)混合(質量比1∶1)，4 ℃反應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同型對照處理的結腸癌患者血漿exosomes獲得方法：exosomes與IgG1同型對照抗體混合(質量比1∶1)，其余操作同前。

2 結果

觀察組、對照組血漿exosomes表面mTGF-β1表達陽性率分別為36.52%±4.26%、9.23%±3.77%，兩組比較P<0.01。與C組比較，B組CD4+T細胞數量及其分泌的TNF-α、IFN-γ水平均降低(P均<0.05)；與B組比較，A組CD4+T細胞數量及其分泌的TNF-α、IFN-γ水平均增加(P均<0.05)。見表1。

表1 三組CD4+T細胞數量及其分泌的TNF-α、IFN-γ水平比較

注：與B組比較，*P<0.05。

3 討論

Exosomes是由多種動物活細胞分泌的小囊泡體，含有大量的蛋白質、脂質、RNA和microRNA等成分，并可將這些成分傳遞給附近或遠處的受體細胞，進而介導一系列的生物學功能[1～3]。研究發現，樹突狀細胞(DCs)來源的exosomes在體內可刺激CD8+T細胞依賴的抗腫瘤效應，導致腫瘤消退[7,8]；腫瘤細胞來源的exosomes在腫瘤細胞侵襲、轉移、耐藥及免疫調節過程中發揮重要作用，亦可通過抑制DCs、NK、CD4+和CD8+T細胞等的抗腫瘤免疫反應以及誘導調節性T細胞(Treg)和骨髓來源的抑制性細胞(MDSCs)在腫瘤免疫逃逸中發揮作用[9～14]。乳腺癌細胞通過攜帶高水平miR-122的exosomes在轉移前微環境中抑制非腫瘤細胞的葡萄糖攝取，為即將轉移的腫瘤細胞提供一個高葡萄糖環境[11]。研究發現，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，例如與肺轉移有關的exosomes表面整合素為α6β4和α6β1，整合素αvβ5則與肝轉移有關[9]。在腎上皮細胞癌中，具有耐藥性的腫瘤細胞會通過exosomes傳遞與耐藥相關的長鏈非編碼RNA(lncRNA)藥物敏感性受體結合，介導腫瘤細胞耐藥[10]。Exosomes能抑制單核細胞分化為DCs和抑制DCs成熟，亦能激活髓系來源的MDSCs的抑制活性[9,14]。腫瘤exosomes含有膜蛋白HSP72，通過Hsp72/TLR2途徑促進MDSCs的免疫抑制功能[15]。Abusamra等[16]研究發現，前列腺癌細胞來源的exosomes表達FasL，與T細胞表面的Fas受體結合，誘導T細胞凋亡。

TGF-β1是具有免疫抑制作用的細胞因子，可抑制T細胞增殖，抑制IFN-γ、IL-12等細胞因子產生，通過抑制機體的抗腫瘤免疫反應促進腫瘤的發生、發展[17]。研究發現，TGF-β1上調殺傷抑制性受體NKG2A表達，抑制殺傷活化性受體NKG2D和NKp30在NK細胞上的表達，從而抑制NK細胞的抗腫瘤活性[18，19]。本研究結果顯示，結腸癌患者血漿中exosomes表面表達mTGF-β1顯著升高；結腸癌患者血漿exosomes能抑制CD4+T細胞增殖以及IFN-γ和TNF-α的產生，而采用TGF-β1阻斷性單抗可顯著抑制exosomes對CD4+T細胞增殖及IFN-γ、TNF-α產生的抑制作用；上述結果提示，結腸癌患者血漿exosomes表面高表達mTGF-β1，mTGF-β1表達升高可能是構成結腸癌免疫抑制環境的重要因素，可促進結腸癌的發生、發展。

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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

國家自然科學基金資助項目(81373149)；江蘇省自然科學基金資助項目(BK20151195)；蘇州市科技計劃項目(SYS201363)。

居頌文(1977-)，男，副研究員，研究方向為腫瘤免疫學。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)