结肠癌患者血浆外泌体表面膜结合型转化生长因子β1表达及其生物学作用

摘要

目的：观察结肠癌患者血浆外泌体（ exosomes ）表面膜结合型转化生长因子β1（ mTGF-β1）表达，探讨其生物学作用。方法选取结肠癌患者20例（观察组），同期体检健康者10例（对照组），提取两组血浆exosomes，采用流式细胞仪分析exosomes 表面mTGF-β1表达。从结肠癌患者肝素抗凝全血中分离外周血单个核细胞（ PB-MCs），分选获得纯化的CD4＋T细胞，采用CD3、CD28诱导细胞增殖，将细胞随机分为三组，A组加入TGF-β1阻断性单抗处理的结肠癌患者血浆exosomes（1μg／mL），B组加入IgG1同型对照抗体处理的结肠癌患者血浆exosomes （1μg／mL），C组加入健康人血浆exosomes（1μg／mL），培养3天时进行台盼蓝染色计数活细胞数；采用ELISA法检测细胞培养上清液中TNF-α和IFN-γ水平。结果观察组血浆exosomes表面mTGF-β1表达阳性率为36．52％±4．26％，高于对照组的9．23％±3．77％（P＜0．01）。与C组比较，B组CD4＋T细胞数量及其分泌的TNF-α、IFN-γ水平均降低（P均＜0．05）；与B 组比较，A组CD4＋T细胞数量及其分泌的TNF-α、IFN-γ水平均增加（P均＜0．05）。结论结肠癌患者血浆exosomes表面高表达mTGF-β1，mTGF-β1表达增加可能是构成结肠癌免疫抑制环境的重要因素，可促进结肠癌的发生、发展。%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-β1 on 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 .Mean-while, 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 1μg/ml exosomes from plasma of patients treated with anti-TGF-β1 monoclonal anti-body ( mAb) , group B was added with 1μg/ml exosomes from plasma of patients treated with control IgG 1 mAb and group C was added with 1μg/mL exosomes from plasma of healthy donors .The cell count was calculated by Trypan blue stainin , and the levels of TNF-αand IFN-γwas detected by ELISA on day 3 after culture.Results The positive rate of mTGF-β1 expres-sion 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-αand IFN-γsecreted in the group B were all lower than those in the group C (all P<0.05).Compared with group B, the number of CD4+T cells and the levels of TNF-αand IFN-γsecreted in the group A were higher (all P<0.05).Conclusion The mTGF-β1 expression is highly expressed on the surface of exosomes in the plasma from patients with colorectal cancer , and the increased expression of mT-GF-β1 may play an important role in the colon cancer immunosuppressive environment , which promotes the occurrence and development of colon cancer .