CD4+CD25highCD127low调节性T细胞促进上皮性卵巢癌转移机制的初步探讨

doi:10.3969/j.issn.1673-8640.2019.02.004

摘要/Abstract

摘要：

目的探讨CD4⁺CD25^highCD127^low调节性T细胞（Treg）在上皮性卵巢癌（EOC）转移中的作用及相关细胞因子涉及的信号机制。方法采用流式细胞术检测34例EOC患者（EOC组）、26例卵巢良性肿瘤患者（卵巢良性肿瘤组）和34名体检健康者（正常对照组）外周血CD4⁺CD25^highCD127^lowTreg的比例。收集所有患者的临床资料,包括年龄、病理类型、国际妇产科联合会（FIGO）分期、组织学分化情况、淋巴结转移情况。建立正常对照者CD4⁺T细胞与EOC细胞株OVCAR3的共培养模型,并采用酶联免疫吸附试验（ELISA）检测白细胞介素10（IL-10）和转化生长因子β1（TGF-β1）水平。采用实时荧光定量聚合酶链反应（PCR）检测OVCAR3细胞中基质金属蛋白酶（MMP）-2 mRNA和基质金属蛋白酶抑制因子（TIMP）-2 mRNA的表达。采用抗IL-10受体抗体和抗TGF-β1受体抗体作为阻断剂,预处理OVCAR3细胞后再进行共培养,并检测MMP-2 mRNA和TIMP-2 mRNA的表达。结果 EOC组外周血CD4⁺CD25^highCD127^lowTreg明显高于卵巢良性肿瘤组和正常对照组（P<0.05）,而卵巢良性肿瘤组与正常对照组之间差异无统计学意义（P>0.05）。淋巴结转移阳性EOC患者外周血CD4⁺CD25^highCD127^lowTreg比例明显高于淋巴结转移阴性患者（P<0.05）,而不同年龄、病理类型、FIGO分期及组织学分化情况的EOC患者之间CD4⁺CD25^highCD127^lowTreg比例差异均无统计学意义（P>0.05）。共培养后CD4⁺T细胞分泌的IL-10和TGF-β1水平明显高于CD4⁺T细胞单独培养（P<0.05）。与OVCAR3细胞单独培养相比,共培养后的OVCAR3细胞中MMP-2 mRNA表达明显升高（P<0.05）,TIMP-2 mRNA明显降低（P<0.05）。采用阻断剂预处理后共培养的OVCAR3细胞MMP-2 mRNA表达下调（P<0.05）、TIMP-2 mRNA表达上调（P<0.05）。结论 CD4⁺CD25^highCD127^lowTreg可增强IL-10和TGF-β1的表达,并作用于EOC细胞的相关受体,可能对EOC侵袭、转移起重要作用。

关键词: 调节性T细胞, 基质金属蛋白酶2, 基质金属蛋白酶组织抑制因子2, 上皮性卵巢癌

Abstract:

Objective To investigate the role of CD4⁺CD25^highCD127^low regulatory T cells（Treg） in the metastasis of epithelial ovarian cancer（EOC）,and to analyze the signaling mechanism of the related cytokines. Methods Flow cytometry was used to determine the percentages of CD4⁺CD25^highCD127^low Treg in peripheral blood of 34 patients with EOC,26 patients with benign ovarian tumors and 34 healthy subjects（healthy control group）. The clinical data of all patients,including age,pathological type,the International Federation of Gynecology and Obstetrics（FIGO） stage,histological differentiation and lymph node metastasis,were collected. A coculture system between EOC cell line OVCAR3 and healthy subjects' CD4⁺ T cells was established. The levels of interleukin 10（IL-10） and transforming growth factor-beta 1（TGF-β1） were determined by enzyme-linked immunosorbent assay（ELISA）. The expression levels of matrix metalloproteinase（MMP）-2 mRNA and tissue inhibitor of metalloproteinase（TIMP）-2 mRNA in OVCAR3 were determined by real-time fluorescence quantitation polymerase chain reaction（PCR）. The levels of MMP-2 mRNA and TIMP-2 mRNA were also analyzed after blocking IL-10 receptor and TGF-β1 receptor in OVCAR3 after the coculture experiment. Results The level of CD4⁺CD25^highCD127^low Treg in EOC group was higher than those in benign ovarian tumor and healthy control groups（P<0.05）,and there was no statistical significance between benign ovarian tumor and healthy control groups（P>0.05）. The percentage of CD4⁺CD25^highCD127^low Treg was higher in lymphatic invasion group than that in non-lymphatic invasion group（P<0.05）. There was no statistical significance for CD4⁺CD25^highCD127^low Treg percentage in EOC group with different ages,pathological types,FIGO stages and histological differentiation（P>0.05）. The levels of IL-10 and TGF-β1 from CD4⁺ T cells were up-regulated after coculturing with CD4⁺ T cells（P<0.05）. The level of MMP-2 mRNA was up-regulated（P<0.05）,and the level of TIMP-2 mRNA in OVCAR3 was down-regulated in the coculture experiment with CD4⁺ T cells（P<0.05）. The level of MMP-2 mRNA was down-regulated,and the level of TIMP-2 mRNA in OVCAR3 was up-regulated after blocking（P<0.05）. Conclusions CD4⁺CD25^highCD127^low Treg can enhance the expression of IL-10 and TGF-β1 and stimulate the related receptors on EOC cells. It plays a role in the invasion and metastasis of EOC.

Key words: Regulatory T cell, Matrix metalloproteinase 2, Tissue inhibitor of metalloproteinase 2, Epithelial ovarian cancer

中图分类号:

R331.1

柯星, 张良, 沈立松. CD4⁺CD25^highCD127^low调节性T细胞促进上皮性卵巢癌转移机制的初步探讨[J]. 检验医学, 2019, 34(2): 110-115.

KE Xing, ZHANG Liang, SHEN Lisong. Preliminary study on the mechanism of CD4⁺CD25^highCD127^low regulatory T cells promoting the metastasis of epithelial ovarian cancer[J]. Laboratory Medicine, 2019, 34(2): 110-115.

图/表 4

表1 CD4+CD25highCD127lowTreg在不同临床特征EOC患者外周血中的比例（%,$bar{x}$±s）

临床特征	例数	CD4⁺CD25^highCD127^lowTreg
年龄（岁）
<50	19	8.17±0.83
≥50	15	8.28±0.71
病理类型
浆液性	22	8.45±0.68
黏液性	12	7.79±0.97
FIGO分期
Ⅰ~Ⅱ期	18	8.21±0.70
Ⅲ~Ⅳ期	16	8.22±0.90
组织学分化
高~中分化	20	7.85±0.66
低分化	14	8.75±0.98
淋巴结转移
阴性	19	7.04±0.62
阳性	15	9.70±0.86^*

图1 CD4+T细胞单独培养及与OVCAR3细胞共培养TGF-β1和IL-10分泌水平的比较注：■CD4+T细胞与OVCAR3细胞共培养;□CD4+T细胞单独培养

图2 OVCAR3细胞单独培养及与CD4+T细胞共培养MMP-2 mRNA和TIMP-2 mRNA表达的比较注：■OVCAR3细胞与CD4+T细胞共培养;□OVCAR3细胞单独培养

图3 受体阻断处理后OVCAR3细胞MMP-2 mRNA和TIMP-2 mRNA表达的变化注：（a）MMP-2 mRNA的表达;（b）TIMP-2 mRNA的表达;与对照比较,*P<0.05;与同型对照isotype IgG1 control抗体比较,#P<0.05

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