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中华普通外科学文献(电子版) ›› 2021, Vol. 15 ›› Issue (03) : 172 -177. doi: 10.3877/cma.j.issn.1674-0793.2021.03.003

所属专题: 文献

论著

结肠癌来源外泌体对自然杀伤细胞miR-18a和NKG2D表达的影响
钟勇辉1,(), 谢福川2, 魏宜胜3   
  1. 1. 516000 惠州市中心人民医院普外科
    2. 516000 惠州市中心人民医院肿瘤放疗科
    3. 510260 广州医科大学附属第二医院胃肠外科
  • 收稿日期:2020-12-30 出版日期:2021-06-01
  • 通信作者: 钟勇辉
  • 基金资助:
    国家自然科学基金资助项目(81672436); 惠州市科技计划项目(2016Y010)

Effect of colon cancer derived exosomes on the expression of miR-18a and NKG2D in natural killer cells

Yonghui Zhong1,(), Fuchuan Xie2, Yisheng Wei3   

  1. 1. Department of General Surgery, Huizhou Central People’s Hospital, Huizhou 516000, China
    2. Department of Tumor Radiotherapy, Huizhou Central People’s Hospital, Huizhou 516000, China
    3. Department of Gastrointestinal Surgery, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China
  • Received:2020-12-30 Published:2021-06-01
  • Corresponding author: Yonghui Zhong
引用本文:

钟勇辉, 谢福川, 魏宜胜. 结肠癌来源外泌体对自然杀伤细胞miR-18a和NKG2D表达的影响[J/OL]. 中华普通外科学文献(电子版), 2021, 15(03): 172-177.

Yonghui Zhong, Fuchuan Xie, Yisheng Wei. Effect of colon cancer derived exosomes on the expression of miR-18a and NKG2D in natural killer cells[J/OL]. Chinese Archives of General Surgery(Electronic Edition), 2021, 15(03): 172-177.

目的

基于miR-18a/NKG2D探究结肠癌来源外泌体(CDEs)对自然杀伤(NK)细胞杀伤作用的影响。

方法

取人结肠癌SW480细胞培养后收集CDEs并进行鉴定;从健康志愿者外周血中分离NK细胞并采用IL-2刺激其增殖,流式细胞仪进行鉴定;将CDEs与NK细胞共培养后采用CCK-8法检测NK细胞活力;流式细胞术分析CD107a表达,酶联免疫吸附法(ELISA)检测肿瘤坏死因子α(TNF-α)和干扰素γ(IFN-γ)的表达,评估NK细胞对SW480细胞的杀伤功能;流式细胞术测定NK细胞表面NKG2D受体表达水平;实时荧光定量PCR(qPCR)检测NK细胞中miR-18a的表达。

结果

电子透射显微镜下CDEs呈圆盘状、双层膜囊泡状结构,Western blotting可检测到外泌体阳性标志蛋白CD63、CD81的表达;IL-2刺激后CD3-、CD56+的NK细胞比例显著增加(P<0.05);CDEs并未明显影响NK细胞生长活力(P=0.999),与阴性对照组相比,经CDEs干预的NK细胞CD107a表达、TNF-α、IFN-γ、NKG2D受体表达水平明显降低(P<0.05),miR-18a表达明显增加(P<0.05),且呈一定的浓度依赖性。

结论

CDEs可能通过上调miR-18a的表达、下调NKG2D表达,抑制NK细胞的杀伤作用来介导结肠癌细胞的免疫逃逸。

Objective

To explore the effect of colon cancer derived exosomes (CDEs) on the killing effect of natural killer (NK) cells based on miR-18a/NKG2D.

Methods

Human colon cancer SW480 cells were cultured, and CDEs were collected and identified. NK cells were isolated from peripheral blood of healthy volunteers and stimulated by IL-2, then the proliferation was identified by flow cytometry. After CDEs were co-cultured with NK cells, the NK cell viability was detected by CCK-8 method. The expression of CD107a was analyzed by flow cytometry, the expression of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) was detected by enzyme-linked immunosorbent assay (ELISA), the cytotoxicity of NK cells to SW480 cells was evaluated. The expression level of NKG2D receptor on NK cells was measured by flow cytometry and miR-18a in NK cells by quantitative PCR (qPCR).

Results

CDEs were disk-shaped and double membranous vesicular structure under electron transmission microscope. Western blotting could detect the expression of positive marker protein CD63 and CD81 in exosome. After IL-2 stimulation, the proportion of CD3-, CD56+ NK cells increased significantly (P<0.05); CDEs did not significantly affect the growth activity of NK cells (P=0.999). Compared with the negative control group, the expression levels of CD107a, TNF-α, IFN-γ and NKG2D receptor in NK cells intervened by CDEs were significantly decreased (P<0.05), the expression of miR-18a was significantly increased (P<0.05), in a concentration dependent manner.

Conclusion

CDEs may mediate the immune escape of colon cancer cells byup-regulating the expression of miR-18a, down-regulating the expression of NKG2D and inhibiting the killing effect of NK cells.

表1 qPCR引物序列
图1 结肠癌SW480细胞来源的外泌体鉴定 A为透射电子显微镜观察结果(×50 000);B为Western blotting检测结果
图2 自然杀伤细胞的鉴定
表2 CDEs对NK细胞活力的影响(±s,%,n=3)
图3 流式细胞术检测NC组和不同浓度CDEs组自然杀伤细胞CD107a的表达情况
表3 CDEs对NK细胞TNF-α、IFN-γ水平的影响(±s,ng/L,n=3)
图4 流式细胞术检测自然杀伤细胞表面NKG2D受体表达
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