国际肿瘤学杂志

国际肿瘤学杂志 ›› 2021, Vol. 48 ›› Issue (5): 275-281.doi: 10.3760/cma.j.cn371439-20210115-00053

• 论著 • 上一篇    下一篇

TGF-β诱导肺脏癌相关成纤维细胞高表达IL-17D并促进MDSC募集

申加兴, 张珊, 陈祥静, 王丽, 孙晓艳, 吕衍民, 宋冠华, 姚成芳()   

  1. 山东第一医科大学基础医学院 山东省医学科学院基础医学研究所,济南 250062
  • 收稿日期:2021-01-15 修回日期:2021-03-05 出版日期:2021-05-08 发布日期:2021-06-09
  • 通讯作者: 姚成芳 E-mail:yaocf9941@163.com
  • 基金资助:
    国家自然科学基金(82074088);国家自然科学基金(81573728)

TGF-β induces high expression of IL-17D in lung cancer-associated fibroblast and promotes recruitment of MDSC

Shen Jiaxing, Zhang Shan, Chen Xiangjing, Wang Li, Sun Xiaoyan, Lyu Yanmin, Song Guanhua, Yao Chengfang()   

  1. Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan 250062, China
  • Received:2021-01-15 Revised:2021-03-05 Online:2021-05-08 Published:2021-06-09
  • Contact: Yao Chengfang E-mail:yaocf9941@163.com
  • Supported by:
    National Natural Science Foundation of China(82074088);National Natural Science Foundation of China(81573728)

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摘要：

目的 探讨TGF-β诱导肺脏癌相关成纤维细胞(CAF)表达IL-17D,并促进骨髓来源的抑制性细胞(MDSC)募集的关键机制。方法 采用C57BL/6小鼠建立B16黑色素瘤细胞肺癌转移模型(肿瘤模型组),另设对照组,每组6只。应用流式细胞术(FACS)检测肿瘤小鼠肺脏CAF及其分泌IL-17D能力和MDSC比例的变化;采用ELISA和RT-qPCR检测肺肿瘤TGF-β水平的改变;FACS分选肺脏成纤维细胞,采用RT-PCR技术检测TGF-β对成纤维细胞分泌IL-17D、CCL2和CCL7的影响;Transwell检测TGF-β处理的肺脏成纤维细胞对MDSC的趋化作用。结果 肿瘤模型组肺脏CAF(CD45-CD326-CD31-)比例显著高于对照组[(28.02±2.23)% vs. (7.35±2.14)%,t=9.956,P<0.001]。肿瘤模型组中CAF分泌IL-17D的能力较对照组显著升高[(38.27±2.93)% vs. (19.04±3.16)%,t=5.995,P=0.001];肿瘤模型组肺脏MDSC比例明显高于对照组[(12.93±1.27)% vs. (8.21±1.40)%,t=4.804,P=0.009]。与对照组相比,肿瘤模型组肺脏TGF-β蛋白水平和转录水平均明显升高[(1 685.07±135.61)ng/L vs. (1 047.98±68.50)ng/L,t=5.051,P=0.002;2.17±0.03 vs. 1.00±0.05,t=51.237,P<0.001]。肺脏成纤维细胞经不同浓度TGF-β(0、5、10 μg/L)分别处理24 h后,其IL-17D mRNA相对表达量分别为0.42±0.01、0.67±0.01、0.84±0.04,CCL2 mRNA相对表达量分别为0.89±0.08、1.08±0.04、1.19±0.01,CCL7 mRNA相对表达量分别为0.53±0.05、0.65±0.04、0.74±0.03,随着TGF-β浓度升高,成纤维细胞IL-17D、CCL2、CCL7表达水平明显升高(F=57.384,P<0.001;F=15.802,P=0.004;F=14.544,P=0.005)。另外,与对照组相比(TGF-β为0 μg/L),肺脏成纤维细胞经10 μg/L TGF-β处理24 h后,可促进肿瘤小鼠脾脏MDSC的迁移[(9.59±0.21)% vs. (2.14±0.24)%,t=6.585,P<0.001]。结论 肿瘤微环境中TGF-β诱导肺脏CAF高表达IL-17D,是促进CCL2、CCL7表达和MDSC迁移的重要因素。

关键词: 肿瘤微环境, 转化生长因子β, 癌相关成纤维细胞, 白细胞介素-17D, 骨髓来源的抑制性细胞

Abstract:

Objective To investigate the key mechanism of transforming growth factor-β (TGF-β) inducing the expression of interleukin-17D (IL-17D) in lung cancer-associated fibroblast (CAF) and promoting the recruitment of myeloid-derived suppressor cells (MDSCs). Methods C57BL/6 mice were established for B16 lung melanoma metastasis model (tumor model group), and control group was set up, 6 mice in each group. Flow cytometry (FACS) was used to detect the lung CAF and the changes of its ability to secrete IL-17D and the proportion of MDSCs in tumor mice. The changes of TGF-β level in lung tumor were examined by ELISA and quantitative real-time PCR (RT-qPCR). Lung fibroblasts were screened by FACS, and the effects of TGF-β on the secretion of IL-17D, C-C motif chemokine ligand (CCL)2 and CCL7 in fibroblasts were detected by RT-PCR. The migration of MDSCs under the condition of TGF-β stimulating fibroblasts was detected by Transwell. Results The proportion of CAF (CD45-CD326-CD31-) in the tumor model group was higher than that in the control group [(28.02±2.23)% vs. (7.35±2.14)%, t=9.956, P<0.001]. The ability of CAF to secrete IL-17D in the tumor model group was significantly higher than that in the control group [(38.27±2.93)% vs. (19.04±3.16)%, t=5.995, P=0.001]. The proportion of MDSCs in the tumor model group was significantly higher than that in the control group [(12.93±1.27)% vs. (8.21±1.40)%, t=4.804, P=0.009]. Compared with the control group, the protein and transcription levels of TGF-β in lung of the tumor model group were significantly increased [(1 685.07±135.61) ng/L vs. (1 047.98±68.50) ng/L, t=5.051, P=0.002; 2.17±0.03 vs. 1.00±0.05, t=51.237, P<0.001]. In vitro, lung fibroblasts were stimulated with different concentrations of TGF-β (0, 5 and 10 μg/L) for 24 hours, the relative expressions of IL-17D mRNA secreted by stimulated fibroblasts were 0.42±0.01, 0.67±0.01 and 0.84±0.04 respectively, the relative expressions of CCL2 mRNA in each group were 0.89±0.08, 1.08±0.04, 1.19±0.01 and CCL7 were 0.53±0.05, 0.65±0.04, 0.74±0.03 respectively. With the increase of TGF-β concentration, the expression levels of IL-17D, CCL2 and CCL7 in fibroblasts were significantly increased (F=57.384, P<0.001; F=15.802, P=0.004; F=14.544, P=0.005). In addition, compared with the control group (0 μg/L TGF-β), fibroblasts treated with 10 μg/L TGF-β for 24 hours could promote the migration of MDSCs in spleen of tumor mice [(9.59±0.21)% vs. (2.14±0.24)%, t=6.585, P<0.001]. Conclusion TGF-β can induce high expression of IL-17D in lung CAF, which is an important factor in promoting the expressions of CCL2 and CCL7 and the migration of MDSCs in tumor microenvironment.

Key words: Tumor microenvironment, Transforming growth factor beta, Cancer-associated fibroblasts, Interleukin-17D, Myeloid-derived suppressor cells