选择性ER调节剂对骨肉瘤细胞增殖及CCL2表达的抑制作用研究

doi:10.3969/j.issn.1673-8640.2022.04.004

检验医学 ›› 2022, Vol. 37 ›› Issue (4): 319-324.DOI: 10.3969/j.issn.1673-8640.2022.04.004

• 新型肿瘤标志分子研究专题 • 上一篇下一篇

选择性ER调节剂对骨肉瘤细胞增殖及CCL2表达的抑制作用研究

孙小丹¹, 许静¹, 刘鷖雯², 何怡青², 杜艳², 张国良², 高锋¹, 杨翠霞¹()

1.上海交通大学附属第六人民医院检验科,上海 200233
2.上海交通大学附属第六人民医院中心实验室,上海 200233

收稿日期:2021-11-29 修回日期:2021-02-22 出版日期:2022-04-30 发布日期:2022-06-07
通讯作者: 杨翠霞
作者简介:杨翠霞,E-mail： dr.steven@163.com。
孙小丹,女,1995年生,硕士,主要从事肿瘤生长及耐药研究;
许静,女,1979年生,学士,主管技师,主要从事临床检验工作。第一联系人：
孙小丹与许静对本研究具有同等贡献,并列为第一作者。
基金资助:
国家自然科学基金资助项目(81672843);国家自然科学基金资助项目(81974445)

Inhibitory effects of antiestrogen on the proliferation and CCL2 expression of osteosarcoma cells

SUN Xiaodan¹, XU Jing¹, LIU Yiwen², HE Yiqing², DU Yan², ZHANG Guoliang², GAO Feng¹, YANG Cuixia¹()

1. Department of Clinical Laboratory,Shanghai Jiao Tong University Affiliated Sixth People's Hospital,Shanghai 200233,China
2. Central Laboratory,Shanghai Jiao Tong University Affiliated Sixth People's Hospital,Shanghai 200233,China

Received:2021-11-29 Revised:2021-02-22 Online:2022-04-30 Published:2022-06-07
Contact: YANG Cuixia

摘要/Abstract

摘要：

目的探讨选择性ER调节剂他莫昔芬对骨肉瘤细胞增殖及CC趋化因子配体（CCL）2表达的影响。方法检测骨肉瘤细胞（143B和U-2OS）及成骨细胞（hFOB1.19）中雌激素受体（ER）的表达量。采用细胞增殖实验和平板克隆形成实验观察他莫昔芬对骨肉瘤细胞增殖的影响。采用荧光定量聚合酶链反应（PCR）检测骨肉瘤细胞中炎症因子IL-6、IL-8、TGF-β、CCL2和CCL5基因的相对表达量。采用酶联免疫吸附试验（ELISA）检测他莫昔芬对骨肉瘤细胞CCL2表达的影响。结果 2种骨肉瘤细胞（143B和U-2OS）的ER mRNA相对表达量均显著低于成骨细胞（hFOB1.19）;他莫昔芬可显著抑制骨肉瘤细胞的增殖及炎症因子IL-6、IL-8、TGF-β、CCL2和CCL5基因的表达,其中对CCL2表达的抑制作用最为显著。结论他莫昔芬可抑制骨肉瘤细胞的增殖及CCL2的表达,提示其或可作为骨肉瘤的辅助化疗药物。

关键词: 雌激素受体, CC趋化因子配体2, 他莫昔芬, 骨肉瘤

Abstract:

Objective To investigate the effects of antiestrogen（tamoxifen）on the proliferation and the expression level of CC chemokine ligand （CCL）2 of osteosarcoma cells. Methods The difference of estrogen receptor（ER） expression between osteosarcoma cells（143B,U-2OS） and osteoblast（hFOB1.19） was observed. CCK8 experiment and plate colony formation assay were used to observe the effects of tamoxifen on the proliferation of osteosarcoma cells. The relative expression levels of inflammatory factors,IL-6,IL-8,TGF-β,CCL2 and CCL5 in osteosarcoma cells were determined by fluorescence quantitative polymerase chain reaction（PCR）. The effects of tamoxifen on CCL2 expression of osteosarcoma cells in cell culture medium were determined by enzyme-linked immunosorbent assay（ELISA）. Results The expression level of ER mRNA in osteosarcoma cells（143B,U-2OS） was lower than that in osteoblast（hFOB1.19）. Tamoxifen could inhibit the proliferation of osteosarcoma cells. Tamoxifen could inhibit the expressions of inflammatory factors,IL-6,IL-8,TGF-β,CCL2 and CCL5,especially the chemokine CCL2 in osteosarcoma cells. Conclusions Tamoxifen could inhibit the proliferation of osteosarcoma cells and the expression of CCL2,suggesting that it might be used as a adjuvant drug for postoperative chemotherapy of osteosarcoma.

Key words: Estrogen receptor, CC chemokine ligand 2, Tamoxifen, Osteosarcoma

中图分类号:

R446.62

孙小丹, 许静, 刘鷖雯, 何怡青, 杜艳, 张国良, 高锋, 杨翠霞. 选择性ER调节剂对骨肉瘤细胞增殖及CCL2表达的抑制作用研究[J]. 检验医学, 2022, 37(4): 319-324.

SUN Xiaodan, XU Jing, LIU Yiwen, HE Yiqing, DU Yan, ZHANG Guoliang, GAO Feng, YANG Cuixia. Inhibitory effects of antiestrogen on the proliferation and CCL2 expression of osteosarcoma cells[J]. Laboratory Medicine, 2022, 37(4): 319-324.

图/表 5

表1 引物序列

基因	正向引物（5'~3'）	反向引物（5'~3'）
ERα	TACTGCATCAGATCCAAGGGAA	CCTCGGGGTAGTTGTACAC
IL-6	AATAACCACCCCTGACCCAAC	ACATTTGCCGAAGAGCCCT
IL-8	AAACCACCGGAAGGAACCAT	CCTTCACACAGAGCTGCAGAAA T
TGF-β	AAGGACCTCGGCTGGAAGTGC	CCGGGTTATGCTGGTTGTA
CXCL1	AAGAACATCCAAAGTGTGAACG	CACTGTTCAGCATCTTTTCGAT
CCL2	AAGATCTCAGTGCAGAGGCTCG	CACAGATCTCCTTGGCCACAA
CCL5	ATGACTCCCGGCTGAACAAG	CGGGTGGGGTAGGATAGTGA
GAPDH	AACGGATTTGGTCGTATTGGG	TCGCTCCTGGAAGA TGGTGAT

图1 骨肉瘤细胞（143B和U-2OS）和成骨细胞（hFOB1.19）ER mRNA表达比较注：与hFOB1.19细胞比较,*P<0.05。

图2 选择性ER调节剂他莫昔芬对骨肉瘤细胞增殖的影响注：（a）CCK-8增殖实验;与DMSO组比较,*P<0.05;（b）平板克隆实验;（c）不同组平板克隆实验细胞数的比较;与DMSO组比较,*P<0.05;（d）骨肉瘤细胞（143B和U-2OS）pAkt表达的免疫印迹图;（e）骨肉瘤细胞（143B和U-2OS）ERK和pERK表达的免疫印迹图。

图3 选择性ER调节剂对骨肉瘤细胞143B炎症因子表达的抑制作用注：（a）IL-6;（b）IL-8;（c）TGF-β;（d）CXCL1;（e）CCL2;（f）CCL5;与DMSO组比较,*P<0.05。

图4 DMSO、E2和他莫昔芬对骨肉瘤细胞（143B和U-2OS）CCL2分泌的影响注：（a）DMSO、E2和他莫昔芬对143B细胞CCL2分泌的影响;（b）DMSO、E2和他莫昔芬对U-2OS细胞CCL2分泌的影响;与DMSO组比较,*P<0.05。

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选择性ER调节剂对骨肉瘤细胞增殖及CCL2表达的抑制作用研究

Inhibitory effects of antiestrogen on the proliferation and CCL2 expression of osteosarcoma cells

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