乳腺癌细胞条件培养基对骨髓间充质干细胞生物学行为的影响

doi:10.11958/20231487

天津医药 ›› 2024, Vol. 52 ›› Issue (5): 454-458.doi: 10.11958/20231487

乳腺癌细胞条件培养基对骨髓间充质干细胞生物学行为的影响

刘丹阳¹(), 李永涛², 张海燕¹, 李林¹, 刘洋³, 沈雷²

1 齐齐哈尔医学院组织学与胚胎学教研室（邮编161006）
2 基础医学科研中心
3 齐齐哈尔市第一医院普外科

收稿日期:2023-10-11 修回日期:2024-01-04 出版日期:2024-05-15 发布日期:2024-05-09
作者简介:刘丹阳（1982），女，讲师，主要从事肿瘤微环境与肿瘤生物学方面研究。E-mail：519110362@qq.com
基金资助:
黑龙江省教育厅科学技术研究项目(2020-KYYWF-0010)

Effect of breast cancer cell conditioned medium on biological behavior of bone marrow mesenchymal stem cells

LIU Danyang¹(), LI Yongtao², ZHANG Haiyan¹, LI Lin¹, LIU Yang³, SHEN Lei²

1 Department of Histology and Embryology, Qiqihar Medical College, Qiqihar 161006, China
2 Medical Research Center, Qiqihar Medical College, Qiqihar 161006, China
3 Department of General Surgery, Qiqihar City First Hospital

Received:2023-10-11 Revised:2024-01-04 Published:2024-05-15 Online:2024-05-09

刘丹阳, 李永涛, 张海燕, 李林, 刘洋, 沈雷. 乳腺癌细胞条件培养基对骨髓间充质干细胞生物学行为的影响[J]. 天津医药, 2024, 52(5): 454-458.

LIU Danyang, LI Yongtao, ZHANG Haiyan, LI Lin, LIU Yang, SHEN Lei. Effect of breast cancer cell conditioned medium on biological behavior of bone marrow mesenchymal stem cells[J]. Tianjin Medical Journal, 2024, 52(5): 454-458.

摘要/Abstract

摘要：

目的探讨MCF-7乳腺癌细胞条件培养基对骨髓间充质干细胞（BMSC）增殖、凋亡和迁移的影响及分子机制。方法正常环境下培养的BMSC为对照组，以MCF-7细胞条件培养基培养的BMSC为MCF-7条件培养基组，向MCF-7条件培养基组添加10 nmol/L GSK690693（Akt抑制剂）为Akt抑制剂组，向MCF-7条件培养基组添加10 μmol/L Reparixin（CXCR1/2抑制剂）为CXCR1/2抑制剂组。MTT实验检测各组BMSC增殖情况，Annexin V-FITC/PI双标记流式细胞凋亡实验检测各组BMSC凋亡率，Transwell细胞迁移实验检测各组BMSC的迁移能力，酶联免疫吸附试验检测两种细胞培养上清液和MCF-7细胞条件培养基中白细胞介素（IL）-8蛋白含量，Western blot检测各组BMSC的蛋白激酶B（Akt）/磷酸化Akt（p-Akt）和哺乳动物雷帕霉素靶蛋白（mTOR）/磷酸化mTOR（p-mTOR）蛋白表达。结果与对照组相比，MCF-7条件培养基组BMSC的细胞增殖水平、迁移数目以及p-Akt和p-mTOR蛋白相对表达量均增高，细胞凋亡率降低（P<0.05）；与MCF-7条件培养基组相比，CXCR1/2抑制剂组和Akt抑制剂组BMSC的细胞增殖水平、迁移数目以及p-Akt和p-mTOR蛋白相对表达量均降低，细胞凋亡率增加（P<0.05）；MCF-7细胞条件培养基和MCF-7培养上清液中IL-8蛋白含量均较BMSC培养上清液中IL-8蛋白含量高（P<0.05）。结论 MCF-7细胞条件培养基通过激活Akt-mTOR信号通路促进BMSC增殖和迁移，抑制BMSC凋亡，其中IL-8-CXCR1/2轴发挥关键作用。

关键词: 乳腺肿瘤, 肿瘤微环境, 细胞增殖, 细胞凋亡, 细胞运动, 骨髓间充质干细胞

Abstract:

Objective To explore effects of MCF-7 breast cancer cell conditioned medium on proliferation, apoptosis and migration of bone marrow mesenchymal stem cells (BMSC) and its molecular mechanism. Methods BMSC cultured under normal environment was used as the control group. BMSC cultured with MCF-7 cell conditioned medium was selected as the MCF-7 conditioned medium group. The MCF-7 conditioned medium group supplemented with 10 nmol/L GSK690693 (Akt inhibitor) was used as the Akt inhibitor group. The MCF-7 conditioned medium group added with 10 μmol/L Reparixin (CXCR1/2 inhibitor) was used as the CXCR1/2 inhibitor group. BMSC proliferation was detected by MTT assay. Annexin V-FITC/PI double labeled flow cytometry was used to detect BMSC apoptosis rate in each group. Transwell cell migration assay was used to detect the migration ability of BMSC in each group. The contents of interleukin (IL)-8 protein in two kinds of cell culture supernatant and MCF-7 cell conditioned medium were detected by enzyme-linked immunosorbent assay. The protein expression levels of protein kinase B (Akt)/phosphorylated Akt (p-Akt) and mammalian target of rapamycin (mTOR)/phosphorylated mTOR (p-mTOR) in BMSC of each group were detected by Western blot assay. Results Compared with the control group, cell proliferation level, migration number and the relative expression levels of p-Akt, p-mTOR protein of BMSC were increased in the MCF-7 conditioned medium group, and the apoptosis rate was decreased (P<0.05). Compared with the MCF-7 conditioned medium group, cell proliferation level, migration number and the relative expression levels of p-Akt, p-mTOR protein of BMSC were decreased in the CXCR1/2 inhibitor group and the Akt inhibitor group, and the apoptosis rate was increased (P<0.05). The contents of IL-8 protein in MCF-7 cell conditioned medium and MCF-7 culture supernatant were higher than that in the BMSC culture supernatant (P<0.05). Conclusion MCF-7 cell conditioned medium promotes BMSC proliferation and migration and inhibits BMSC apoptosis by activating Akt-mTOR signaling pathway, in which IL-8-CXCR1/2 axis plays a key role.

Key words: breast neoplasms, tumor microenvironment, cell proliferation, apoptosis, cell movement, bone marrow mesenchymal stem cell

中图分类号:

R737.9

图/表 6

表1 各组BMSC实验结果的比较（n=3，$\bar{x}±s$）

Tab.1 Comparison of BMSC experimental results between the four groups

组别	OD值	凋亡率/%	迁移数目/（个/视野）
对照组	0.73±0.01	8.52±0.45	151.40±9.01
MCF-7条件培养基组	1.01±0.02^a	3.38±0.62^a	290.80±25.31^a
CXCR1/2抑制剂组	0.82±0.02^b	6.15±0.54^b	205.80±23.80^b
Akt抑制剂组	0.77±0.02^b	6.59±0.34^b	169.20±9.43^b
F	119.400^＊＊	36.030^＊＊	44.600^＊＊

图1 Annexin V-FITC/PI双标记流式细胞凋亡实验检测各组BMSC凋亡率

Fig.1 The apoptosis rate of BMSC detected by Annexin V-FITC/PI double labeled flow cytometry in each group

图2 各组BMSC细胞迁移的典型图像（结晶紫染色，标尺=50 μm）

Fig.2 Typical images of BMSC cell migration in each group (Crystal violet staining，Scale bar=50 μm)

图3 两种细胞培养上清液和MCF-7细胞条件培养基中IL-8蛋白含量比较 A：BMSC培养上清液；B：MCF-7细胞条件培养基；C：MCF-7培养上清液；n=3，a与BMSC培养上清液比较，P<0.05。

Fig.3 Comparison of IL-8 protein content between two kinds of cell culture supernatant and MCF-7 cell conditioned medium

图4 Western blot检测各组BMSC的Akt/p-Akt和mTOR/p-mTOR蛋白表达 A：对照组；B：MCF-7条件培养基组；C：Akt抑制剂组；D：CXCR1/2抑制剂组。

Fig.4 Expression levels of Akt/p-Akt and mTOR/p-mTOR protein in BMSC of each group detected by Western blot assay

表2 各组BMSC的Akt、p-Akt、mTOR、p-mTOR蛋白相对表达量比较（n=3，$\bar{x}±s$）

Tab.2 Comparison of relative expression levels of Akt, p-Akt, mTOR and p-mTOR of BMSC between the four groups

组别	Akt	p-Akt	mTOR	p-mTOR
对照组	0.85±0.02	0.54±0.04	0.84±0.03	0.50±0.09
MCF-7条件培养基组	0.91±0.02	0.89±0.01^a	0.89±0.02	0.86±0.04^a
CXCR1/2抑制剂组	0.86±0.01	0.76±0.04^b	0.86±0.03	0.60±0.02^b
Akt抑制剂组	0.89±0.06	0.49±0.02^b	0.88±0.01	0.48±0.02^b
F	1.569	88.590^＊＊	1.957	22.820^＊＊

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乳腺癌细胞条件培养基对骨髓间充质干细胞生物学行为的影响

Effect of breast cancer cell conditioned medium on biological behavior of bone marrow mesenchymal stem cells

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