乳腺癌细胞培养上清液促进人脂肪间充质干细胞迁移和增殖的机制探讨

doi:10.11958/20230255

天津医药 ›› 2023, Vol. 51 ›› Issue (11): 1153-1157.doi: 10.11958/20230255

• 细胞与分子生物学 • 下一篇

乳腺癌细胞培养上清液促进人脂肪间充质干细胞迁移和增殖的机制探讨

刘丹阳¹(), 王璐璐², 何军², 姜杨², 李永涛², 张晓东², 李鹏辉², 沈雷²^,^△()

1.齐齐哈尔医学院组织胚胎学教研室（邮编161006）
2.齐齐哈尔医学院基础医学科研中心（邮编161006）

收稿日期:2023-02-27 修回日期:2023-06-02 出版日期:2023-11-15 发布日期:2023-11-07
通讯作者: ^△E-mail：shenlei815@qmu.edu.cn
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资助(2020-KYYWF-0010);齐齐哈尔市科技计划联合引导项目(LHYD-2021002)

The mechanism of breast cancer cell culture supernatant promoting migration and proliferation of human adipose mesenchymal stem cells

LIU Danyang¹(), WANG Lulu², HE Jun², JIANG Yang², LI Yongtao², ZHANG Xiaodong², LI Penghui², SHEN Lei²^,^△()

1. Department of Histology and Embryology, Qiqihar Medical College, Qiqihar 161006, China
2. Medical Research Center, Qiqihar Medical College, Qiqihar 161006, China

Received:2023-02-27 Revised:2023-06-02 Published:2023-11-15 Online:2023-11-07
Contact: ^△E-mail：shenlei815@qmu.edu.cn

刘丹阳, 王璐璐, 何军, 姜杨, 李永涛, 张晓东, 李鹏辉, 沈雷. 乳腺癌细胞培养上清液促进人脂肪间充质干细胞迁移和增殖的机制探讨[J]. 天津医药, 2023, 51(11): 1153-1157.

LIU Danyang, WANG Lulu, HE Jun, JIANG Yang, LI Yongtao, ZHANG Xiaodong, LI Penghui, SHEN Lei. The mechanism of breast cancer cell culture supernatant promoting migration and proliferation of human adipose mesenchymal stem cells[J]. Tianjin Medical Journal, 2023, 51(11): 1153-1157.

摘要/Abstract

摘要：

目的探讨MDA-MB-231乳腺癌细胞培养上清液对人脂肪间充质干细胞（hAdMSC）迁移、增殖和凋亡的影响及分子机制。方法将MDA-MB-231细胞上清液和不含胎牛血清的RPMI-1640培养基以1∶4的体积比混匀后培养的hAdMSC为MDA-MB-231上清液组。向MDA-MB-231上清液组添加10 μmol/L Reparixin（CXCR1/2抑制剂）为CXCR1/2抑制剂组；向MDA-MB-231上清液组添加10 nmol/L GSK690693（Akt抑制剂）为Akt抑制剂组。无任何刺激进行培养的hAdMSC为对照组。细胞划痕实验检测各组hAdMSC的迁移能力，CCK-8实验检测各组hAdMSC增殖情况，Annexin V-FITC/PI双标记流式细胞凋亡实验检测各组hAdMSC凋亡率，Western blot检测各组hAdMSC的mTOR/磷酸化mTOR（p-mTOR）和Akt/磷酸化Akt（p-Akt）蛋白表达。结果与对照组相比，MDA-MB-231上清液组hAdMSC的24 h和48 h细胞划痕闭合面积、细胞增殖水平以及p-Akt和p-mTOR蛋白相对表达量均增加，细胞凋亡率降低（P<0.05）；与MDA-MB-231上清液组相比，Akt抑制剂组和CXCR1/2抑制剂组hAdMSC的48 h细胞划痕闭合面积、细胞增殖水平以及p-Akt和p-mTOR蛋白相对表达量均降低，细胞凋亡率均增加（P<0.05）。结论 MDA-MB-231乳腺癌细胞培养上清液通过激活Akt-mTOR信号通路促进hAdMSC迁移和增殖，抑制hAdMSC凋亡，其中IL-8-CXCR1/2轴发挥关键作用。

关键词: 乳腺肿瘤, 细胞迁移, 细胞增殖, 肿瘤微环境, 脂肪间充质干细胞

Abstract:

Objective To explore the effect of MDA-MB-231 breast cancer cell culture supernatant on migration, proliferation and apoptosis of human adipose mesenchymal stem cell (hAdMSC) and its molecular mechanism. Methods hAdMSC cultured from MDA-MB-231 cell supernatant and RPMI-1640 medium without fetal bovine serum were mixed at a volume ratio of 1∶4 to form the MDA-MB-231 supernatant group. CXCR1/2 inhibitor group was added with 10 μmol/L Reparixin (CXCR1/2 inhibitor) to the MDA-MB-231 supernatant group. The Akt inhibitor group was added with 10 nmol/L GSK690693 (Akt inhibitor) to the MDA-MB-231 supernatant group. hAdMSC cultured without any stimulation was used as the control group. The migration ability of hAdMSC in each group was detected by cell scratch assay. hAdMSC proliferation was detected by CCK-8 assay. Annexin V-FITC/PI double labeled flow cytometry was used to detect hAdMSC apoptosis rate in each group. The protein expression levels of mTOR/phosphorylated mTOR (p-mTOR) and Akt/phosphorylated Akt (p-Akt) in hAdMSC of each group were detected by Western blot assay. Results Compared with the control group, the 24 h and 48 h scratch closure area, cell proliferation level and relative expression levels of p-Akt, p-mTOR protein of hAdMSC were increased in the MDA-MB-231 supernatant group, and the apoptosis rate was decreased (P<0.05). Compared with the MDA-MB-231 supernatant group, the 48 h cell scratch closure area, cell proliferation level and relative expression levels of p-Akt, p-mTOR proteins of hAdMSC were decreased in the Akt inhibitor group and the CXCR1/2 inhibitor group, and the apoptosis rate was increased (P<0.05). Conclusion MDA-MB-231 breast cancer cell culture supernatant promotes hAdMSC migration and proliferation and inhibits hAdMSC apoptosis by activating Akt-mTOR signaling pathway, in which IL-8-CXCR1/2 axis plays a key role.

Key words: breast neoplasms, cell migration, cell proliferation, tumor microenvironment, adipose derived mesenchymal stem cells

中图分类号:

R737.9

图/表 7

表1 各组hAdMSC的细胞划痕闭合面积比较（n=3，×104 μm2，$\bar{x}±s$）

Tab.1 Comparison of cell scratch closure area of hAdMSC between the four groups

组别	24 h	48 h
对照组	6.57±1.05	20.53±0.69
MDA-MB-231上清液组	39.54±1.98^a	61.19±0.21^a
Akt抑制剂组	35.93±0.48	43.00±0.26^b
CXCR1/2抑制剂组	37.68±0.95	45.23±0.32^b
F	318.300^＊＊	3 240.000^＊＊

图1 各组hAdMSC细胞划痕的典型图像（标尺=100 μm）

Fig.1 Typical images of hAdMSC cell scratches in each group （Scale bar=100 μm）

图2 各组hAdMSC的A450比较柱状图 A：对照组；B：MDA-MB-231上清液组；C：Akt抑制剂组；D：CXCR1/2抑制剂组；n=3，a与对照组比较，b与MDA-MB-231上清液组比较，P<0.05。

Fig.2 Histogram of hAdMSC A450 in each group

图3 各组hAdMSC凋亡率比较的柱状图 A：对照组；B：MDA-MB-231上清液组；C：Akt抑制剂组；D：CXCR1/2抑制剂组；n=3，a与对照组比较，b与MDA-MB-231上清液组比较，P<0.05。

Fig.3 Histogram of hAdMSC apoptosis rate in each group

图4 Annexin V-FITC/PI双标记流式细胞凋亡实验检测各组hAdMSC凋亡率的典型图像

Fig.4 Typical images of hAdMSC apoptosis rate detected by Annexin V-FITC/PI double labeled flow cytometry

图5 MDA-MB-231上清液促进hAdMSC表达p-Akt和p-mTOR蛋白 A：对照组；B：MDA-MB-231上清液组；C：Akt抑制剂组；D：CXCR1/2抑制剂组。

Fig.5 MDA-MB-231 supernatant promoted expression levels of p-Akt and p-mTOR proteins in hAdMSC

表2 各组hAdMSC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 the relative expression levels of Akt, p-Akt, mTOR and p-mTOR hAdMSC between the four groups

组别	Akt	p-Akt	mTOR	p-mTOR
对照组	1.11±0.06	0.43±0.02	1.06±0.09	0.59±0.05
MDA-MB-231 上清液组	1.12±0.01	1.03±0.03^a	1.10±0.04	0.94±0.02^a
Akt抑制剂组	1.02±0.05	0.48±0.03^b	1.03±0.02	0.71±0.03^b
CXCR1/2抑制剂组	1.09±0.01	0.77±0.04^b	0.95±0.03	0.73±0.02^b
F	2.622	144.000^＊＊	2.890	86.400^＊＊

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乳腺癌细胞培养上清液促进人脂肪间充质干细胞迁移和增殖的机制探讨

The mechanism of breast cancer cell culture supernatant promoting migration and proliferation of human adipose mesenchymal stem cells

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