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

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10μmol/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。

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 10μmol/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.