Objective: To investigate the effect of mesenchymal stem cells (MSCs) on apoptosis of breast cancer cell line MCF-7 induced by cisplatin (DDP), MSCs derived from breast cancer (BC-MSCs) or adjacent non-cancerous tissues (BN-MSCs) were isolated, cultured and identified. Methods: BC-MSCs and BN-MSCs were isolated and cultured by tissue adherent method. The differentiation potential of BC-MSCs was detected by osteogenic and adipogenic induction, and cell surface markers of BC-MSCs and BN-MSCs were evaluated by flow cytometry. MCF-7 cells were co-treated with DDP and conditioned medium (CM) collected from BC-MSCs and BN-MSCs after being cultured for 48 hours, respectively. Inhibition rate of cell proliferation was evaluated by MTT. Cell apoptosis and viability were detected by MUSE cell analyzer. Cytokines in MSC-CM were detected by Luminex liquid chip. Interleukin 6 (IL-6) mRNA expressions in MCF-7 cells with different treatment were detected by RT-PCR. Results: The morphology of BC-MSCs and BN-MSCs successfully isolated and cultured was uniform fibroblast-like clusters under the microscope. These cells expressed high levels of CD29 and CD44, but neither CD14 nor CD34 were detected. MSCs could also differentiate into osteoblasts and adipocytes after specific induction. After treatment with 2.5, 5, 10, 20, 40 and 80 μmol/L DDP, the inhibitory rates of proliferation of MCF-7 cells in DDP group were (17.33±2.00)%, (22.37±0.73)%, (30.77±1.23)%, (44.93±1.27)%, (62.03 ±1.97)% and (73.93±1.10)%, respectively. While the inhibitory rates of DDP+ BC-MSCs group were (8.27±0.63)%, (11.50±1.30)%, (20.57±0.93)%, (32.60 ±1.90)%, (52.27±0.73)% and (62.13±2.17)%, respectively. The inhibitory rates of DDP+ BN-MSCs group were (12.90±1.60)%, (16.53±2.87)%, (25.90±1.50)%, (39.40±2.40)%, (57.40±0.70)% and (69.03±1.07)%, respectively. The inhibitory rates of DDP+ BC-MSCs group were significantly lower than those of DDP group (P<0.05). The apoptotic rates of MCF-7 cells in DDP group, DDP+ BC-MSCs group and DDP+ BN-MSCs group were (47.77±1.98)%, (29.20±2.12)% and (37.92±2.21)%, respectively. The apoptotic rates of DDP group was significantly higher than that of DDP+ BC-MSCs group (P<0.05). The cell viabilities of MCF-7 in DDP group, DDP+ BC-MSCs group and DDP+ BN-MSCs group were 0.52±0.02, 0.72±0.02 and 0.64±0.02, respectively. The cell viability of DDP group was significantly lower than that of DDP+ BC-MSCs group (P<0.05). The result of Luminex liquid chip analysis showed that, the level of IL-6 in BC-MSCs group increased 2.50±0.68 fold when compared with BN-MSCs group (P<0.05). The relative expressions of IL-6 mRNA in DDP group and DDP+ BC-MSCs group were 1.02±0.10 and 7.58±0.55, respectively, with a statistically significant difference (P<0.01). The apoptotic rates of MCF-7 cells in DDP+ BC-MSCs group with or without IL-6 neutralizing antibody were (27.41±1.95)% and (42.45±2.87)%, respectively, with a statistically significant difference (P<0.05). The cell viabilities of MCF-7 cells in DDP+ BC-MSCs group with or without IL-6 neutralizing antibody were (72.40±2.60)% and (59.76±3.89)%, respectively, with a statistically significant difference (P<0.05). Conclusions: BC-MSCs and BN-MSCs have been isolated and cultured successfully. Compared with BN-MSCs, BC-MSCs could attenuate the effect of DDP on MCF-7 cells, evidently decrease the apoptosis and increase the proliferation and vitality in an IL-6 dependent manner.
目的： 分离、培养、鉴定乳腺癌来源间质干细胞(BC－MSCs)及癌旁组织来源间质干细胞(BN－MSCs)，探讨其对顺铂(DDP)诱导的人乳腺癌MCF－7细胞凋亡的影响。 方法： 采用组织贴壁法分离培养BC－MSCs和BN－MSCs，成骨、成脂诱导法检测BC－MSCs的分化能力，流式细胞术检测BC－MSCs和BN－MSCs的表面标记。收集BC－MSCs和BN－MSCs 48 h培养上清液，分别与DDP共同处理MCF－7细胞，四甲基偶氮唑蓝(MTT)实验检测不同处理组MCF－7细胞增殖抑制率，Muse全能细胞分析仪检测MCF－7细胞凋亡和活力情况，Luminex液态芯片技术检测MSCs培养上清中白细胞介素6(IL－6)水平，逆转录聚合酶链反应(RT－PCR)检测不同处理组MCF－7细胞中IL－6 mRNA的表达水平。 结果： 成功分离培养出BC－MSCs和BN－MSCs,镜下呈纤维样长梭形，高表达CD29、CD44，不表达CD14、CD34，诱导后可分化为脂肪细胞和成骨细胞。2.5、5、10、20、40、80 μmol/L DDP作用MCF－7细胞48 h后，DDP组的细胞抑制率分别为(17.33±2.00)%、(22.37±0.73)%、(30.77±1.23)%、(44.93±1.27)%、(62.03±1.97)%和(73.93±1.10)%, DDP＋BC－MSCs组的细胞抑制率分别为(8.27±0.63)%、(11.50±1.30)%、(20.57±0.93)%、(32.60±1.90)%、(52.27±0.73)%和(62.13±2.17)%, DDP＋BN－MSCs组的细胞抑制率分别为(12.90±1.60)%、(16.53±2.87)%、(25.90±1.50)%、(39.40±2.40)%、(57.40±0.70)%和(69.03±1.07)%, DDP＋BC－MSCs组的细胞抑制率明显低于DDP组，差异有统计学意义(P<0.05)。DDP组、DDP＋BC－MSCs组和DDP＋BN－MSCs组的细胞凋亡率分别为(47.77±1.98)%、(29.20±2.12)%和(37.92±2.21)%，DDP＋BC－MSCs组与DDP组差异有统计学意义(P<0.05)。DDP组、DDP＋BC－MSCs组和DDP＋BN－MSCs组的细胞活力比值分别为0.52±0.02、0.72±0.02和0.64±0.02，DDP＋BC－MSCs组与DDP组差异有统计学意义(P<0.05)。Luminex液态芯片分析结果显示，BC－MSCs组的IL－6水平是BN－MSCs组的(2.50±0.68)倍，差异有统计学意义(P<0.05)。DDP组和DDP＋BC－MSCs组的IL－6 mRNA相对表达量分别为1.02±0.10和7.58±0.55，差异有统计学意义(P<0.01)。DDP＋BC－MSCs组和DDP＋BC－MSCs＋IL－6中和抗体组的细胞凋亡率分别为(27.41±1.95)%和(42.45±2.87)%，差异有统计学意义(P<0.05)。DDP＋BC－MSCs组和DDP＋BC－MSCs＋IL－6中和抗体组的细胞活力分别为(72.40± 2.60)%和(59.76±3.89)%，差异有统计学意义(P<0.05)。 结论： 成功地分离、培养出BC－MSCs和BN－MSCs。与BN－MSCs比较，BC－MSCs能够改善DDP对MCF－7细胞的作用，显著减少DDP诱导MCF－7细胞的凋亡，促进增殖与活力，且与其分泌的IL－6有关。.
Keywords: Apoptosis; Breast neoplasms; Cisplatin; Interleukin-6; Mesenchymal stem cells.