ABSTRACT
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.
ABSTRACT
Objective To investigate the molecular mechanism of bone marrow mesenchymal stem cells (BM-MSCs) in repairing cisplatin-induced acute renal injury.Methods The rats were injected 6 mg/kg of cisplatin intraperitoneally,and bone marrow mesenchymal stem cells (BM-MSCs group) or PBS (PBS group) were injected respectively via tail vein after 24 hours.The rats without injecting cisplatin were selected as a normal control group.The repair effect of BM-MSCs on renal injury was observed by HE staining and immunohistochemistry.In addition,NRK-52E cells were cultured in vitro and treated with cisplatin for 6 hours.Then,NRK-52E cells were continued to culture for 48 hours or co-cultured with BM-MSCs for 48 hours,and NRK-52E cells untreated with cisplatin were used as a control.The expression levels of miR-92b and its target gene PTEN were detected by qRT-PCR,and the expression level of p-Akt by western blot.Results HE staining showed that the tubular protein casts in BM-MSCs group were significantly less than that in PBS group,and that the renal tubular structure was significantly improved in BM-MSCs group.Immunohistochemical staining indicated that the number of cells expressing proliferating cell nuclear antigen (PCNA) in BM-MSCs group (131.0 ± 14.4) was significantly higher than that in PBS group (42.2 ±6.1,t =11.28,P ＜0.01).qRT-PCR results showed that in the vivo experiment,compared with the expression level of miR-92b and PTEN in the normal control group (1.11 ± 0.78,1.01 ± 0.21),PBS group were (4.64 ± 1.06) and (0.61 ± 0.2),respectively (all P ＜ 0.05);BM-MSCs group were (2.27 ± 0.81) and (1.1 ± 0.1),respectively (all P ＜ 0.05).In vitro experiment,compared with the expression level of miR-92b and PTEN in the negative control group (1.12 ± 0.77,1.02 ± 0.13),cisplatin group were (7.64 ± 0.72) and (0.58 ± 0.2),respectively (all P ＜ 0.05),cell group were (4.38 ± 0.50) and (1.15 ± 0.23),respectively (all P ＜ 0.05).Western blot results showed that compared with the expression level of p-Akt in cisplatin group (0.96 ± 0.18),p-Akt expression in cell group was (2.11 ± 0.11,P ＜ 0.01).Conclusion BM-MSCs may repair the cisplatin-induced acute renal injury via down-regulating the expression level of miR-92b.
