Abstract
Objective To investigate the effects of bilirubin on human bone marrow mesenchymal stem cell (BMMSC) and to provide the evidence for selecting patients with liver function failure suitable for BMMSC transplantation therapy. Methods Bilirubin of different concentration (0, 10, 20, 30, 40 and 50 μmol/L) was separately added into human BMMSC cell culture medium. The cell proliferation was detected by cell counting kit(CCK)-8 assay. The cell apoptosis rate was observed by Annexin-Ⅴ/ propidium iodide(PI)labeling and flow cytometry. Pro-apoptosis protein of B-cell lymphoma-2(Bcl-2)family and caspase-3 was measured by Western blotting. And the change of mitochondrial membrane potential after bilirubin treatment was determined by JC-1 staining. Student′s t test was performed for comparison between two groups. Spearman correlation analysis was also used. Results After treated with higher concentration of bilirubin (30, 40 and 50 μmol/L), the proliferation of BMMSC showed down trend compared with that of BMMSC treated with lower concentration of bilirubin (10 and 20 μmol/L), but the differences at the same time period in cell proliferation were not statistically significant among groups with different bilirubin concentration (all P>0.05). Treated by bulirubin 0, 10, 20, 30, 40 and 50 μmol/L for 24 hours, the apoptosis rates of BMMSC were (3.20±1.20)%, (16.80±2.70)%, (19.20±4.20)%, (22.60±2.80)%, (28.40±5.20)% and (34.20±3.50)%, respectively. The cell apoptosis rate was positively correlated with bilirubin concentration (r=0.682, P=0.032). The expression of Bad, Bak, Bax, BID, Bik and Bim, caspase-3 at protein level increased as the concentration of bilirubin increased. The results of JC-1 mitochondrial membrane potential staining showed that along with the concentration of bilirubin increasing, cell fluorescent changed from red to green, which indicated the decrease of mitochondrial membrane potential. Conclusions Bilirubin could induce BMMSC apoptosis by mitochondria depend Bcl-2 and caspase-3 pathway. The level of bilirubin might be one of the parameters in judging patients with liver function failure suitable for BMMSC transplantation therapy. Key words: Bilirubin; BMSCs; Apoptosis; Bcl-2; Caspase-3; Mitochondria
Published Version
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