肝细胞损伤条件培养基对大鼠骨髓间充质干细胞肝向分化的促进作用

doi:10.13481/j.1671-587x.20160613

Abstract

Abstract:

Objective: To prepare the hepatocellular injury-conditioned medium (CM), and to simulate the microenvironment of injured tissue in vitro, and to explore the effect of CM on hepatic differentiation of bone marrow mesenchymal stem cells (BMMSCs) in the rats.Methods: The BMMSCs from 1-week-old rats were isolated and cultured by differential adherence and differential gradient method combined with optimized gradient centrifugation and culture time for adherent fluid exchange.The rats aged 6-8 weeks were injected intraperitoneally with 60% carbon tetrachloride solution (0.75 mL·kg^-1) to prepare the hepatocyte injured tissue homogenate supernatant (CM1) and hepatocyte injured serum (CM2). The third passage (P3) BMMSCs were randomly divided into 0%CM group (control group, cultured with 10%FBS L-DMEM), 10%CM1 group, 20%CM2 group, and 10%CM1+10%CM2 group. The morphology of cells was observed under inverted microscope;the expressions of cell-surface marker CD105 in BMMSCs and the positive expression rates of albumin (ALB) in cells in various groups were detected by immunohistochemistry method;the positive rate of lipid droplet formation in the pocess of adipogenic differentiation was detected by oil red O staining; the expression levels of alpha-fetoprotein (AFP) and ALB in the cells in various groups were detected by RT-PCR method;the positive rate of glycogen synthesis was detected by periodic acid-shiff staining; the hepatic differentiation ability of BMMSCs in various groups were identified and compared. Results: After primary culture of bone marrow cells for 72 h, the adherent cells showed round or spindle-shaped, covered with the bottom of the fibrous shuttle,and showed uniform and orderly shape, and the P3 BMMSCs surface markers CD105 showed positive expression.The P3 BMMSCs were induced f or 4 d by dexamethasone, insulin, 3-isobutyl-1-methylxanthine and indomethacin (DX+IBMX+IS+ID), the formation of lipid droplets was observed under microscope. After P3 BMMSCs were induced by CM, the expressions of AFP and ALB were positive,and the staining of cell glycogen was positive. After induced for 7 d, the cytoplasmic contents of P3 BMMSCs were abundant and the cells were round or triangular or liver board-like arrangement in CM groups. After induced for 7, 14 and 21 d, compared with control group, the positive expression rates of ALB in BMMSCs in CM groups were significantly increased (P<0.01); the positive expression rates of ALB in BMMSCs in 10% CM1+10% CM2 group were higher than those in 10%CM1 group and 20%CM2 group (P<0.05). After induced for 7 and 14 d by CM, compared with control group, the positive rates of glycogen synthesis in BMMSCs in CM groups were significantly increased (P<0.01);the positive rates of glycogen in 10%CM1+10%CM2 group were higher than those in 10% CM1 group and 20% CM2 group (P<0.05).Conclusion: CM1 and CM2 could induce the differentiation of BMMSCs into hepatocyte-like cells,and the lower concentrations of CM1 and CM2 can achieve the same effect.The microenvironment provided by CM1 and CM2 is more beneficial to the differentiation of BMMSCs into hepatocytes, which could induce cell differentiation more efficiently.

Key words: proliferation, conditioned medium, hepatic differentiation, bone marrow mesenchymal stem cells

CLC Number:

R657

ZHAO Meizi, WANG Dandan, YU Zhenjing, DUAN Yanli, MA Lina. Promotive effect of hepatocellular injury-conditioned medium on hepatic differentiation of bone marrow mesenchymal stem cells in rats[J].Journal of Jilin University Medicine Edition, 2016, 42(06): 1108-1115.

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Promotive effect of hepatocellular injury-conditioned medium on hepatic differentiation of bone marrow mesenchymal stem cells in rats

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