Study On Bioactivity And Preliminary Clinical Application Of Peripheral Blood Hematopoietic Stem Cells (PBHSC) In Patients With Acute-on-chronic Liver Failure

Stem cells have been applied to replacement therapy, gene therapy and tissue engineering for its capacity of self-renewal and multi-lineage differentiation. Liver failure is a life-threatened serious disease with many complications and high mortality rate. The application of stem cells in treatment of liver failure has received more and more attention.AIM:To investigate the bioactivity of the peripheral blood hematopoietic stem cells (PBHSC) in patients with acute-on-chronic liver failure, the mechanism of the PBHSC in treatment of liver injury, and the effect of improvement of internal environment on the PBHSC.Method:We isolated CD34+ cells from peripheral blood of the patients with acute-on-chronic liver failure and healthy controls. After cultured it in serum-free medium (SFEM), We studied the bioactivity of CD34+ cells by observing the morphology, recording growth curve, detecting cell cycle and cell apoptosis. CD34+ cells and culture solution were collected at the time points of 3,5,7,10,12 and 14 days. We detected HGF, MMP-9, TNF-a and IL-6 in culture solution of varying time points by ELISA. Expression of PK-M2 (a marker of immature hepatocytes), Integrin-β1 (a marker expressed in the development of liver) and L-PK (a marker of mature hepatocytes) were examined by RT-PCR and immunofluorescence. We cultured CD34+ cells of the patients with acute-on-chronic liver failure in medium with serum collected before or after plasma exchange. The growth curves were recoreded. Expression of PK-M2, Integrin-β1 and L-PK were examined by RT-PCR and immunofluorescence. The peripheral blood monouclear cells (PBMC) of patients with acute-on-chronic liver failure labeled with CFSE were transplanted into rats of liver-damage. We observed the PBMC in rats’liver. Compared the pathology of rats’liver tissue with control group.Result:Bioactivity of CD34+ cells from patients with acute-on-chronic liver failure is as good as that of CD34+ cells from healthy controls. HGF, MMP-9, TNF-α and IL-6 were found in cell culture medium. RT-PCR and immunofluorescence indicated that PK-M2, Integrin-β1 expressed on CD34+ cells of patients with acute-on-chronic liver failure. The CD34 cells in medium with serum collected after plasma exchange grew better than The CD34+ cells in medium with serum collected before plasma exchange (p<0.05). PK-M2 and Integrin-β1 expressed on CD34+ cells of both groups. In transplantation group, PBMCs labeled with CFSE were found in rats’ liver, the liver damage were repaired well.Conclusion:This study indicated that bioactivity of CD34+ cells of patients with acute-on-chronic liver failure was good, CD34+ cells of patients with acute-on-chronic liver failure can secrete HGF, MMP-9, TNF-α and IL-6, which can promote the growth of hepatocytes, and CD34+ cells of patients with acute-on-chronic liver failure can differentiate along a direction to hepatocyte lineage. Improvement of internal environment by plasma exchange helps to sustain the bioactivity of CD34+ cells. PBMCs of patients with acute-on-chronic liver failure migrated to the injured liver in rats of liver-damage, and repaired the injured liver.