Effect Of Bone Marrow Mesenchymal Stem Cells On Liver Regeneration After Major Hepatectomy In Rats

Objectives: (1) To investigate labeling and tracing methods of bone marrow mesenchymal stem cells (MSCs), and optimize tracer technique; (2) To observe the homing capacity and the effects on liver regeneration of bone marrow mesenchymal stem cells in the rat remnant liver after major hepatectomy by different transplantation approach.Methods: (1) Rat bone marrow MSCs were isolated and cultured in vitro.The surface antigens (CD29,CD34, CD45, CD90) of MSCs were identified by flow cytometry. MSCs were labeled with BrdU, DAPI and GFP, respectively. The labeling efficiency of BrdU was calculated with immunocytochemistry, and those of DAPI and GFP were observed under fluorescence microscope.The advantages and disadvantages of three tracer techniques were compared. (2) Bone marrow mesenchymal stem cells (about 1.5×106 each rat) labeled with DAPI were injected into the caudal or portal vein of rat with 70% major hepatectomy. And"hepatectomy only"rats were used as control group. Serum was drawn to inspect liver function indexes 3 d and 9 d after the hepatectomy. And liver samples were collected 9 d after the hepatectomy.Immunohistochemistry was used to detect the expression of Ki-67 and BrdU positive cells. Fluorescence microscopy was used to observe the migration status of the cells by different approach.Results: (1) Flow cytometry showed that MSCs expressed CD29 and CD90 but not CD34 or CD45. Three kinds of markers showed no significant toxicity on the cells. For BrdU labeling, the optimal dosage and timing were 10μmol/L and 48 hours respectively; for DAPI labeling, the optimal dosage and timing were 1μg/ml and 12 hours respectively. When MOI(multiplicity of infection) was 8, the infection efficiency of lentivirus-GFP was above 90% after infection 12h around. (2) ALB of the two cell transplantation groups were significantly higher than those in control group at 9 d (both p < 0. 05).The rate of Ki-67(103.30±40.44,95.82±26.67 vs 60.29±35.71/ HPF×400) and BrdU positive cells of the two cell transplantation groups(15.92±3.06,16.01±3.32 vs 11.63±3.74/ HPF×400) were significantly higher than those of hepatectomy control group(all p< 0. 05).The cells labeled with DAPI of rat remnant liver were significantly higher in group received transplantation via portal veins than via caudal veins(18.1±3.4 vs 7.6±2.0 / HPF×400)Conclusion: (1) Bone marrow mesenchymal stem cells transfected by GFP gene is a clear, stable method for labeling cells, which is important to adult stem cells tracing. (2) Bone marrow mesenchymal stem cells injected into the caudal or portal vein can promote liver regeneration after major hepatectomy in rats. But the MSCs into the liver are less via the tail vein than via the portal vein.