The Interaction Between Rat Bone Marrow Mesenchymal Stem Cells And CBRH-7919Hepatoma Cells

PARTI The effect of rat bone marrow mesenchymal stem cells paracrine substance on the proliferation and invasion of CBRH-7919hepatoma cellsObjectiveTo explore the effect of bone marrow mesenchymal stem cells paracrine substance on the proliferation and invasion of CBRH-7919hepatoma cells.MethodsSeparated the BMSC of Wistar rats, cultured in vitro after identification, prepared bone marrow mesenchymal stem cells-conditioned medium (BMSC-CM), MTT assayed the effect of different concentration of BMSC-CM on the proliferation of7919hepatoma cells, flow cytometry tested the cell cycle of7919hepatoma cells, ELISA detected the AFP secreted by7919hepatoma cells. Transwell chamber test the effect of BMSC-CM on the invasion of CBRH-7919hepatoma cells.Results1.BMSCs were isolated by density gradient centrifugation and adherent cell separation. The passage3BMSCs show fibroblast-like. arranged a certain direction. high expression of CD29and CD44,1ow expression of CD34and CD45.2.MTT assay showed that the absorbance value(A value) of CBRH-7919he patoma cells in25%、50%、75%and100%BMSC-CM group were0.395±0.050,0.459±0.079、0.501±0.084and0.566±0.099, all higher than0%BMSC-CM(0.277±0.064)(P<0.05),with the increase of BMSC-CM’s concentration, the A val ue increased, showing concentration dependency.3.Flow cytometry showed that the cell division and proliferation index(PI) of CBRH-7919hepatoma cells in25%、50%、75%、100%BMSC-CM group w ere20.773±1.342、21.838±1.368、23.503±1.568�'�26.486±2.124, all higher than that of0%BMSC-CM group(16.653±0.847)(P<0.05). PI increased with the el evation of BMSC-CM.4.ELISA showed that the AFP secreted by CBRH-7919hepatoma cells in25%,50%,75%and100%BMSC-CM group were12.562±0.071,14.660±0.106,16.397±0.058and23.196±0.137.all higher than that in0%BMSC-CM group(3.1 43±0.033)(P<0.05). With the increase of BMSC-CM’s concentration, the AFP v alue also increased.5.Transwell chamber invasion experiment showed that the number of CBRH-7919hepatoma cells on the membrane of the chamber in25%,50%,75%and100%BMSC-CM group were22.0000±1.309,56.750±1.669,70.000±1.309,99.620±1.847.more than that in0%BMSC-CM group (9.375±1.061)(P<0.05).Conclusions1.Bone marrow mesenchymal stem cells can be successfully isolated by de nsity gradient centrifugation and adherent cell-based screening method, The cell phenotype by flow cytometry were CD29+CD44+CD34-CD45-2.Rat bone marrow mesenchymal stem cells-conditioned medium can prom ote the proliferation and the invasion of CBRH-7919hepatoma cells. PART2The differentiation of bone marrow mesenchymal stem cells into vascular endothelial-like cells induced by rat CBRH-7919hepatoma cells in vitroObjectiveTo explore the effect of CBRH-7919hepatoma cells inducing the bone marrow mesenchymal stem cells (BMSCs) to differentiate into vascular endothelial-like cells and the mechanism.MethodsThere were3groups:single BMSC group,CBRH-7919hepatoma cells and BMSC co-culture group and containing anti-VEGF antibody CBRH-7919hepatoma cells and BMSC co-culture group, a co-culture system containing BMSC and CBRH-7919hepatoma cells based on transwell chamber indirect model was established.After48h and72h,immunofluorescence technique tested the expression of VEGFR2and CD31on BMSCs, semi-solid medium tested the in vitro angiogenesis of BMSCs.ResultsIn single BMSC group,there were no CD31and VEGFR2expression, and no capillary-like structure. In the co-culture group,after48h, BMSC expressed CD31and VEGFR2positive rate was:(11.50±1.87)%and (12.33±1.37)%respe ctively, and could also formed characteristic capillary-like structures(8.00±0.05)/high power field, after72h, the expression of CD31and VEGFR2increased si gnificantly (24.43±2.23)%and (24.86±0.69)%, the characteristic capillary-like st ructures also increased(22.00±0.02)/high power field.In containing anti-VEGF an tibody group, there were no CD31and VEGFR2expression and capillary-like structure.ConclusionsBone marrow mesenchymal stem cells can differentiated into vascular endothelial-like cells after cocultured with CBRH-7919hepatoma cells.This process may be induced by VEGF secreted by CBRH-7919hepatoma cells.