Experimental Study On The Isolation, Culture And Induced Differentiation Of Rat Bone Marrow Stromal Stem Cells

Objective Bone marrow stromal stem cells (BMSCs) arise from the complex array of supporting structures in marrow. BMSCs can be harvested easily from bone marrow,and it can duplicate themselves. BMSCs have a high proliferative potential in culture, also they are capable of differentiating to a wide variety of cell types, and when autologous transplanted, there exist no immunorejection. So BMSCs can be used to treat hepar dieases.We studied the isolation, culture and induced differentiation of rat BMSCs, especially the proper condition and mechanism of hepatocytes differentiation in vitro and in vivo,and identified the hepatocytes to provide an experimental groundwork for the therapy of hepatic diseases using BMSCs transplantion.Methods BMSCs were isolated from bone marrow of rats,then cultured in low-glucose DMEM with 20% NCS for expanding,and observed the growth and biology characterization of BMSCs.We investigated the optimal condition of culture BMSCs using MTT method,and the optimal condition of differentiation into multiple cells such as osteoblast and adipocytes ;We provided BMSCs the hepatic microenvironment using some factors from liver such as G and CM,and compared with the different culture factors such as HGF,NGF,and RA by immunocytochemistryjWe identified the hepatocytes using ICG,and proved it having the function of hepar by PAS response;We injected BMSCs labelled with BrdU into the tail vein of mice to test by immunohistochemistry whether BMSCs could differentiate into hepatocytes and express hepatocellular phenotypes in vivo.Results (l)The MTT activity of BMSCs xompared with control groups(low-glucose DMEM with 10% newborn calf serum),that of 20% serum groups increased(P<0.01),and that of RPMI-1640 groups decreased(P<0.01),while the difference between high-glucose and low-glucose group is not notable. (2) The BMSCs have the multipotentiality to differentiate into osteoblast,adipocytes and hepatocytes. (3) Immunocytochemistry revealed that the BMSCs induced by G, CM, HGF,NGF,and RA all express alpha-fetoprotein and albumin compared with control group. (4)The cytoplasm of BMSCs take on different degree of Purplish red color in different groups on PAS...