Isolation, Culture, Identification And Tracing Study In Vitro Of Bone Marrow Mesenchymal Stem Cells Of T2DM

Objective: To explore isolate, culture, identification and tracing with BrdU, DAPI, SPIO in virto of mesenchymal stem cell (MSCs) from bone marrow of T2DM.Method: MSCs from bone marrow of the T2DM were isolated and cultured by density gradient centrifugation and combined with attachment culture method. Then, morphous of MSCs were observed by inverted microscope, identified by flow cytometry and osteogenic, chondrogenic, Adipogenic induction. MSCs were labeling by BrdU, DAPI, SPIO in vitro, then caculted the ratio of labeling.Result It was efficiently method to isolate and culture MSCs by density gradient centrifugation and combined with attachment culture. Flow cytometry examination showed that MSCs, cultured in vitro, were found anti-CD45negative and anti-CD44, anti-71, anti-105 positive. MSCs could induced osteogenic, chondrogenic, Adipogenic cell .There is no significant difference of MSCs between DM and normal.MTT showed there is no significant difference of OD in generation 1—6.(P>0.01) HGF,VEGF secrete by control group were (13316±1421) pg/106,(1582±131 )pg/106. HGF,VEGF secrete by T2DM group were (l3321±1552) pg/106,(1433±142) pg/106.Compare of two group ,no statistical differences(P<0.05), VEGF secrete by MSCs of T2DM group lower than control group.(P<0.05)。Conclusion1,Perocll density gradient centrifugation and adherent screening are effective ways which get higher purity and activity of bone marrow-derived mesenchymal stem cells.2,MSCs heavy proliferate under cultured in vitro, so it achieves substantial amplification,and meets clinical need: The experiment confirmed on behalf of 10 before the MSC are able to maintain their stem cell characteristics which can transform into osteoblast phenotype and secrete bone-specific matrix in a specific culture medium induced by researching the morphology of MSCs, in vitro growth kinetics,differentiation potentials and other characteristics of cell biology.3,The experiment successfully isolated and purifying MSCs from bone marrow. This was a practical, feasible method to long-time culture MSCs in vitro and could use for stem cell transplantation. BrdU, DAPI, SPIO labeling was a safe, efficiently, convenient method to tracing stem cell in vitro.