Effect Of IGF-1 At Different Doses On Proliferation And Differentiation Of Human Umbilical Cord Mesenchymal Stem Cells

Objective 1,To explore the conditions of isolation, purification, proliferation and culture of umbilical cord mesenchymal stem cells (hUCMSCs) in vitro, and study its biological characteristics; 2,To verify the effects of IGF-I at different doses on cell proliferation and its dose-effect relationship; 3,To observe the change of OD of hUCMSCs during differentiating into the adipocytes in the condition of IGF-1 with different concentrations,to elucidate the suitable concentration of IGF-1 in adipocyte induction.Methods 1,After stripping off arteries and veins, maining parts of umbilical cord were cut into small sections and plated in 100mm culture dish.Adherent cells coming out of fragments.The morphology of hUCMSCs were observed by inverted phase contrast microscope, The growth curves of them were drawn and the surface antigens of hUCMSCs were detected by flow cytometry.To induce the cells to differentiate into adipose and bone,and the potentiality of its osteogenic and adipogenic differentiation were detected by chemistry staining; 2,The 2nd generation hUCMSCs were seeded onto 96-well culture platend divided into 7 groups(G1,G2..., G7). The former 5 groups were cultured in 0.02 volume fraction FCS culture medium containing IGF-Ⅰat doses of 40,60,80,100,120μg/L; the sixth group was cultured in 0.05 volume fraction FCS culture medium as positive control, and the seventh group was cultured in 0.02 volume fraction FCS culture medium as negative control. The effects of IGF-Ⅰon cell proliferation were observed with MTT test; 3,hUCMSCs were separated and cultivated in vitro..hUCMSCs were induced to adipocytes.The induced culture medium was interfered with different IGF-1 concentration.MTT.Results 1,4-6 days after primary culture, adherent cells cames out of frgements and these cells could be maintained for 10 passages without obvious changes in morphology or growth pattern.Flow cytometry analysis revealed that CD29,CD44 and HLA-ABC were expressed on the cell surface.CD34,CD45 and HLA-DR were negative or weakly expressed.The induction in vitro demonstrated the cells exhibited multi-potential differentiation into osteogenic and adipogenic cells; 2,There was no difference among the G1and G2groups and G7group on days 3and 6(P>0.05); There was no difference among the G3, G4and Gsgroups (P>0.05).③On the second day, the proliferative rate of hUCMSCs in the positive G6group was significantly higher than that in G1and G2groups but lower than in G3, G4and G5groups(P<0.000 or< 0.006). On the fourth day, the proliferative rate of hUCMSCs in the G6group was significantly higher than that in each other group (P<0.000 or<0.006).④On days 3 and 6, the proliferative rate of hUCMSCs in G1and G2groups was significantly lower than that in G3, G4 and G5groups(P<0.000); the G6 group was remarkably higher than the G7 group (P<0.000).3,There is not significant between different concentrations of IGF-1 and adipocytes induction.However, there is a significant between the number of lipids and different IGF-1 concentrations.The number of lipids of 80ng/ml,100ng/ml and 120ng/ml group is more than other two groups(P< 0.01),and there was no difference among the 80ng/ml,100ng/ml and 120ng/ml group (P>0.05)。Conclusion 1,hUCMSCs could be cultured and proliferated in vitro and also have the similar biological characteristics with bone marrow mesenchymal stem cells;2,Exogenous IGF-Ⅰpromotes the proliferation of hUCMSCs in a dose-dependent manner.100μg/L is found to be the most effective concentration,which provides an optimal culture condition for rapid proliferation of hUCMSCs in vitro.3,80ng/ml is the proper concentration using IGF-1 to effect the adipocyte induction.