The Ability Of Adipogenic Differentiation Of Diabetic Mouse Bone Mesenchymal Stem Cells And Its Intervention With EGb761

Part one The ability of adipogenic differentiation of bone mesenchymal stem cells in diabetic mouseObjectives:To compare the ability of cell differentiation from BMSCs to adipogenic cells in vitro in db/db mouse and db/m mouse.Methods:BMSCs were isolated and purified from eight-week-old db/db mouse and db/m mouse.The marker of cell surface was detected by FCM (Flow Cytometry). CCK-8 assay was used to assess the ability of mesenchymal stem cells proliferation. Oil Red O staining was performed on the 21st day of adipogenic induction. RT-PCR was used to evaluate the gene expression of PPARy and C/EBPa. Western Blot was used to evaluate the protein expression level of PPARy and C/EBPa which were related to adipogenic differentiation.Results:1. BMSCs were high positive for CD29 and CD44, low positive for CD34 and CD45(the positive rates for BMSCs in db/m mouses were 99.5%,99.1%,2.7% and 2.8%, respectively; the positive rates for BMSCs in db/db mouses were 94.6%, 97.4%,3.1% and 4.1%, respectively).2.The ability of mesenchymal stem cells proliferation in db/db mouse was a little lower than that in non-diabetic db/m mouse (P>0.05).3. Oil Red O staining showed the number of bone fat droplets in db/db mouse were much more than that in db/m mouse, and the area of bone fat droplets in db/db mouse were larger than that in db/m mouse.4. RT-PCR showed the gene expression of PPARy and C/EBPa was much higher in db/db mouse than that in db/m mouse on the 21st day of adipogenic induction (P<0.05).5.Western Blot showed the protein expression level of PPARy and C/EBPa was much higher in db/db mouse than that in db/m mouse on the 21st day of adipogenic induction (P<0.05).Conclusions:1. The BMSCs of both groups were high activity and high purification. There was no significant difference in proliferation capacity between db/db mouse and db/m mouse; 2. The ability of adipogenic cell differentiation from mesenchymal stem cells in db/db mouse was higher than that in db/m mouse.Part two Effect of EGb761 on the osteogenic differentiation of bone mesenchymal stem cells in diabetic mouseObjectives:To investigate the effect of EGb761 on cell differentiation from BMSCs to osteoblasts in db/db mouse.Methods:BMSCs were extracted from eight-week-old db/db mouse. CCK-8 was used to screen the cell toxicity of BMSCs in db/db mouse. Then the optimal concentration was selected to intervene the osteogenic differentiation of BMSCs in db/db mouse. Alizarin red staining to determine the alkaline phosphatase activity was performed on the 14th day. RT-PCR was used to detect gene expression of ALP andRUNX2. Western Blot was used to detect protein expression of ALP and RUNX2.Results:1. The concentration of less than 1000mg/L had no significant cytotoxicity on cell growth and proliferation. And optimum stimulus concentration was 10mg/l, which was selected in this study.2. Alizarin red staining showed, compared with the induction group, the EGb761 intervention group had larger mineralized nodules, increased number and dyed darker, which rendered as a reddish-brown. The ALP activity of EGb761 intervention group was stronger than induction group.3. Compared with the normal bone formation induction group, the gene expression levels of ALP and RUNX2 were significantly increased in EGb761 intervention group (P<0.05).4.Compared with the normal bone formation induction group, the protein expression levels of ALP and RUNX2 were significantly increased in EGb761 intervention group (P<0.05)Conclusions:EGb761 could promote BMSCs differentiate into osteoblasts in db/db mouse.Part three Effect of EGb761 on the adipogenic differentiation of bone mesenchymal stem cells in diabetic mouseObjectives:To investigate the effect of EGb761 on cell differentiation from BMSCs to osteoblasts in db/db mouse and explore the effect of EGb761 on adipogenic differentiation and its possible mechanisms.Methods:BMSCs were extracted from eight-week-old db/db mouse. And EGb761 was used to intervene the process of adipogenic differentiation. On the 21st day, Oil Red O staining was performed. RT-PCR was used to detect gene expression of PPARy and C/EBPa, and Western Blot was used to detect protein expression of PPARy, C/EBPα and β-catenin.Results:1. Oil Red O staining showed the number of fat droplets in the EGb761 intervention group was much less than that in the normal induction group, and the fat droplets was also smaller than the normal induction group.2. On the 21st day, PR-PCR showed, compared with the normal induction group, the gene expression levels of PPARy and C/EBPa were significantly lower in EGb761 intervention group (P<0.05) and the gene expression levels of β-catenin were significantly higher in EGb761 intervention group (P<0.05).3.Western blot showed the protein expression levels of PPARy and C/EBPa were significantly lower in EGb761 intervention group (P<0.05) and the protein expression levels of β-catenin were significantly higher in EGb761 intervention group (P<0.05).Conclusions:EGb761 could inhibit BMSCs from differentiating into adipocytes in db/db mouse. One of the possible mechanism was EGb761 could regulate part of the gene expression level related of Wnt pathway in the process of BMSCs adipogenic differentiation.