| OBJECTIVE:To screen the best concentration of magnesium ion to promote the vascular differentiation of SD rat bone marrow mesenchymal stem cells(BMSCs);To explore whether magnesium ion promotes the vascular differentiation of BMSCs through Notch signaling pathway.METHODS:BMSCs were isolated by adherent method;BMSCs were identified by flow cytometry;according to the concentration of magnesium ion,the experiment was divided into 6 groups(control,2,5,10,20,40 mmol/L).The proliferation ability of BMSCs in each group was detected by CCK-8.The toxicity of BMSCs was analyzed by Calcein-AM/PI living/dead cell double staining.After BMSCs were induced by vascular induction medium for 7 days,the expression of angiogenesis related mRNA was detected by RT qPCR and the tubule formation was detected by matrix glue tube forming assay.After obtaining the optimum concentration of magnesium ion,the experiment was divided into five groups:BMSCs with normal medium control group(Group A),BMSCs with vascular endothelial induction medium group(Group B),BMSCs with 5 mmol/L magnesium ion vascular endothelial induction medium group(Group C),BMSCs with 2μmol/L DAPT and vascular endothelial induction medium group(Group D),BMSCs with 2 μmol/L DAPT and 5 mmol/L magnesium ion vascular endothelial induction medium group(Group C),Group E was induced by magnesium ion.RT-qPCR and Western Blot were used to detect the expression of mRNA and protein related to each component of blood vessels,scratch test and Transwell test were used to detect the cell migration ability and matrix glue tube forming test was used to detect the tubule formation in each group.RESULTS:(1)CD29 and CD90 were highly expressed in the isolated BMSCs,while CD45 and CD34 were lowly expressed.(2)CCK-8 showed that on day 1,3 and 5,BMSCs in 2,5 and 10 mmol/L Mg2+groups increased more than those in the control group,and that in 5 mmol/L Mg2+group was the strongest(p<0.05).The proliferation of BMSCs in 20 and 40 mmol/L Mg2+group was weaker than that in control group.(3)There was no significant difference in the number of dead cells between 2,5,10 mmol/L Mg2+ and control groups.The number of dead cells in 20 and 40 mmol/L Mg2+group was significantly higher than that in control group(p<0.05).(4)Compared with the control group,the expression of vascular related mRNA and the number of tubules in 2,5 mmol/L magnesium ion group were increased,and the increase in 5 mmol/L magnesium ion group was more obvious than that in other groups(p<0.05).(5)Compared with Group A,the expression of mRNA and protein related to blood vessels,the number of blood vessels and the expression of endothelial cell surface antigen in Group B,C,D and E were more than those in Group A,and the migration ability of cells was stronger,and it was better on day 14 than on day 7(p<0.05).(6)Compared with Group D and E,Group B and C had more mRNA and protein expression related to vascular differentiation,more number of vascular formation and stronger cell migration ability,which was better on day 14 than on day 7(p<0.05).(3)Compared with Group B,Group C had more mRNA and protein expression related to vascular differentiation,more number of vascular formation and stronger cell migration ability,which was better on day 14 than on day 7(p<0.05).(4)there was no significant difference in angiogenic indexes between Group D and Group E.CONCLUSION:(1)2 mmol/L,5 mmol/L and 10 mmol/L magnesium ions can promote the proliferation of BMSCs,and 5 mmol/L magnesium ions may have the strongest effect on the proliferation and differentiation of BMSCs.(2)Magnesium ion enhance the angiogenesis differentiation of BMSCs and enhance the migration ability of BMSCs through Notch signaling pathway. |
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