Experimental Study On Mobilizating Of Bone Marrow EPCs In Diabetic Mice By AMD3100 Combined With G-CSF

Objective:Endothelial progenitor cells (EPCs) have play a important role in angiogenesis in the ischemic area. Blood vessel disease caused by diabetes will lead to the lack of blood supply to the organization has been recognized by everyone. So, it is of great significance to study how to mobilize bone marrow endothelial progenitor cells to peripheral circulation in order to improve blood supply and promote wound healing in diabetic patients.The experiment use diabetic male mice (db/db male mice) as the research object, to compare the mobilization effects of AMD3100 combined with G-CSF on bone marrow endothelial progenitor cells with use G-CSF individually, and observe the difference in the number of endothelial progenitor cells at different points in the peripheral circulation of diabetic mice. Through the optimization of mobilization methods and time, to find a method which can effectively mobilize the bone marrow vascular endothelial progenitor cells under the diabetic state, to provide a new idea and theoretical basis for the treatment of diabetic wound with autologous bone marrow endothelial progenitor cells.Methods:Using AMD3100 (Selleck, Cat No.S3013, Lot.03) and recombinant human granulocyte colony stimulating factor injection (rhG-CSF, Kyowa Hakko kirin China Pharmaceutical Company, production number:S20140010) for mobilizate the bone marrow vascular endothelial progenitor cells in diabetic mice by the dosage (the injection dose of AMD3100 was 6mg/kg*d-1, and the injection dose of G-CSF was 100ug/kg*d-1)and method (the diabetic mice were injected subcutaneously for 6 days) which were introduced by the literature at home or abroad. Selecting 60 ten tp eleven weeks old male diabetic mice (to avoid the effects of estrogen on EPCs), choosing the method of random number table, and dividing them into three groups:Group A (AMD3100+G-CSF mobilization group), Group B (AMD3100+NS mobilization group) and Group C (NS control group), each group had 20 mice. At each group, setting up four time points:3d、5d、7d、10d, and selecting 5 mice randomly. We taking their peripheral blood from heart to immunofluorescence staining and detecting the proportion of CD133/Flk-1 double fluorescent positive cells by flow cytometry, the results were used x±s to express, and were contrasted and analyzed by statistics.Results:Testing the proportion of CD133/Flk-1 double fluorescent positive cells in peripheral blood of diabetic mice in each group by Flow Cytometry, the results by statistical analysis are:①For Group C (NS control group), there was no significant difference in the proportion of CD133/Flk-1 double fluorescent positive cells at four time points:3d,5d,7d and 10d (p>0.05).②For Group A (AMD3100+G-CSF mobilization group) and Group B (AMD3100+NS mobilization group), after mobilization, the proportion of CD133/Flk-1 double fluorescent positive cells in peripheral blood of diabetic mice have a significant difference (p<0.05), and show a certain change:the proportion of CD133/Flk-1 double fluorescent positive cells increased gradually form 3d to 7d and peaked at 7d, then decreased gradually.③ The proportion of CD133/Flk-1 double fluorescent positive cells in three groups shows difference at four time points (p<0.05):Compared with Group B, the CD133/Flk-1 double fluorescence positive cell percentage of Group A increased (p<0.05); Compared with Group C, the CD133/Flk-1 double fluorescence positive cell percentage of Group B increased (p< 0.05).Conclusion:Whether using AMD3100 combined with G-CSF or G-CSF individually can effectively mobilize the bone marrow vascular endothelial progenitor cells under diabetic state; Using the two mobilization methods of AMD3100 combined with G-CSF or G-CSF individually, the proportion of vascular endothelial progenitor cells in the diabetic mice peripheral blood showed some changes, namely, the proportion of vascular endothelial progenitor cells was gradually increased at 3d,5d,7d after mobilization, and reached the peak at 7d, then decreased gradually. The effect of mobilization of bone marrow endothelial progenitor cells by AMD3100 combined with G-CSF was significantly stronger than that of using AMD3100 individually.