| Objective:To investigate the role of AMD3100 and granulocyte colony-stimulating(G-CSF)used alone or in different sequences in the mobilization of bone marrow endothelial progenitor cells(EPCs)in diabetic mice(db/db mice),and to observe its effect on the healing and vascularization of refractory wounds.It is providing a new idea that obtains diabetes patients' EPCs for cell therapy.Methods:Phase I:With reference to the dose and methods described in related articles in China and foreign countries,AMD3100 and recombinant human granulocyte colony-stimulating(rhG-CSF)injection were used for the mobilization of bone marrow EPCs.A totle of 80 male db/db mice aged 5-6 weeks were randomly divided into group A(AMD3100+NS mobilization group),group B(AMD3100+G-CSF mobilization group),group C(G-CSF+AMD3100 mobilization group)and group D(NS control group).A totle of 80 male db/b+ mice were chosen as the corresponding control group,and were divided into groups A',B',C' and D'.Peripheral blood samples were collected from the heart on days 3,7,10 and 14 after mobilization,and flow cytometry was uesd to measure the the proportion of CD34+/CD133+/Flk-1+ cells.The results were used mean ± standard deviation(%)to express,and comparison and analysis were performed after statistical analysis.Phase II:Two full-thickness wounds were generated by 10 mm diameter circular excisions on the shaved back of 30 male db/db mice,which aged 9-12 weeks.Those mice were randomly divided into group E(full-thickness wound+NS control group),group F(full-thickness wound+mobilization group).The mobilization plan selected the best mobilization plan obtained by the db/db mice in the first stage of the experiment and the mobilization began at once after wound formation.The wound healing rate of each group was observed and calculated at 3,7,10,14 days after mobilization until the wound was completely covered by epithelium in one group.HE staining was performed to observe pathological morphological changes and immunohistochemical detection of CD31 expression on the wound at 3,7,10,14 days after mobilization until the wound was completely covered by epithelium.The results were used mean±standard deviation(%)to express,and comparison and analysis were performed after statistical analysis.Results:Phase ?:?There was no difference in the proportion of CD34+/CD133+/Flk-l+cells between group D and group D' at the time points 3 d,7 d,10 d and 14 d after mobilization(P>0.05),but the proportion of CD34+/CD133+/Flk-1+cells in group D' was higher than that in group D(P<0.05).?For group A,the proportion of CD34+/CD133+/Flk-1+cells increased gradually from 3 d to 7 d and peaked at 7 d,then decreased slightly.Group B and Group C reached the peak at 10 d,then decreased gradually and the proportion of CD34+/CD133+/Flk-1+cells at the peak was significantly different from the other three time points(P<0.05).On the 10 d5 the proportion of CD34+/CD133+/Flk-1+cells in the peripheral blood of db/db mice in group B and group C was higher than that in group A and group D(P<0.05),but there was no significant difference of the proportion of CD34+/CD133+/Flk-l+cells in the peripheral blood of db/db mice in group B and group C(P>0.05).?For Group A',Grouq B' and Group C',after mobilization,the proportion of CD34+/CD133+/Flk-1+ cells increased gradually from 3 d to 7 d and peaked at 7 d,then decreased gradually,and was significantly different from the other three time points(P<0.05).On the 7th day after the peak,the proportion of CD34+/CD133+/Flk-1+ cells in Group C' was higher than that in Group A',Group B' and Group D'(P<0.05).Phase ?:?On days 7,10 and 14 after mobilization,the wound healing rate of group F was higher than that in group E(P<0.05).Wounds reached complete closure on day 17 after mobilization in group F,while wounds of group E reached complete closure on day 20 after mobilization.The healing time of group F was three days earlier than that of group E.?On day 3 after mobilization,the collagen fibers of the wound tissue were seen under the microscope to swell and fuse,forming homogeneous red-stained hyaline substances and inflammatory cell infiltration,and a number of erythrocytes were observed in some vascular lumens.However,the swelling degree of collagen fibers in group F was less than that in group E,and inflammatory cell infiltration and hyaline degeneration in group F were less common than group E.On day 7 after mobilization,there were increased epidermal cells and new capillaries in the wound of group F,and a large number of collagenous fibers with disordered arrangement were observed at the edge of the wound in group F,while inflammatory cell infiltration and a small number of epidermal cells were observed under microscope in group E.On day 10 after mobilization a small of epidermis was observed in group F.On day 14 after mobilization,the surface of the wound was basically covered by epidermal cells with good continuity and disordered arrangement of collagen fibers in group,while the surface of the wound was not completely covered by epidermal cells in group E,and inflammatory cells were still infiltrated under the scab.On day 17 after mobilization,the epidermis of group F was thickened and keratinized well,with visible glandular formation and neat arrangement of collagen fibers.The epidermal cells of group E basically covered the wound surface,and the collagen fibers tended to be an orderly arrangement.?The expression level of CD31 positive cells in the F,group was higher than that in the E group at 7,10,14 and 17 days after mobilization(P<0.05).Conclusions:AMD3100 combined with G-CSF has a better effect in the mobilization of bone marrow EPCs than AMD3100 alone.In non-diabetic conditions,G-CSF followed by the addition of AMD3100 has a better mobilization effect,but in diabetic conditions,AMD3100 followed by the addition of G-CSF has a better mobilization effect AMD3100 combined with G-CSF mobilizes bone marrow EPCs to promote the healing of diabetic wounds by promoting angiogenesis,and reducing inflammatory cell infiltration and edema. |
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