| Objective：The aims of our study were to confirm and to compare the effectiveness of human umbilical cord mesenchymal stem cells (UCMSCs) and human umbilical cord blood-derived endothelial colony-forming cells (ECFCs) or co-transplantation via animal study using a lower limb ischaemia model of diabetic mouse,and to preliminarily approach the mechanism of angiogenesis of them.Methods:1.To establish a diabetic mouse model:We induced diabetes mellitus in sixty6-week-old male C57BL/6J mice by intraperitoneal injection of streptozotocin(STZ)(150mg/kg).The successful establishment of a diabetic mouse model is definited if the blood glucose of the mouse is higher than16.7mmol/1, and eventually we induced fifty diabetic mice.2.Diabetic mice were divided randomly into five groups,group A:to transplant UCMSCs(n=10),group B:to transplant ECFCs(n=10),group C:to co-transplant UCMSCs and ECFCs(n=10), group D:ontrol group,which is injected PBS (n=10),group E:sham operated group(n=10).3.To establish lower limb ischemia model of mouse:The parts of branches of femoral arteries and veins of mice of group A,B,C and D were ligated and excised including the superficial femoral artery and vein, the popliteal artery and vein,the saphenous artery and vein,to build a lower limb ischaemia mouse model with diabetes mellitus.4.Index of observation:Gross appearance of the skin and wound healing of ischemic limb were observed, skin temperature and transcutaneous oxygen pressure(TcPO2) of ischemic limb were messured before and after established lower limb ischemia model of mouse and on21days after transplantation of stem cells,capillary density of gastrocnemius muscle of ischemic limb was detected with immunohistochemical staining,the homing and migration of the transplantation cells was observed by using CM-Dil to trace, then protein level of vascular endothelial growth factor (VEGF),Akt and Erkl/2protein level were messured by western blotting.Results:l.Aftter establishment of lower limb ischemia model, lower limb of mice present appearance of ischemia,such as darkness of the skin,toe,and toenail of the mice.Comparing with group D, there was improvement of ischemia and recovery of blood flow in the lower limb of mice of group A,B and C.2.The time of wound healing of mice in group A, B and C was significantly shorter than group D(P<0.05)，and the time of wound healing of group A and C was more shorter than group B (P<0.05) while there was no significant difference between group A and C(P>0.05).Skin temperature and TcPO2of ischemic limb were significantly decreased after establish lower limb ischemia model in group A,B,C and D (P<0.05),and they were significantly increased in group A,B and C (P<0.05) while there was no change in group D (P>0.05)on21days after transplantation of stem cells.Compared with group A and B,Skin temperature and TcPO2of ischemic limb of group C mice were higher on21days after transplantation of stem cells (P<0.05) while there was no significant difference between group A and B(P>0.05).3.UCMSCs and ECFCs could home and migrate to muscle fibers.UCMSCs and ECFCs which were traced could surrounding vessels.4.Capillary density in the gastrocnemius of mice in group A(P<0.05),B(P<0.05) and C(P<0.01) significantly increased when compared with group D and E,and the highest capillary density is group C (P<0.05).5.Compared with group D and E,VEGF,Akt and Erkl/2level of group A,B and C were significantly increased(P<0.05). The level of Akt and Erkl/2protein almost had no significant difference between group A and B(P>0.05).The level of VEGF protein was higher in group B than group A.The level of VEGF,Akt and Erkl/2protein were higher in group C than group A and B.Conclusion:1.This preliminary study suggests that UCMSCs,ECFCs or co-transplantation of them could effectually improve lower limb ischaemia with diabetes mellitus and promoted neovascularization. 2.There was synergetic effect by co-transplantation of UCMSCs and ECFCs.The angiogenesis induced by the synergistic dual-cell system was higher than found in transplantation of either UCMSCs or ECFCs alone. |
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