| Objective:Using Astragalus polysaccharide(APS)intervene the rat bone marrow derived Endothelial progenitor cells(EPCs)in vitro,we transplanted it in muscle of sciatic nerve area in Diabetic peripheral neuropathy(DPN)model rats to observe its influence on blood flow velocity,vascular endothelial growth factor(VEGF)?nerve growth factor(NGF)of sciatic nerve in DPN model rats.Methods:1.After isolating the rat bone marrow mononuclear cells by using density gradient centrifugation,we cultured them with the DMEM medium(containing 20%fetal bovine serum)7 days in vitro.Extracted some of cells to identification by applying for double fluorescent staining and flow cytometry,then we cultured the EPCs in the DMEM medium(containing 0.4 mg/mL APS)for 24 hours in vitro.Finally,we collected all cells for another purpose.2.Fed by high sugar and fat diet,injected intraperitoneal one-time through 1%Streptozotocin solution(STZ),the DPN model rats were established.Measured all the Motor nerve conduction velocity of rat sciatic nerve(MNCV)before and after modeling by applying noninvasive electromyography.All rats were divided into the following five groups:the normal group(normal rats 10),the DPN group(10),the PBS group(10),the EPCs group(10),the APS intervention group(10).3.The DPN model rats were treated by local transplantation in anesthesia,and the parts of transplantation treatment were the muscle nearby of the sciatic nerve.The APS intervention group rats were transplanted by the EPCs through APS intervention for 24 hours.The EPCs group rats were transplanted by the EPCs cultured in vitro.The PBS group rats were transplanted using the PBS buffer.There was no transplantation treatment in the DPN group and normal group.Before and after transplantation therapy,we measured the blood flow velocity changes of all rats in sciatic nerve area.Applying for the Western blot and the Real-Time PCR technique to detect the expression of VEGF and NGF of all rats sciatic nerve.Results:(1)The blood flow velocity variety of the sciatic nerve areathe blood flow velocity of sciatic nerve area in normal rats was 100.06±7.57cm/s.After modeling successful,the blood flow velocity of the DPN model rats was slowed significantly to 40.25±3.00cm/s,significant difference before and after modeling,statistically significant(P<0.05).Compared with the DPN group,the PBS transplantation could not improve the blood flow velocity of DPN model rats in sciatic nerve area.However,the APS group and EPCs group could increased it obviously,and significant difference,statistically significant(P<0.05).After the transplantation treatment,the blood flow velocity of DPN model rats in sciatic nerve area of the APS group were increased to 76.46±3.20cm/s,comparing with EPCs group,significant difference(P<0.05).(2)The expression of VEGF protein and VEGFmRNA in sciatic nerve of all rats.In normal rats group,the expression of VEGF protein and VEGFmRNA in sciatic nerve was low.Compared before modeling with after modeling,the expression of them were increased significantly after modeling successful,two groups were different(P<0.05).PBS transplantation treatment could not improve the expression of VEGF protein and VEGFmRNA.However,through the transplantation treatment,the APS group and EPCs group could reduce them to some extent,compared with those of DPN group rats,significant difference and statistically significant(P<0.05).Compared with the EPCs group,the APS group could reduce the expression of VEGF protein and VEGFmRNA in sciatic nerve obviously,and significant difference(P<0.05).(3)The expression of NGF protein and NGFmRNA in sciatic nerve of all rats after transplantation treatment.Compared with the normal rats group,the expression of NGF protein and NGFmRNA in DPN rats'sciatic nerve were declined obviously,significant difference,statistically significant(P<0.05).After the transplantation treatment of the pure EPCs and the EPCs through APS intervention,the expression of them was increased obviously comparing with the DPN rats,and the difference was statistically significant(P<0.05).Compared with the DPN rats group,the transplantation treatment of PBS buffer could not improve their expression,there was no difference(P>0.05).The transplantation treatment of the EPCs through APS intervention could increasing more the expression of the NGF protein and NGFmRNA in sciatic nerve than the transplantation treatment of the pure EPCs,and significant difference,statistically significant(P<0.05).Conclusion:(1)After the EPCs transplantation treatment,the blood flow velocity in sciatic nerve area of the DPN model rats was improved,the expression of NGF protein and NGFmRNA in sciatic nerve was increased,meanwhile,the expression of VEGF protein and VEGFmRNA was decreased.(2)APS intervention therapy could increase the effect of EPCs transplantation treatment.Applying for transplantation therapy of the EPCs through APS intervention in vitro,the blood flow velocity in sciatic nerve area of the DPN model rats was improved dramatically,meanwhile,the expression of NGF protein and NGFmRNA in sciatic nerve was increased obviously,the' expression of VEGF protein and VEGFmRNA was decreased significantly. |
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