| Objective: To investigate the effect of platelet-rich plasma on the recovery of blood flow in ischemic lower limbs and its possible mechanism in the model of ischemic hindlimbs in diabetic mice and the effect of PAI-1 on muscle fibrosis.Methods: 1.The preparation of PRP: After the mice were anesthetized,blood was taken from the vena cava and PRP was obtained by a second gradient centrifugation method.2.ELISA test of PRP: By using ELISA to detect the content of VEGF-A,PDGF-BB in PRP.3.Establishment of diabetic mouse model: Choose about eight weeks of general C57/BL16 mouse for the experiment.Injecting Streptozotocin(STZ)solution into mice abdominal cavity for five days,and then high fat,sugar feeding for 2weeks,measure their tail vein blood sugar levels every week.4.Established model of hindlimb ischemia:After the mouse was anesthetized,cut open the skin of the left inguinal position.The femoral artery of the left lower limb of the mouse was ligated by the lower margin of the femoral deep artery and the upper edge of the popliteal artery,and then the femoral artery was cut between two ligations.After rewarming,the blood flow in the lower part of the abdomen and the lower limbs of the mouse were scanned by laser doppler scanner,recording its perfusion recovery.Scanned and recorded the blood flow recovery of pre-and post-artery ligation and 3,7,11,14 days after the surgery.5.Experimental grouping: On the second day after operation,the mice were randomly divided into three groups,(1)control group(gastrocnemius injection of normal saline 50μL);(2)C57 PRP group(C57 source PRP 50μL injected into gastrocnemius muscle);(3)PAI-1-KO PRP group(PRP 50μL isolated from plasminogen activator inhibitor-1 knockout mice injected in the gastrocnemius muscle),six mice in each group.6.Specimen collection: After scanning on the 14 th day,the mice were sacrificed,bilateral adductor muscle and gastrocnemius muscle were harvested,partially fixed with 4% paraformaldehyde and then sent to the pathology section for embedding,partially frozen in liquid nitrogen and preserved in-80℃.7.Pathological examination:(1)HE staining: observe the muscle regeneration after ischemia and capillary distribution;(2)Immunofluorescence: The vascular endothelial cells were labeled with CD31,vascular smooth muscle cells were labeled with α-SMA,angiogenesis and pericyte coverage were observed,and angiogenesis and stability were evaluated;(3)Masson staining: observe the collagen deposition in the muscle.8.RT-PCR:The expression of VEGF-A,PDGF-BB,TGF-β,Type I collagen,Type III collagenin muscle tissues were analyzed by Real time PCR.9.Statistical methods: Data are presented as the mean±SEM and were analyzed by ANOVA and by unpaired two-tailed Student’s t test.P <0.05 was regarded as statistically significant.Results: 1.ELISA test of PRP: The level of VEGF-A and PDGF-BB in PRP from C57 mice and PAI-1 knockout mice had no significantly different.2.Establishment of a diabetic mouse model: After 5consecutive days of STZ injection,mice were fed with high-sugar and high-fat diet for 2 weeks.All the mice became diabetic mice and the model was successfully established.3.The effect of PRP on blood flow recovery of hindlimb ischemia model in mice: Compared with control group,PAI-1-KO PRP group and C57 PRP group increased blood flow in the lower limb ischemia area,but there was no significant difference between the two groups.4.HE staining showed that capillary distribution and muscle regeneration of PAI-1-KO PRP and C57 PRP group were better than that of control group.5.Immunofluorescence results showed that the positive expression of CD31 andα-SMA in the ischemic area of C57 PRP group and PAI-1-KO PRP group was higher than that of the control group,suggesting that PRP promoted the formation of microvessels and the stability of blood vessels.6.Masson staining results showed that all the three experimental groups showed different degrees of collagen deposition,and fibrosis of PAI-1-KO PRP group was improved compared with C57 PRP group.7.The results of RT-PCR showed that the expressions of VEGF-A,PDGF-BB and TGF-β in the ischemic side of PRP group were higher than those in the control group,but there was no significant difference in non-ischemic side.The expressions of type I collagen and type III collagen in the ischemic lateral gastrocnemius muscle of three groups had significantly different.The expression level of C57 PRP group was higher than that of control group,and the expression level of PAI-1-KO PRP group was lower than that of control group.Conclusions: 1.PRP can promote the recovery of blood perfusion in the ischemic lower limbs of mice so that it has a positive effect on the treatment of hind limb ischemia,while it’s better for diabetic mice.2.PRP can up-regulate expression of VEGF-A,PDGF-BB,TGF-β.3.PRP can promote the formation and maturity of blood vessels.4.The PRP of the PAI-1 gene knockout mice can improve the ischemia of lower limb and reduce the formation of fibrosis of muscle. |
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