The Role Of As-microRNA-205-5p Modified Mesenchymal Stem Cells In The Treatment Of Diabetic Foot

Purpose:Diabetic foot(DF),one of the most serious complications of diabetes mellitus(DM),is the leading cause of non-traumatic amputation.The pathogenesis of DF is still far from being full elucidated.However,numerous studies have demonstratedthat the foot ulcers resulted from neuropathy and vasculopathy,including reduced neovascularization,was the main cause of DF under the chronic long-term hyperglycemia condition.Thus,it is very significance to study how to effectively promote the angiogenesis of foot ulcers in the treatment of diabetic foot.Mesenchymal stem cells(MSCs)have high self-renewal ability and multi-directional differentiation potential,and hold great clinical value in cell replacement therapy and tissue engineering repair.Meanwhile,emerging studies indicate that autologous MSCs transplantation is the newest and potential treatment ofDF.Transplanted MSCs improvelocal angiogenesis and blood flow through self-differentiation,releasing vascular endothelial growth factor(VEGF)and so on,which resulting in attenuating effects of DF.However,the function of autologous MSCs is affect by long-term hyperglycemia.The number and quality of endogenous MSC in diabetic patients also showed a downward trend,and function was also affected.Non-coding microRNAs(miRNAs)are endogenous non-coding single-stranded small RNAs with post-transcriptional regulatory activity,high conserved size and 19-25 bp in size,and are linked to the target gene 3’-untranslated region(3’-untranslated region)-UTR),a miRNA can regulate the expression of multiple genes,and the same target gene can be regulated by multiple miRNAs.It has been found that miRNAs are involved in the development and progression of diabetes mellitus and its chronic complications.At the same time,miRNAs play an important role in the regulation of MSCs function.Therefore,it is very important to study how to improve the function of mesenchymal stem cells for the treatment of diabetic foot.Method:1.Screen the miRNAs candidates associated with the pathogenesis of DF1.1 Using WB and PCR to screen the significantly difference expression protein between the normal and diabetic food tissues.Isolating MSCs.Healthy volunteers draw bone marrow,MSCs were isolated and identify by detecting cell surface antigen and cell differentiation potential;take the identified MSCs in culture.The cells were identified by PCR and WB,and it was clear that miRNAs were regulated by transcriptional regulation of VEGF in mesenchymal stem cells.The miRNA-miR-205-5p,which could regulate VEGF,was screened by bioinformatics technique:Targetcan.All predicted results were integrated to select miRNA-mir-205-5p,which may regulate VEGF expression.(The study has passed approval of The First Affiliated Hospital of Nanchang University Medical Research Ethics Committee)2.Investigate the potential role of miR-205-5p in the pathogenesis of DF in vitro and in vivo2.1 Investigated VEGF expression in MSCs using as-miR-205-5pto loss function of miR-205-5p.2.2 Establishment of diabetic foot model in NOD/SCID miceThe reasons for the use of immunodeficient mice were to avoid the effects of xenograft rejection(mice on human source MSCs)on the experiment.10-weeks-old male immune-dificient NOD/SCID mice was used to establish the diabetic model by intraperitoneal injection of streptozotocin(STZ)at 130 mg/kg.One week later,blood glucose was measured and the blood was taken from the tail to measure the blood glucose level.Blood glucose>ll.1mmo/L-16.7mmo/L(200mg/dl-300mg/dl).Foot skin ulcer injury mouse model,ulceration area is 2 mm~2.Mice were received intradermal injection of 10~6 MSCs(null and as-miR-205-5p transfected MSCs)at the site of skin ulcer.The wound gaps were measured with vernier caliper.Imunostaining was used to measure the vascularization around the wound.2.3 The effects of down-regulation of mi R-205-5p in MSCs on DF in vivoPeritoneal injection of as-miR-205-5p-modified mesenchymal stem cells or control group mesenchymal stem cells was administered subcutaneously around the lesion site to the diabetic foot mouse model.As-miR-205-5p modified MSCs or non-specific modified control MCSs was subcutaneous injected around the in diabetic foot lesions.After 4 weeks,the wound healing,and the change of blood vessel density in the ulcer lesion were observed.After four weeks to observe the healing of ulcer wounds,Letin staining ulcer treatment of vascular density changes.Result:1,The expression of VEGF was decreased in DF tissues,and regulated by miR-205-5p1.1 Compared with normal human foot tissue,Through ELISA experiments,WB detection,the expression of VEGF in diabetic foot tissue was significantly decreased(P<0.05).1.2 In vitro separation of mesenchymal stem cells,The expression of CD29,CD34,CD44,CD45,CD166 and HLA-DR were detected by flow cytometry.MSCs were identified by in vitro differentiation experiments:Alcian blue staining,Von Kossa staining,oil red O color identification mesenchymal Stem cell differentiation function.1.3 Using the bioinformatics data analysis(TargetScan),reverse sequencing analysis data,we identified that miR-205-5p was expressed in MSCs,and couldbind to the 3’-UTR region of VEGF,which suggested that VEGF was a potential target of miR-205-5p.Further study confirmed that VEGF was regulated by miR-205-5p.Further amplification of miR-205-5p by PCR,WB and luciferase reporter gene could inhibit the expression of VEGF protein.2.MiR-205-5p attenuated DF through regulating VEGF expression in MSCs2.1 In vitro experiments shown that loss function of miR-205-5p using as-mi R-205-5P did not affect the function of MSCs.However,transfection of as-mi R-205-5p up-regulated VEGF expression in MSCs.2.2 NOD/SCID mice received STZ by ntraperitoneal injection and followed skin injury and by silicone ring around the wound were successful to induce diabetic and DF model.DF mice received either null-MSCs or as-miR-205-5p-MSCs did not alter hyperglycemia and beta cell mass quantification.However,as-miR-205-5p-MSCs injection significantly improved wound healing and increased vessel density.Conclusion:Taking together,our data suggest that miR-205-5p suppression in MSCs may improve their therapeutic effects on DF,seemingly through augmentation of VEGF-mediated vascularitziton.This study revealed that as-miR-205-5p modified MSCs were effective in the treatment of diabetic foot.And this study provided a new target and direction for clinical treatment of diabetic foot.