| Research background and objective:The long-lasting unhealing foot ulcer is one of the serious complications that patients with diabetes mellitus(DM)are susceptible to suffer from.This kind of foot ulcer characterized by peripheral nerve damage and ischemia is clinically called diabetic foot.Studies have shown that the proportion of diabetic patient complicated with diabetic foot ranges from 15%to 20%,approximately 20%of these patients require amputation,and the amputation is a double blow to the patients`psychology and physiology.Delayed wound healing of diabetic ulcer involves a variety of complex mechanisms.At present,the pathological studies on diabetic foot ulcers are mainly focused on some pathogenic factors,such as microbial invasion,re-epithelialization disorder,persistent inflammatory response,impaired immune function and so on.Blood flow disorder is another potential pathogenic factor that accompanies the formation of all diabetic ulcers,which can seriously affect the normal healing of wounds.Many studies have emphasized the importance of adequate blood supply and angiogenesis in tissue repair and considered that local angiogenesis disorder and reduced blood supply is one of the important pathological changes of delayed chronic wound healing in diabetes.Therefore,the treatment strategy of strengthening the wound angiogenic response may effectively promote the healing of chronic diabetic wounds.Angiogenesis is a dynamic process coordinated by endothelial cells(ECs),perivascular supporting cells(vascular smooth muscle cells and pericytes)and immune cells.Angiogenesis disorder has been considered as a common pathological change in a variety of diseases,including cancer,peripheral arterial disease,diabetic vascular disease,rheumatoid arthritis and inflammatory bowel disease.The growth factor-mediated signal transduction and the alteration of extracellular components both have a regulatory effect on the angiogenesis process,and they are in a coordinated and balanced state.The vairous types of cells,including endothelial cells,smooth muscle cells,fibroblasts,platelets,immune cells and stem/progenitor cells,can participate in the coordination of this process by secreting relevant growth factors,among which basic fibroblast growth factor(b FGF),transforming growth factor?,transforming growth factor?,tumor necrosis factor?,vascular endothelial growth factor(VEGF)and phospholipase C-?(PLC?1),etc play a major role.Bone marrow mesenchymal stem cells(BM-MSCs)have the functions of differentiation,paracrine and immune regulation,and are considered to play an important role in the process of angiogenesis.In addition,Shin et al found that the impaired function of endogenous mesenchymal stem cells(MSCs)and the significant decreased MSCs migrated to the wound in diabetic animal model is one of the important factors leading to the difficulty of wound healing.At present,several mechanisms of MSCs have been found to accelerate wound healing by promoting angiogenesis,including secretion of angiogenic factors,differentiation into endothelial cells and/or pericytes to promote angiogenesis,recruitment of endogenous stem/progenitor cells,and regulation of extracellular matrix production or remodeling and immunosuppressive effects.In recent years,the therapeutic effect of exogenous MSCs in animal models of a variety of diseases,especially in the treatment of trauma and ischemic diseases has received widespread attention.A number of animal experiments have shown that exogenous MSCs injection therapy can accelerate the healing of chronic wounds such as diabetic wounds by promoting wound basal angiogenesis.However,the biological function of primary MSCs obtained from tissue decreased significantly after in vitro culture and expansion,resulting in a lower therapeutic effect than expected.Therefore,it is urgent to find a way to enhance the repair function of long-term cultured BM-MSCs,and further detect its effect on diabetic wound healing through animal experiments.c-Casitas b-lineage lymphoma(c-Cbl)is a proto-oncogene in cells,which is a member of the Cbl family.The protein it encodes is considered to be an E3 ubiquitin ligase and contains a characteristic RING Finger(RNF)domain,which can regulate a variety of signals mediated by cell membrane receptors.Experiments have confirmed that in the pathological process of tumor and retinal angiogenesis,inhibition of c-Cbl can enhance endothelial cell proliferation and angiogenesis induced by VEGF,thereby promoting this process.In addition,the study also found that in diabetic mouse model,knocking out the c-Cbl gene can improve obesity and insulin resistance induced by a high-fat diet.These results suggest that c-Cbl can affect the cellular functional changes by regulating signal molecular transduction and then participate in the process of angiogenesis,and c-Cbl gene plays an important role in diabetic animal model.However,whether and how c-Cbl plays a role in the functional changes of BM-MSCs after long-term expansion and culture is still unclear.Therefore,in this article,the SD rats were intra-peritionelly injected with streptozotocin(STZ)and were screened based on blood glucose standard to select type?diabetic rats for establishing diabetic wound model.The role of c-Cbl in the functional changes of BM-MSCs cultured in vitro for long-term expansion was studied,and observed the effect of regulated BM-MSCs on promoting diabetic wound healing in animal models.The research results are of great significance for the application of MS Cs in the treatment of diabetic and other chronic wounds,and provide a theoretical basis for improving the reduced functions of MSCs after expansion and culture in vitro.In addition,exploring the regulatory effect of c-Cbl on the angiogenic function of MSCs can better clarify the relationship between the functional changes of MSCs and angiogenesis,which has a certain reference value.Methods:1.The rat BM-MSCs were expanded and cultured in vitro and the activation level and interaction between c-Cbl and protein kinase B(Akt)were detected.The purchased Sprague-Dawley(SD)rat BM-MSCs were expansioned and cultured in vitro,and the protein expression levels of Y731c-Cbl/c-Cbl and S473-Akt/Akt in passage 3(P3),passage 6(P6)and passage 10(P10)cells were detected by western blotting(WB).The expression of c-Cbl was inhibited by locked nucleic acid modified antisense oligonucleotide gapmers(LNA Gapmers)in P10 cells,and the protein expression of S473-Akt/Akt was detected again.2.To observe the effect of c-Cbl on angiogenic factor paracrine and migration ability of long-term cultured BM-MSCs.The levels of vascular endothelial growth factor A(VEGFA),hepatocyte growth factor(HGF)and basic fibroblast growth factor(b FGF)secreted by P3,P10 and c-Cbl inhibited P10 BM-MSCs were determined by enzyme linked immunosorbent assay(ELISA).At the same time,the tube formation test was employed to detect the alterations of vascular-like structures formed by endothelial cells in the conditioned medium of each cell groups.Beyond that,the migration function of cells in differnt group was examined by transwell assay.3.To observe the effect of c-Cbl on the proliferation and senescence of BM-MSCs after long-term cultured in vitro.P3,P10 and c-Cbl inhibited P10 MSCs were stained with?-galactosidase staining kit to detect the ratio of senescent cells.Cell counting kit-8(CCK-8)test was used to detect the cell proliferation rate of each group at 24,48,72 and 96 hours,respectively.4.The establishment of rat diabetic wound model.First of all,SD rats were given by intraperitoneal injection of STZ at a dose of 65mg/kg to induce the type I diabetic rat model.Then,two symmetrical round full-thickness skin wounds were made on the back of rats with the diameter of 1.8 cm.One day after operation,BM-MSCs were injected around the wound sites for therapeutic intervention.5.To observe the effect of c-Cbl on the angiogenesis of diabetic wound promoted by long-term cultured BM-MSCs.The negative control P10 BM-MSCs(transfected with scramble LNA Gapmers),c-Cbl down-regulated P10 BM-MSCs(transfected with c-Cbl LNA Gapmers)and the same volume of phosphate buffer saline(PBS)were injected around the wound tissue of diabetic rats,respectively.Immunohistochemical assay was used to detect the expression of platelet endothelial cell adhesion molecule-1(PECAM-1/CD31)and VEGFA in the wound tissue at7 and 14 days after operation,in order to determine the angiogenesis level of the wound.6.To observe the effect of c-Cbl on the promotion of diabetic wound healing by long-term cultured BM-MSCs.Diabetic rats were injected with PBS,P10 and c-Cbl inhibited P10 MSCs around the wound tissue respectively,and the wound healing rate was calculated at 0,3,7 and 14 days after surgery.In addition,the paraffin sections of the wounds were collected 14 days after operation for hematoxylin and eosin(HE)staining and Masson staining,and the pathological scores were calculated.Results:1.In the BM-MSCs cultured by long-term expansion in vitro,the phosphorylation level of c-Cbl was significantly increased while the phosphorylation level of Akt was inhibited.The c-Cbl LNA Gapmer transfection could down-regulate the protein expression level of c-Cbl,and after the down-regulation of c-Cbl,the phosphorylation level of Akt was increased,indicating that c-Cbl mediates the phosphatidylinositol 3-kinase(PI3K)/Akt signal inhibition of BM-MSCs induced by long-term expansion in vitro to a certain extent.2.The paracrine angiogenic factor and migration ability of BM-MSCs,were inhibited by long-term expansion in vitro,and the paracrine and migration ability could be improved by down-regulating the c-Cbl expression.3.The proliferation ability of long-term cultured BM-MSCs decreased and showed a higher cellular senescence rate,while down-regulation of c-Cbl expression could enhance its proliferation ability and reduce the senescence rate to a certain extent,but it did not over-activate the proliferative activity of long-term cultured MSCs.4.The type I diabetic rats induced by STZ were successfully constructed,and on this basis,the wound model of diabetic rats was made.Besides,the model construction has a high success rate and strong stability.5.Down-regulation the expression of c-Cbl can improve the therapeutic effect of long-term cultured BM-MSCs on promoting angiogenesis of diabetic wounds.Compared with the control group,local injection of c-Cbl down-regulated BM-MSCs can significantly promote the early expression of VEGFA in wound tissue,and ultimately promote the angiogenesis process of diabetic wounds.6.Down-regulation the expression of c-Cbl can improve the therapeutic effect of the long-term cultured BM-MSCs on promoting diabetic wound healing rate.In addition,the histopathological scores analyzed by HE and Masson staining showed that the wound score after treatment with c-Cbl down-regulated BM-MSCs was significantly higher than that in the control group.Conclusions:1.Down-regulating the expression of c-Cbl can improve the inhibition of Akt phosphorylation and the reduction of angiogenic-related functions of BM-MSCs after long-term expansion and culture,indicating that the expression level of c-Cbl is related to the Akt signal and functional changes of BM-MSCs to a certain extent.2.Down-regulating the expression of c-Cbl can improve the therapeutic effect of long-term cultured BM-MSCs on angiogenesis and healing of wound in diabetic rats. |
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