| Purpose: According to statistics,the prevalence of diabetes is increasing year by year.At present,1 out of 11 adults in the world has diabetes,and about25% of diabetic patients can develop and become diabetic foot patients.The annual incidence of diabetic foot in my country has reached 8.1%.As one of the most serious chronic complications of diabetic patients,diabetic foot is mainly caused by the pathological changes of nerves and blood vessels in the foot,which leads to the prolonged and unhealed wound of diabetic foot.The previous treatment methods for diabetic foot,such as continuous dressing change,continuous closed vacuum suction on diabetic foot wounds,non-surgical treatments such as leech,and surgical treatments such as debridement,skin grafting,vascular reconstruction,and nerve decompression,but the curative effect is not good.The treatment cycle of diabetic foot is long and the prognosis is poor.It has been plagued by doctors and patients for a long time.How to treat severe diabetic foot to avoid amputation has become a world problem.In recent years,Tibia Transverse Transport TTT has achieved good clinical effects in the treatment of diabetic foot.The results of the study show that TTT surgery can effectively induce the regeneration of the microvascular network of the diabetic foot wound.However,the related mechanism is not yet fully understood.The purpose of this project is to explore the mechanism of TTT treatment of diabetic foot to promote vascular regeneration.Method: This study is divided into three parts.The first part: TTT treatment of diabetic foot to promote blood vessel regeneration phenomenon analysis.Collect preoperative and postoperative imaging data and tissue specimens of clinical patients.CTA and CT perfusion were used to compare the vascular conditions of the diabetic foot before and after the operation,and the local tissue samples of the foot ulcer wound before the operation and 1 month after the operation were collected for HE staining and blood vessel-related markers such as CD31 and SMA-α and other immunohistochemical analysis.The second part: TTT activates endothelial cells and the increase of chemokines promotes angiogenesis.Collect the peripheral blood of clinical patients before and 1 month after the operation for ELisa to detect the related growth factors that promote the activation of endothelial progenitor cells and flow cytometry to detect the content of endothelial progenitor cells in the peripheral blood,and collect the local wound tissue for immunohistochemistry Detection.ELisa was used to detect growth factors that promote angiogenesis and factors that activate endothelial progenitor cells such as VEGF,PDGF-BB,TGF-β1,etc.in plasma.At the same time,flow cytometry is used to detect endothelial progenitor cells(key cells that promote angiogenesis)markers such as CD34,CD133,CD309,etc.Immunohistochemistry was used to detect the local content of growth factors that promote vascular regeneration,such as VEGF,PDGF-BB,and TGF-β1.The third part: TTT promotes angiogenesis through the Ras/Raf/MEK/ERK signaling pathway.Collect the local tissue samples of patients before and 1 month after the operation and use immunohistochemistry to detect the content of key factors Ras,Raf,MEK/ERK in the pathway.Western-blot was used to detect the contents of Ras,Raf,and MAPK6 in fresh tissue samples from the edge of the diabetic foot wound 1 month after the TTT operation and before the operation.Results: 1.This experiment collected 20 eligible patients who completed the follow-up,including 13 males(65%),7 females(35%),age(58±10)years old,and diabetes course(18±8)years,Diabetic foot course(1.4±0.8)years,Texas staging(2B: 0 cases(0%),2C: 4 cases(20%),2D: 8 cases(40%),3B: 4cases(20%),3C: 2 cases(10%),3D: 2 cases(10%))Diabetic foot comprehensive classification(Type I: 2 cases(10%),Type II: 9 cases(45%),Type III: 6 cases(30%),type IV: 3 cases(15%)).The 20 patients selected this time all had tissue samples and imaging data before and after surgery,and completed follow-up.2.After undergoing TTT surgery,CTA and CT perfusion of the feet of diabetic foot patients found increased blood vessels.The relevant data has been published in English journals.3.HE staining was performed on the marginal tissues of the local wound of diabetic foot before TTT operation and 1 month after operation.HE scored the three aspects of angiogenesis,granulation tissue formation,epidermal and dermal regeneration,preoperative angiogenesis(1.000 ± 0.000),postoperative angiogenesis(3.050± 0.887),the difference was statistically significant(P < 0.05).Preoperative granulation tissue formation(1.000±0.000)and postoperative granulation tissue formation(3.000±0.794),the difference was statistically significant(P<0.05).Preoperative epidermal and dermal regeneration(1.500±0.513),postoperative epidermal and dermal regeneration(2.450±0.510),the difference was statistically significant(P<0.05).It can be seen that postoperative angiogenesis,granulation tissue formation,and epidermal and dermal regeneration are significantly increased than before.4.The immunohistochemical detection of blood vessel-related markers on the local wounds of diabetic feet showed that the preoperative CD31 positive cell rate was(0.409±0.046)% and postoperatively(1.205±0.049)%,the difference was statistically significant(P<0.05).The positive rate of SMA-α before operation was(3.1205 ± 0.942)%,and after operation(15.550±1.505)%,the difference was statistically significant(P<0.05).The blood vessels in the local tissues increased significantly after surgery.5.The Ilisa experiment detects the changes in the levels of VEGF,TGF-β1,and PDGF-BB in the peripheral blood plasma of diabetic foot patients before and 1month after the operation.The preoperative VEGF concentration was(282.8±19.79)pg/ml,and the postoperative concentration was(1089±29.55)pg/ml,the difference was statistically significant(P<0.05).The concentration of TGF-β1 before operation was(8.702±0.948)pg/ml,and the concentration after operation was(33.85±5.463)pg/ml,the difference was statistically significant(P<0.05).The concentration of PDGF-BB before operation was(344±45.94)pg/ml,and the concentration after operation was(3153±971.7)pg/ml,the difference was statistically significant(P<0.05).The concentrations of VEGF,TGF-β1,and PDGF-BB in the blood were significantly increased one month after TTT operation.6.The contents of VEGF,TGF-β1 and PDGF-BB were detected by immunohistochemistry on the local wound tissues of patients before and 1 month after operation.The positive cell rate of VEGFR-2 was(0.495±0.037)% before operation and(2.819±0.264)% after operation,the difference was statistically significant(P<0.05).The positive rate of TGF-β1cells before operation was(3.063±0.167)%,and after operation(7.045±0.141)%,the difference was statistically significant(P<0.05).The positive cell rate of PDGF-BB was(2.912±0.131)% before operation and(6.886±0.146)% after operation,the difference was statistically significant(P<0.05).The levels of VEGF,TGF-β1,and PDGF-BB in the local tissues of diabetic foot after TTT were significantly increased compared with those before operation.7.Perform immunohistochemical detection of the endothelial cell marker CD34 on local tissues.The positive cell rate was(1.036 ± 0.138)%before operation and(4.596 ± 0.710)% after operation,the difference was statistically significant(P < 0.05).Flow cytometry was used to detect the content of endothelial progenitor cells in peripheral blood,and the markers CD34/CD133/CD309 were used to detect CD34(0.813±0.066)% before surgery and(4.047±0.232)% after surgery,the difference was statistically significant Significance(P<0.05).CD34+,CD133+,and CD309+ were(0.047±0.007)%before operation and(0.522±0.042)% after operation,the difference was statistically significant(P<0.05).The content of local endothelial cells after TTT was significantly higher than that before surgery,and the content of endothelial progenitor cells in peripheral blood was also significantly higher than that before surgery,indicating that TTT can activate the proliferation and migration of endothelial progenitor cells,and promote the formation of diabetic foot vascular network and wound healing heal.8.Perform immunohistochemistry on local tissues to detect the content of Ras,Raf,MEPK6/ERK3,the key factors of Ras/Raf/MEK/ERK signaling pathway in local tissues.The results showed that the preoperative positive rate of Ras was(1.655±0.674)%,and the postoperative positive rate was(13.195±3.093)%,the difference was statistically significant(P<0.05).The preoperative positive rate of Raf was(2.103±1.029)%,and the postoperative positive rate was(11.322±1.674)%,the difference was statistically significant(P<0.05).The preoperative positive rate of MEPK6/ERK3 was(1.591±0.548)%,and the postoperative positive rate was(16.515±2.336)%,the difference was statistically significant(P<0.05).Western-blot was used to detect the contents of Ras,Raf,MAPK6/ERK3,the key factors of Ras/Raf/MEK/ERK signaling pathway in fresh tissues of local wounds.The results showed that the contents of Ras,Raf,and MAPK6/ERK3 were(0.231±0.021),(0.253±0.022),(0.288±0.017)before operation,and(1.536±0.058),(1.811±0.091),(1.723±0.107),the difference was statistically significant(P<0.05).It can be concluded that TTT can activate the Ras/Raf/MEK/ERK signaling pathway to promote the regeneration and repair of the vascular network of the diabetic foot wound.Conclusion: 1.TTT is used for the treatment of diabetic foot patients.CTA and CT perfusion confirmed the regeneration of the vascular network at the edge of the ulcer wound.The increased levels of local vascular markers CD31 and SMA-α further indicate the increase of local blood vessels in the diabetic foot and promote wound healing.2.TTT can promote the increase of growth factors VEGF,PDGF-BB and TGF-β1,promote angiogenesis and activate the proliferation and migration of endothelial progenitor cells.3.After TTT operation,the endothelial progenitor cells in the peripheral blood of patients increased significantly,and the local endothelial cells also increased.This indicates that endothelial progenitor cells may be activated to promote vascular regeneration and repair of diabetic foot wounds.4.The key factors of Ras/Raf/MEK/ERK signaling pathway after TTT increase in the margins of diabetic foot wounds,indicating that TTT surgery may promote blood vessel regeneration through the cascade of Ras/Raf/MEK/ERK signaling pathways. |
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