| BACKGROUNDDiabetic foot ulcer is one of chronic complications of diabetes,and it is one of important reasons for amputation,which gave great stress to the patients,and brought economic burden to the society and the family of patients.According to reports,the lower limb skin ulcer patients accounted for 4%to 10%of patients with diabetes.And the risk of re-amputation of diabetic foot ulcer was 50%.With the improvement of our living standard,there will be more and more diabetes patients in our society.It is important for us to thorough research the mechanism of diabetic foot ulcer.The repair of injury is a complex but orderly biology process,including inflammation,granulation multiplies and moulding of injury and so on.There are many cells,extra cellular matrix,cytokine and tissue participating in every period of the repair of injury.There are many factors to have influence on the repair of injury and cause un-healing.It is important to research the change of the skin and foot ulcer of diabetes patients.Some studies showed that there were many factors which had relationship with injury repair of diabetic foot ulcer,including(1) cell metabolism disorder caused by low insulin level(2) disorder of inflammatory reaction;(3) influence of AGEs in skin of diabetes patients.Inflammatory reaction is the first step of injury repair;including acute stage and chronic phase.In acute stage of inflammatory,the main reactions are vascular reaction and infiltration of inflammatory cell,including neutrophilic granulocyte.The functions of these cells include phagocytizing bacterium,excreting cytokine and chemotaxis to other inflammatory cell.In chronic phase,the main cells in area are macrophage and lymphocyte.The functions of these cells are excreting cytokine, antigen presentation,promoting angiopoiesis and lymphangiogenesis.If damaged in acute stage or chronic phase,it would be delayed in repairing of injury.Recent study showed that delay and disorder of inflammatory reactions were the main reasons of impaired of diabetic foot ulcer.Macrophage is very important for injury repair in chronic phase of inflammatory.The number and function of macrophage have influence on process of injury repair.In chronic phase of inflammatory,macrophage can secret a lot of chemotatic factors,such as TNF-αand IL-1,which can attract inflammatory cells to injury area.In the stage of granulation,macrophage can secret factors,such as VEGFC,which participates in angiopoiesis and lymphangiogenesis.In the stage of moulding,macrophage can promote proliferation and cell division of fibroblasts.So those from above,the number and function of macrophage are correlated with all the stage of inflammatory.In the past studies,angiopoiesis was important for injury repair.But in recent studies,lymphangiogenesis was earlier than angiopoiesis,and lymphangiogenesis of VEGFR3 positives was the preparer for angiopoiesis.And in other studies,the author found that the function of lymphatic vessel was not only draining but also transporting dendritic cell to the injury area.So lymphangiogenesis was the same important for injury repair as angiopoiesis.So whether decrease of lymphatic vessel formation in diabetic foot ulcer or not? And what was the reason for it? All of these are not report in past study.There are lymphatic vessel ancestral cell in peripheral blood,and the mark is CD34~+/CD133~+/VEGFR3~+.The ancestral cell will form lymphatic vessel in injury area under VEGFC.It will be decreased of lymphatic vessel endothelial cell because of less VEGFC or reduced ancestral cell in bone marrow.But we did not know the change of VEGFR3~+ cells in peripheral blood of diabetes.And we did not know the relation between the number of VEGFR3~+ cells and diabetic foot ulcer.And we also did not know the influence of lymphatic vessel ancestral cell in diabetes patiens.All of this was not reported in past study.In normal skin,macrophages located in the basilar part between epidermis and dermis.The macrophages in basilar part are the first line of defense of our skin.If skin was damaged,the macrophages would transmigrate to the damaged area and produced a marked effect.In a past study,the number of macrophage in the skin of db/db mice was decreased.And the author thought it was the reason for un-healing. But there was not a study show what was the reason of macrophage damaging.Based on above,we divided our design into three parts.The aim was to explore the changes of number and function of macrophages and lymphatic vessel endothelial cells in diabetic foot ulcer skin,non-diabetic foot skin and normal skin tissue and the cause of those.We also wanted to explore the secretion of VEGFC in macrophages treated by glucose and AGEs. Chapter 1 Decreased macrophage number and activation and reduced lymphatic vessel formation in non-anemic diabetic foot ulcer skin tissueOBJECTIVETo investigate the number and function of macrophage and lymphangiogenesis in diabetic foot ulcer skin tissue,non-diabetic foot ulcer skin tissue and normal skin tissue.METHODS1.Diabetic foot ulcer skin tissue,non-diabetic foot ulcer skin tissue,matched with age,gender and the time of ulceration.,were experimental groups,and normal skin tissue was control group.2.Investigate the basic histological change of skin tissue in three groups.3.Detected protein of CD68,VEGFR3,LYVE-1,and VEGFC in skin tissue.4.Gene expression of CD68,VEGFR3,LYVE-1,VEGFC in skin tissue detected by RT-PCR.5.Detected protein of CD68 and LYVE-1 by confocal microscopy and investigate the relation between CD68 and LYVE-1.RESULT1.Age among DF group,NDF group and NC group were not significantly different (P＞0.05),and time of ulceration between DF group and NDF group was not significantly different(P＞0.05).2.Typical histological changes could be seen in skin tissue of DF group and NDF group,including thickening of epidermis,derangement of dermis and so on. Thickness of epidermis were increased in NDF group and DF group,1.761±0.983mm, 1.790±0.102mm than that of in NC 0.970±0.052mm(P＜0.001).Thickness of dermis were decreased in NDF group and DF group,1.471±0.063mm,1.434±0.062mm than that of in group NC 2.787±0.109mm(P＜0.001).3.Protein of CD68,VEGFR3,LYVE-1 and VEGFC located in basilar part of NC group but disappeared in skin tissue of DF group and NDF group.In inflammatory area of dermis,expression of CD68 in NDF group was more than that of in DF group. The VEGFR3 positive cells,LYVE-1 positive cells were located in the borderline between dermis and epidermis in NDF group,but less in DF group.VEGFC was located around collagen in dermis in NDF group,but less in DF group.Protein of CD68 in epidermis in group NC,NDF and DF were 77.200±2.588%; 33.800±4.147%;19.400±1.140%.Protein of CD68 in dermis in group NC,NDF and DF were 9.400±1.140%;79.800±1.304%;57.600±2.074%.Protein of LYVE-1 in epidermis in group NC,NDF and DF were 87.600±1.140%;16.600±1.673%; 9.600±1.342%;Protein of LYVE-1 in dermis in group NC,NDF and DF were 92.000±0.707;187.000±3.033:76.600±1.673.Protein of VEGFR3 in epidermis in group NC,NDF and DF were 81.400±1.140%;16.600±1.140%;8.800±1.483%; Protein of VEGFR3 in dermis in group NC,NDF and DF were 82.800±1.643; 179.400±2.608;71.800±3.347.Protein of VEGFC in epidermis in group NC,NDF and DF were 79.200±1.304%;22.600±1.140%;9.200±2.168%;Protein of VEGFC in dermis in group NC,NDF and DF were 115.400±2.702;193.000±3.162; 103.000±2.915.4.Gene expression of CD68 in group DF was significantly less than group NC (P＜0.001).Gene expression of CD68 in group NDF was significantly higher than group NC(P＜0.001).Gene expression of CD68 in group DF was significantly less than group NDF(P＜0.001).Gene expression of VEGFR3 in group DF was significantly less than group NC(P＜0.001).Gene expression of VEGFR3 in group NDF was significantly higher than group NC(P＜0.001).Gene expression of VEGFR3 in group DF was significantly less than group NDF(P＜0.001).Gene expression of LYVE-1 in group DF were significantly less than group NC(P＜0.001). Gene expression of LYVE-1 in group NDF was significantly higher than group NC (P＜0.001).Gene expression of LYVE-1 in group DF was significantly less than group NDF(P＜0.001).Gene expression of VEGFC in group DF was significantly less than group NC(P＜0.001).Gene expression of VEGFC in group NDF was significantly higher than group NC(P＜0.001).Gene expression of VEGFC in group DF was significantly less than group NDF(P＜0.001).5.CD68 positive cells were in basilar part of epidermis,and LYVE-1 positive cells were in borderline between epidermis and dermis.CD68 positive cells and LYVE-1 positive cells were not overlapping after built up by confocal microscopy.CD68 positive cells were disappeared in epidermis in group NDE neither been of LYVE-1. But CD68 positive cells and LYVE-1 positive cells could be conduplicate in lympgatic vessel endothelial cell by confocal microscopy,and the main signal was CD68.CD68 positive cells and LYVE-1 positive cells were disappeared in epidermis in group DF.In inflammation area of dermis,we found CD68 positive cells and LYVE-1 positive cells,but we could not find stacked signal in lymphatic vessel endothelial cell.CONCLUTIONS1.It was more thickening of epidermis in diabetic foot than in non-diabetic foot.And it was more thinning of dermis in diabetic foot than in non-diabetic foot.And it was absent and disorder of collagen in dermis in DF and NDF.It maybe had relations with débridement and chronic inflammation. 2.It was decreased of the number,function of macrophages in the skin tissue of DF. And it was decreased of the lymphatic vessel formation in the skin tissue of DF.But it was increased of the number,function of macrophages in the skin tissue of NDF.And it was increased of the lymphatic vessel formation in the skin tissue of NDF We supposed that it was related with impaired wound healing.3.The macrophages were appeared in the lymphatic vessel in NDF.And it was negative in DF.We supposed that the macrophages in NDF skin tissue could change into lymphatic vessel endothelial cell.,but couldn't in DF.Chapter 2 Expression of VEGFR3 and number of VEGFR3 positive cells in peripheral blood leukocyte of non-ischemia diabetic foot ulcer patientsOBJECTIVETo investigate gene expression of VEGF3 and number of VEGFR3~+ cells in peripheral blood leukocyte of diabetes patients and non-ischemia diabetic foot ulcer patients.METHODS1.Leukocytes were from four groups,group DF,group DM1(HbA1C＜7%),group DM2(HbA1C≥7%) and group NC.2.Leukocytes were from anticoagulated blood 5ml. 3.Gene and protein expression of VEGFR3 of leukocyte in every group was detected by RT-PCR and by immunofluorescence.4.Analysis relation between gene expression of VEGFR3 and clinical data,such as FBG,TG,CH,SBP,DBP,HbA1C,CP and so on.5.Number of VEGFR3 positive leukocyte was detected by immunofluorescence.RESULTS1.Gene expression of VEGFR3 in group DF,in group DM1 and in group DM2 were significantly less than that of in group NC(P＜0.001).Gene expression of VEGFR3 in every group were group DF(Mean Rank=8.50),group DM2(Mean Rank=12.50), and group DM1(Mean Rank=25.50).2.Gene expression of VEGFR3 had relation with FBG(Spearman's r=-0.518 P＜0.01);with SBP(Spearman's r=-0.551 P＜0.01);with DBP(Spearman's r=-0.391 P＜0.05) and with HbA1C(Spearman's r=-0.633 P＜0.01).3.Protein of VEGFR3 of group DM1 and group DF were significantly less than that of in group NC(P＜0.001).And Protein of VEGFR3 of group DM2 was significantly less than that of in group NC(P＜0.001).4.Number of VEGFR3 positive leukocyte in group DF,in group DM1 were significantly less than that of in group NC(P＜0.001).CONLUSION1.Gene expression of VEGFR3 of diabetes patients was less than that in normal control group,and the higher of HbA1C,the less of gene expression of VEGFR3. Gene expression of VEGFR3 had no relation with HbA1C in non-ischemia diabetic foot ulcer patients. 2.There were positive correlations among gene expression of VEGF3 and FBG, HbA1C,SBP and DBP.3.Number of VEGFR3 positive cells in group DF,DM2 and DM1 was significantly less than that of in group NC,and it was a possible reason for impaired diabetic foot ulcer.Chapter 3 Basal secretion of VEGFC and phagocytosis ability in U937 cell line influenced by high Glucose and/or AGEOBJECTIVETo explore basal secretion of VEGFC and phagocytosis ability in U937 cell line influenced by high Glucose and/or AGE.METHORDS1.Detected the basal secretion of VEGFC influenced by Glucose and/or AGEs in different time and/or in different concentration.2.We divided U937 cell into four groups,glucose group(15mmol/L G,24H), AGEs group(25ug/mlAGE,24H),combination group(15mmol/L G,24H and 25ug/mlAGE,24H) and NC group.3.Cytoactive was detected by trypan blue staining.4.Gene expression of VEGFC was detected by RT-PCR.5.Protein of VEGFC was detected by immunofluorescence.6.Ability of phagocytosis was detected by Indian ink phagocytosis test. RESULTS1.There were four groups through preliminary experiment,include G group (15mmol/L G,24H),AGE group(25ug/mlAGE,24H),combination group(15mmol/L G,24H and 25ug/mlAGE,24H) and NC group.2.Cytoactive was the same in every group.There was no significant difference in every group.3.Gene expression of VEGFC in all group was significantly less than that of in group NC(P＜0.001).Gene expression of VEGFC in group combination and group AGE were significantly less than that of in group NC(P＜0.001).4.Result of immunofluorescence was that we selected 5 high power field randomly and calculated gray scale.Experimental group was decreased of protein VEGFC than group NC.Group G was decreased of protein VEGFC than group AG. Group GAG was decreased of protein VEGFC than group GG.Group GAG was decreased of protein VEGFC than group AG.5.Ability of phagocytosis in group combination was significantly lower than that of in group NC(P＜0.001).Ability of phagocytosis in group G was significantly lower than that of in group NC(P＜0.01).Ability of phagocytosis in group AGE was significantly lower than that of in group NC(P＜0.05).CONCLUSION1.High glucose,AGE and combination of glucose and AGE had no influence on the activity of U937 cell line.2.Gene expression of VEGFC in group combination was the least,the second was group G,and the third is group AGE.3.Protein of VEGFC was the same as gene expression of VEGFC. 4.Ability of phagocytosis in group combination was the least,the second was group G and the third was group AGE.
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