The Expression Of PDGFR-? In Granulation Tissue Of Diabetic Foot Wound

Objective: Diabetic foot is one of the most serious complications of diabetes,which is most persistent and is mainly caused by vascular disease,neuropathy,infection,critical lower limb ischemia and the abnormality of local microenvironment.The mechanism of its delayed healing is unclear till now,so this issue,is to observe the expression of platelet derived growth factor receptor ?(PDGFR-?)in granulation tissue of diabetic foot wound and non-diabetic wound,which to investigate the reasons of persistence of diabetic foot wound further.Methods:1 From January 2015 to June 2016,10 patients met the inclusion criteria,who were suffered from diabetic foot ulcers.On the contrast,10 patients with non-diabetic ulcers as a control group.2 All of patients were received of routine debridement after admission for10 to 15 days.Fresh granulation tissue were collected from the skin tissues both groups,which were divided into two-part: one was fixed by 4%paraformaldehyde,then was undergoing HE staining and immunocytochemical staining,the other was put into liquid nitrogen immediately,for its rapidly cooling.Then the tissue specimens were stored in the refrigerator-80? quickly,for Western blot analysis and Real-time PCR detection.3 To observe the structure of granulation tissue in HE sections under the optical microscope(400×)and select 10 views for each HE section randomly.The quantity of capillaries was counted and the average value was used to observe the growth of granulation tissue.4 Immunohistochemical staining was performed on paraffin slice and the qualitative analysis of the expression of PDGFR-? was made.Firstly,theexpression of PDGFR-? was observed under light microscope(400 ×),the brown yellow or brown granules was defined as positive staining.The digital medical image analysis system(MoticMedical 6)was used to analyze the PDGFR-? optical density of 10 hot spots fields on each slice which selected randomly.The average optical density was used to semi quantitative analysis of PDGFR-?.5 To detect the expression of PDGFR-? on protein level with Western blot.The protein bands of interest which expressed as grey density were analyzed by Image J software on both groups and the GAPDH was used as endogenous reference.The relative expression of PDGFR-? in each band was expressed by the ratio of the grey density of PDGFR-? and the corresponding GAPDH.The expression of PDGFR-? was made by protein quantitative analysis.6 Real-time PCR was applied to observe the expression of PDGFR-? on gene level in granulation tissue.PCR was used to collect the fluorescence signal.We got the relative quantity of each target on gene expression,that is,the quantity of PDGFR-? mRNA.The expression of PDGFR-? was made by gene quantitative analysis.Results:1 The observation of granulation tissue: the granulation tissue was observed in two groups,the tissue was fresh which was bright red,granular growth,Soft touch and hemorrhaging in the control group.The granulation tissue of experimental group was aging,which showed that the granular growth was not obvious and the feeling was rather tough.2 HE staining of granulation tissue: HE stained slices were observed under microscope both group.On the control group,the granulation tissue showed with dense capillaries and numbers of fibroblasts infiltration.The granulation tissue capillaries of experimental group were sparse and the distribution of fibroblasts were scattered compared with the control group.The quantity of mean capillary were input to statistical analysis,we found that the quantity of capillaries in diabetic foot group was significantly lower than thatin control group(P < 0.01).3 The observations of immunohistochemical staining under the Microscope: a certain number of brown yellow or brown particles were observed on both groups,in which more brown yellow particles on control group.Compared with the control group,there was a significant decrease of brown granules in the granulation tissue on experimental group.PDGFR-? in granulation tissue was made by semi quantitative analysis and the average optical density was represented as the expression of PDGFR-?.The average optical density in granulation tissue of diabetic foot group was significantly lower than the control group(P < 0.05).4 The results of Western blot analysis showed that: the expression of PDGFR-? was input into quantitative analysis on both groups.The relative protein expression of PDGFR-? in granulation tissue of control group and was0.61±0.13,the relative protein expression of PDGFR-? in granulation tissue of diabetic foot group was 0.33±0.10,which indicated that the relative protein expression of PDGFR-? in granulation tissue of diabetic foot group was lower than control group(P < 0.01).5 The results of Real-time PCR showed that: PCR was used to detect the gene expression of PDGFR-? which were made by statistical analysis on both groups.On the control group,PDGFR-? relative gene expression value was1.32±0.49 and on diabetic foot group was 0.79±0.16.Compared with the control group,the content of PDGFR-? mRNA in granulation tissue of diabetic foot group was significantly lower(P<0.01).Conclusions:1 On the control group,the granulation tissue shows hyperplasia,numbers of capillary formation,a large amount of fibroblast infiltration.Compared with the control group,the number of capillary in the granulation tissue on diabetic foot group was less,the distribution of fibroblasts cells were scattered and the granulation tissue was dysplasia.2 Compared with the control group,the content of PDGFR-? in granulation tissue on diabetic foot group was significantly lower.It may berelated to the ischemia and hypoxia of the wound which was caused by vasculopathy and neuropathy,so that PDGFR-? can not play its biological role,which may contribute to the delayed healing of diabetic foot.