| Objective: By analyzing the expression difference of the transforming growth factor-?1(TGF-?1)of diabetic foot wound granulation tissue between which treated by the negative pressure wound therapy(NPWT)and which treated by conventional gauze therapy,to investigate how NPWT effect the expression of TGF-?1 of diabetic foot wound granulation tissue on both protein and mRNA level and provide theoretical basis of NPWT on diabetic foot.Methods:1 A total of 40 patients with diabetic foot wounds hospitalized in the department of endocrinology of Bethune International Peace Hospital of PLA from November 2013 to March 2015 and in accordance with the inclusion criteria and exclusion criteria were enrolled in this clinical trial.Communicate with the patient who signed informed consent voluntarily.All the 40 volunteers were randomly divided into NPWT group and control group through the computer randomized algorithms,with 20 cases in each group.2 Patients in NPWT group were treated with negative pressure wound therapy,while patient in the control group were treated with conventional gauze therapy.Granulated tissue was harvested on the day before treatment(day 0)and the 7th day of the treatment(day 7)from each patient of NPWT group and control group.Each biopsy was divided into three parts and labeled as part A part B and part C respectively.3 To observe the formation of granulation tissue,biopsy of part A was fixed in 4% paraformaldehyde immediately after it was obtained from the living and preserved at 4 ? for paraffin embedding until all samples were collected to complete.4 Slice part of the tissue of group A which was paraffin embedded for HE staining and observe the growth of granulation under the optical microscope(Magnification,400×).5 Slice part of the tissue of group A which was paraffin embedded for TGF-?1 staining with immunohistochemistry and examined in a optical microscope(Magnification,400×).Brown color denotes positive staining.Quantitative analysis of cellular TGF-?1 expression was evaluated by measuring positively stained areas in wound biopsy sections(5 random fields for each sample)using a digital medical image analysis system(MoticMedical6.0)and expressed as mean optical density(OD).6 To further determine the protein levels of TGF-?1,biopsy of part B was stored in the refrigerator of-80? until all samples were collected to complete for Western blot analysis.The ?-actin was used as an endogenous loading control.The protein bands of interest were qualified by Image J software and expressed as grey density.TGF-beta 1 content of each band was finally represented by the ratio of the grey density of TGF-beta 1 and the grey density of the corresponding ?-actin.7 Biopsies of part C was preserved in liquid nitrogen immediately and sent to the laboratory for real-time PCR(RT-RPR).The upstream and downstream PCR primers for TGF-?1 were designed as 5?CCCACAACGAA ATCTATGACA3? and 5?AGAGCAACACGGGTTCAG3?.All experimental work was carried out under RNase-free environment.GAPDH was served as an internal control.The mRNA expression levels were calculated using the2-CT method,and were quantified with normalization to GAPDH.According to the calculation formula of RQ=2-CT,we got relative quantitative value(RQ value),which was used to represent the gene expressionof TGF-?1 for statistical analysis.Results: 1 Aging ranulation was witnessed in biopsy samples before treatment from both group characterized by dull color and hard tactility with little bleeding;Yet scarcely any fresh granulation tissue or micro vascular were observed when microscope was used,with no obvious fibroblasts and inflammatory cells infiltration.Biopsy samples from both group after treatment of 7 days showed fresh ranulation characterized by bright red color,soft and moist tactility with more bleeding,which was obvious in the NPWT group.By microscopic observation,neonatal granulation tissue was observed in specimens from both NPWT group and control group after treatment,which is more significant in specimens from NPWT group presented by more intense inflammatory cells,fibroblasts and neoformation of vessels compared with the control group;2 Signal of positive staining for TGF-?1 was weak displayed as light yellow in specimens from both NPWT group and control group on day0.while afther treatment of 7days,we found stronger signal of positive staining for TGF-?1 which was more obvious in the NPWT group displayed as deep yellow or dark brown than which in the control group display as yellow or light brown.Immunohistochemical data revealed increase of protein expression of TGF-?1 before(day 0)and after treatment(day 7)in specimens from both NPWT group and control group after treatment(NPWTgroup t=8.250,p<0.01,?=0.05;control group t=3.204,p<0.01,?=0.05),which is more obverous in specimens from NPWT group compaired with the control group(t=5.96,p<0.01,?=0.05);3 What Western blot analysis showed was consistent with Result 2: protein expression of TGF-?1 in specimens from both NPWT group and control group performed up-regulation after treatment(day 7)(NPWTgroup t=5.648,p<0.01,?=0.05;control group t=10.734,p< 0.01,?=0.05),which is more significantly boverous in NPWT group(t=5.96,p<0.01,?=0.05);4 Similarly,real-time PCR revealed the change of mRNA expression of cellular TGF-?1 before and after treatment in specimens from both NPWT group and control group after treatment(NPWTgroup t=11.321,p<0.01,?=0.05;control group t=9.671,p<0.01,?=0.05),,which is more significant in specimens from NPWT group compaired with the control group(t=8.02,p<0.01,?=0.05).Conclusions:Both NPWT and conventional gauze therapycan up-regulate the expression of TGF-?1 of diabetic foot wound granulation tissue and facilitate granulation formation in diabetic foot wounds so as to promote the wound healing.Compared with conventional gauze therapy,NPWT can up-regulate the expression of TGF-?1 of diabetic foot wound granulation tissue and facilitate granulation formation in diabetic foot wounds so as to promote the wound healing more significantly obvious. |
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