| Objective:Diabetic foot wound is chronic non-healing wound and its pathogenesis is complex.Transforming growth factor-beta 1(TGF-?1)is a multifunctional bioactive agents,which plays an important role in wound healing.Studies have confirmed that supplied with exogenous TGF-?1 can make up for the inadequate secretion in diabetic foot wound,thus can shorten the wound healing time.However,the effectiveness of TGF-?1 is not so good in clinical trials.The possible cause is the abnormal expression of TGF-?receptor.To study the role of transforming growth factor-beta receptor type?(TGF-?R?)in the wound healing of diabetic foot,and to provide a new idea for wound healing mechanism and treatment of diabetic foot,we investigate the expression of TGF-?R? in granulation tissue of diabetic foot and non diabetic wound.Methods:1 According to the admission criteria and exclusion criteria,in diabetic foot group,we collected 10 diabetic foot patients from department of endocrinology;10 patients without diabetes mellitus from the burn wards were chosen as control group.All those were inpatients of Bethune International Peace Hospital of PLA,from January 2015 to June 2016.The general data of two groups were collected and statistically analyzed,which were age,gender,fast blood glucose(FBG),glycosylated hemoglobin(HbA1C),ankle-brachial index(ABI),alanine aminotransferase,creatinine,etc.2 All patients of diabetic foot group and control group received appropriate debridement.Granulation tissues were collected on the 10th-15 th day after debridement,then each sample was divided into three parts and marked as part A,part B and part C.3 Samples of part A were dipped in 4% paraformaldehyde fixation,andsaved in 4 degree refrigerator.All samples of part A routinely were embedded in the paraffin finally.4 Some of part A were stained with HE,observed the morphology and cell composition of granulation tissue under optical microscope(Magnification,400×).The capillary density was counted and statistically analyzed.5 The other of part A were stained with immunohistochemistry.Expression of TGF-?R? was observed under optical microscope(Magnification,400×).We selected 10 random positive staining areas of each sections and analyzed the expression of TGF-?R? by digital medical image analysis system(Image-pro-plus 6.0).Optical density(OD)represented the expression of TGF-?R?.Compare the expressions of two groups.6 Samples of part B were collected for Western blot.The grey densities of TGF-?R? and GAPDH were measured by Quantity One software.The ratio of TGF-?R? and GAPDH denoted amount of TGF-?R? expression.7 Samples of part C were collected for Real-time PCR.The first sample of the control group was set as standard 1 to obtain the Ct values of the other sample.According to the formula of RQ=2-??Ct,we got relative quantitative value(RQ).RQ indicated the gene expression of TGF-?R?.Results:1 There were no significant differences in age,gender,alanine aminotransferase,creatinine and other indicators between diabetic foot group and control group(P>0.05).Compare with control group,FBG and HbA1 C were higher,but ABI was lower in diabetic foot group(P<0.01).2 Granulation tissue of diabetic foot group was dark red in color,hardy to touch,and less bleeding.Meanwhile granulation tissue of control group was red in color,soft to touch,moist,easy to bleeding.Observe HE stained section under light microscope(Magnification,400×),granulation tissue of diabetic foot group was with less vascularization,with less fibroblasts and full of intense inflammatory cells.Granulation tissue of control group was with more vascularization,full of fibroblasts and with less inflammatory cells.Compare with control group,the number of capillaries was less in diabetic foot group(P<0.01).3 Observe immunohistochemical stained section under light microscope(Magnification,400×),TGF-?R? was positively expressed in both in diabetic foot group and control group.Compared with control group,the expression of TGF-?R? was significantly decreased in diabetic foot group(P<0.01).4 Western blot band analysis showed,TGF-?R? was positively expressed in both in diabetic foot group and control group.Compared with control group,the expression of TGF-?R? was significantly decreased in diabetic foot group(P<0.01).5 Real-time PCR results further indicated TGF-?R? was positively expressed in both groups.But compared with control group,the expression of TGF-?R? was significantly decreased in diabetic foot group(P<0.01).Conclusions:1 Granulation tissue of diabetic foot wound is with less vascularization,and severely aged.2 The protein expression and mRNA expression of TGF-?R? are significantly decreased in granulation tissue of diabetic foot wound.3 The lack of TGF-?R? maybe one of reasons of impaired granulation tissue formation,and non-healing wounds in diabetic foot. |
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