Study On The Expression Of Myofibroblasts In Granulation Tissue Of Diabetic Foot

Objective: Myofibroblasts are the main cell component of granulation tissue which promotes wound healing.they are derived from fibroblasts.α-smooth muscle action(α-SMA)is a characteristic,contractile product coming from differentiation process of fibroblasts turning into myofibroblasts,and it is an important material involved in wound healing.So compared with fibroblasts,myofibroblasts have a more important role in wound healing.However,no relevant studies have been found on the formation of myofibroblasts in diabetic foot granulation tissue.In this paper,we analyzed the expression of α-SMA-the specific protein of myofibroblasts in gene and protein levels in diabetic foot granulation tissue and non-diabetic wound granulation tissue.After separating the two groups of fibroblasts,α-SMA staining was performed,we observed the formation of myofibroblast in diabetic foot granulation tissue in tissue and cell levels,and discussed the mechanism of delayed wound healing in diabetic foot.Methods:1 Filtered through inclusion criteria and exclusion criteria,a total of 10 diabetic foot patients who were hospitalized in the Bethune international peace hospital from January 2016 to June 2017 were selected as the experimental group,10 patients with non-diabetic wound who in the same time period were treated as the control group.10 non-diabetic patients who were admitted to the burns department for treatment and met the admission criteria in the same time period were treated as the control group.2 After admission,the two groups were given routine dressing,and we removed the necrotic tissue,every other day at a time,the local fresh granulation tissue was taken after 10-15 days.We divided the granulation tissue into three parts.1 part was fixed with 4% paraformaldehyde fixed fluid,and then it was buried in the paraffin for immunohistochemical staining.We put part 2 into the liquid nitrogen to make it cool quickly,and then transferred it to-80 ℃ temperature refrigerator for preservation,and then we performed Western blot and Real-time PCR detection.With the third part,we cultured myofibroblasts and performed immunohistochemical staining.3 Immunohistochemical staining: In the two groups of granulation tissue,the expression of α-SMA was analyzed semi-quantitatively.In the light microscope(400 x),the brownish yellow granule was observed to be positive for α-SMA staining.In each dyed slice,10 positive areas were randomly selected,then we took pictures and performed α-SMA optical density value detection using digital medical analysis system(6.0)MoticMedical,and then we used the average optical density value(optical density,OD)semiquantitative analyzed the expression of α-SMA.4 Western blot: The expression of α-SMA was analyzed in protein level.Image J software was used to analyze the grayscale values of two groups ofα-SMA.the α-SMA’s expression in each stripe is expressed by the ratio ofα-SMA and GAPDH ’s grayscale values,and then the protein quantitative analysis was performed on the α-SMA.5 real-time PCR: The expression of α-SMA was detected in the gene level.That is,the expression of α-SMA mRNA.And the gene quantitative analysis was performed on α-SMA.6 Cells culture: We put the specimen in saline for a short time.Then we took it out and cut it with scissors and digested it with type I collagenase.Using the method of “adherentting to lummox with different times”,the cells were detached and purified,then we got fibroblasts.Then we cultured the cells until 3 to 4 days,more than 80% fibroblasts were stretched and they are fixed.7 Immunohistochemical staining on cells: The fixed cells were stained.Those that are stained positive are myofibroblasts.We observed and calculated the percentage of positive cells in the field.Results:1.Immunohistochemical stained slices was observed under the micro-scope: The brownish yellow granules were observed in both groups.However,compared with the control group,the brown-yellow particles with positive staining in the experimental group were sparse.The average optical density is used for semi – quantitatively of α-SMA.The results showed that the expression of α-SMA in the experimental group was significantly lower than that in the control group(P < 0.01).2.The results of Western blot of the two groups of granulation tissue showed: The ratio of α-SMA and GAPDH bands’ in the experimental group and the control group were statistically analyzed.The expression of α-SMA was quantitatively analyzed.The relative protein expression value of α-SMA in the experimental group is 0.32 ± 0.09,and the relative protein expression value of α-SMA in the control group is 0.58±0.15,in the experimental group,the expression quantity of α-SMA is significantly lower than that in the control group(P < 0.01).3.Real-time PCR analysis results: In the experimental group,the relative gene expression value of α-SMA is 0.83± 0.27,The relative gene expression of α-SMA in the control group is 1.29±0.30,the expression of α-SMA mRNA in the granulation tissue of diabetic foot group was lower than that in the control group(P < 0.01).4.Immunohistochemical staining of myofibroblasts: Observed in the light microscope(400 x),the staining ratio of the brown-yellow myofibroblasts in the diabetic foot group was significantly lower than that in the control group.Conclusion:Compared with non-diabetic foot granulation tissue,the formation of myofibroblasts in diabetic foot granulation tissue decreased significantly.The reduction of myofibroblast can cause obstacles on local contraction of wound,so we hypothesized that the reduction of myofibroblast may be one of the reasons for the difficulty of diabetic foot healing.