Expression Of MIF And EMT-related Protein Fn In Keloid Tissue And Its Correlation With Postoperative Recurrence

Purpose:At present,the pathogenesis of keloid(Keloid,KD)is not clear,which has resulted in a variety of ways to treat keloid,but the effective treatment is very rare,which also led to a high recurrence rate of keloid after surgery.Therefore,it is necessary to find the protein related to its prognosis.Current research has confirmed that the process of epithelial-mesenchymal transition(EMT)is closely related to the formation of keloids.When the EMT process occurs,epithelial cells lose their normal cell polarity,and with the break of intercellular connections,epithelial cells gain invasion and migration,and eventually become mesenchymal cells.In this process,in addition to the breaking of the intercellular connection and the rearrangement of the cytoskeleton,the expression of mesenchymal markers(fibronectin,Fn)increased,and the shape of fibroblasts changed from rectangle to spindle,which also enhanced the migration and invasion ability of fibroblasts.Macrophage migration inhibitory factor(MIF)is an endocrine immune substance,which mainly plays a role in limiting or regulating the activity of macrophages in the body.Current research has confirmed that there is abnormal expression of macrophage migration inhibitory factor in some autoimmune diseases,chronic inflammatory diseases and malignant tumor diseases.Through relevant data analysis,it is found that MIF is related to the occurrence,development and even prognosis of the above related diseases.Interestingly,the biological characteristics of the above diseases are similar to keloid;At the same time,MIF can affect the expression of related factors by regulating related signal pathways,thus delaying cell apoptosis and promoting the excessive growth and proliferation of fibroblasts.Therefore,MIF can promote the growth and proliferation of keloid fibroblasts.In this study,immunohistochemical staining and Elisa detection were used to detect the expression of epithelial stromal transformation marker protein Fn and MIF in normal tissues and keloids.At the same time,patients with keloids were followed up to record the recurrence,and to explore the expression of Fn and MIF in keloid tissues and the correlation with the prognosis after surgery.Method:1.Collect 60 clinical keloid specimens after surgical excision,and 30 normal skin specimens as control.The samples were fixed with 10% neutral formalin,dehydrated with conventional gradient alcohol,embedded in paraffin,and sectioned for immunohistochemical staining.The sections were observed under the light microscope.Two attending physicians in the department of pathology observed and counted under the same microscope with the same magnification,randomly selected five fields of vision,calculated the percentage of positive cells in each field of vision,and took the average value.2.Collect the venous blood of 60 patients with keloid,and collect the venous blood of 30 normal patients before surgery as control.The venous blood serum was extracted and the expression of Fn and MIF was detected with Elisa kit.3.The patients with keloid were followed up regularly(the 1st,3rd,6th and 12 th months after operation)and the recurrence was recorded.According to the follow-up,patients with keloid were divided into two groups,recurrence group and non-recurrence group.The expression levels of Fn and MIF in the two groups were analyzed for survival.Result:1.Immunohistochemical staining results1.1 Control group(normal tissue)1.1.1 Histomorphology under H-E staining light microscope: the specific layer of epidermis can be observed in normal skin tissue,the junction between the stratum corneum and the hyaline layer is not obvious and is eosinophilic,the shape of basal cells is mostly columnar,and the keratinocytes are round or oval;The fiber bundles of subcutaneous tissue are arranged neatly,and the cell shape is round or oval,without obvious nodules.1.1.2.Immunohistochemical staining results: Compared with the expression in dermal tissue,MIF and Fn positive particles are mainly concentrated in the epidermis,and the relevant skin appendages such as hair follicles and sebaceous glands are deeply stained.1.2 Experimental group(keloid tissue)1.2.1 Histomorphology under the light microscope of H-E staining: the epidermal layer in keloid tissue is flat and atrophic,and the transparent layer and the cuticle are thinner than the normal tissue,showing eosinophilic;The collagen fibers of the subcutaneous tissue are crisscrossed,accompanied by different degrees of hyaline degeneration.Under the light microscope,homogeneous eosinophilic staining of the leaf-like structure can be seen;In addition,thicker fiber bundles can be seen.1.2.2 Immunohistochemical staining results: MIF and Fn are expressed in a wide range and with a high density,in which dermal cells are darker in color,and in addition,the cytoplasm of fibroblasts and positive particles around microvessels are concentrated.2.Elisa resultCalculate the linear regression equation of the standard curve according to the standard concentration and the corresponding OD value provided in the Elisa kit manual,and then calculate the corresponding MIF and Fn concentrations on the regression equation according to the OD value of the measured sample.2.1 The expression of Fn in keloid tissue was 1.53 ± 0.18(ng/ml);The expression of MIF was 1.44 ± 0.22(ng/ml);2.2 The expression of Fn in normal tissues is 0.60 ± 0.25(ng/ml);The expression of MIF was 0.44 ± 0.10(ng/ml).Compared with normal tissues,the expression level of Fn and MIF in keloid tissue was higher,and there was a statistical difference between the two groups(P<0.05).3.Follow-up resultsAccording to the follow-up of keloid patients after surgery,they can be divided into two groups: recurrence group and non-recurrence group,of which 13 are in the recurrence group and 17 are in the non-recurrence group.The positive rate of MIF and Fn in recurrent group was 90.48% and80.95% respectively;The positive rate of MIF and Fn in non-recurrent group was 61.54% and53.84% respectively.4.Statistical analysis results4.1 Expression of MIF and Fn in experimental group and control group4.1.1 The expression of MIF and Fn in the experimental group and the control group was detected by immunohistochemical staining.The results showed that the positive rate of MIF in the experimental group was 71.67%,and the positive rate of MIF in the control group was 26.67%;The positive rate of Fn was 63.33% in the experimental group and 36.67% in the control group.The difference was statistically significant(P<0.05).4.1.2 Elisa resultsThe absorbance values(OD values)of MIF and Fn at 450 nm were measured,and the expression content(concentration)of MIF and Fn was calculated according to the standard curve regression equation.4.2 According to the follow-up results,keloid patients were divided into recurrent group and non-recurrent group.Combined with the immunohistochemical results,survival analysis was carried out by Kaplan-Meier method.The results showed that patients in Fn(-)&MIF(-)group had the best prognosis,while those in Fn(+)&MIF(+)group had the worse prognosis.Conclusion:1.Fn and MIF were expressed in both keloid tissue and normal tissue,but the positive rate and quantity of expression were more obvious in keloid tissue,with statistical significance(P<0.05).2.The expression of Fn and MIF in the patients with keloid in the recurrence group was more obvious than that in the non-recurrence group,and the difference was statistically significant(P<0.05).It is suggested that Fn and MIF can be used as biological indicators to predict the recurrence of keloid after surgery.