Expression And Analysis Of Leptin In Pathologic Scars And Keloid Fibroblasts

Abstract:Objective:⑴To study the expression of leptin in pathologic scars,⑵to study the expression of leptin at different times,different concentrations of human keloid fibroblasts（HKF）and human skin fibroblasts（HSF）in the supernatant.It gave further understanding whether leptin involved in the formation of pathological scar and provided some new theoretical basis for the prevention and treatment of pathological scars.Subjects and Methods:1.Experimental specimens including the keloid,hypertrophic scar and normal skin tissue were originated from the department of dermatologic surgery and the department of burn surgery in Sichuan Provincial People’s Hospital（from February 2015 to November 2015）.All experimental specimens were divided into keloid group（n=20）,the hyperplastic scar group（n=20）and normal skin tissue（n=10）.Permissions had been obtained by the patients before collection.The age of patients range from16 to 50 years old.There were 18 cases of men,22 cases of women,with duration of 6 months to 6 years.They had no radiotherapy,local injections or drugs such as silicone for a year before this operation.Normal skin tissues were from skin grafting originally from the department of dermatologic surgery in Sichuan Provincial People’s Hospital,patients aged 16-50 years old.By immunohistochemistry（SP method）,3 section specimen were cut after paraffin embedding.The first section were stained by HE,then read section to determine the specimen for normal skin tissue or pathological scar.Then antibody（rabbit anti-human leptin）was dropped in the second section,and PBS was dropped in the third section as a negative contrast.Based on the analysis method of Shimizu^[1],scoring criteria mainly depends on the number of positive cells being shaded:0 point for uncolored,1 point for light color（light yellow）,2 points for deep colored（yellowy brown）,3 points for brown;0 point if the percentage of stained cells were less than 5%,1 point if less than33%,2 points between 33%and 66%,3 points for 75%and larger.If the sum of above count less than 1 point,i t shows negative（-）.It represents positive（+）for 2⁴ points.It represents strongly positive（+）for 5 or more points.Then we used statistical software SPSS20.0 to analyze experimental data.2.We used tissue culture method for primary culture of scar fibroblasts.We observed cell morphology,take pictures,and cultured cells.Finally,according to Immunohistochemistry（SP method）,we identify them as human keloid fibroblasts（HKF）.Experiments employing human skin fibroblasts（HSF）were from the Kunming Cell Bank,Chinese Academy of Sciences.3.We inoculated HKF and HSF with concentrations of 6×10⁴/ml and 9×10⁴/ml separately in six-well plates.We collected the cell culture supernatant at 24h,48h and 72h after inoculation to measure leptin OD value by using enzyme-linked immunosorbent assay（ELISA）.The standard concentration gradient and OD value were used to calculate the curve equation.Then according to the standard curve,the corresponding cell culture supernatant in leptin concentrations can be counted.At the end SPSS20.0 statistical software were used to analyze data.Results:1.Leptin’s expression in normal skin,hypertrophic scars and keloids:⑴By contrasting different expression of leptin in normal skin group,hypertrophic scars and keloid group’s dermis,we can find^?2=34.074,P<0.001,which has statistical significance.By pairwise comparison of the above three groups:leptin’s expression in keloid group was stronger than normal skin group,which was statistical Z=-5.161,P<0.001;the expression of hypertrophic scar group was stronger than that in normal skin group,which was statistical Z=-4.603,P<0.001;the expression of keloid group was stronger than that in hypertrophic scars group,which was statistical Z=-2.054,P=0.04.⑵Leptin’s expression in normal skin group,the group of hypertrophic scars and keloids group’s epidermis:The expression of leptin in the three groups had no significant difference.The difference between statistics was^?2=1.633,P=0.442,which was insignificant.2.Primary culture of keloid fibroblasts:by immunohistochemistry（SP）method to identify HKF,the results showed that vimentin was positive.The cell was identified as HKF.3.ELISA test HKF,HSF culture supernatant of leptin expression:⑴In the concentration of 6×10⁴/ml,leptin OD value mean±standard difference,at 24h,48h and 72h after HKF inoculation,were 0.083±0.009,0.089±0.016,0.090±0.009;In the concentration of 6×10⁴/ml,leptin OD values mean±standard difference,at 24h,48h and 72h after HSF inoculation,were0.065±0.015,0.068±0.013,0.073±0.013;⑵In the concentration of 9×10⁴/ml,leptin OD mean±standard deviation,at 24h,48h and 72h after HKF inoculation,were 0.091±0.011,0.098±0.014,0.098±0.009;In the concentration of 9×10⁴/ml,leptin OD values mean±standard deviation,at 24h,48h and 72h after HSF inoculation,were 0.075±0.010,0.077±0.017,0.086±0.004;⑶By using independent sample t-test for statistical analysis,at the 24h,48h,72h the cells in a concentration of 6×10⁴/ml,their leptin concentrations of HKF were higher than those of HSF,the differences had statistical significance（P<0.05）;⑷when the cell concentration was 9×10⁴/ml,leptin concentrations of HKF were also higher than those of HSF,the differences had statistical significance（P<0.05）;⑸In HKF,when the cell concentration was 9×10⁴/ml,leptin concentrations through all the periods were always higher than observation results of cell concentration of 6×10⁴/ml at the72th hour.This indicated that the cell concentration could increase the expression of leptin,which was more significant than the effect of time.The phenomenon also existed in HSF.Conclusion:1.High expression of leptin in pathological scar may be related to pathological scar occurrence and development.2.Scar fibroblasts is involved in leptin participation in the pathogenesis of pathological scar,which is related to the incubation time and concentration of scar fibroblasts.3.By monitoring the expression of leptin protein in scar tissue,this may help determine its development and outcomes.4.This study provides a theoretical basis to study leptin signaling pathway in the pathogenesis of pathological scar and looking for leptin targets to hypertrophic scar.