Expression Of α-SMA And Integrin-β₁ In Human Hypertrophic Scar Mechanical Model

Objective Using the established mechanical model of human hypertrophic scar to study the expression and significance ofα-SMA and Intergin-β₁ in the process of normal human fibroblasts transforming into hypertrophic scar fibroblasts by giving continuous mechanical tension in different time periods.The results will provide a theoretical basis for further study on the effect ofα-SMA and Intergin-β₁ expression on hypertrophic scar formation.Methods Scar tissue and normal dermal tissue were obtained from hypertrophic scar patients after scar resection.Hypertrophic scar fibroblasts（HSFB）and normal skin fibroblasts（NSFB）were cultured in vitro by tissue block culture method,and the 4th to6th generation cells were obtained by enzyme digestion method for related experimental study.These two kinds of cells were divided into mechanical stretch group（experimental group）and non-stretch group（control group）.Using the multi-channel stress loading system,the experimental groups were given the periodic mechanical stretch tension within 0.1hz,10%stretch amplitude and sinusoidal waveform.The daily stretch time was4h,and the time period was set as³d,⁵d and⁷d.The control groups without mechanical strain were cultured by the same time under normal conditions.When the experimental groups and control groups were cultured to 3 d、5 d、7 d,the cells of each group were respectively for the following tests:CellI Immunochemical staining（ICC）to detect the expression of Proliferating Cell Nucleus Antigen（PCNA）and Cell Immunofluorescence staining（IF）detection ofα-Smooth Muscle Actin（α-SMA）and Intergin-β₁ protein expression;Protein expression levels ofα-SMA and Integrin-β1 were determined by Western Blot（WB）.Results 1)PCNA expression:PCNA protein expression in NSFB and HSFB cells under mechanical stretch in different time periods was found that NSFB（NC group）showed a general proliferation state within 7 days,while NSFB（NS group）showed a certain proliferation activity under mechanical stretch.With the continuous influence of stretch,the expression of PCNA increased,and the cells in the NS group began to proliferate actively.The expression of PCNA was similar to that in the normal HSFB（HC group）on the fifth day of tension.PCNA expression of HSFB（HS group）was increased,indicating that cells in HS group showed active proliferation.2)α-SMA expression:The expression ofα-SMA in NSFB of NC group was less in each period,but the expression ofα-SMA in NSFB of NS group increased with the increasing tension time.From the results of fluorescence expression,the increasing trendency ofα-SMA expression of HSFB in HC group was similar with that of NSFB in NS group.Proteins were extracted from four groups of cells at different time periods and then semi-quantitatively detected by western-blot.The following results were obtained:HC group showed statistically significant difference from NC group,and HS group showed statistically significant difference from NS group（P<0.05）.The difference in expression level ofα-SMA was statistically significant（P<0.05）in each treatment group（3 and 7 days）after 4h tension loading each day.Compared with HC group,the 5 days NS group had no statistically significant difference in protein expression（P>0.05）.3)Integrin-β₁ expression:The expression of Integrin-β₁ increased incrementally in cell culture at various periods in NC、NS、HS groups,and the same cell line showed different expression levels under the influence of stimulation signals;HC group showed no obviously growth tendency.From the results of fluorescence expression,the increasing trend of Integrin-β₁ expression of HSFB in HC group was similar to that of NSFB in NS group.Proteins were extracted from four groups of cells at different time periods and then semi-quantitatively detected by western-blot.The following results were obtained:HC group showed statistically significant difference from NC group,and HS group showed statistically significant difference from NS group（P<0.05）.The difference in expression level of Integrin-β₁ was statistically significant（P<0.05）in each treatment group（3 and 7days）after 4h tension loading each day.Compared with HC group,the 5 days NS group had no statistically significant difference in protein expression（P>0.05）.Conclusion The expression levels ofα-SMA and Integrin-β₁ are closely related to the proliferation status of cells.There was a positive correlation between the proliferation and differentiation of fibroblasts and the transformation of normal skin tissue into hypertrophic scar during the scar hyperplasia period.