The Expression And Functions Of HtrA4 In Heat-injured Fibroblasts

Objective: To investigate the expression and functions of HtrA4 in denatured dermis and heat-injured fibroblasts.Methods: The denatured dermis and corresponding normal skin tissues of 3 burn patients were collected clinically.The tissue structure was observed by hematoxylin and eosin stain,and the m RNA and protein expression of HtrA4 in denatured dermis were detected by q PCR and WB assay.The primary fibroblasts from discarded prepuce were heat injured in a water bath at 53 ℃for 50 s to construct a heat-injured fibroblast model,and then the changes of cell morphology were observed.The repair of heat-injured fibroblasts was observed by CCK8 assay to detect cell activity at 6h,24 h,48h,72 h,96h and 120 h respectively.The cells were infected with HtrA4 overexpression virus and treated with heat injury at 53 ℃for 50 s.The changes of HtrA4 m RNA and protein expression were detected by q PCR and WB assay,the changes of fibroblast proliferation were observed by CCK8 assay,and the changes of fibroblast migration ability were observed by Wound scratch assay and Transwell assay.Results:1.HE staining showed the destruction of the epidermis,swelling of the dermis,hyaline degeneration of collagen,and infiltration of inflammatory cells of the denatured dermis;q PCR and WB results showed that the m RNA and protein expression of HtrA4 in the degenerated dermis increased.2.After the primary cultured fibroblasts were heat injured at 53℃for 50 s,the cell volume was enlarged and blunt,some cell membrane boundaries were unclear,and some cytoplasm appeared translucent;within 24 h of heat injury,the cell activity decreased and the proliferation rate decreased significantly,and reached the lowest point at 24 h.After24h,the activity of heat-injured cells recovered,and the proliferation rate gradually increased.After 48 h,the proliferation trend of heat-injured fibroblasts was similar to that of normal fibroblasts.The m RNA expression level of HtrA4 gradually increased within 48 h after heat injury,and reached a peak at 48 h.With the prolongation of time,the m RNA expression level of HtrA4 gradually decreased,and reached the normal level at 7d after heat injury.3.After fibroblasts were infected with HtrA4 virus,they were heat-injured at 53°C for 50 s.The results of q PCR and WB showed that the m RNA and protein expression of HtrA4 increased significantly after infection with HtrA4 virus.Significant;CCK8 assay showed that the proliferation ability of fibroblasts increased significantly after HtrA4 overexpression,and the proliferation ability of fibroblasts increased more significantly after heat injury;Wound scratch assay and Transwell assay showed that the migration ability of fibroblasts increased significantly after HtrA4 overexpression,and the migration ability of fibroblasts increased more significantly after heat injury.Conclusion: HtrA4 expression was upregulated in denatured dermis and heat-injured fibroblasts,and high expression of HtrA4 significantly promoted proliferation and migration of heat-injured fibroblasts and primary fibroblasts.