| It is well established that skin wound healing can be temporally divided into three phases, the early acute exudative, fibro - prolifera-tive and tissue remodeling phases. Inflammatory cell such as neutro-phils, macrophages and fibroblasts migrate to and proliferate in the exudative and fibro - proliferative phases. Recent study has suggested that apoptosis occurs and plays an important role in achieving a decrease in cellularity during skin wound healing. Recently, more and more study indicated the family of caspase protease produce a marked effect in apoptosis. Being the important execute factor, caspase -3, - 6, - 7 no doubt have the very important value.To investigate if caspase - 3, - 6, -7 play an important role in inducing neutrophil, macrophage and fibroblast apoptosis during skin wound healing and explore the applicability of caspase - 3, - 6, - 7 to determination of wound age , Immunohistochemical study on the expression of caspase - 3, - 6, - 7 were perform on 33 mice skin incised wounds at different posttraumatic intervals.Materials and MethodsA total of 36 adult mice, each weights 35 ~ 40g. A 1 - cm - long incision was made with a scalpel in the skin layer on the central dor-sum. After wounding, each mouse was individually housed in a cageand given sterilized chow and redistilled water to prevent bacterial infection. 1.5x1 cm2 specimens were taken from the wounded sites after the animal were sacrificed by cervical dislocation after anaestheti-zation at 0h,1h, 3h, 6h, 12h, 1d, 3d, 5d, 7d, 10d, 14d (3 mice in each group) post - wounding. The remaining 3 mice were used as controls. Immunohistochemistry SABC method was used to investigate the protein expression of caspase -3, -6, -7. After inhibition of endogenous peroxidase activity (3% hydrogen peroxide PBS solution for 20 minutes) , antigen retrieval was then performed with heating in a microwave oven for lOminutes. After serum incubated, sections were incubated with polyclonal antibodies against caspase - 3 , - 6, -7 at 25C for 2 hours at a dilution of 1:200. Goat and - rabbit IgG -biotiny incubated at 25C for 20 minutes (caspase -6, -7 incubated with rabbit anti - goat IgG - biotiny). The slides were incubated with SABC at 25C for 20minutes. Visualization of antibody binding was the DAB method. Finally, the sections were counterstained with hematoxylin, dehydrated and coverslipped.Positive cells of caspase - 3 , - 6, - 7 were identified and counted in 10 randomly selected microscope fields in three sections of eath group at a 400 - fold magnification, counted the ratio of positive cell of caspase -3, -6, -7. The data analysis were performed using SPSS for Windows 10. 0 with a significant level of P <0. 05.ResultsThe expression of caspase - 3, - 6, - 7 in skin wound healing : in the control specimens, the expression of caspase - 3, - 6, - 7 site in the basal cell layer, spinous layer, granular layer and sweat glandepithelium, sebaceous gland epithelium. Expression of caspase - 3,-6, -7 was detectable in polymorphonuclear cells (PMNs) in thewound specimens aged 3 hour, and in the wound specimens aged from6 ~ 24 hour after wound caspase - 3, - 6, - 7 was identified in a large number of infiltrating PMNs and part of mononuclear cells (MNCs). Afterwards, the MNCs and fibroblastic cells (FBCs) accounted in the most part of the caspase - 3, - 6, - 7 - positive cells.The ratio of positive cell of caspase - 3, - 6, - 7: The ratio of positive cell of caspase - 3 is low in the wound specimens aged 1 ~ 3 hour (4. 53± 6. 53% ) , and maximized in the wound aged 3 day (62. 66±4. 84% ) , thereafter, the ratio decreased and minimized in the specimens aged 14 day (21. 56±4. 54% ). The ratio of positive cell of caspase - 6 is low in the wound specimens aged 1 ~ 3 hour ( 3.05±4. 80% ) , and maximized in the wound aged 3 day (55.33± 3. 65% ) , thereafter, the ratio decreased and minimized in the specimens aged 14 day (21. 26±4. 71%). The ratio of positive cell of caspase -7 is low in the wound specimens ag...
|
PDF Full Text Request