Biomarkers For Vital Reaction In Skin Contusions

BackgroundsThe indication of injury time,whose key is to distinguish pre-life and postmortem injuries and infer the survival time after injury by diagnosing the existence of vital reaction,is one of the main tasks of forensic pathology.Traditional diagnostic methods are mainly based on morphological changes,including hemorrhage and microscopic histopathology.However,it is sometimes difficult to identify vital reaction due to the lack of morphological evidence especially when the time of injury and death is very close,which brings great trouble to the actual prosecution.More and more studies have shown that the expression of some cytokines in tissues changes rapidly after injury.With the development of molecular biology techniques such as high throughput sequencing,these changes can be detected more easily.Therefore,more and more attention has been paid to the screening of meaningful molecular markers that can be used as diagnosis of vital reaction by these techniques.ObjectivesPotential markers for vital reaction in skin contusions were preliminarily explored by RNA-seq technique,and the feasibility of C-X-C chemokine ligand 1(CXCL1)and its receptor C-X-C chemokine receptor 2(CXCR2)as biomarkers was validated and evaluated.MethodsExperiment 1:A total of 16 adult male BALB/c mice were randomly divided into two groups:control group and contusion group.The control group was not treated with trauma.In the contusion group,a 5mm X 5mm subcutaneous hemorrhage occurred on the back skin after 10 seconds of compression with flat-mouth forceps.Then all mice were killed 0.5 hours after injury and the injured skin was collected as the contusion group.The same body's non-injured abdominal skin was collected as the negative control group.The contents of detection were as follows:Differential expression genes(DEGs)were screened by RNA-seq;Bioinformatics analysis was carried out;Results of RNA-seq were validated by Real-time fluorescence quantitative PCR(RT-qPCR).Experiment 2:A total of 30 adult male BALB/c mice were randomly divided into three groups:control group(3 mice),Antemortem injury group(24 mice)and postmortem injury group(3 mice).The antemortem injury group was randomly divided into eight groups according to eight different time points of collecting skin samples(three mice in each group).The control group was not treated with injury.Mice in the antemortem injury group were compressed with flat-mouth forceps for about 10 seconds,resulting in a 5mm X 5mm subcutaneous hemorrhage of the back skin.All the mice were killed at 0.5 hours after injury,and the injured skin was collected as the antemortem injury group at 8 time points of 0,6,12,24,48,72,96 and 120 hours respectively.Mice in the postmortem injury group were killed directly,and the back skin of the mice was injured by the same method at 0.5 hours after death.Skin samples were collected from the injured area.The above samples were collected for subsequent detection.The contents of detection were as follows:The expression of CXCLI and CXCR2 proteins in tissues was detected by Western blotting;The expression of CXCL1 and CXCR2 mRNA in tissues was detected by RT-qPCR.ResultsResults 1:The results of RNA-seq showed that 659 genes were up-regulated and 996 genes were down-regulated in contusion skin of mice compared with the control group.These differentially expressed genes could be clustered into corresponding classifications by further bioinfomatic analysis,and some of them could be verified by RT-qPCR in mice and human tissuesResult 2:Compared with the control group,the protein expression levels of CXCL1 and CXCR2 in mice and human tissues did not change significantly in the injured group.But the mRNA expression levels of CXCL1 and CXCR2 increased significantly,and the increasing trend was stable,which could be detected even when the samples were degraded.ConclusionsConclusion 1:RNA-seq is a powerful tool to reveal potential markers for vital reaction.Conclusion 2:The changes of CXCL1 and CXCR2 at the level of mRNA,rather than protein,are potential markers of vital reaction in skin contusions.