Associativity Study Between ICAM-1 And NF-κB MRNA Expression And Dating Of Wound After Skin Contusion Of Rat

Objective:To examine the expression trend of ICAM-1 mRNA and NF-κBmRNA in the skin and muscle after skin contusion of rat by real-time fluorescent quantitative RT-PCR and their relation with the wound ages.In order to explore the application for estimating the early time of skin contusion in forensic medicine aspect.Methods:The 48 SD rats were divided into control group(contain 6 rats) and experimental group(contain 42 rats) randomly.The skin contusion was performed on the right hind leg of experimental animal by using a gravitational force of a free falling weight.The skin and muscle tissue of 30mg were cut respectively at 0.5h,1h,6h,12h,18h,24h,30h(6 rats in each subgroup) and then the samples were stored in liquid nitrogen.Rats in control group were dealt as same as rats in experimental group except for gravitational force.Total RNA was isolated from each sample using SV Total RNA Isolation System and reverse transcript to cDNA,then their reaction outcome were quantitated by SYBR GreenⅠreal-time fluorescent quantitative PCR.The standard curves were made for ICAM-1 and NF-κB to housekeeping gene (rpL32) with cDNA.The results were assessed by statistic analysis.Results:(1) The purity,concentration and integrity all can suffice the next experiments by UV-Visible Spectrophotometer and 2100 bioanalyzer(lab-on-a-chip).(2)The amplification effect of ICAM-1 and NF-κB to housekeeping gene(rpL32) were concordant by standard curve, it indicated that the housekeeping gene and primers were suitable to the experiment. (3)ICAM-1 mRNA expression arrived the peak value at 0.5h in skin after skin contusion of rats, fell to 7folds of the control group at 24h and then increased.In muscle tissue ICAM-1 mRNA expression was remarkably increased at 0.5h(P＜0.001) after injury and arrived the peak value at 6h,fell to the lowest at 18h and then increased.(4)NF-κB mRNA expression was obviously increased at 0.5h(P＜0.05) after injury and arrived the peak value at 6h,fell at 18h under the control group and then increased slightly.In muscle tissue NF-κB mRNA expression was remarkably raised at 0.5h(P＜0.001) and arrived the peak value at 1h after injury,then fell but another peak appear at 18h.Conclusion:The time-dependent expression of ICAM-1 mRNA and NF-κBmRNA in the skin and muscle tissue after skin contusion of rat could be applied in dating of wound for skin contusion.Real-time fluorescent quantitative RT-PCR is accurate and sensitive and refer to forensic study.