Expression Of ASCT2mRNA In Rat Skeletal Muscle After Contusion

ObjectiveTo investigate the expression of ASCT2mRNA in the contused muscle of rats and the relationship between the ASCT2expression and the wound age, aimed to give some advice to wound age estimation.MethodsThe animal we took were96Sprague-Dawley rats in this study. The rats were divided into3parts:control group (n=6) and4,8,12,16,20,24,36,40,44, and48h (n=72) contusion groups, and the others18rats were contused after death and divided into6,12, and18h groups. After general anesthesia and shaving right posterior limb of rats,we got a weight of250g fall upon the right posterior limb from150cm’s high through a cylinder.Whereafter,we extracted the RNA from contused skeletal muscle of three groups and synthesized cDNA by reverse transcription polymerase chain reaction(RT-PCR).Sequence-specific primers were used to conduct real-time PCR to analyze the expression of ASCT2mRNA that was quantified using rpl13intrinsic fluorescent assay and compared by the2＾-△△Ct method with control samples.Results(1)We use the quotient of OD260/OD280given by Ultraviolet spectrophotometer to estimate the purity of the total RNA. The results proved that the total RNA we extrated was acceptable.(2)The standard curve showed that the amplification efficiency of the two genes was89.5%and90.1%respectively and the correlation coefficient is much higher than0.98.The result indicated that the linearity of the standard curve was nearly linear.(3)The outcome of experiment show that the expression of ASCT2mRNA up-regulated at196.40%,189.15%,182.54%and136.65%(P<0.05) in the points of12,16,36and40h after contusion compared to control group,yet there was no significant change in the expression of ASCT2mRNA in the range of4to8h,20to32h and44to48h(P>0.05) after contusion.(4)There was no significant change in the expression of ASCT2mRNA between sample-contusion and the sample-control within normal skeletal muscle (P>0.05).(5)The expression of ASCT2mRNA in the contused muscle of postmortem rats were90%around compare with the control group (P<0.05) and there was no significant change in the different time point of the postmortem contusion group(P>0.05).Conclusion(l)The result show that the expression of ASCT2mRNA in the contused muscleincreased obviously compared with the sample-control in the range of12to16and36to40h after contused (P<0.05)and that means the non-contused muscle from the same contused rat could be evaluated the contused muscle.(2)Compared to the control group, a significant degradation of ASCT2mRNA in the postmortem samples was detected.However, there was no significant change in the different time point of the postmortem samples after injury indicated that the degradation of RNA didn’t make the negative effect between6to18h after death.(3)It is suggested that ASCT2mRNA analysis may be useful to estimate the wound age for its time-related expression in contused muscle.