| Objective:Based on differentially expressed genes from Pre-researches, the expression of SFRP5 m RNA was detected on injured muscle in rat by Real-time PCR. To explore whether SFRP5 m RNA is another appropriate marker for wound age estimation in forensic pathology and forensic daily casework, we analyzed the levels of SFRP5 m RNA during adult muscle regeneration, and investigated whether the SFRP5 m RNA expression was affected by types of injury or degrees of contusion. In addition, we explored the function of SFRP5 m RNA during skeleton muscle wound repair.Methods:(1) Animal groups: 132 adult Sprague-Daley rats were selected and assigned to 6 groups according to the principle of random assignment as follows respectively: Normal control group(n = 6); Injury groups which at different time points over muscle regeneration(A total of 12 groups : 4h, 8h, 12 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h, 40 h, 44 h and 48h)(n = 6); Uninjured groups or contralateral muscles which was drawn from the same rats on injury groups(at the time points: 24 h, 36 h and 48h); Different degree injury groups which were inflicted various degree of injury(A total of four groups: minor injured group at 4h and 8h and serious injured group at 4h and 8h)(n = 6); Postmortem groups which at different time points after the death(A total of 3 groups: After the death of 12 h, 18 h and 24h) and incised wound groups(A total of 2 groups: 4 h and 8h)(n = 6).(2) Preparation of contusion model: 72 rats were fixed to the board and their hairs faded after general anesthesia. The gravity hammer fell free to hit the right hind limb of the thigh muscles, which resulting in the skeletal musclecontusion. The damaged rats were sacrificed by the way of cervical dislocation at a certain time, and then the muscle lesions were drawn and frozen; Simultaneously, the uninjured muscles of left hind limbs have also been drawn from the same injured rats at the time points of 24 h, 36 h and 48 h, which taken as contralateral groups; Different injury severity groups: Gravity Hammers were free fall from a height of 200 cm or 130 cm to the rats, and then the injured muscles were drawn and frozen quickly at 4 hours and 8 hours after injury separately; Postmortem groups: The rats were sacrificed by the way of cervical dislocation before placed in artificial climate chamber, and then the muscles of the right hind limbs were drawn and frozen at setting times; Incised wound models: Incised the full-thickness skin of the right hind limbs, and the rats were sacrificed at 4 hours and 8 hours after injury by the way of cervical dislocation before injured materials were drawn and frozen.(3) Invitrogen TRIzol Reagent Kit was used to extract total RNA from the injured and uninjured muscles according to the kit instructions. After that we used the UV spectrophotometer to get the ratio of OD260 / OD280 of total RNA and purity of the nucleic acids, and then evaluated it.(4) c DNA was synthesized by reverse transcription of total RNA, after that the expression of SFRP5 m RNA was detected by real-time PCR of sequence-specific primers, and was analyzed by the 2-△△Ct method using RPL13 m RNA as intrinsic fluorescent assay.Results:(1)The ratios of OD260/OD280 of the total RNA that extracted by TRIzol Teagent reagent were between 1.8 ~ 2.2, which conformed that the total RNA were acceptable and could be used in the followed experiments.(2)The standard curve showed that the amplification efficiencies of the two genes were 110.5% and 120.2%, and their correlation coefficient were 0.995 and 0.982 respectively, which implicated that RPL13 m RNA could be taken as a reference gene of SFRP5 m RNA.(3) The expression of SFRP5 m RNA was down-regulated within 48 hours after contusion, and was decreased slowly before increased with time. The lowest expression was at the time point of 20 h. Compared to the control, the expression levels of SFRP5 m RNA after muscle contusion at 4,8,12,16 and 20 hours were 65.0%,44.6%, 36.1%, 31.4%, 22.3%, and at 24、28、32、36、40、44 and 48 hours were 26.3%、 36.3%、 37.2 %、47.3 %、50.4%、 61.8 %、68.8%, respectively. The expression levels were ranging 22.3%-68.8% compared to the control group.(4) By comparison with the normal group, the expression of SFRP5 m RNA in the contralateral uninjured muscles were no significant difference(P>0.05), which implicated that the undamaged muscles from the same injured individuals maybe could be taken as control in relative quantitative experiments when SFRP5 m RNA used as marker for wound age estimation;(5) Compared to the control, the expression of SFRP5 m RNA were decreased both at the serious and minor injured groups. However, there is no statistical difference between the level of SFRP5 m RNA in serious and in minor contusion groups no matter at 4h or 8h after contusion(P>0.05), which implicated that the expression levels of SFRP5 m RNA were not affected by the degree of injury.(6) Compared with the control, the expression of SFRP5 m RNA at each time point after death had no statistical difference(P> 0.05), which indicated that the expression of SFRP5 m RNA levels remained relatively stable within 24 hours postmortem, and that the SFRP5 m RNA could be used as a stable marker for dating of wound within 24 hours after death.(7) Compared with the control, the expression of SFRP5 m RNA in the incised group was significantly reduced. There were significantly differences between incised and contusion groups at the four hours after injury, however, there were no significantly difference at the eight hours.Conclusions:The expression levels of secreted frizzled-related protein 5 m RNA(SFRP5 m RNA) were decreased before increased only in damaged muscles. In our present study, there was no evidence for the expression level of SFRP5 m RNA affected by the degree of wound, while whether the SFRP5 m RNA expression was affected by the types of damage(incised wound or contusion) remains unclear. In addition, the expression of SFRP5 m RNA has no significant degradation within 24 hours after death. Therefore, SFRP5 m RNA is an appropriate candidate marker for wound age estimation. |
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