大鼠肌肉挫伤后肌钙蛋白I mRNA表达与损伤时间关系的研究

发布时间：2018-09-07 14:23

【摘要】： 目的:应用实时荧光定量PCR技术检测大鼠肌肉挫伤后骨骼肌肌钙蛋白I(skeletal troponin I,sTnI)mRNA表达,探寻其时序性表达规律,探讨sTnI mRNA表达变化应用于损伤时间推断的可行性,旨在为法医学早期损伤时间的推断提供科学依据。方法:选取健康成年雄性SD大鼠63只,随机分为正常对照组(6只)、死后损伤组(9只)和生前损伤组(48只)。均实施麻醉、褪毛后,利用改进的自由落体装置,250g重力锤自150cm高度垂直自由落下,造成大鼠右后肢大腿内侧肌群挫伤。损伤后0.5h、1h、6h、12h、18h、24h、30h、36h八个时间点(每个时间点6只)取挫伤处肌肉组织50mg,置于液氮中保存备用;正常对照组大鼠未打击造伤;死后损伤组脱颈处死后,自由落体打击同一部位,其余处理与实验组相同。以膜吸附技术为基础的SV Total RNA Isolation System提取肌肉组织中的总RNA,逆转录合成cDNA第一条链,梯度浓度稀释cDNA原液分别制作sTnI和参比基因的相对定量标准曲线;选取管家基因核糖体蛋白L32(Ribosomal Protein L32,RPL32)为参比基因,利用SYBR Green I嵌合荧光法实时定量PCR检测sTnI mRNA逆转录合成cDNA的相对表达量,分别与损伤时间进行统计学分析。结果:(1)Agilent 2100芯片生物分析仪和2%甲醛变性琼脂糖凝胶电泳检测总RNA纯度、浓度及完整性较好,均可满足下一步实验的要求。 (2)sTnI与参比基因RPL32扩增效率分别为103.6%和100.5%,一致性较好,说明参比基因选择正确;扩增曲线拐点清楚,基线平而无上扬现象,说明引物设计特异性强、反应性能良好;sTnI基因融解温度为88°C,RPL32基因融解温度为84°C,两者融解曲线均为单一峰型,基底窄而峰高,表明并无引物二聚体等非特异性扩增产生。 (3)大鼠骨骼肌挫伤后sTnI mRNA表达明显下调(P0.05),挫伤后0.5h、1h、6h sTnI mRNA表达量分别为正常组的78.17%、41.58%、32.13%且差异具有统计学意义(P0.05),6h~36h sTnI mRNA表达量与对照组相比差异无统计学意义(P0.05)。 (4)为了比较生前损伤组正常肌肉与损伤肌肉中sTnI mRNA的表达水平,选取挫伤后18h组大鼠左后肢同一部位取材。结果表明正常肌肉中sTnI mRNA表达与正常对照组相比无差异(P0.05),而同一个体损伤肌肉中sTnI mRNA表达与正常肌肉中sTnI mRNA表达存在差异(P0.05)。 (5)与正常对照组相比,死后损伤组双后肢sTnI mRNA的量约下降了30%(P0.05),死后损伤组各时间点sTnI mRNA的量无差异(P0.05)。结论:(1)生前损伤组正常肌肉中sTnI mRNA表达与正常对照组相比无差异(P0.05),而同一个体损伤肌肉中sTnI mRNA表达与正常肌肉中sTnI mRNA表达存在差异(P0.05), sTnI mRNA的表达仅在损伤肌肉中发生下调,同一个体未遭受损伤部位的肌肉可以作为评价损伤肌肉的对照。 (2)大鼠肌肉挫伤后36h内sTnI mRNA呈时序性表达下调趋势,与损伤时间有一定的关系,其时序性变化可望作为骨骼肌早期损伤时间推断的指标之一,服务于法医学实践。 (3)实时荧光定量PCR技术检测分子水平的变化敏感而且准确,适合法医学研究及检案的需要。
[Abstract]:AIM: To detect the expression of skeletal troponin I (sTnI) mRNA in skeletal muscle of rats after muscle contusion by real-time fluorescence quantitative PCR, and to explore the regularity of its sequential expression, and to explore the feasibility of applying the changes of sTnI mRNA expression to the estimation of injury time, so as to provide scientific basis for forensic early injury time estimation.
Methods: 63 healthy adult male SD rats were randomly divided into normal control group (6 rats), postmortem injury group (9 rats) and prenatal injury group (48 rats). Fifty mg of muscle tissue was taken from the contusion site at eight time points (six at each time point) at 2h, 18h, 24h, 30h and 36h, and stored in liquid nitrogen for reserve; rats in the normal control group were not injured by percussion; rats in the postmortem injury group were executed after neck removal by free fall, and the rest of the treatments were the same as those in the experimental group. Total RNA was extracted from muscle tissues and the first strand of the cDNA was synthesized by reverse transcription. The relative quantitative standard curves of sTnI and reference genes were made by gradient dilution of the original cDNA. The housekeeping gene ribosomal protein L32 (RPL32) was selected as the reference gene and the sTnI mRNA inversion was detected by SYBR Green I chimeric fluorescence real-time quantitative PCR. Results: (1) Agilent 2100 microarray Bioanalyzer and 2% formaldehyde denatured agarose gel electrophoresis were able to detect the purity, concentration and integrity of total RNA, which could meet the requirements of the next experiment.
(2) The amplification efficiencies of sTnI and RPL32 were 103.6% and 100.5% respectively, which indicated that the selection of reference genes was correct; the inflection point of amplification curve was clear and the baseline was flat without rising phenomenon, indicating that the primer design was specific and the reaction performance was good; the melting temperature of sTnI gene was 88 C, and the melting temperature of RPL32 gene was 84 C. For a single peak type, the basal and narrow peaks showed no primer and no specific amplification of primers such as two dimers.
(3) The expression of sTnI mRNA was significantly down-regulated after skeletal muscle contusion in rats (P 0.05). The expression of sTnI mRNA was 78.17%, 41.58%, 32.13% in the normal group at 0.5 h, 1 h and 6 h after contusion, and the difference was statistically significant (P 0.05).
(4) In order to compare the expression of sTnI mRNA in normal and injured muscles of prenatal injury group, the left hind limbs of rats in 18 h post-contusion group were taken from the same site. The results showed that there was no difference in the expression of sTnI mRNA between normal muscle and normal control group (P 0.05), but the expression of sTnI mRNA in injured muscles of the same body was higher than that in normal muscle (P 0.05). There were differences (P0.05).
(5) Compared with the normal control group, the amount of sTnI mRNA in both hind limbs of postmortem injury group decreased by about 30% (P 0.05), and there was no difference in the amount of sTnI mRNA at each time point of postmortem injury group (P 0.05).
Conclusion: (1) The expression of sTnI mRNA in normal muscle of prenatal injury group was not different from that of normal control group (P 0.05), but the expression of sTnI mRNA in injured muscle of the same body was different from that in normal muscle (P 0.05). The expression of sTnI mRNA was down-regulated only in injured muscle, and the muscles of the same body could act as injured muscle. To evaluate the control of injured muscles.
(2) The expression of sTnI mRNA was down-regulated sequentially within 36 hours after muscle contusion in rats, which was related to the time of injury. The time-series changes of sTnI mRNA may serve as one of the indicators for estimating the time of early skeletal muscle injury and serve forensic practice.
(3) Real-time fluorescence quantitative PCR is sensitive and accurate in detecting the changes of molecular level, which is suitable for forensic research and case detection.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：D919

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