大鼠骨骼肌挫伤后ASCT2 mRNA时序性表达研究

发布时间：2018-08-12 10:23

【摘要】：目的：应用实时定量PCR技术检测大鼠骨骼肌挫伤后细胞内ASCT2mRNA表达,分析其与挫伤的时间关系,旨为法医学早期损伤时间的推断探寻新的思路。方法： (1)选取成年雄性Sprague-Dawley大鼠96只,并且将其随机分成3组,包括：生前损伤组(72只)、正常对照组(6只)、死后损伤组(18只)。生前损伤组动物经麻醉、固定于鼠板和褪毛后,将重力锤自由落下打击右后肢大腿内侧肌群致骨骼肌挫伤,分别于伤后4、8、12、16、20、24、28、32、36、40、44、48小时迅速颈椎脱臼处死,并取材。在生前损伤12、24、36小时组动物左后肢大腿内侧骨骼肌取材,以比较正常肌肉和损伤肌肉之间ASCT2mRNA表达水平。死后损伤组动物按照处死并打击致挫伤之后的时间间隔不同,分别6、12、18小时三个时间点取材。正常对照组动物未经打击,直接颈椎脱臼处死；(2)使用Invitrogen TRIzol Reagent试剂盒提取肌肉组织中总RNA,并且使用紫外分光光度计对总RNA纯度进行检验,并根据紫外分光光度计所测得OD260/OD280的比值来评价核酸纯度；(3)将RNA逆转录合成cDNA,再以cDNA为模板、RPL13为内参基因进行Real.time PCR,采用2＾-△△CT法比较其与正常肌肉组织中ASCT2mRNA的相对表达量。结果： (1)根据紫外分光光度计所测得结果提示：总RNA完整性较理想,符合后续实验的要求；(2)目的基因与内参基因的扩增效率分别为89.5%和90.1%,相关系数均大于理想值0.98,表明直线性较好,定量准确,扩增效率较高且较为一致,表明RPL13作为ASCT2mRNA的内参基因较为合适；(3)损伤组肌肉组织中ASCT2mRNA在挫伤后12、16、36、40小时表达量分别为正常组的196.40%、189.15%、182.54%和136.65%,损伤后4、8、20、24、28、32、44、48小时ASCT2mRNA表达量与正常组相比无统计学差异(P0.05),说明ASCT2mRNA在损伤12至16小时其表达量迎来第一个高峰,随后又降至正常水平,而到了损伤后36至40小时表达量又迎来第二个高峰,之后回落并趋于稳定；(4)生前损伤组正常肌肉ASCT2mRNA的表达相对于正常对照组,二者并无差异(P0.05)；(5)正常对照组与死后损伤组的各个时间点之间的ASCT2mRNA表达量并不存在差异(P0.05)。结论： (1)ASCT2mRNA表达水平在生前损伤组正常肌肉和正常对照组肌肉之间不存在差异,说明同一个体未受损伤的肌肉组织可以作为评价挫伤肌肉组织的参照检材；(2)与正常组相比,死后损伤组的RNA尽管出现了降解,但是在死后的6-18小时内ASCT2mRNA的表达水平趋于稳定,说明不影响实验结果。(3)大鼠骨骼肌挫伤后48小时内ASCT2mRNA呈时序性表达,有希望成为推断骨骼肌损伤时间的新指标。
[Abstract]:Objective: to detect the expression of ASCT2mRNA in rat skeletal muscle contusion cells by real-time quantitative PCR, and to analyze the relationship between the expression of ASCT2mRNA and the time of contusion, so as to find a new way to infer the time of early injury in forensic medicine. Methods: (1) 96 adult male Sprague-Dawley rats were randomly divided into 3 groups: antemortem injury group (72 rats), normal control group (6 rats) and postmortem injury group (18 rats). After anaesthesia, fixation on the rat plate and hair loss, the injured animals in the antemortem group dropped the gravity hammer freely and hit the medial thigh muscle group of the right hind limb to cause contusion of skeletal muscle. The contusion of skeletal muscle in the right hind limb was carried out in 48 hours after the injury, and the cervical vertebrae was dislocated and executed for 48 hours. The medial thigh skeletal muscle of the left hind limb was collected from the animals in the group of 12 ~ 24 ~ 36 hours of antemortem injury to compare the expression level of ASCT2mRNA between the normal muscle and the injured muscle. In postmortem injury group, the time interval after death was different, and the time interval was 612 hours and 18 hours, respectively. The normal control group was killed without attack. (2) Invitrogen TRIzol Reagent kit was used to extract total RNAs from muscle tissue, and ultraviolet spectrophotometer was used to test the purity of total RNA. The purity of nucleic acid was evaluated according to the ratio of OD260/OD280 measured by UV spectrophotometer. (3) reverse transcription of RNA was used to synthesize cDNA. then cDNA was used as internal reference gene for Real.time PCR. The relative expression of ASCT2mRNA was compared with that in normal muscle tissue by 2 ^ -CT. Results: (1) according to the results of ultraviolet spectrophotometer, the integrity of total RNA is ideal, which meets the requirements of subsequent experiments; (2) the amplification efficiency of target gene and internal reference gene were 89.5% and 90.1%, respectively, and the correlation coefficient was higher than that of ideal value 0.98, which indicated that the linearity was better, the quantity was accurate, the amplification efficiency was higher and the amplification efficiency was consistent, which indicated that RPL13 was more suitable as the internal reference gene of ASCT2mRNA. (3) the expression of ASCT2mRNA in muscle tissue of the injured group was 182.54% and 136.65% of the normal group at 12 ~ 1616 ~ 36 ~ 40 hours after contusion, respectively. The expression of ASCT2mRNA in the muscle tissue of the injury group was not significantly different from that of the normal group at 48 hours after injury (P0.05), which indicated that the expression of ASCT2mRNA reached the first peak at 12 to 16 hours after injury. Then it decreased to normal level, and then reached the second peak 36 to 40 hours after injury, then decreased and tended to stabilize; (4) the expression of ASCT2mRNA in normal muscle in antemortem injury group was not different from that in normal control group (P0.05). (5) there was no difference in ASCT2mRNA expression between normal control group and postmortem injury group (P0.05). Conclusion: (1) there is no difference in the expression of ASCT2mRNA between normal muscle and normal muscle in antemortem injury group, indicating that the muscle tissue without injury in the same body can be used as a reference material for evaluating contusion muscle tissue. (2) compared with the normal group, the expression of RNA in postmortem injury group tended to be stable within 6-18 hours of postmortem injury, indicating that it did not affect the experimental results. (3) the expression of ASCT2mRNA in rat skeletal muscle was time-sequentially expressed within 48 hours after contusion of skeletal muscle. Hopefully, it will be a new indicator for estimating the time of skeletal muscle injury.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：D919.4

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