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目的应用实时定量PCR技术检测大鼠骨骼肌挫伤后细胞中性氨基酸载体ASCT2 mRNA表达,分析其与挫伤时间的关系。方法建立大鼠骨骼肌挫伤模型,分别取伤后4、8、12、16、20、24、28、32 h及对照组检材。提取总RNA,逆转录合成第一链的cDNA,以RPL13为内参基因进行荧光定量检测,采用2-△△Ct法比较其与对照组肌肉组织中ASCT2 mRNA的相对表达量。结果损伤组肌肉组织中ASCT2 mRNA在挫伤后12、16h表达量分别为对照组的196.40%和189.15%,损伤后4、8、20、24、28、32h ASCT2 mRNA表达量与对照组相比无统计学差异(P>0.05),说明ASCT2 mRNA在损伤12～16h其表达量较正常组及其他损伤时间组要高,而在损伤20h以后ASCT2 mRNA表达量又降至正常水平。结论大鼠骨骼肌挫伤后32h之内ASCT2 mRNA的表达有一定的时间规律性,可望作为推断骨骼肌损伤时间的指标之一。
Objective To detect the expression of cell-neutral amino acid carrier ASCT2 mRNA after contusive contusion in rats by real-time quantitative polymerase chain reaction (PCR-RT-PCR), and to analyze its relationship with contusion time. Methods The rat model of skeletal muscle contusion was established. The rats were sacrificed at 4, 8, 12, 16, 20, 24, 28, 32 h after injury and the control group. The total RNA was extracted and cDNA of the first strand was reverse transcribed. RPL13 was used as the internal control gene to detect the fluorescence quantitatively. The relative expression of ASCT2 mRNA in the muscle tissue of the control group was analyzed by 2- △△ Ct method. Results The expression of ASCT2 mRNA in injured muscle tissue at 12 and 16 hours after contusion was 196.40% and 189.15% respectively. Compared with the control group, the expression of ASCT2 mRNA at 4, 8, 20, 24, 28 and 32 h after injury (P> 0.05). The results indicated that the expression of ASCT2 mRNA was higher than normal group and other injury time groups at 12 ~ 16h after injury, while the expression of ASCT2 mRNA decreased to normal level after 20h injury. Conclusion The expression of ASCT2 mRNA within 32h after skeletal muscle contusion in rats has a certain time regularity, which is expected to be one of the indexes to infer skeletal muscle injury time.