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目的探讨大鼠死后骨骼肌细胞核DNA降解与晚期死亡时间的关系。方法 54只SD大鼠随机均分为0d、1d、3d、7d、21d、28d、35d、42d时间点组,采用断颈法处死后,在相应时间点采用常规HE染色观察和采用Hoechst33258荧光染色法检测DNA浓度、进行看家基因GAPDH PCR半定量,对骨骼肌DNA降解情况进行分析。结果 HE染色结果显示,随着死后时间的延长,骨骼肌的自溶程度增加,在死后7d,细胞核HE切片中细胞核基本消失。Hoechst 33258荧光定量和GAPDH PCR半定量分析显示,大鼠死后21d以内,DNA浓度及GAPDH的表达量迅速下降,期间各时间点之间均存在显著性差异(P=0.000),而在之后至42d期间,数值维持在一个较低的水平,组间无显著性差异。上述实验结果可相互印证。结论大鼠骨骼肌组织DNA含量死后21d内呈明显下降趋势,其变化规律可尝试用于晚期死亡时间推断。
Objective To investigate the relationship between postmortem interval and nuclear DNA degradation in skeletal muscle of rats. Methods 54 Sprague-Dawley rats were randomly divided into 4 groups: 0d, 1d, 3d, 7d, 21d, 28d, 35d and 42d. The rats were sacrificed by cervical dislocation and stained with Hoechst33258 at the corresponding time points. Method to detect DNA concentration, housekeeping gene GAPDH PCR semi-quantitative analysis of skeletal muscle DNA degradation. Results HE staining showed that with the extension of postmortem interval, the degree of autolysis of skeletal muscle increased. At 7 days after death, the nucleus of HE in the nucleus almost disappeared. Hoechst 33258 fluorescence quantitative PCR and GAPDH PCR semi-quantitative analysis showed that the DNA concentration and GAPDH expression decreased rapidly in rats within 21 days after death, there were significant differences (P = 0.000) between the time points and During 42d, values remained at a lower level with no significant difference between groups. The above experimental results can be confirmed with each other. Conclusion The content of DNA in skeletal muscle of rats shows a decreasing trend within 21 days after death, and its variation may try to infer the late death time.