大鼠死后脑组织RNA降解与死亡时间推断的研究

A pilot study on cerebral RNA degradation and its applicability to estimation of postmortem interval in rats

目的探讨SD大鼠死后脑组织中甘油醛-3-磷酸脱氢酶(GAPDH)mRNA、18s rRNA含量的比值与死亡时间(PMI)的关系。方法SD大鼠处死后分别置于15℃、20℃环境中,采用TRIzol试剂提取SD大鼠在死后即刻、1、2、3、5、7d时脑组织中的总RNA,采用RT-PCR方法与凝胶图像分析技术检测18s rRNA和GAPDH mRNA的含量,并将二者的比值(GAPDHmRNA/18s rRNA)与PMI进行统计学回归分析。结果SD大鼠死后脑组织中GAPDH mRNA在2d时降解不明显,但到3、5、7d时降解程度显著增加,且随着温度的升高,降解速度增加;而18S rRNA降解缓慢,直到7d仍无显著性降解变化,且降解速度几乎不受温度的影响。对GAPDHmRNA/18s rRNA值(Y)与PMI(X)进行回归相关分析:20℃时,Y=0.62-0.004X(R=-0.981,P<0.001);15℃时,Y=0.965-0.001X(R=-0.709,P<0.001)。结论SD大鼠死后脑组织中GAPDHmRNA和18s rRNA基因的相对稳定性即GAPDHmRNA/18s rRNA值与PMI存在明显相关性。

Objective To study the time-dependent changes of GAPDH mRNA and 18s rRNA after different postmortem interval in rat brain. Methods SD rats were killed by CO2 asphyxia and placed at ambient temperature 15℃ and 20℃. respectively. TRIzol reagent was used to isolate total cerebral RNA at postmortem intervals of Oh, l d,2d,3d,5d,7d. The ratio of GAPDH mRNA and 18s rRNA was detected by RT-PCR technique and gel image analysis system. The ratios of GAPDH mRNA to 18s rRN at different postmortem intervals were statistically assessed by regression analysis. Results No evident degradation of GAPDH mRNA was detected at 2d, which enhanced at 3d, 5d, and 7d postmortem and accelerated at higher ambient temperature. However, 18S rRNA degraded slowly. No significant degradation of 18S rRNA was noted at 7d postmortem, which was not markedly affacted by ambient temperature. By correlative regression analysis between ratio（Y） of GAPDH mRNA to 18s rRNA and postmortem interval（X）, Y = 0.62 - 0.004X （R= -0.981,P〈0.001） , at20℃. Y=0.965-0.001X（R= -0.709,P〈0.001）at 15℃. The correlation of postmortem interval with ratio of GAPDH mRNA to 18s rRNA was statistically significant. Conclusion The ratio of GAPDH mRNA to 18s rRNA in the cerebral is a useful parameter for estimation of postmortem interval.