3种方法检测HCMV先天性感染对老年小鼠大脑皮质细胞凋亡的比较

The comparison of the three methods detecting apoptosis in cerebral cortex with HCMV congential infection in aged mouse

目的分析TdT介导的dUTP缺口末端标记技术(TUNEL)、流式细胞术和透射电镜3种细胞凋亡检测方法的优缺点。方法在已建立的HCMV先天性感染小鼠中枢神经系统模型基础上,选用鼠龄为24个月模型鼠进行试验。试验分为3组(每组6只)HCMV先天性潜伏感染老年小鼠组(A组)、HCMV先天性潜伏再激活感染老年小鼠组(B组)和正常对照老年小鼠组(C组)。采用TUNEL法和流式细胞仪检测各组小鼠大脑皮质细胞凋亡情况,采用透射电镜观察凋亡细胞的超微结构变化。结果TUNEL法和流式细胞术检测发现C组凋亡细胞最少,A组凋亡细胞比率较C组有所升高,而B组凋亡细胞最多;检测结果经T检验分析发现A组、B组与C组各组间凋亡率差异均有显著性。透射电镜检测发现,同C组比较,A组和B组部分神经元细胞核染色体固缩,核物质碎片化,凋亡小体形成等特征性凋亡改变。结论HCMV先天性感染可致老年小鼠大脑皮质细胞凋亡。TUNEL法和流式细胞术可用于凋亡的定量检测,但TUNEL法易受主观因素的影响,而透射电镜是凋亡定性检测的可靠方法。

Objective To compare the three methods detecting apoptosis： TUNEL, FCM and electron microscopy. Methods Firstly,the mouse model aged 24 months of HCMV congenital latent infection in cerebral tissue was established. There were total 18 mice which were classified into three groups： group A for HCMV congenital latent infection, group B for HCMV reactivated infection, and group C for control. TUNEL and FCM were adopted for the apoptosis detection in cerebral cortex. The electron microscope was used for observing the changes of the ultramicrostructure in apoptotic cells. Results Through TUNEL and FCM, the apoptosis cells in control group C were the least , the number in group A were more than group C, while the number in group B were the most in the three groups. The results were analysed by T test and the differences were significant（P 〈0. 01 ）. By electron microscopy, we found that some chromosome in cerebral cortex shrinking, nucleus fragments and apoptotic body in group A and B, but in the group C we seldom found these characteristic changes. Conclusion On the basis of these results, HCMV congential latent infection can induce apoptosis in mouse cerebral cortex when aged. TUNEL and FCM can be used for quantitative analysis, but TUNEL is easily affected by subjective factors, and electron microscope is the reliable method for apoptosis detection.