新生小鼠巨细胞病毒肝炎模型的建立

A study on the establishment of neonatal mice model of cytomegalovirus hepatitis

目的探讨建立鼠巨细胞病毒(MCMV)感染致新生小鼠肝炎模型的可行性。方法48只日龄24 h内新生BALB/c小鼠随机分为实验组和对照组,实验组每只小鼠腹腔注射MCMV Smith株20μl TCID50(104.31/0.1 ml),对照组注射无菌生理盐水20μl,注射第3、7、14天取血清和肝脏,检测血清ALT水平,肝脏组织HE染色后用光镜检查组织病理损害,同时提取肝脏组织的DNA,应用MCMV及β-actin引物行PCR扩增,然后跑电泳和测序。结果实验组小鼠ALT水平(U/L)在3天时即明显升高,7天达高峰,14天时有所下降,与对照组比较,差异均有统计学意义[3天:(58.7±11.5)比(25.0±10.6),7天:(169.6±57.4)比(25.1±8.4),14天:(157.3±15.5)比(26.5±9.4),P均<0.01]。实验组小鼠肝组织内均可见肝细胞气球样变性,点灶状坏死,门管区炎性细胞浸润,肝细胞核内病毒包涵体,7天时最严重;对照组肝细胞无相似病理变化。实验组MCMV-DNA PCR电泳全部出现阳性条带,阳性条带测序结果与MCMV基因序列的同源性完全相符。结论 MCMV能侵袭BALB/c新生小鼠引起肝炎,这种模拟人类巨细胞病毒肝炎的新生小鼠模型的建立为该病动物实验研究提供了可能。

Objective To study the feasibility of establishing neonatal mice hepatitis model with murine cytomegalovirus （MCMV） infection. Methods Total of 48 newborn BALB/c mice less than 24 h after birth were randomly assigned into the MCMV and control groups. The MCMV group was inoculated intraperitoneally with MCMV Smith （20 ±1 TCID50 （10431/0. 1 ml）, while the control group was inoculated with sterile saline. The mice were sacrificed at 3,7,14-day separately, and the serums, liver tissues were harvested immediately. The alteration change of liver function was assessed by serum ALT level, histopathological damage were examined by light microscopy, and MCMV-DNA of the liver were extracted for PCR analysis and sequencing. Results Compared with the control group, serum ALT level of MCMV group raised significantly on day 3, reaching peak on day 7, and declined on day 14 [ day 3 ： （58.7±11.5） vs. （25.0 ±10.6）, day7： （169.6±57.4） vs. （25.1 ±8.4）, day 14：（157.3 ± 15.5） vs. （26.5 ±9.4）, P 〈0.01]. Pathological changes in MCMV group included ballooning degeneration of the liver cell, focal necrosis, inflammatory cells infiltration within the portal area, viral inclusion bodies within the nucleus of the liver cell. The most severe pathological changes were presented on day 7. Positive bands displayed from the MCMV group through MCMV- DNA PCR electrophoresis, and confirmed using MCMV gene homology sequencing method. Conclusions The research implicated that MCMV can attack newborn BALB/c mice inducing hepatitis, and the establishment of the BALB/c mice hepatitis＇ model made it possible for further animal studies of CMV hepatitis.