急性胆道感染诱发肝细胞凋亡的实验研究

Experimental study on hepatocyte apoptosis induced by acute biliary tract infection

目的研究急性胆道感染过程中是否存在肝细胞凋亡,并探讨肝细胞凋亡的可能机制。方法 30只实验大鼠适应喂养7 d后随机分为5组,每组6只,其中1组为对照组,余下4组为实验组。实验组通过胆总管注入大肠埃希菌,建立急性胆道感染模型,并于注入大肠埃希菌6、12、24及48 h后处死。对照组动物胆总管内注入1 ml生理盐水,6 h后处死。取各组肝组织,制作切片。HE染色,光镜下观察;Tunnel染色,计数调亡肝细胞,免疫组织化学试验检测bcl-2及Bax的表达,各组数据组间比较采用方差分析,进一步两两比较采用LSD-t检验以及Dunnett's T3法。结果对照组肝组织无明显病理变化,而实验组各组可见肝细胞凋亡改变。各实验组与对照组相比,Tunnel染色阳性表达差异均有统计学意义(P<0.05),24及48 h组Tunnel染色阳性表达较12h组明显增加,差异有统计学意义(P<0.05)。对照组与实验组间Bax表达差异有统计学意义(P<0.05),在实验组间随着时间延长表现出增多的趋势,但差异无统计学意义(P>0.05)。而Bcl-2在对照组和6及12 h实验组间差异无统计学意义(P>0.05),但随着时间的延长,24及48 h组与12 h组比较差异有统计学意义(P<0.05)。结论急性胆道感染后肝细胞在短时间内即会出现细胞凋亡,并且贯穿于急性胆道感染始终,基因层面表现为Bax表达增强,Bcl-2表达减弱。Bax与Bcl-2等凋亡相关基因表达的改变与肝细胞凋亡存在时间上的一致性,表明肝细胞凋亡可能与Bax与Bcl-2等基因有关。

Objective To examine the occurrence and possible mechanism of hepatocyte apoptosis in acute biliary tract infection.Methods After adaptive feeding for seven days,30 male rats were randomly divided into five groups （n=6 each）,i.e.,one control group injected with 0.2 ml of saline through the common bile duct and four experimental groups injected with an equal volume of Escherichia coli cell sus-pension to build an acute biliary tract infection model.Animals of the control group were killed 6 h after injection of saline,and those of the four experimental groups were killed respectively at the time points of 6,12,24,and 48 h after injection of E.coli.Liver tissue specimens were taken from all the rats to prepare tissue sections.Pathological features were examined by hematoxylin-eosin staining and light micros-copy.Hepatocyte apoptosis was examined by cell counting after TUNEL staining and calculation of the apoptotic index.The expression of ap-optosis-related proteins （Bcl-2 and Bax）was measured by immunohistochemistry.Data comparison between groups was performed by a-nalysis of variance,and pairwise comparisons were further conducted using the LSD-t test and Dunnett′s T3 test.Results Tissue speci-mens of the control groups had no significant pathological changes,while those of the experimental groups showed hepatocyte apoptosis. Compared with the control group,the four experimental groups had significantly greater values of apoptotic index （P〈0.05）.Additionally, the values of apoptotic index in the 24 and 48 h experimental groups significantly increased over time compared with that of the 12 h experi-mental group （P〈0.05）.Bax protein expression level significantly differed between the control group and the experimental groups （P〈0.05）,showing a slight increasing trend over time in the latter four groups （P〉0.05）.Bcl-2 protein expression level had no significant differences between the control group and the 6 or 12 h experimental groups （P〉0.05）.Over time,however,significant differences were observed in Bcl-2 expression level between the 12 h experimental group and the 24 or 48 h experimental group （P〈0.05）.Conclusion Hepatocyte apoptosis occurs within a short time after acute biliary tract infection.It persists in the whole process of the infection,with in-creased expression of Bax （inducer of apoptosis）and decreased expression of Bcl-2 （inhibitor of apoptosis）.The changes in the expression of apoptosis-related genes coincide with hepatocyte apoptosis,suggesting that hepatocyte apoptosis may be associated with Bax and Bcl-2 genes.