肝病大鼠创伤弧菌攻击后肝细胞损伤和血清细胞因子检测

The hepatocyte injury and detection of serum cytokines after hepatopathy rats attacked by Vibrio vulnificus

目的:探讨创伤弧菌(Vibrio vulnificus,Vv)所致肝病大鼠肝细胞损伤、血清细胞因子的变化及其相关性.方法:采用四氯化碳制备慢性肝病大鼠18只,分Vv攻击和肝病大鼠对照组(n=9),Vv致死性感染多器官损伤肝病大鼠模型(5.0×107cfu/只),在观察肝细胞超微结构改变的同时检测大鼠血清中的前炎症因子(TNF-α、IL-1 β、IL-6)和抗炎症因子(IL-10)的含量变化,还与接受相同菌量攻击、盐水攻击的正常大鼠(n=9)相比较.结果:在接受Vv攻击后4 h肝病大鼠组仅存活1只,5 h后全部死亡(0/9,0%),较之正常大鼠Vv攻击组5h存活8只(8/9,88.9%)差异有显著性(P＜0.01);血清细胞因子的含量检测提示,肝病大鼠血清IL-10(32.5 pg/ml)显著低于正常大鼠Vv攻击组(71.8 pg/ml,P＜0.05),而TNF-α(54.3 pg/ml)、IL-1 β(421.1 pg/ml)和IL-6(740.9 pg/ml)则分别明显高于正常大鼠Vv攻击组(40.7、255.4和352.5 pg/ml)差异有显著性(P＜0.01,P＜0.01,P＜0.01).超微结构提示,肝病Vv攻击组肝细胞线柱体嵴减少、肿胀、空泡样改变,提示肝细胞损伤严重,而正常大鼠以同样Vv菌量攻击后未见同样改变,提示肝病大鼠Vv攻击后的死亡率、肝细胞损伤程度以及TNF-α、IL-1 β、和IL-6的含量均较非与肝病大鼠Vv攻击组明显升高,而IL-10则显著低于正常大鼠Vv攻击组.结论:本结果提示肝病大鼠Vv攻击后血清前炎症因子(TNF-α、IL-1 β、IL-6)以及抗炎症因子(IL-10)的浓度与大鼠的死亡率、肝细胞损伤程度相关.

Objective： To study hepatocyte injury and changes of serum cytokines of hepatopathy rats and their correlation after Vibrio vulnificus （Vv） attack and provide experimental evidence for the researches of Vv infection. Methods： Eighteen chronic hepatopathy rats which were prepared by carbon tetrachloride were divided into Vv attack group and control group to make multiple organ dysfunction hepatopathy rat models of Vv lethal infection （5.0 ×10^7cfu each）. The changes of ultrastructure of hepatocyte were observed and the concentration of serum pro-and anti-inflammatory cytokines TNF-α, IL-1β, IL-6 and IL-10 were also detected. In addition, the results were also compared with normal rats attacked by the same amount of bacteria （n=9）. Results： After 4 h of Vv inoculation, only one hepatopathy rat survived, and 5 h later, all died （0/9, 0%）. Eight normal rats survived 5 h after Vv attack （8/9,88.9%）. There was significant difference between the survival rate of hepatopathy group and normal group after they were attacked by the same amount of Vv （P〈0.01）. The result of cytokines detection showed that serum IL-10 level of hepatopathy rats （32.5pg/ml） was significantly lower than that of normal rats （71.8 pg/ml, P〈 0.05）, however, TNF-α （54.3 pg/ ml）,IL-1β （421.1 pg/ml） and IL-6 （740.9 pg/ml ） level of hepatopathy rats were significantly higher than those of normal rats which were 40.7 pg/ml （P〈 0.01）, 255.4 pg/ml （P〈 0.01） and 352.5 pg/ml （P〈0.01）respectively,after they were attacked by the same amount of Vv and there was significant difference. The observation of ultrastructure showed that hepatopathy, mitochondrial crista decrease, swollen and vacuole changes occurred, suggesting that hepatocytes were severely damaged. However,those changes were not observed in the normal rats group, which was attacked by the same amount of Vv respectively. Conclusions： In this study, the serum proand anti-inflammatory cytokines, TNF-α , IL-1β , IL-6 and IL-IO concentrations of Vv attack hepatopathy group correlate with the mortality rate, and the damage degree of the hepatocytes.