【摘要】 目的探讨内毒素(即脂多糖)诱导D-半乳糖胺致敏大鼠急性肝衰竭肝细胞凋亡情况及肝损伤发生机制。方法48只Wistar大鼠进行随机对照分组实验,分为6 h、24 h和48 h取材3大组(各16只),每个大组再分为处理组和对照组(各8只)。处理组大鼠以脂多糖(50μg／kg)+D-半乳糖胺(300 mg／kg),用1 ml无菌生理盐水溶解后腹腔内注射,对照组动物仅腹腔内注射1 ml生理盐水。在相应时间点,门静脉或下腔静脉采血查丙氨酸氨基转移酶(ALT);肝组织切片分别行透射电镜检查和脱氧核糖核苷酸末端转移酶介导的缺口末端标记分析(TUNEL分析);基因表达通过逆转录。聚合酶链反应(RT-PCR)的方法检测。结果所有处理组大鼠ALT水平均显著高于对照组(66 U／L±3 U／L),而6 h组ALT水平(399 U／L±83 U／L)显著低于24 h组(3178 U／L±63 U／L)和48 h组(1506 U／L±56U／L)。48 h组ALT水平则低于24 h组。透射电镜检查见正常对照组肝组织内罕有凋亡细胞,脂多糖／D-半乳糖胺处理6 h组凋亡的肝细胞明显较多,且多表现为早期细胞凋亡的特征,24 h和48 h组的肝细胞凋亡明显多于6 h组,并多表现为晚期凋亡的特征。肝组织的TUNEL分析显示,对照组可见少量凋亡的肝细胞(凋亡指数为2．6％±1．1％),6 h组凋亡的肝细胞明显增多(凋亡指数为7．3％±1.5％),24 h和48 h组肝组织内则可见大量肝细胞凋亡(凋亡指数分别为71．8％±10．3％和68．2％±11．9％)。iNOS mRNA在正常对照组无表达,脂多糖／D-半乳糖胺作用后早期(6 h)有高水平的表达,24 h和48 h则显著较低;p53基因在对照组和6 h组有低水平表达,在24 h和48 h组表达明显较高;p21waf1/cip1基因在对照组无表达,6 h出现低水平表达,24 h mRNA表达水平达峰值,在48 h则迅速下降到0。结论小剂量脂多糖可诱导D-半乳糖胺致敏大鼠发生急性肝衰竭;细胞凋亡是其重要的病理形态学改变;肝衰竭肝损伤的发生与iNOS基因早期高水平的表达有密切关系。更多还原

【Abstract】 Objective To investigate the situation of hepatocellular apoptosis in D-galactosamine (D-GalN)-sensitized rats with lipopolysaccharide (LPS)-induced acute liver failure and the mechanisms of liver injury therein. Methods Forty eight Wistar rats were randomly divided into 6 equal groups to be injected peritoneally with LPS (50μg/kg) and D-GalN (300 mg/kg) (treatment groups) or normal saline of the same volume (control groups) , and then were killed 6, 24, or 48 hours later. Blood samples were collected from the portal vein or vena cava inferior to detect the contents of serum alanine aminotransferase (ALT), livers were take out to detect the hepatocellular apoptosis by TUNEL assay or ultrastructral observations, and the expressions of iNOS, p53, and p21wafl/cipl gene were detected by reverse transcription polymerase chain reaction (RT-PCR). Results The ALT levels of the treatment groups were all significantly higher than those of the corresponding control groups, with the peaks 24 hours after treatment Transmission electron microscopy showed that apoptotic cells were rare in the control subgroups, but were abundant in the liver tissues of the treatment subgroups. The apoptotic indices of liver cells of the 6, 24, and 48 hours treatment subgroups were 7. 3%±1. 5% , 71. 8%±10. 3% , and 68. 2%±11. 9% respectively, all significantly higher than those of the control groups (2. 6%±1.1%, all P <0. 05). The apoptotic index increased gradually along with the time, however, the apoptotic indices of the 24 and 48 hours treatment subgroups were not significantly different (P > 0. 05). The mRNA expression levels of iNOS gene of the control subgroups, 6 hours treatment subgroup, 24 hours treatment subgroups, and 48 hours treatment subgroup were 0, 0. 53±0. 11, 0. 36±0. 08, and 0. 15±0. 04 respectively with a significant difference among different subgroups, and with a peak 6 hours after treatment. The p53 expressions of the control subgroups, 6 hours treatment subgroup, 24 hours treatment subgroups, and 48h treatment subgroup were 0. 031±0. 006, 0. 022±0. 008, 0. 49±0. 11, and 0. 39±0. 17 respectively, being low in both control subgroups and 6h treatment subgroup and significantly upregulated in the 24 and 48 hours treatment groups. Expression of p21wafl/cipl was not detected in the control subgroups and 48 hours treatment subgroup, but was found in the 6 hours and 24 hours treatment subgroups, with a peak in the 24 hours treatment subgroup. Conclusion Acute liver failure can be induced by low dose LPS in D-GalN-sensitized rats, which may be associated with the early high expression of iNOS gene; Apoptosis is the important morphological feature in this process.更多还原