静脉注射携带人白介素-10的腺病毒载体对肝硬化大鼠肝缺血再灌注损伤的保护作用

Intravenous injection of adenoviral vector carrying human IL-10 gene against hepatic ischemia-reperfusion injury in rats with biliary hepatocirrhosis

目的探讨静脉注射携带hIL-10腺病毒载体对淤胆性肝硬化大鼠肝常温缺血再灌注损伤的保护作用。方法结扎SD大鼠的胆总管建立淤胆性肝硬化模型,4周后通过每只尾静脉注射Ad-hIL10-EGFP1×109PFU/ml,体内转染72h后,进行常温肝缺血再灌注损伤实验(缺血15min,再灌注60min)。经静脉取血,进行肝功能生化检测,再通过酶联免疫吸附法(ELISA)检测大鼠血清内hIL-10的表达情况。并取肝脏冰冻切片,用倒置荧光显微镜观察肝组织中EGFP的表达,石蜡切片HE染色观察肝脏的组织形态变化;免疫组化方法观察肝脏内hIL-10的表达情况;Tunel法检测肝脏内细胞凋亡情况。结果治疗组的肝脏冰冻切片,荧光显微镜下可见EGFP的表达;同时免疫组化有较多的hIL-10染色;ELISA检测血清中hIL-10的表达量为(723·8±301·7)ng/ml。相对应的是,治疗组的肝功能较对照两组明显改善,组织病理改变较对照两组减轻,肝细胞凋亡数量明显减少,差异有显著意义(P<0·05)。结论hIL-10肝脏基因治疗可以减轻肝硬化大鼠的肝缺血再灌注损伤,其机制可能是减少肝缺血再灌注损伤过程中肝细胞凋亡的数量。

Objective To investigate the protecting effects of intravenous injection of adenoviral vector containing human IL-10 gene （hIL 10） on hepatic ischemia-reperfusion injury in rats with biliary hepatocirrhosis. Methods The model of biliary cirrhosis was established in SD rats by ligating the bile duct. Four weeks later, AD-hIL10-EGFP （1.0 × 10^9 plaque forming units/ml） was delivered to the liver of rats by intravenous injection. Seventy-two hours after the transfection, hilar area of rat liver was clamped for 15 min and released for 60 min. Blood samples were collected from the inferior vena cava. T-Bil, ALT and AST were tested, and the serum level of hIL-10 was determined with ELISA. Fresh frozen sections of rat liver were made, and the expression of EGFP was observed with fluoroscopy. Immunohistochemical method was used to study the expression of hIL 10 in rat liver. The paraf- fin-embedded sections were dyed with HE to observe the histo-morphologlcal changes of liver tissue af ter ischemia reperfusion. The apoptosis of hepatocytes was determined with TUNEL assay. Results Immunohistochemistry and ELISA showed that the rats pretreated with Ad hIL10-EGFP could express hIL-10 in liver and hIL-10 circulated at a high level. Meanwhile, the values of T-Bil, ALT and AST, the histological changes of the liver and the number of apoptotic cells significantly decreased （P 0.05）. Conclusions The pretreatment with adenoviral vector carrying hIL 10 can attenuate hepatic ischemia-reperfusion injury in rats with biliary hepatocirrhosis. The mechanism of which might be that hIL-10 can reduce the number of apoptotic liver cells during the period of hepatic ischemia/reperfusion.