构建四氯化碳诱导的家兔肝纤维化模型

背景：四氯化碳诱导的大鼠肝纤维化模型常用于抗肝纤维化药物的筛选和评价，但目前很少有四氯化碳诱导兔肝纤维化模型的报道。 目的：建立家兔肝纤维化动物模型，并观察造模过程中动物的肝脏功能和组织病理学变化。 设计、时间及地点：随机对照动物实验，于2007-12/2008-04在解放军成都军区昆明总医院实验动物中心完成。 材料：40只普通级日本大耳家兔，雌雄各半，体质量1.75~2.25 kg，用于建立家兔肝纤维化动物模型。 方法：40只家兔采用随机数字表法分为实验组(n=30)和对照组(n=10)。实验组腹腔注射40 g/L 四氯化碳橄榄油溶液，对照组腹腔注射等量生理盐水。1~3周四氯化碳剂量为0.1~0.2 mL/kg，根据动物的情况每周注射2次；3~6周四氯化碳剂量调整为0.3~0.4 mL/kg；6~8周为0.4~0.5 mL/kg。于注药后4，8，12周分别留取肝组织和血清标本，进行苏木精-伊红染色病理观察和生化检测。 主要观察指标：实验家兔肝脏大体及病理学改变，静脉血生化指标的变化。 结果：纳入日本大耳家兔40只，造模20 d 时，实验组家兔死亡4只，原因主要为急性肝坏死及对药物的耐受性差。40 d 时死亡1只；50 d 腹腔注射因药物误入血管死亡1只。①对照组家兔肝脏外观呈暗红色，病理切片示：肝小叶结构完整，肝细胞围绕中央静脉呈放射状排列。实验组8周时，肝脏呈暗紫色，表面有轻微粟粒样改变，病理切片示：肝细胞点状及点灶状坏死，汇管区炎症细胞浸润，呈早期纤维化症状。12周时，肝脏呈灰褐色，有较明显粟粒样改变。病理切片示：肝小叶结构破坏，肝索排列紊乱，肝细胞脂肪变性，间质纤维组织增生，有炎性细胞浸润，为明显肝纤维化症状。③家兔血生化指标随着注药时间的延长，白蛋白含量及白球蛋白比值逐渐下降，球蛋白、间接胆红素、直接胆红素逐渐升高，谷丙转氨酶、谷草转氨酶前期明显升高后期又有所下降。 结论：长期给予四氯化碳可导致家兔的肝纤维化形成，并有较明显的阶段性变化。

Establishment of rabbit models of carbon tetrachloride-induced liver fibrosis

BACKGROUND: Rat models of hepatic fibrosis induced by carbon tetrachloride (CCL4) can be commonly used in screening and evaluating drugs against hepatic fibrosis. However, there are few studies on rabbit models of hepatic fibrosis induced by carbon tetrachloride. OBJECTIVE: To establish rabbit models of hepatic fibrosis, and to observe liver function and histopathological changes during model establishment. DESIGN, TIME AND SETTING: The randomized, controlled animal study was performed at the Animal Experimental Center of Kunming General Hospital of Chengdu Military Area Command of Chinese PLA from December 2007 to April 2008. MATERIALS: Forty ordinary Japanese rabbits with big ears, (50% male and 50% female), weighing 1.75-2.25 kg, were used for rabbit hepatic fibrosis model establishment. METHODS: Totally 40 rabbits were randomly assigned into a experimental group (n=30) and a control group (n=10). Rabbits in the experimental group were injected with 40 g/L CCL4 and olive oil suspension into the abdominal cavity, whereas those in the control group were treated with the same volume of saline. At 1-3 weeks, the injection frequency was 0.1-0.2 mL/kg, twice every week. At 3-6 weeks, according from diet reaction and body variation of rabbits, the quantity was 0.3-0.4 mL/kg. At 6-8 weeks, the quantity was adjusted to 0.4-0.5 mL/kg. At 4, 8 and 12 weeks after drug injection, hepatic tissue and serum specimen was subjected to hematoxylin-eosin staining for pathological observation and biochemical index. MAIN OUTCOME MEASURES: General and pathological changes in the liver; Biochemical indicator changes in venous blood. RESULTS: Forty Japanese rabbits were included. At 20 days of model establishment, 4 rabbits died of acute hepatic necrosis and bad tolerance to drugs. At 40 days, 1 rabbit died; At 50 days, 1 died of error injection of drugs into blood vessels in the experimental group. The liver of the control group looks kermesinus. Pathological sections exhibited that the structure of hepatic lobule was integrity; central veins radiated arranging to hepatic cells. At 8 weeks, the liver in the experimental group developed purple, seems wrapping with millets. Pathological sections showed punctiform or lamellar cellular necrosis, inflammatory cell infiltration around liver canaliculus, displaying early symptoms of liver fibrosis. At 12 weeks, the liver looks gray, seems wrapping with many types of millet. Pathological sections exhibited that the structure of hepatic lobule was destroyed, hepatic cord was in disorder, hepatic cells exhibited fatty degeneration, some of which showing interstitial fiber fibroplasias and inflammatory cell infiltration. These were significant hepatic fibrosis symptoms. Accompany with time prolonged, albumin levels and ratio of albumin to globulin decreased rapidly, globulin, indirect bilirubin and direct bilirubin contents gradually increased. Alanine aminotransferase and aspartate aminotransferase contents significantly increased, but decreased later. CONCLUSION: Injecting CCL4 for a long time can result to liver fibrosis in rabbit models, and have significant difference in different time.