卡介苗联合脂多糖诱发大鼠原代培养肝细胞免疫性损伤模型的构建

目的：联合卡介苗（BCG）和脂多糖（LPS）构建大鼠原代培养肝细胞免疫性损伤模型，用于护肝药物的初步筛选。方法：预注BCG 12d的大鼠制备原代肝细胞，用10mg/L LPS诱发肝细胞损伤。用联苯双酯（DDB）、马洛替酯（MLT）、水飞蓟宾（SB）和甘利欣（GRZ）进行防护，同法进行整体动物实验，以论证该模型的适用性和可靠性。测定血清和上清液中AST、LDH和一氧化氮（NO）；计算肝脾指数；进行肝脏病理检查。结果：体外实验中，BCG＋LPS组上清液AST、LDH和NO明显高于正常对照组（P＜0.01)，且呈时间依赖性增加，12h达稳态。DDB、MLT、SB和GRZ均可明显抑制12h的AST、LDH和NO。整体实验中，BCG和LPS处理6h后血清AST、NO及肝脾指数明显高于正常对照组（P＜0.01)，LDH增加但无统计学差异（P＞0.05)；肝脏病理损伤明显。DDB、MLT、SB和GRZ对上述指标和病理损伤均有不同程度抑制作用。结论：BCG联合LPS诱发的大鼠原代培养肝细胞免疫性损伤模型可望用于护肝药物活性的初步筛选。

Establishment of BCG combined LPS-induced hepatocyte immunotoxicity model to assess liver protective effects

Objective: To establish a hepatocyte immunotoxicity model for screening of liver protective medications. Methods: Cytotoxicity was induced by coincubating BCG pretreated rat hepatocytes in vivo and with 10 mg/L LPS in vitro . Biphenyldimethylesterate (DDB), malotilate(MLT), silybin(SB) and glycyrrhizin (GRZ) were coincubated along with LPS to prevent the hepatocyte injury and verify the applicability and reliability of the model . AST, LDH and nitric oxide (NO) were measured in both the serum and supernatant. The liver and spleen index were calculated and the liver histopathologic changes were examined microscopically. Results: Supernatant AST, LDH and NO in the BCG combined LPS group were increased in comparison with the control group ( P < 0.01 ). This increase was attenuated by the addition of DDB, MLT, SB and GRZ ( P <0.05). The serum AST, NO and liver and spleen index were also increased significantly compared with the control group ( P <0.01). Microscopic exam revealed serious histopathologic changes in the BCG combined LPS group. Hepatoxicity with associated liver enzyme elevation but histopathologic changes were attenuated by DDB, MLT, SB and GRZ. Conclusion: BCG combined LPS induced hepatocyte immunotoxicity in an in vitro rat model may be a useful technique to assess the effectiveness of liver protective medications.