对乙酰氨基酚对大鼠原代肝细胞及BRL-3A细胞的毒性作用比较

目的 建立大鼠原代肝细胞提取鉴定体系,比较对乙酰氨基酚(APAP)对大鼠原代肝细胞和永生化细胞BRL-3A的毒性作用。方法 采用胶原酶原位两步灌流法提取大鼠原代肝细胞,通过过碘酸雪夫染色(PAS)和肝细胞双核结构进行鉴定;CCK 8法测定APAP对大鼠原代肝细胞及BRL-3A细胞毒性作用的IC₅₀;光学显微镜透射电镜观察APAP对2种细胞的损伤情况;全自动生化分析仪测定细胞上清AST、ALT、LDH、ALP、ALB、BUN、TP、GLU 8项生化指标的变化。结果 PAS糖原染色鉴定获取的大鼠原代肝细胞,双核结构,细胞存活率浮动在80%~95%;最佳接种密度为60000/cm²,在第3~5天为对数生长期;APAP作用于大鼠原代肝细胞24 h的IC₅₀为18.03 mmol/L,95%置信区间为(17.28~18.81)mmol/L,作用于BRL-3A的IC₅₀为20.05 mmol/L,95%置信区间为(18.99~21.17)mmol/L;透射电镜结果显示,在30 mmol/L APAP作用下,2种细胞细胞器肿胀,核膜破裂,细胞膜边界模糊不清;与对照组比较,大鼠原代肝细胞分泌的天冬氨酸氨基转移酶(AST)、尿素氮(BUN)、葡萄糖(GLU)、碱性磷酸酶(ALP)、乳酸脱氢酶(LDH)随着APAP浓度增加产生显著变化,而BRL-3A细胞几乎所有的酶学指标变化均差异不显著。结论 与永生化细胞BRL-3A比较,大鼠原代肝细胞更能体现药物的肝脏毒性作用,但其体外培养存活时间较短;BRL-3A细胞缺少肝脏重要酶类,增加细胞内肝脏酶类是提升其作为肝脏毒性筛选模型的更好手段之一。

Comparison on toxic effects of acetaminophen on primary rat hepatocytes and BRL-3A cells

Objective To establish a rat primary hepatocytes isolation and identification system, and carry out studies in rat primary hepatocytes and BRL-3A cells for liver toxicity characteristic of early drug evaluation. Methods Rat primary hepatocytes were isolated by two-step in situ collagenase perfusion method, and then were identified by PAS staining and its dual-nuclei structure; The IC₅₀ values, which was hepatocelluar toxicity of APAP in rat primary hepatocytes, was evaluated by CCK8 assay; Damage to the two kinds of cells from the drug was observed using inverted phase contrast microscope, hematoxylin eosin (HE) staining, and transmission electron microscopy (SEM); Automatic biochemical analyzer was used to detect the contents of ALT, AST, ALP, LDH, TP, ALB, GLU, and BUN changes in cell supernatants after administration. Results PAS staining shows positive involvement of two nuclei in some cells, cell viability was ranged between 80% and 95%. Rat primary hepatocytes grew best with a cell density of 60000/cm², 3-5 d was ogarithmic growth phase; Primary rat hepatocytes BRL-3A were exposed to concentration of APAP showed that IC₅₀ values were 18.03 mmol/L, 95%CI=(17.28, 8.81) mmol/L and 20.05 mmol/L, 95%CI=(18.99, 21.17) mmol/L. In the high- dose groups, transmission electron microscopy revealed that cells were organelles swelling, nuclear membrane rupture and the nucleus were almost invisible. Meanwhile, compared with control group, with drug concentration increased, the values of aspartate aminotransferase (AST), urea nitrogen (BUN), glucose (GLU), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) were significantly changed, while other targets were no significantly changed in BRL-3A cells. Conclusion Compared with immortalized BRL-3A cells, primary hepatocytes can be more reflective model with the liver toxicity. However, primary hepatocytes cannot live for a long time and lack of extensive metabolic enzymes. Thus, increasing the kinds of liver enzymes of immortalized cells is a better means to boost BRL-3A cells as liver toxicity screening model.