大鼠原代培养肝细胞作为早期肝毒性筛选模型的研究

目的研究大鼠原代培养肝细胞作为早期肝毒性的筛选模型的生物学特征。方法采用二步灌流法分离提取大鼠肝细胞进行培养;采用荧光倒置显微镜观察原代培养肝细胞的生长情况;采用MTT法绘制肝细胞生长曲线;采用全自动生化分析仪检测细胞上清液的ALT、AST、ALP、CK、LDH、TP、ALB、GGT值;采用爬片技术进行HE检查;采用Elisa法检测细胞上清液中细胞色素P450(CYP450)的总量,并采用肝细胞毒性药物对乙酰氨基酚对肝细胞作为早期肝毒性筛选模型进行验证。结果肝细胞培养4 h后开始贴壁,24 h后绝大多数肝细胞贴壁,体积明显增大;48 h后肝细胞贴壁牢固,维持至第8日,细胞开始逐渐脱落。肝细胞的对数生长期为第2至第3日,ALT、AST、LDH、CK在提取4 h后增高,第1日大幅降低后趋于平稳至第7日。TP、ALB、GGT、ALP提取4 h降低,后趋于平稳至第7日,肝细胞CYP450第1日至第5日分泌呈上升趋势,第6⁷日,轻微下降。HE结果显示提取的肝细胞与肝组织的HE涂片基本一致。对乙酰氨基酚对肝细胞的生长具有抑制作用,IC50为20.5 mmol·L-1,能使肝细胞肿胀,细胞核萎缩,使肝细胞分泌ALT、ALP、LDH增多,对肝细胞分泌CYP450的功能具有抑制作用。结论系统全面的阐述肝细胞的生物学特征及在早期毒性评价中的应用,肝细胞作为肝早期毒性筛选模型敏感的评价指标,对于原代培养肝细胞用于药物的早期毒性筛选研究起到了一个示范作用。

Primary culture hepatocytes in drug early toxicity screening model in rats

Objective To systematically study the biological character of rat primary cultured hepatocytes in drug early toxic- ity screening model. Methods Rat hepatocytes were isolated by 2 step perfusion method and cultured in CO2 incubator; the growth was observed by fluorescence microscopy; the growth curve was drawn by MTT method; the contents ofALT, AST, ALP, CK, LDH, TP, ALB, and GGT were detected by automatic biochemistry analyzer; HE examination was done by climbing slice technique; the content of P450 was detected by ELISA and the hepatocelluar toxicity of acetaminophen was validated in early toxicity screening model. Results The hepatocytes began to paste the wall after 4 h, most hepatocytes attached the wall after 24 h and the volume increased significantly. Hepatocytes pasted firmly after 48 h, maintained up to D8, and the cells began to fall offgradually. The Log growth period ofhepatocytes was D2 - D3; ALT, AST, LDH and CK increased after 4 h, and began to stablize on D1, and continued until D7. TP, ALB, GGT and ALP decreased after 4 h, and began to stablize, and continued until D7. CYP450 seretion increased on D1 - D5, and slightly decreased on D6 - D7. HE showed that the HE photo of hepatocytes was consistent with the HE photo of liver tissue. Acetaminophen inhibited the growth of hepatocytes, the IC5o 20.5 was mmol~ L- 1, which induced the swelling of liver cell and liver cell nuclei atrophy. The secretion of ALT, ALP and LDH increased, the secretion of P450 on liver cell function was inhibited, consistent with literature review. Conclusion Biological characteristics of primary culture hepatocytes are systematically studied. Sensitive evaluation indexes of primary culture hepatocytes in early toxicity screening model are found, providing a reference for in vitro drug toxicity evaluation.