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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6~7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。
Objective To study the biological characteristics of rat primary cultured hepatocytes as a screening model for early hepatotoxicity. Methods Two-step perfusion method was used to isolate and culture rat hepatocytes. The growth of primary cultured hepatocytes was observed by fluorescence inverted microscope. The growth curve of hepatocytes was drawn by MTT method. The growth of hepatocytes was detected by automatic biochemical analyzer ALT, AST, ALP, CK, LDH, TP, ALB and GGT were detected by HE staining. HE staining was performed by using slide technique. The total amount of cytochrome P450 (CYP450) in the cell supernatant was detected by Elisa method. Paracetamol was used to validate hepatocytes as an early hepatotoxicity screening model. Results After 4 hours, the hepatocytes began to adhere to the wall, and most of the hepatocytes adhered to the wall 24 hours later. The volume of the hepatocytes increased significantly. After 48 hours, the hepatocytes adhered firmly and maintained until the 8th day. The logarithmic growth phase of hepatocytes was from day 2 to day 3, and the levels of ALT, AST, LDH and CK increased after 4 h of extraction. After the first day, the levels of ALT, AST and LDH tended to be stable until the 7th day. TP, ALB, GGT, ALP extraction 4 h decreased, then stabilized to the 7th day, liver cell CYP450 secretion increased from day 1 to day 5, the first 6 to 7, decreased slightly. HE results showed that the extracted liver cells and liver tissue HE smears were basically the same. Acetaminophen has an inhibitory effect on the growth of hepatocytes with an IC50 of 20.5 mmol·L-1, which can cause hepatocyte swelling and atrophy of the nucleus, increase the secretion of ALT, ALP and LDH, and has the effect on the secretion of CYP450 by hepatocytes Inhibition. Conclusion The biological characteristics of hepatocytes and their application in early toxicity evaluation are systematically expounded systematically. Hepatocytes as a sensitive indicator of liver early toxicity screening model play an important role in the early toxicity screening of primary cultured hepatocytes for drugs Demonstration role.