大鼠体外肝星状细胞活化过程中代谢活化相关 CYP450亚型的变化

摘要

OBJECTIVE To investigate the role of several xenobiotic metabolism-associated CYP450 isoforms in the auto-activation of hepatic stellate cells(HSCs) invitro. METHODS HSCs were isolated from adult Wistar rats and cultured on plastic as an in vitroauto-activation model. Positive expression of α-smooth muscle actin(α-SMA)was used as an activated marker of HSCs and detected by immunocytochemical staining in HSCs cultured for 1,2,5 or 11 d. The expressions of CYP450 isoforms were analyzed by real-time RT-PCR in the HSCs. RESULTS The immunocytochemical stai-ning showed no α-SMA expression in HSCs cultured for 1 d,while its expression gradually increased during culture since day 2. ln terms of α-SMA expression,HSCs cultured for 1,2,5,and 11 d were classified as the quiescent,early,middle and later stages of activation,respectively. The RT-PCR results revealed that CYP1B1,CYP2B1/ 2,and CYP2E1 mRNA were expressed at high levels in the early stage of HSCs activation(at day 2),which were 2.1-,1.6-,and 23.9-fold those in the quiescent HSCs(day 1),respectively. Further study revealed that mRNA expressions of these up-regulated CYPs in the early stage of activation were diminished at the subsequent two stages. The levels of CYP1A1 and CYP1A2 mRNA decreased constantly throughtout the activation process. CONCLUSION Multiple xenobi-otic metabolism-associated CYP450 isoforms might be involved in the auto-activation of rat HSCs invitro.%目的：观察大鼠肝星状细胞（HSC）活化不同时期外源活化相关 CYP450亚型CYP1A1， CYP1B1，CYP1A2，CYP2B1/2和 CYP2E1 mRNA 表达水平的变化规律。方法分离大鼠 HSC，接种在无包被塑料培养瓶中作为 HSC 自氧化活化模型，免疫细胞化学法检测不同培养时间 HSC 活化标志物α-平滑肌肌动蛋白（α-SMA）表达。实时定量 RT-PCR 法测定 HSC 自氧化激活的不同时期多种外源物活化相关 CYP450亚型 mRNA 的表达变化。结果免疫细胞化学结果表明，在塑料培养瓶中培养1 d 的 HSC 并不表达α-SMA，但随着培养时间的延长，α-SMA 表达逐渐增强，培养11 d 的 HSC 处于完全活化状态，能表达典型肌丝条纹和较强α-SMA，说明培养1，2，5和11 d 的 HSC 分别处于未活化期、活化早期、中期和晚期。实时定量 RT-PCR 结果显示，在未活化的 HSC（培养1 d）中，有 CYP1A1， CYP1B1， CYP1A2， CYP2B1/2和 CYP2E1的 mRNA 表达；在 HSC 活化早期（培养2 d），CYP1B1，CYP2B1/2，CYP2E1 mRNA 表达最高，分别为未活化 HSC 的2.1，1.6（P﹤0.05）和23.9（P﹤0.01）倍；而在活化中期（培养5 d）及晚期（培养11 d），这些 CYP 亚型表达显著降低甚至测不出。然而，CYP1A1和 CYP1A2在整个活化过程均持续下降。结论多种外源物活化相关 CYP450亚型在 HSC 自氧化激活过程发生了明显改变，其中CYP1B1，CYP2B1/2和 CYP2E1的 mRNA 表达在活化早期显著升高，这些变化可能与 HSC 的早期激活增殖有关。