van Pelt F N, Knight T L, Kenna J G
Department of Pharmacology and Toxicology, St Mary's Hospital Medical School, (Imperial College of Science, Technology and Medicine), Norfolk Place, London W2 1PG, UK.
Toxicol In Vitro. 1993 Jul;7(4):311-5. doi: 10.1016/0887-2333(93)90020-6.
Previous investigations have implicated an immune response to trifluoroacetylated proteins (TFA-proteins) in the pathogenesis of halothane hepatitis. The objective of this study was to establish conditions for generation of TFA-proteins in hepatocytes exposed to halothane in vitro. Monolayer cultures of rat hepatocytes were incubated in sealed flasks with or without added halothane, then subcellular fractions were prepared by differential centrifugation and analysed by immunoblotting for reactivity with anti-TFA antiserum. The specificity of the antiserum was verified by hapten inhibition with N--TFA-l-lysine. TFA-proteins were generated when hepatocytes were cultured with halothane, but not when hepatocytes were cultured without halothane, and were concentrated in the microsomal fraction. Generation of TFA-proteins was greater when hepatocytes were exposed to an initial halothane concentration of about 0.17 mm-halothane than when hepatocytes were exposed to higher concentrations (0.6 mm and 1.4 mm). The molecular masses of the major TFA-proteins produced in vitro (100, 80 and 60 kDa) were very similar, if not identical, to the molecular masses of the major TFA-proteins produced in livers of rats treated ip with halothane in vivo, as were the kinetics of TFA-protein formation and turnover.
以往的研究表明，对三氟乙酰化蛋白（TFA蛋白）的免疫反应与氟烷性肝炎的发病机制有关。本研究的目的是建立体外培养的肝细胞暴露于氟烷时产生TFA蛋白的条件。将大鼠肝细胞单层培养物置于密封烧瓶中，添加或不添加氟烷进行孵育，然后通过差速离心制备亚细胞组分，并通过免疫印迹分析其与抗TFA抗血清的反应性。通过用N-三氟乙酰-L-赖氨酸进行半抗原抑制来验证抗血清的特异性。当肝细胞与氟烷一起培养时会产生TFA蛋白，而在没有氟烷的情况下培养肝细胞时则不会产生，并且TFA蛋白集中在微粒体组分中。当肝细胞暴露于初始氟烷浓度约为0.17 mmol/L的氟烷时，TFA蛋白的产生量比肝细胞暴露于更高浓度（0.6 mmol/L和1.4 mmol/L）时更大。体外产生的主要TFA蛋白的分子量（100、80和60 kDa）与体内腹腔注射氟烷的大鼠肝脏中产生的主要TFA蛋白的分子量非常相似（如果不是完全相同的话），TFA蛋白形成和周转的动力学也是如此。
