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目的:通过比较未经灌流处理的重肝血浆及处理过的重肝血浆对CYP3A的影响,阐明C3A细胞安定代谢变化的原因,为未来C3A细胞的改造与功能优化奠定基础。方法:制备血浆和培养C3A细胞;实验分为4组:正常胎牛血浆(NFBP)组,正常人血浆(NHP)组,体外灌流慢性重型肝炎患者血浆(HPP)组,体外未灌流慢性重型肝炎患者血浆(CSHP)组。用分光光度法测定红霉素N-脱甲基酶(ERD)活性即CYP4503A的活性,Western blot法测定CYP4503A4的表达。结果:CSHP组ERD活性低于NFBP组(P<0.05)及NHP组(P<0.05),HPP组ERD活性也低于NFBP组(P<0.05)及NHP组(P<0.05),HPP组ERD活性高于CSHP组(P<0.05);CSHP组CYP4503A4的表达低于NFBP组(P<0.05)及NHP组(P<0.05),HPP组CYP4503A4的表达也低于NFBP组(P<0.05)及NHP组(P<0.05),HPP组CYP4503A4的表达高于CSHP组(P<0.05)。结论:经慢性重型肝炎患者血浆培养的C3A细胞,CYP4503A活性及蛋白表达降低。与之相比,经过体外灌流的慢性重型肝炎患者血浆其C3A细胞CYP4503A活性及蛋白表达有所增加,CYP4503A活性增加可能是导致安定代谢变化的原因。
OBJECTIVE: To elucidate the reasons for the changes in the stability and metabolism of C3A cells by comparing the effect of non-perfusion heavy hepatic plasma and treated severe hepatic plasma on CYP3A, so as to lay the foundation for the future transformation and functional optimization of C3A cells. Methods: The preparation of plasma and culture of C3A cells were divided into four groups: normal fetal bovine plasma (NFBP) group, normal human plasma (NHP) group, perfused chronic severe hepatitis patient plasma (HPP) group, Patient plasma (CSHP) group. The activity of erythromycin N-demethylase (ERD), namely CYP4503A, was measured spectrophotometrically and the expression of CYP4503A4 was determined by Western blot. Results: The ERD activity in CSHP group was lower than that in NFBP group and NHP group (P <0.05). The ERD activity in HPP group was also lower than that in NFBP group (P <0.05) and NHP group (P <0.05) (P <0.05). The expression of CYP4503A4 in CSHP group was lower than that in NFBP group and NHP group (P <0.05). The expression of CYP4503A4 in HPP group was also lower than that in NFBP group (P <0.05) NHP group (P <0.05). The expression of CYP4503A4 in HPP group was higher than that in CSHP group (P <0.05). Conclusion: The activity and protein expression of CYP4503A in C3A cells from patients with chronic severe hepatitis are decreased. In contrast, plasma CYP4503A activity and protein expression in C3A cells of patients with chronic severe hepatitis were significantly increased after in vitro perfusion. The increase of CYP4503A activity may be responsible for the change of diazepam metabolism.