胶原夹心培养大鼠肝细胞及其细胞色素P450酶活性的测定

目的　自制大鼠尾腱Ⅰ型胶原培养原代大鼠肝细胞 ,测定肝细胞中细胞色素P450 (cytochromeP450 ,CYP)酶活性。方法　制备无菌大鼠尾腱Ⅰ型胶原 ,比较单层胶原铺底法 (collagencoated ,CC)及胶原夹心法 (collagensandwichsys tem ,CSS)培养大鼠肝细胞的生长及功能维持情况。CSS法培养肝细胞 ,分别加入泼尼松龙 (10 0 μmol·L- 1,3d)、尼莫地平 (50 μmol·L- 1,2d)和利福平 (50 μmol·L- 1,2d) ,测定CYP1A、CYP2E1及CYP3A酶活性。结果　CSS法培养 6d后 ,肝细胞生长良好。CC法培养 3d后细胞开始脱落 ,此后持续增多 ;6d后 ,培养上清液中ALT、AST与LDH活性升高 ,均高于同期CSS法培养细胞上清液中的水平。加入经CYP3A代谢药物 ,CSS培养肝细胞CYP1A活性无明显改变 ;尼莫地平使CYP3A活性升高 3 3 % ,而CYP2E1活性下降 45% (P <0 0 5) ;利福平使CYP3A活性升高为对照组的1 94倍 (P <0 0 5) ,对CYP2E1活性无作用。结论　同为CYP3A底物 ,泼尼松龙、尼莫地平和利福平对CYP亚型的影响不同 ,但不改变肝细胞CYP1A对致癌物的活化。CSS培养法可作为体外模型用于药物代谢研究

Culture of rat hepatocytes in collagen sandwich system and its cytochrome P450 activity measurement

AIMTo establish a collagen sandwich system (CSS) wit h self-prepared collagen for rat hepatocytes culture[FQ(14*2。46,X-WZ]in vitro, and study the effect of CYP3A substrates on cyt o chrome P450 activities of hepatocyte cultured by CSS. METHODSS terilized type Ⅰ collagen was prepared from rat tail tendons. The morphology of hepatocyte cultured by collagen coated (CC) and CSS method was investigated and compared. Activities of ALT, AST, and LDH in aliquots of extracellular medium w ere measured by biochemical assay. CYP1A, CYP2E1 and CYP3A activities of hepatoc ytes were also determined. RESULTSBy CSS method, the shape of hepatocytes was maintained, and the structure of cells was integrated after cult ivated for 6 d. However, in CC method after 3 d, hepatocytes dropped from the co llagen matrix, and the activities of ALT, AST and LDH in aliquots were increased . As compared with control group, in CSS cultivated hepatocytes, the activities of CYP3A increased 33%, and CYP2E1 decreased 45% by nimodipine, and CYP3A was in duced 1 94-fold (P<0 05) by rifampicin, but CYP2E1 was unchanged. All the se CYP3A substrates had little effect on CYP1A activities. CONCLUSION As the same CYP3A substrates, the effects of prednisolone, rifampicin, a nd nimodipine on CYP subtypes were different, and they didn't interfere CYP1A wh ich mediated the activation of precarcinogens. The primary hepatocytes cultured by CSS method could be used as a effective model for the research of drug metabo lism in vitro.