新鲜分离的与预培养的成年肝细胞对胆红素代谢和细胞活性的差异比较

Differences in bilirubin metabolism between fresh-separated and pre-incubated hepatocytes from adult animals

目的探讨新鲜分离的成年动物肝细胞(adult animal hepatocytes,AAH)与预培养24h的AAH对人体胆红素代谢活性的差异,为体外生物人工肝支持系统(extracorporeal bioartificial liver support system,EBLSS)寻求最佳的肝细胞材料应用方案。方法福建省龙岩市第二医院采用机械分离法获得3个月龄的雄性小白鼠肝细胞,设新鲜分离组与预培养组,两组培养液均用含20%胎牛血清、5U胰岛素/ml的高糖型DMEM培养基。新鲜分离组在获取肝细胞后即加入胆红素血清作共培养3h后离心取上清液送检,预培养组对肝细胞作预培养24h再加入胆红素血清继续共培养3h后离心取上清液送检;设不含肝细胞的胆红素血清作对照;用贝克曼全自动生化分析仪检测两组总胆红素、直接胆红素、间接胆红素的变化;以相同的方法作3次重复实验后分析评估。结果新鲜分离肝细胞组使总胆红素、间接胆红素分别下降33.91%、39.29%,而直接胆红素变化不大;预培养组使总胆红素、间接胆红素分别下降8.00%、5.40%。两组总胆红素变化的比较P<0.01(t=3.707),间接胆红素变化的比较P<0.01(t=4.734)。以上实验在完全相同的条件下重复3次,实验结果均显示新鲜分离的AAH对胆红素的代谢活性优于预培养的AAH。结论新鲜分离的AAH对胆红素的代谢活性优于预培养的AAH,在EBLSS使用时以新鲜分离的AAH为佳。

Objective To find out the optimal procedures and suitable hepatocytes for the extracorporeal bioartificial liver support system （EBLSS）, we observed the differences in bilirubin metabolism between fresh-separated hepatocytes and pre-incubated cells for 24 hours from adult animals Methods Hepatocytes were obtained by mechanical separation from male mice of 3 months age, suspended in high glucose DMEM culture medium containing 20% fetal bovine serum and 5 U/ml insulin, and divided into fresh-separated and pre-incubated groups. For hepatocytes in fresh-separated group, they were cultured immediately after separation in medium containing bilirubin serum for 3 hours. For hepatocytes of prencubated group, the cells were first cultured for 24 hours, and then cultured for 3 hours in medium containing bilirubin serum. Bilirubin serum in medium without hepatocytes was used as the control. Total, direct and indirect bilirubin concentrations were determined by using an auto-analyzer （Backman）. Determina- tions were repeated in wiplicate. Results Total bilirubin and direct bilirubin reduced by 33.91% and 39.29%, respectively, in fresh-separated group, and reduced by 8.0% and 5.40%, respectively, in pre-incubated group. There were statistically differences in changes of total bilirubin （P 〈 0.01, t =3.707） and indirect bilirubin （P 〈 0.01, t = 4.734） between the two groups. Conclusion Fresh-separated hepatocytes from adult animals have the better activity of bilirubin metabolism than the pre-incubated cells, and are recommended in EBLSS.