人肝细胞离线混合生物人工肝支持系统的构建与实验研究

摘要

目的 构建人肝细胞离线混合生物人工肝支持系统,探讨其治疗肝衰竭患者离体血浆的可行性,为开展离线生物人工肝治疗肝衰竭患者奠定基础.方法 采用转染人肝再生增强因子(hALR)基因的Hep G2细胞为生物材料,将细胞培养于费森尤斯血浆滤过器(Psu-2S)中,构建生物反应器.利用慢性乙型病毒性肝炎基础上发生的慢加急性肝衰竭患者血浆置换后离体血浆作为治疗对象,先行2h血浆胆红素吸附治疗,然后应用构建的生物人工肝系统对其进行实验性治疗.结果 生物反应器建立过程顺利,无菌技术达到要求,细胞培养液离心检测未见细胞漏出,生物反应器外腔液中细胞活力平均达95.6%,细胞增殖旺盛.肝衰竭患者血浆在胆红素吸附治疗前后分别为(176.19±54.14)μmol/L,(50.10±16.85)μmol/L,差异有统计学意义(t=8.32,P=0.0002),白蛋白、尿素氮、血糖等指标在胆红素吸附前后差异无统计学意义.血浆在生物人工肝治疗前后,胆红素分别为(50.10±16.85)μmoL/L,(30.27±15.02)μmol/L,差异有统计学意义(t=13.19,P=0.000);同时尿素氮(UERA)明显升高(t=15.4,P=0.000);血糖(GLU)明显降低(t=5.67,P=0.0013);白蛋白治疗前后分别为(6.13±2.04)g/L及(7.19±2.42)g/L,差异无统计学意义(t=1.73,P=0.134).结论 自行构建的混合生物人工肝支持系统细胞繁殖活性良好,对慢加急性肝衰竭患者血浆胆红素有较好的吸附及代谢作用并具有一定的蛋白合成功能.%Objective To construct an off-line hybrid bioartificial liver supporting system with human liver cell line,and study it's effect on the plasma from patients with liver failure.Methods We established the bioreactor using Psu-2s(Fresenius)cultured with Hep G2 cell transfected with human augmenter of liver regeneration (hALR) gene,then constructed a hybrid bioartificial liver supporting system,at last using the bioartificial liver support system to purify the plasma treated 2 hours with serum bilirubin absorbent,separated from acute on chronic liver failure patients infected by hepatitis B virus.Results Bioreactor was successful constructed.The cell viability in perigastrum of bioreactor is 85.2% and cell propagated rapidly.Before and after treating with bilirubin absorbent,serum total bilirubin was(176.19 ±54.14)μmol/L and(50.1±1 6.85)μmol/L respectively(P=0.0002).While there were no signifcance difference in the level of albumin.urea and glucose.At the begin and end of treatment with bioartificial liver,serum total bilirubin was(50.10±16.85)μmol/L and(30.27±15.02)μmol/L respectively(P=0.000),the urea and albumin increased,urea has significantly difference,but the change of albumin hasn't.Conclusion The off-line hybrid bioartificial liver supporting system with human liver cell line were builded successfully and have synthesis and metabolism functions for acute on chronic liver failure patients.