摘要目的 设计一种新型混合型生物人工肝(HBAL),通过对模拟肝衰竭血清的净化作用评价其疗效,探讨其临床应用的可行性.方法 选用中国人肝细胞系-1(CL-1)作为肝细胞供体.在微重力环境下,肝细胞微载体共培养5d,细胞总量约4.0×109个,细胞密度约为4.0×107/ml.然后将其在无菌环境下灌入自制生物反应器中,制成生物部分.非生物部分采用血液灌流+胆红素吸附.生物部分和非生物部分通过两套聚乙烯胶管构成一个封闭的环路.监测模拟肝衰竭血清中未结合胆红素(UBD)、鹅去氧胆酸(CDCD)、胆酸(CA)、血氨(AA)经过混合型生物人工肝循环后浓度的变化以及生物反应器中肝细胞功能、形态及肝细胞活性的变化.结果 在整个治疗过程中,循环后24 h,模拟肝衰竭血清中UBD、CDCD、CA和AA的浓度分别从(335.3±6.0)μmol/L、(395.0±5.6) μmol/L、(155.7±4.5)μmol/L、(39.0±2.6)μmol/L降至(106.0±10.9)μmol/L、(131.8±28.7) μmol/L、(42.2±7.3)μmoL/L、(3.5±1.0).μmol/L,与0h比较差异有统计学意义(P＜0.05);24 h后浓度下降趋于平稳;循环48 h后,CL-1细胞功能发生变化,生物反应器中模拟肝衰竭血清中的丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、乳酸脱氢酶(LDH)分别从(25.9±4.2)IU/L、(22.0±3.6) IU/L、(0.28±0.09) μmol/L升高至(31.0±2.6) IU/L、(31.6±8.0) IU/L、(0.41±0.12)μmoL/L,与0h比较差异有统计学意义(P＜0.05).反应器中肝细胞的数量和活力在循环48 h后亦显著下降(P＜0.05).结论 (1)我们构建了一种新型灌流型生物反应器.在这种生物反应器中CL-1细胞能保持较高的活性和良好的功能.(2)新型混合生物型人肝可以显著降低模拟肝衰竭血清中的UBD、CDCD、CA、AA浓度,具有清除这些毒性物质的功能,提示其具有明显的肝功能支持作用.

abstractsObjective To design a new type of hybrid bioartificial liver (HBAL), evaluate its efficacy in vitro, and explore the feasibility in clinical application.Methods CL-1 human hepatocytes were cultured on microcarriers for 5 days, when cell count reached about 4.0 × 109 with cell density of about 4.0 × 107/ml.CL-1 cells cultured on microcarriers in home-made bioreactor constitute the biological part of the HBAL.The abiotic part included blood perfusion and bilirubin adsorption, and blood pump was employed as the circulation driver, which were parts of HBAL.The changes of the concentrations of indirect bilirubin (UBD), chenodeoxycholic acid (CDCD), cholic acid (CA), blood ammonia (AA), AST, ALT and LDH were observed under the condition of in vitro circulation.Meanwhile, the function, morphology and the cell activity of CL-1 cells were also observed.Results After in vitro circulation for 24 h, the concentrations of UBD, CDCD, CA and AA significantly decreased from (335.3 ± 6.0) μmol/L, (395.0 ± 5.6) μmol/L, (155.7 ± 4.5) μmol/L, (39.0 ± 2.6) μmol/L at 0 h to (106.0 ± 10.9) μmol/L, (131.8 ± 28.7) μmol/L, (42.2 ± 7.3) μmol/L, (3.5 ± 1.0) μmol/L, respectively.At 48 h, ALT, AST and LDH significantly increased from (25.9 ± 4.2) IU/L, (22.0 ± 3.6) IU/L, (0.28 ± 0.09) μmol/L to (31.0 ± 2.6) IU/L,(31.6 ± 8.0) IU/L, (0.41 ± 0.12) μmol/L, meanwhile the count and vitality of CL-1 cells were significant declined.Conclusions (1) In the new HBAL system, CL-1 cells can keep its viability and function in vitro;and (2) the HBAL appears to be effective in purifying the serum in liver failure simulation model by clearing out non-conjugated bilirubin, chenodeoxycholic acid, cholic acid and ammonium chloride, which seems to be a promising therapeutic option.