高原猪肝细胞生物反应器的生物学活性

BIOLOGICAL ACTIVITY OF THE HOLLOW FIBER BIOREACTOR WITH CULTURED PORCINE HEPATOCYTE AT HIGH ALTITUDE

目的了解在高原环境下,培养球形聚集体猪肝细胞中空纤维型生物反应器的生物学活性特征,为构建高原体外生物人工肝提供实验依据。方法采用已适应本地高原环境的长白仔猪,并分离其肝细胞,经体外球形聚集体培养2 d后,移入中空纤维型生物反应器外腔中,内腔以培养液循环,继续培养10 d,观察培养细胞形态,检测外腔培养液中的总蛋白、白蛋白、尿素和乳酸脱氢酶水平变化。结果经过2 d培养后,肝细胞形成了大量的结构较为稳定的球形聚集体;中空纤维反应器培养前,外腔培养液中总蛋白、白蛋白及尿素水平分别为（16.2±0.3）g/L、（5.9±0.3）g/L和（2.5±0.2）mmol/L,培养2 d后均呈现明显升高,差异有显著性（P〈0.05）,4 d达高峰,分别为（22.7±2.4）g/L（、7.8±0.4）g/L和（4.6±0.5）mmol/L,然后逐渐下降,培养8~10 d时明显低于4 d时水平;乳酸脱氢酶水平培养6 d时明显升高,差异有显著性（P〈0.05）,8~10 d时进一步升高,差异有显著性（P〈0.05）。结论在高原环境条件下,以培养球形聚集体猪肝细胞2~6 d时制备的中空纤维型生物反应器的生物学活性良好,为进一步应用于构建高原环境下生物人工肝奠定了基础。

Objective To understand the characteristic of biological activity of the hollow fiber bioreactor with cultured porcine hepatocytes at high altitude,so as to provide experimental basis for making up bioartificial liver at altitude.Methods Hepatocytes isolated from the porcine liver were cultured for 2 ds to form spheroid,then moved into the outside cavity of hollow fiber reactor and cultured for 10 ds.The shape of cultured cells was observed,and the total protein,albumin,urea and lactate dehydrogenase in culture supernatants were determined.Results After spheroid culture for 2 ds,the cells gathered closly to form spheroids.And the number of spheroid was increased,but the shape of cultured cells did not change.After culture in hollow fiber reactor for 2 ds,the levels of total protein,albumin,and urea in culture supernatants were significantly increased,reached the peak on 4th d（（22.7±2.4）g/L,（7.8±0.4）g/L,and（4.6±0.5）mmol/L respectively）,which were higher than those before culture（（16.2±0.3）g/L,（5.9±0.3）g/L,and（2.5±0.2）mmol/L respectively,P0.05）,then gradually decreased and significantly lower on 8～10 th d than those on 4th d（P0.05）.The level of lactate dehydrogenase significantly rose from 6th d（P0.05）,and that on 8～10th d was further increased.Conclusion The hollow fiber bioreactor,which contains the porcine hepatocyte spheroids cultured for 2～6 ds,has good biological activity,and it may be used for further study of making up bioartificial liver at high altitude.