改良原代大鼠肝细胞的体外分离培养和功能研究

目的建立经济有效且功能良好的原代肝细胞体外分离培养系统。方法采用0．02％胶原酶和Percoll分离液分离纯化大鼠肝细胞．光镜和电镜下观察HepatoZYME-SFM培养的肝细胞形态，自动生化仪定期检测培养肝细胞功能，RT—PCR半定量方法检测白蛋白mRNA，高效液相色谱法（high performance liquid chromatography，HPLC）分析安定代谢能力。结果分离纯化的大鼠肝细胞总数（2-3）×10^8 cells／整肝，活力和纯度大于95％，生长良好形态正常。培养3d后ALT、AST显著下降，加入NH4Cl后8d内BUN保持相对稳定高水平，白蛋白合成在第3-4天达到高峰，培养的2～5d内肝细胞可有效清除安定。结论通过使用低浓度胶原酶灌流、Pereoll分离液纯化以及HepatoZYME—SFM无血清培养基培养，肝细胞可有效保持良好形态结构和一定的生物合成代谢能力。

Research on the Function and Morphometrics of Cultured Primary Rat Hepatocytes

Objective To develop a isolation and culture system of mature hepatocytes in vitro with high production rate, satisfactory function and low cost. Method Rat primary hepatocytes were isolated by 0.02% collagenase perfusion method, and then were purified by Percoll gradient. ALT, AST, albumin and urea levels in the supernatant were measured. Semi-quantitative RT-PCR technique was used to detect the expression of albumin mRNA in periodic interval. The quantitative determination of the remnant diazepam was analyzed by high performance liquid chromatography （HPLC） method. Result （2-3）×10^8 cells per whole liver could be obtained with viability and purity above 95%. Hepatocytes cultured in HepatoZYME-SFM grew well showing normal hepatocyte morphometrics. ALT, AST levels in the supernatant decreased after 3 days culture. Albumin secretion and urea synthesis were maintained at high levels in 8 days. The elimination of diazepam was observed in 2-5 days. Conclusion Percoll gradient method can be used to increase the viability and purity of freshly isolated rat hepatoeytes. Hepatocyte morphometrics and their biological function could be effectively maintained in HepatoZYME-SFM medium.