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目的:建立肝脏细胞的共培养和研究同一个体不同细胞的代谢,深入探讨肝纤维化的细胞机制。方法与结果:采用胶原酶肝脏原位灌流,消化肝脏将细胞分散,通过离心初步分得肝实质细胞和非实质细胞,肝实质细胞采用49.2％淋巴细胞分离液行密度梯度离心,获得精制肝细胞;非实质细胞经进一步酶消化,再用18％Nycodenz行密度梯度离心,得到纯化的贮脂细胞。肝细胞得率为5×10~7/肝～10×10~7/肝,存活率>85％,贮脂细胞得率为2×10~7/肝～4×10~7/肝,存活率>98％,纯度>95％。经本方法同时分得的两种细胞在得率,纯度和活率方面与单独分离的细胞相近,且具经济、简便、稳定的特点。结论:建立了同时分离培养肝细胞和贮脂细胞的方法。
OBJECTIVE: To establish a co-culture of liver cells and study the metabolism of different cells in the same individual, and to further explore the cellular mechanism of liver fibrosis. Methods and Results: The hepatocytes were digested with collagenase in situ and the cells were digested by digestion. The primary hepatic parenchymal cells and nonparenchymal cells were separated by centrifugation. The hepatic parenchyma cells were separated by density gradient centrifugation using 49.2% lymphocyte separation liquid to obtain purified hepatocytes ; Non-parenchymal cells were further digested with enzyme and then subjected to 18% Nycodenz line density gradient centrifugation to obtain purified fat-storing cells. The yield of hepatocytes was 5 × 10 ~ 7 / liver ~ 10 × 10 ~ 7 / liver, the survival rate was> 85%, and the storage rate of fat storage cells was 2 × 10 ~ 7 / liver ~ 4 × 10 ~ 7 / liver. Rate> 98%, purity> 95%. The two kinds of cells which are divided at the same time by this method are similar to those isolated separately in terms of yield, purity and viability, and are economical, simple and stable. Conclusion: The method of simultaneous separation and cultivation of hepatocytes and fat storage cells was established.