28℃条件下短期培养新生广西巴马小型猪肝细胞的初步观察

A preliminary study of short-term incubation of Guangxi BAMA suckling mini-pig hepatocytes at 28 ℃

目的 对2 8℃条件下短期培养猪肝细胞的可行性进行初步评价。方法 将刚分离的新生广西巴马小型猪肝细胞采用2 8℃条件下的短期培养1～3d后,恢复3 7℃继续培养,观察升温前后肝细胞的形态及LDH、AST的释放量,检测2 8℃短期培养后肝细胞的氨清除率和尿素合成量。结果 在2 8℃短期培养的肝细胞,用倒置相差显微镜观察到典型的形态特征,细胞未见明显增殖,恢复3 7℃继续培养后,肝细胞迅速增殖并维持典型的肝细胞形态;经2 8℃短期培养后的肝细胞和未经2 8℃短期培养的肝细胞均保持较强的尿素合成和氨清除能力,但在2 8℃培养2d和3d后的肝细胞尿素合成量较在2 8℃培养1d后的肝细胞尿素合成量显著降低(P <0 0 1) ;培养期内LDH、AST漏出量较少。结论 2 8℃短期培养能较好地维持猪肝细胞的形态学特征和功能,有望成为一种有效的保存和运输生物人工肝细胞材料的方法。

Objective To observe the feasibility of short-term incubation of Guangxi BAMA suckling mini-pig hepatocytes at 28 ℃. Methods Freshly isolated porcine hepatocytes were incubated at 37 ℃ for 24 h, then divided into 4 groups: control (continued to culture at 37 ℃), group A, B, C (cultured at 28 ℃ for 24, 48, 72 h respectively), finally returned to culture at 37 ℃ for 24 h. Cellular morphological characteristics, the content of lactate dehydrogenase (LDH) and aspartate aminotransferase (AST), and ammonia clearance rate and urea synthesis were examined after all hepatocytes recultured at 37 ℃ for 24 h. Results The typical cellular charactisterics of cultured hepatocytes could be observed in control group and groups A, B, C. The proliferation of hepatocytes was inhibited in groups A, B, C, but proliferative ability was restored quickly as the temperature changed to 37 ℃. Ammonia clearance and urea synthesis of groups A, B, C were close to those of control group. While the activity of urea synthesis gradually decreased as the hypothermic incubation at 28 ℃ prolonged. A slight release of LDH and AST into the culture medium was found during the culture of hepatocytes. Conclusion The liver-specific functions and morphological characteristics of hepatocytes can be kept unchanged by short-term incubation at 28 ℃, which would be an effective method for preservation and delivery of hepatocytes for a bioartificial liver system.