斜带石斑鱼肝细胞分离及原代培养方法的建立

本研究以斜带石斑鱼肝细胞为实验对象,在不同培养条件下进行原代培养,旨在探讨稳定可靠的斜带石斑鱼肝细胞分离及原代培养方法。采用组织块分离法和胰蛋白酶(含EDTA)消化法分离肝细胞,并通过密度梯度离心法分离纯化肝细胞,细胞悬液于DMEM/F-12、M199和L-15培养液中培养;细胞活力及数量采用血球计数板计数,并通过MTT法测定细胞增殖率;同时,测定不同时间培养上清液中乳酸脱氢酶(LDH)活性、白蛋白(ALB)和尿素氮(BUN)的含量,以分析肝细胞生长状态。结果表明,组织块方法不适于斜带石斑鱼肝细胞的培养,未见细胞从组织块中迁出,而胰蛋白酶消化法获得良好稳定的培养效果,细胞产量达到1.6×108个/g肝重,活细胞数达到95%;L-15培养基细胞生长明显优于DMEM/F-12和M199培养基;启动原代培养的48~72 h阶段肝细胞生长代谢旺盛,培养上清液中LDH活性显著降低,ALB和BUN含量显著升高。结果显示,0.25%的胰蛋白酶常温消化法适合斜带石斑鱼肝细胞的分离,斜带石斑鱼肝细胞原代培养的最适培养基为L-15培养基,肝细胞在启动原代培养的48~72 h生长代谢旺盛。

Study on the isolation and primary culture of hepatocytes from liver of grouper (Epinephelus coioides)

In order to explore the stable and reliable methods for isolation and primary culture of hepatocytes from Epinephelus coioides, we selected the hepatocytes of E. coioides and then cultivated them under different conditions. E. coioides hepatocytes were isolated by tissue separation and trypsin (0.25% with EDTA) digestion. The hepatocytes could be separated and purified by density gradient centrifugation. The harvested hepatocytes were then suspended in DMEM/F-12, M199, or L-15 (cultured with 5% CO₂). The yield was determined by a hemocytometer. The viability was assessed by Trypan blue exclusion test. Proliferation of hepatocytes was tested by MTT assay. Function of hepatocytes was examined according to the levels of lactic acid dehydrogenase (LDH), albumin (ALB), and urea nitrogen (BUN)of supernatant at different time, respectively. Trypsin digestion method was better than tissue culture. Using 0.25% warm trypsin digestion, the cell yield was 1.6×10⁸ cells per g(liver weight) and the viability was more than 95%. The cells growth were better cultured in L-15 medium than in DMEM/F-12 and M199 media. The liver function index showed that lactic acid dehydrogenase (LDH) significantly decreased, urea nitrogen (BUN) and albumin (ALB) significantly increased during 48-72 h with a strong proliferation. This study indicates that the best method of isolation was pancreatin digestion and the best medium was L-15. During 48 to 72 h in culture, the growth and metabolism of hepatocytes were thriving.