高温对罗非鱼肝细胞原代培养的影响

Characteristic of anti-hyperthermia of tilapia hepatocytes

目的观察温度对罗非鱼肝细胞原代培养的影响。方法胶原酶冷消化法分离罗非鱼肝细胞,以含多种辅助因子的培养基培养。台盼蓝拒染法测细胞活力。倒置光显微镜观察罗非鱼肝细胞的形态,透射电镜观察超微结构。按不同培养温度分为A(28℃)、B(37℃)和C(39℃)三组。比较各组不同时期培养上清液中自蛋白和谷丙转氨酶含量。结果新分离的罗非鱼肝细胞形态完整。37℃下与28℃下培养,生长良好、可存活2～3周。39℃下培养,可存活5～7 d。A组和B组培养上清液中自蛋白和谷丙转氨酶含量比较差异均无统计学意义(P＞0．05),C组和A、B组比较差异均有统计学意义(P＜0．05)。结论胶原酶冷消化法是一种较好的鱼肝细胞分离方法。罗非鱼肝细胞体外培养可耐受37～39℃高温。

Objective To investigate the effect of temperature on primarily cultured tilapia hepatocytes. Methods Tilapia hepatocytes were isolated by the method of collagenase digestion and cultured in medium with some supplementing factors. They were divided into three groups： The hepatocytes in groups A, B and C were cultured at 28℃, 37℃and 39℃ respectively. The viability of the cells was assessed by trypan blure exclusion. The morphologic changes of cultured hepotocytes were observed under light and electron microscopy. The concentrations of albumin, urea, GPT and LDH in supernatant at different culture time points were determined. Results The fresh isolating hepatocytes were round or elliptic. The tilapia hepatocytes cultured at 28℃and 37℃ could be lived during 2-3 weeks, but 5-7 days when cultured at 39℃. At the same culture time, the concentrations of albumin and GPT in group A was same as those in group B （P〉0.05）, but in group C theywere significandy different from those in group A and B （P〈0.05）.Conclusion The tilapia hepatocytes cultured at 28℃ and 37℃ have a good function at the 7th day. The tilapia hepatocyte has the characteristic of anti-hyperthermia.