金钱鱼肾细胞系的建立及生长特性研究

金钱鱼(Scatophagus argus)是一种重要的广盐性海水养殖鱼类,可直接在海水、淡水、咸淡水等不同环境中正常生长。为研究金钱鱼独特的渗透调节机制,本文研究了金钱鱼肾细胞的原代和传代条件及其生长特性,结果表明:原代肾细胞在含有20%胎牛血清(FBS)的L-15培养基里贴壁和生长较好,添加10ng/mL的碱性成纤维细胞因子(basic fibroblast growth factor)bFGF能明显促进细胞增殖,传代细胞采用含有10%胎牛血清的L-15培养基,细胞生长迅速,3—4d即可传代。金钱鱼肾细胞为成纤维样细胞,命名为SK细胞,目前已传至22代。扩增第11代SK细胞的细胞色素氧化酶I(COI)基因,比对结果证明此细胞系来源于金钱鱼。采用CCK-8法检测第12代细胞在低渗(95,137,200mmol/kg),等渗(330mmol/kg)和高渗(430,550mmol/kg)中的增殖情况,结果发现肾细胞在137—430mm/kg的渗透压范围内均可增殖,说明金钱鱼肾细胞对渗透压的耐受性较强。本试验首次建立了金钱鱼肾细胞系,并初步证明了肾细胞对盐度有较强的耐受性,为今后渗透压调节的深入研究奠定了基础。

ESTABLISHMENT AND CHARACTERIZATION OFA NEW EURYHALINE FISH KIDNEY CELL LINE OF SPOTTED SCAT SCATOPHAGUS ARGUS

Spotted scat Scatophagus argus is a euryhaline fish and they could live in seawater, freshwater, or brackish water. We established and characterized a new kidney cell line (SK) of the fish, and successfully subcultured SK to the 22th generations at present. The results showed that the primary cells grew well in L-15 medium with 20% fetal bovine serum (FBS), and adding 10 ng/mL basic fibroblast growth factor (bFGF) could increase significantly the growth rate. Subcultured cells were maintained in L-15 medium with 10% FBS and could be passaged in 3-4 days. SK cells were observed fibroblastic. PCR amplification of l cytochrome oxidase subunit I (COI) for the cells of 11th generation confirmed the origin of this cell line. The CCK-8 method was used to detect the proliferation rate for the twelfth SK cells in hypotonic (95, 137, 200 mmol/kg), isotonic (330 mmol/kg), and hypertonic (430, 550 mmol/kg) conditions. Therefore, the SK cells were able to proliferate in the wide range from 137 to 430 mm/kg osmotic pressure.