剑尾鱼脑细胞系的建立及细胞色素P4501A的诱导表达

Development of a brain cell line from swordtail fish (Xiphophorus helleri) and the inducible expression for cytochrome P4501A

为了构建剑尾鱼脑细胞系并探讨其细胞色素P4501a(CYP1A)基因的诱导效应,实验通过胰蛋白酶消化法对剑尾鱼脑组织进行体外培养,经连续继代培养,建立了可稳定传代的脑细胞系,命名为SFB。SFB最适培养液为含有15%胎牛血清(FBS)的DMEM/F-12和L-15等比混合培养液,培养条件为27℃,5%CO2。生长特性研究表明,第65代细胞的群体倍增时间为43.048h,显示出旺盛的生长和分裂能力。染色体分析发现,培养细胞的染色体众数为48条,SFB核型公式为2n=2st+46t,臂指数(NF)=48,与剑尾鱼一致。诱导实验表明,SFB在10-8～10-5mol/L的苯并(a)芘诱导下,CYP1A mRNA表达量显著提升,且表现出良好的剂量效应关系。脑细胞系的建立为剑尾鱼的毒理学评价研究提供了便利,也为其系统的生态毒理学应用打下基础。

The aim of this study was to establish the swordtail fish,Xiphophorus helleri,brain cell line SFB （ swordtail fish brain cell line） and to study its CYP1A mRNA inductive effect. Primary brain cell culture of swordtail fish was initiated by trypsin digestion methods, and a stable brain cell line was obtained successfully after culture and proliferation. The optimal culture medium for SFB was Leibovitz-15 to blend DMEM/FI2 medium with 1：1 supplemented with 15% fetal bovine serum, and the optimal culture conditions for SFB were 27 ℃ and 5 % CO2. The SFB grew actively under the optimal medium and culture conditions,and the doubling time is 43. 048 hours at 65thpassage. And chromosome analysis showed that the SFB exhibited chromosomal diploidy with a modal chromosome number of 48, the karyotype formula was 2n = 2st ＋ 46t, NF = 48, which are consistent with the swordtail fish. The cells maintained their original shape and high viability after being cryopreserved and resuscitated. The induction experimental results showed that, the expression of CYP1A mRNA in B（a） P-treated（ 10^-8 -10^-5mol/L）SFB cells increased significantly, and increased along with the increasing dosage of B （a） P. A dose-dependent relationship between the levels of CYP1A mRNA expression and doses of B （a）P was observed. Thus, the establishment of this cell line will provide convenience for the toxicological evaluation and a basis for its application to ecological toxicology.