草鱼体外培养细胞中β-萘黄酮对CYP1A基因表达的诱导

CYP1A gene expression in β-naphthoflavone-induced grass carp (Ctenopharyngodon idellus) cell lines

为了建立以CYP1A cDNA为探针的水环境毒理学细胞模型,以β-萘黄酮(BNF)作为诱导剂,通过半定量PCR技术研究CYP1A在草鱼不同细胞系及其对应组织中的诱导表达情况。在半定量PCR反应参数研究中,对关键的退火温度和循环次数进行了优化,结果显示退火温度为57℃,循环次数为30次较为合适,该条件下Gauss迹量的CYP1A/ACT比值能更准确的反映CYP1A的表达水平。对照组和诱导组草鱼细胞中ACT和CYP1A cDNA扩增结果表明,细胞中CYP1A的基础表达量较低,而BNF诱导使GCL、CIK和CO细胞中CYP1A的表达水平得到显著的提高。GCL、CIK和CO细胞三者之间诱导后CYP1A的表达水平有显著差异(P<0.05),诱导表达量大小顺序为GCL>CIK>CO。草鱼细胞系的相应组织中ACT和CYP1A cDNA扩增以及电泳的结果与细胞中较为相似,组织中CYP1A诱导表达量大小顺序也与相应的细胞相同:肝>肾>卵巢。比较分析的结果表明,CYP1A在体内与体外的诱导表达水平具有一定的相关性。

Cytochrome P450 1A （CYP1A）cDNA is used as a probe for evaluation of environmental contaminant. Grass carp （ Ctenopharyngodon idellus ） liver cell （ GCL ）, kidney cell （ CIK ）, blastula cell （ GCB ）, snout fibroblast cell （PSF） and ovary cell （CO） were exposed to β-naphthoflavone （BNF） to induce CYP1A. The potency of induction was assessed by the ratio of CYP1A cDNA to β-actin （ACT） cDNA derived from semiquantitive PCR. PCR reaction parameters were optimized so that annealing temperature was chosen to be 57 ℃ and cycle to be 30 times. Under such conditions, the CYP1A expression level could be quantitated by Gauss trace ratio of CYP1A/ACT. PCR amplification product of ACT and CYP1A cDNA showed that CYP1A expression level was very low in control grass carp cells while increased in BNF-treated cells significantly. The expression level of CYP1A in BNF-treated GCL, CIK and CO cells had significant difference （ P 〈 0.05 ）, which was sorted as GCL 〉 CIK 〉 CO. But that of GCB or PSF cell could not yet be detected. The state of CYP1A expression in grass carp cells was similar to homologous tissues, and the level in homologous tissues of BNF-treated grass carps was sorted as liver 〉 kidney 〉 ovary. The results indicated CFP1A expression level in vitro had certain correlation with that in vivo. This study illustrates the high potential of fish cell lines in ecotoxicology.