三氧化二砷体外诱导人HepG2细胞铁蛋白基因表达的研究

Upregulation of ferritin H(L)chain gene in cultured human HepG2 cells induced by arsenical trioxide

目的了解低剂量三氧化二砷诱导的HepG2细胞的差异表达基因,揭示慢性砷中毒对肝细胞损伤的作用机制。方法在体外用三氧化二砷(5μmol/L)处理HepG2细胞,同时以PBS处理的相同细胞系作为对照;24h后制备细胞裂解液,提取mRNA并逆转录为cDNA,经RsaⅠ酶切后,将实验组cDNA分成2组,分别与2种不同的接头衔接,再与对照组cDNA进行2次消减杂交及2次抑制性聚合酶链反应(PCR),将产物与T/A载体连接,构建cDNA消减文库,并转染大肠杆菌JM109进行文库扩增,随机挑选克隆PCR扩增后进行测序及同源性分析。结果成功构建三氧化二砷处理HepG2细胞差异表达基因的cDNA消减文库。文库扩增后得到109个白色克隆,进行菌落PCR分析,均得到200～1000bp插入片段。挑取含有插入片段的36个克隆进行测序,通过生物信息学分析发现铁蛋白重链和轻链在三氧化二砷诱导的肝HepG2细胞正向消减cDNA文库中显著上调,共有8个阳性克隆子。结论低剂量三氧化二砷刺激细胞后可诱导铁蛋白的表达上调,暗示铁蛋白在砷诱导的肝细胞损伤中起作用。

Objective In order to understand the differentially expressed genes and explore the effects on mechanism of gene expression induced by arsenic trioxide. Methods The mRNA was isolated from human HepG2 cells treated with arsenic trioxide( 5μmol/L ) and DMSO, respectively, then cDNA was synthesized. After restriction enzyme Rsa Ⅰ digestion, small sizes cDNA were obtained. Then tester cDNA was subdivided into two portions and each was ligated with different cDNA adaptor. After tester cDNA was hybridized with driver cDNA twice and underwent nested polymerase chain reaction (PCR) twice, the DNA fragment was subcloned into T/A plasmid vectors to set up the subtractive cDNA library. Amplification of the library was carried out with E. coli strain JM109. The cDNA was sequenced and analyzed in GenBank with Blast search after colony PCR. Results The forward subtracted cDNA library from HepG2 cell line induced by arsenic trioxide was successfully constructed. The sequencing analysis showed that there were eight clones contained ferritin H(L) chain in the library. Conclusion Arsenic trioxide can induce the up expression of ferritin H(L) chain protein in HepG2 cells, indicated that the ferritin H(L) chain may play certain role in the mechanism of anti-arsenical cytotoxicity in liver.