论文部分内容阅读
目的 鉴定K5 6 2细胞中氯化血红素 (hemin)诱导性表达基因。方法 分别提取经hemin诱导培养的K5 6 2细胞 (tester)和未加诱导剂培养的K5 6 2细胞 (driver)mRNA ,逆转录合成双链cDNA。采用抑制性消减杂交 (SSH)技术构建正向消减cDNA文库。筛选出阳性克隆。PCR扩增阳性克隆插入片段。反向Northern点杂交确定hemin诱导后表达上调的cDNA片段并测序后与GenBank序列进行Blast同源性比较分析。结果 发现 15个cDNA片段在hemin诱导后表达上调 ,其中大部分与GenBank中已知功能蛋白质的mRNA序列同源 ,包括珠蛋白epsilon 1(globinε1) ,类谷胱甘肽巯基转移酶Omega(GSTTLp2 8) ,含硒蛋白X1(SEPX1) ,磷酸丙糖异构酶 (TPI1) ,核糖体蛋白L7(RPL7) ,核糖体蛋白S13(RPS13) ,铁蛋白轻链 (ferritinLpolypeptide) ,珠蛋白gammaA(globinAγ) ,RAD5 1同系物C(RAD5 1C) ,铁蛋白重链 (ferritinHpolypeptide) ,X盒结合蛋白 (XBP1)。受hemin诱导性表达的基因克隆中的部分cDNA片段则同源于GenBank中已登录的mRNA序列 ,但相应蛋白质的功能尚不清楚 ,包括cDNADKFZp434I116 ,假定蛋白HSPC0 14及NOL1R2蛋白质。结论 hemin主要诱导K5 6 2细胞红系分化、蛋白质合成及代谢相关基因表达 ,这些发现为hemin诱导造血祖细胞红系分化的差异基因
Objective To identify hemin induced genes in K562 cells. Methods The mRNA of K562 cells cultured in hemin and K562 cells cultured without inducer were extracted and double-stranded cDNA was reverse transcribed. Suppression subtractive hybridization (SSH) was used to construct forward subtracted cDNA library. Positive clones were screened. PCR amplification of positive cloned insert. Reverse Northern blotting confirmed the hemin-induced up-regulated cDNA fragment and sequenced and compared with GenBank Blast homology analysis. Fifteen cDNA fragments were found to be up-regulated after hemin induction. Most of them were homologous to the mRNA sequences of known functional proteins in GenBank, including globinε1, glutathione S-transferase Omega (GSTTLp28 (SEPX1), triosephosphate isomerase (TPI1), ribosomal protein L7 (RPL7), ribosomal protein S13 (RPS13), ferritin Lpolypeptide, globin A gamma , RAD5 1 homolog C (RAD5 1C), ferritinHypeolypeptide, X box binding protein (XBP1). Some of the cDNA fragments in hemin-induced gene clones are homologous to the registered mRNA sequences in GenBank, but the function of the corresponding proteins is unclear, including cDNADKFZp434I116, putative proteins HSPC014 and NOL1R2 proteins. Conclusion Hemin mainly induces erythroid differentiation, protein synthesis and metabolism-related gene expression in K562 cells. These findings indicate that hemin induces differentially expressed erythroid differentiation of hematopoietic progenitor cells