摘要目的 为提高诱导γ珠蛋白(γ globin)基因激活的效率,研究DNA羟甲基化酶(DNA双加氧酶)基因Tet2在诱导γ珠蛋白激活表达过程中的作用,为β地中海贫血病的治疗提供理论依据.方法 在人红白血病细胞系K562中用短发夹RSNA(shRNA)干扰的方法下调Tet2表达后,用酶联免疫吸附测定(ELISA)法检测干扰后细胞基因组5-羟甲基胞嘧啶(5hmC)水平的变化,并用亚硫酸盐测序法检测γ珠蛋白上CG位点羟甲基化状态,最后用荧光定量实时PCR检测药物5-氮胞苷诱导γ珠蛋白激活表达量和γ珠蛋白相关转录因子Nfe4和Klf1表达量的变化.结果 不做处理的对照组K562细胞基因组5hmC水平为0.14％,sh-Tet2组(Tet2下调后)约为0.03％;被5-氮胞苷激活后,Tet2下调组(即sh-Tet2+5-氮胞苷组)γ珠蛋白的表达量为未下调组(5-氮胞苷组)的55％左右,但γ珠蛋白上CG位点的羟甲基化状态没有发现明显变化.同时,γ珠蛋白相关转录因子Nfe4和Klf1的表达量变化显著,sh-Tet2组(Tet2下调后)Nfe4为对照组的约20％,Klf1为对照组的3.5倍.结论 Tet2可维持K562基因组5hmC水平,对γ珠蛋白的激活表达起到一定的调控作用,其作用机制有可能通过调控其上游转录因子来间接发挥调节作用,由此推测Tet2是一个潜在的β地中海贫血的治疗靶点.

abstractsObjective To study the function of ten-eleven translocation 2 (Tet2) in γglobin gene expression in patients with β-thalassemia.Methods Gamma globin expression was induced by 5-azacytidine and Tet2 gene expression was knocked down by short hairpin RNA (shRNA) in a human immortalized myelogenous leukemia K562 cell line.The global 5-hydroxymethylcytosine (5hmC) level was measured by an ELISA kit.5hmC level of γglobin gene was quantified by sulfite sequencing.The mRNA level of Tet2,γglobin,and related transcription factors Nfe4 and Klfl were quantified by real-time PCR.Results Tet2 knockdown resulted in a decreased global 5hmC level from 0.14％ to 0.03％ as of the control group in K562 cells.The expression of γ globin was enhanced after 5-azacytidine treatment in vitro.However,γglobin mRNA level in Tet2 knockdown cells was only 55％ as that in control group.The CG sites on γ globin gene were unmethylated.As Tet2 was down-regulated,the expression levels of Nfe4 and Klf1 decreased by about 80％ and increased to 3.5 folds,respectively.Conclusions Tet2 appears to maintain 5hmC level and facilitates γ globin gene activation.Moreover,Tet2 more likely regulates γglobin expression via affecting transcription factors rather than the gene itself.Thus,Tet2 could be a potential therapeutic target for β thalassemias.