Ahi-1基因导入Jurkat细胞中表达及功能的研究

Expression and Function of Ahi-1 Gene in Jurkat Cells

本研究目的是探讨Ahi-1基因及其编码蛋白在人T淋巴细胞白血病Jurkat细胞中的表达、细胞内定位以及Ahi-1基因对Jurkat细胞生长的调控作用。分别应用Northern blot、Werstern blot检测Ahi-1基因的mRNA及蛋白表达水平。构建Ahi-1的真核表达质粒并导入Jurkat细胞,用G418筛选获得稳定过表达Ahi-1基因的Jurkat-A细胞,用XTT法检测细胞的增殖活性,半固体培养法检测细胞集落形成能力。结果表明:正常人外周血淋巴细胞(PBL)、Jurkat细胞以及HUT78细胞均表达6.5、4.2以及2kb的Ahi-1基因mRNA转录本。PBL及Jurkat细胞均表达140kD的Ahi-1蛋白且主要定位于细胞浆内,但在细胞核内未见表达。PBL尚表达120kD的Ahi-1蛋白,主要存在于细胞核内,Jurkat细胞中120kD的Ahi-1蛋白表达水平低下。甲异靛、阿糖胞苷(Ara-C)、高三尖杉酯碱(HHT)、甲氨喋呤(MTX)以及鬼臼乙叉甙(VP16)可诱导Jurkat细胞核内140kD的Ahi-1蛋白表达增强,甲异靛降低细胞浆内Ahi-1蛋白表达。与转染质粒对照Jurkat细胞(Jurkat-C)相比,高表达外源性Ahi-1的Jurkat-A细胞的生长及集落形成能力显著低于Jurkat-C细胞;细胞总c-myb蛋白表达水平无改变,但Jurkat-A细胞中磷酸化c-myb蛋白表达增强;细胞中AKT磷酸化水平无明显变化。结论:Jurkat细胞表达6.5、4.2以及2kb的Ahi-1转录本;140kD的Ahi-1蛋白主要定位于细胞浆中,多种化疗药物可诱导细胞核内140kD的Ahi-1蛋白表达增强;过表达外源性全长Ahi-1可抑制Jurkat细胞的生长及集落形成能力,并诱导c-myb蛋白磷酸化。

This study was aimed to investigate the expression, protein localization and function of Ahi-1 gene and its encoding protein in Jurkat ceils. The expression of Ahi-1 mRNA and protein were measured by Northern and Western blot respectively. The plasmid containing prototype Ahi-1 was constructed and transfected into Jurkat cells. The Jurkat-A and Jurkat-C cells which either over-expressed the prototype Ahi-1 or not were obtained by selection with G418. The proliferation of the cells was assayed by XTT. The colony formation potential of the leukemia cells was checked by semisolid agarose culture. The results showed that three different transcripts of Ahi-1 （6.5,4.2 and 2 kb） were expressed in peripheral blood lymphocytes （PBLs）, Jurkat and HUT78 cells. Ahi-1 protein with 140 kD localized in the cytoplasm majorly while traceless in the nucleus of Jurkat cells and PBLs from normal donor. Ahi-1 protein with 120 kD could be detected in the nucleus fraction of PBLs. Very low level of Ahi-1 protein with 120 kD was expressed in Jurkat cells. Upregulating expression of Ahi-1 protein with 140 kD in the nucleus was found in Jurkat cells after exposure to meisoindi go, cytarabine, homoharringtonine, methotrexate and etoposide, down-regulating expression of Ahi-1 with 140 kD in the cytoplasm was observed after treatment with meisoindigo. The growth and colony formation potential were inhibited in the Jurkat-A cells, as compared to Jurkat-C cells. Total c-myb and phosphorylated AKT protein were continuously ex- pressed in the Jurkat-C and Jurkat-A cells at similar level, but more phosphorylated c-myb was observed in Jurkat-A ceils. It is concluded that three different transcripts of Ahi-1 at 6.5,4.2 and 2 kb are detected in Jurkat ceils; the Ahi-1 protein with 140 kD majorly expresses in the cytoplasm fraction and exposure to multiple chemotherapeutic compounds increased its expression in nucleus fraction. Over-expression of exogenous Ahi-1 can not only inhibit the growth and col- ony formation protential of Jurkat cells, but also induce the phosphorylation of c-myb in Jurkat cells.