过刊浏览

SHIP 基因诱导白血病细胞株K562 凋亡及其机制

杨琳, 罗建民^*, 刘小军, 温树鹏, 杜行严, 姚丽, 杨敬慈

河北医科大学第二医院血液科，河北省血液病重点实验室，石家庄 050000

摘要

肌醇5’ 磷酸酶(src homology 2 domain-containing inositol-5-phosphatase, SHIP)是继PTEN 之后发现的又一肌醇磷酸 酶，对造血细胞增殖有关键负调控作用。目前国内外对SHIP 基因与人类肿瘤抑制关系方面的研究报道较少。本研究利用 携带野生型SHIP 基因的慢病毒表达载体稳定感染K562 细胞，应用实时荧光PCR、Western blot 检测转染前后细胞内SHIP 基因mRNA 和蛋白水平的变化，MTT 测定细胞增殖水平的变化，ELISA 检测相关激酶活性，TUNEL、Hoechst 33342 检 测细胞凋亡的变化，从而探讨SHIP 基因诱导K562 凋亡的机制，分析SHIP 基因在白血病发病中的意义。结果如下： (1) K562 细胞SHIP 蛋白表达阴性；(2)携带野生型SHIP 基因的慢病毒表达载体转染使K562 细胞表达SHIP mRNA 和蛋白； (3)与对照组相比，转染野生型SHIP 基因导致K562 细胞生长受抑，并出现明显的凋亡征象；(4)转染SHIP 基因的K562 细 胞Akt 磷酸化水平降低；NF-κB 和bcl-xL 表达降低；同时细胞促凋亡基因bad、p27 表达和caspase-9、caspase-3 活性明显 增高。上述结果提示SHIP 通过抑制PI3K/Akt 路径中Akt 的磷酸化，促进其下游bad、p27 表达和caspase-9、caspase-3 的 活化，抑制bcl-xL，最终诱导白血病细胞凋亡；另外，NF-κB 表达下调也是SHIP 抑制白血病细胞增殖并促进细胞凋亡的 重要作用机制。

关键词： SHIP; 慢病毒表达系统; 细胞增殖; 细胞凋亡

分类号：R733.7；R730.231

[The mechanism for SHIP gene to induce the apoptosis of human leukemia cell line K562.] [Ariticle in Chinese]

Yang Lin, LUO Jian-Min^*, LIU Xiao-Jun, WEN Shu-Peng, DU Xing-Yan, YAO Li, YANG Jing-Ci

Province Key Laboratory of Hematology, Department of Hematology, the Second Hospital of Hebei Medical University, Shijiazhuang 050000, China

Abstract

The src homology 2 (SH2)-domain containing inositol-5-phosphatase (SHIP) is another recently identified lipid phosphatase after phosphatase and tensin homology deleted on chromosome ten gene (PTEN). It plays an important role in negatively regulating the proliferation of hematopoietic cells. The relationship between SHIP and the inhibition of tumor proliferation is rarely reported. The purpose of this study is to evaluate the apoptosis induced by SHIP gene in K562 cell line and to explore the involved signaling pathway. The K562 cells were transfected with human SHIP gene by using the lentiviral vector containing SHIP, and the transfection was verified by fluorescent quantitative PCR (FQ-PCR) and Western blot. Then the effects of SHIP protein expression on cell growth and apoptosis were measured. The levels of p-Akt, bcl-2 family, caspase and the activity of NFκB were assayed by Western blot and ELISA, respectively. The results are as follows: (1) Human leukemia cell line K562 was SHIP-negative; (2) Transfection with SHIP gene led to the re-expression of SHIP mRNA and protein in K562, as shown by FQ-PCR and Western blot; (3) The expression of SHIP protein inhibited cell growth and significantly increased apoptosis in K562 cells; (4) Compared to that in control group, the expression level of p-Akt-308 and p-Akt-473 in SHIP-expressing cell group decreased significantly (P<0.01); SHIP activated caspase-9, caspase-3, up-regulated protein levels of bad, p27, down-regulated expression of bcl-xL, while it had no effect on the expression of bcl-2 and bax. Furthermore, the inhibition of NF-κB was achieved along with the inactivation of Akt. These data suggest that SHIP gene has potential abilities to inhibit K562 leukemic cell proliferation and induce its apoptosis via inactivating PI3K/Akt pathway. The loss of SHIP might be the explanation of aberrant high-level p-Akt in human leukemia. It may be at least one of the mechanisms by which the loss of SHIP expression contributes to leukemia progression.

Key words: gene, SHIP; lentivirus expression system; cell proliferation; cell apoptosis

收稿日期：2008-08-18　　录用日期：2009-01-12

通讯作者：罗建民　　E-mail: luojm315@yahoo.com.cn

引用本文：

杨琳, 罗建民, 刘小军, 温树鹏, 杜行严, 姚丽, 杨敬慈. SHIP 基因诱导白血病细胞株K562 凋亡及其机制[J]. 生理学报 2009; 61 (2): 146-154.

Yang Lin, LUO Jian-Min, LIU Xiao-Jun, WEN Shu-Peng, DU Xing-Yan, YAO Li, YANG Jing-Ci. [The mechanism for SHIP gene to induce the apoptosis of human leukemia cell line K562.] [Ariticle in Chinese] . Acta Physiol Sin 2009; 61 (2): 146-154 (in Chinese with English abstract).