[慢病毒载体介导SHIP基因转染对白血病K562细胞增殖的抑制作用及PI3K/Akt信号通路调控]

[Inhibitory effect of lentiviral vector-mediated SHIP gene transfection on proliferation of leukemia K562 cells and PI3K/Akt pathway regulation].

作者信息

Yang Lin, Luo Jian-Min, Liu Xiao-Jun, Wen Shu-Peng, Du Xing-Yan, Yao Li

机构信息

Department of Hematology, The Second Affiliated Hospital, Hebei Midical University, Shijiazhuang, Hebei, 050000, PR China.

出版信息

Ai Zheng. 2009 Apr;28(4):366-72.

PMID:19622295

Abstract

BACKGROUND AND OBJECTIVE

The hemopoietic-restricted Src homology 2-containing inositol 5'-phosphatase (SHIP) acts as a negative regulator for the proliferation and survival of hematopoietic cells by hydrolysing the phosphoinositide 3-kinase (PI3K)-generated second messenger, PtdIns(3,4,5)-P3 (PI-3,4,5-P3) to PtdIns(3,4)-P2 (PI-3,4-P2). This study was to investigate the biological function of SHIP gene in pathogenesis of leukemia cells by lentiviral vector-mediated SHIP transfection.

METHODS

Ectopic SHIP gene was transfected into leukemia K562 cells by the mediation of lentiviral vector. The mRNA level of SHIP was detected by fluorescent quantitative reverse transcription-polymerase chain reaction (FQ-PCR). The expression of SHIP, Akt, and phosphorylated Akt (p-Akt) was detected by Western blot. The proliferation and morphology of K562 cells before and after SHIP gene transfection were compared.

RESULTS

The proliferation of K562 cells was inhibited after transfection: the proliferation inhibition rate was increased from (9.9+/-1.5)% on Day 3 to (40.6+/-2.3)% on Day 5. K562 cells were SHIP-negative but expressed high level of p-Akt which was down-regulated from 0.533 to 0.245 (P<0.01) after SHIP transfection. Apoptotic characteristics were showed in K562 cells after SHIP transfection. The early apoptosis rate was significantly higher in K562-wtSHIP-FIV-G cells than in K562-FIV-G cells and untransfected K562 cells [(38.3+/-4.3)% vs. (8.2+/-0.9)% and (7.7+/-0.8)%, P<0.05].

CONCLUSIONS

SHIP gene can inhibit cell proliferation and promote cell apoptosis via inactivating PI3K/Akt pathway. Loss of SHIP might activate PI3K/Akt pathway and promote the proliferation of K562 cells.

摘要

背景与目的

造血特异性含Src同源2结构域的肌醇5'-磷酸酶（SHIP）通过将磷酸肌醇3激酶（PI3K）产生的第二信使磷脂酰肌醇(3,4,5)-三磷酸（PtdIns(3,4,5)-P3，PI-3,4,5-P3）水解为磷脂酰肌醇(3,4)-二磷酸（PtdIns(3,4)-P2，PI-3,4-P2），对造血细胞的增殖和存活起负调节作用。本研究旨在通过慢病毒载体介导的SHIP转染，探讨SHIP基因在白血病细胞发病机制中的生物学功能。

方法

通过慢病毒载体介导，将SHIP基因异位转染至白血病K562细胞。采用荧光定量逆转录-聚合酶链反应（FQ-PCR）检测SHIP的mRNA水平。通过蛋白质免疫印迹法检测SHIP、Akt及磷酸化Akt（p-Akt）的表达。比较SHIP基因转染前后K562细胞的增殖及形态变化。

结果

转染后K562细胞的增殖受到抑制：增殖抑制率从第3天的（9.9±1.5）%增至第5天的（40.6±2.3）%。K562细胞SHIP阴性，但p-Akt表达水平较高，转染SHIP后其表达水平从0.533降至0.245（P<0.01）。SHIP转染后的K562细胞呈现凋亡特征。K562-wtSHIP-FIV-G细胞的早期凋亡率显著高于K562-FIV-G细胞及未转染的K562细胞[（38.3±4.3）% 对（8.2±0.9）% 和（7.7±0.8）%，P<0.05]。