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背景与目的:SHIP基因主要表达于造血细胞,通过降解PIP3抑制PI3K/Akt信号通路而在造血细胞增殖和存活中起重要负调控作用。本研究通过慢病毒载体介导SHIP基因转染K562细胞,探讨SHIP基因改变及功能丢失与白血病发病的关系。方法:将携带SHIP基因的慢病毒感染K562细胞,FQ-PCR法检测SHIP转录水平,Westernblot法检测转染后SHIP蛋白表达及Akt磷酸化水平的变化;比较SHIP基因表达前后细胞增殖、形态的变化。结果:K562细胞中SHIP蛋白阴性。以携带SHIP基因的慢病毒载体转染K562细胞后,细胞增殖抑制率升高,K562-wtSHIP-FIV-G细胞的增殖抑制率由转染第3天的(9.9±1.5)%升到第5天的(40.6±2.3)%;伴有Akt磷酸化水平明显减弱;转染后,K562-wtSHIP-FIV-G组细胞p-Akt的表达水平由0.533降低到0.245(P<0.01),细胞增殖明显被抑制。此外,SHIP蛋白还能使K562细胞出现凋亡特征,Hoechst33342染色结果转染wtSHIP基因组K562细胞第5天早期凋亡率[(38.3±4.3)%]明显高于K562-FIV-G组细胞[(8.2±0.9)%]和未转染组K562细胞[(7.7±0.8)%]。结论:SHIP基因具有重要的抑制细胞增殖和促进凋亡的能力,SHIP基因缺失导致K562细胞PI3K/Akt信号途径失调,促进K562细胞增殖。
BACKGROUND & OBJECTIVE: SHIP gene is mainly expressed in hematopoietic cells and plays an important negative regulatory role in the proliferation and survival of hematopoietic cells by degrading PI3K / Akt signaling pathway. In this study, lentiviral vector-mediated SHIP gene transfected K562 cells, SHIP gene changes and loss of function and the incidence of leukemia. Methods: K562 cells were infected with lentivirus carrying SHIP gene. The transcription level of SHIP was detected by FQ-PCR. The expression of SHIP protein and Akt phosphorylation were detected by Western blotting. The proliferation and morphological changes of SHIP gene were compared . Results: SHIP protein was negative in K562 cells. After transfected K562 cells with lentiviral vector carrying SHIP gene, the proliferation inhibition rate of K562-wt SHIP-FIV-G cells increased from (9.9 ± 1.5)% on day 3 of transfection to 5 (40.6 ± 2.3)% of the mice in the K562-wt SHIP-FIV-G group decreased significantly from 0.533 to 0.245 (P <0.01) after transfection. The level of Akt phosphorylation was significantly decreased Obviously suppressed. In addition, the SHIP protein also induced apoptosis in K562 cells. The apoptotic rate of K562 cells transfected with wtSHIP K562 cells at the fifth day after Hoechst33342 staining was significantly higher than that of K562-FIV-G cells ([(38.3 ± 4.3)%] [ 8.2 ± 0.9%] and untransfected K562 cells [(7.7 ± 0.8)%]. Conclusion: The SHIP gene has an important inhibitory effect on cell proliferation and apoptosis, and the deletion of SHIP gene leads to the imbalance of PI3K / Akt signaling pathway in K562 cells and the proliferation of K562 cells.