摘要
【目的】探讨SHIP基因对IL-3诱导K562细胞系增殖的抑制作用,阐明SHIP对K562细胞作用的机制。【方法】将慢病毒质粒pReceiver-Lv31-FIV和pReceiver-Lv31-SHIP转染K562细胞;转染细胞与IL-3共培养,Western blot法检测K562细胞Akt磷酸化;观察转染野生型SHIP基因对K562细胞增殖的影响;末端脱氧核苷酸转移酶介导的dUTP缺失末端标记(TUNEL)法检测细胞凋亡。【结果】重组慢病毒载体pReceiver-Lv31-FIV和pReceiver-Lv31-SHIP转染K562细胞,GFP阳性率为74.6%。野生型SHIP基因阻断了IL-3对K562细胞的增殖作用,对照组(K562/FIV)细胞增殖抑制率(3.26%)明显低于转染SHIP基因组细胞增殖抑制率(26.42%,P<0.05);集落形成实验显示SHIP能显著抑制K562细胞集落形成能力[IL-3作用组K562/SHIP细胞形成集落为60.3±6.6,明显低于IL-3诱导的K562/FIV组(91.7±4.2)](P<0.01)。细胞形态观察发现凋亡增加,Western blot检测发现转染SHIP基因组前凋亡酶pro-caspase-3降解增加,TUNEL法证实SHIP蛋白促进细胞凋亡。IL-3作用不同时间段(3h,6h,12h),转染野生型SHIP组细胞增殖明显减弱,且Akt磷酸化水平均低于对照组(P<0.05)。【结论】SHIP基因负调控生长因子(IL-3)介导的K562细胞增殖及其蛋白激酶活化。
[ Objective ] To explore the inhibitory effects of SHIP on IL-3 stimulated K562 cell proliferation, and to elucidate the mechanism of SHIP functioning on K562 cells. [Methods] pReceiver-Lv31-FIV and pReceiver-Lv31-SHIP plasmids were transfected into K562 cells, and the K562 cells were cultured with IL-3. Phosphorylated Akt of K562 cells was examined by Western blot. The proliferation of K562 cells transfected with SHIP or empty vector were examined. The apoptosis of K562 cells was examined by TUNEL and Western blot. [Results] 74.6% GFP positive cells were obtained in SHIP group, 3.26% in FIV group （P 〈 0.05）. SHIP protein inhibited the colony formation retroviral vector SHIP-FIV transfected K562 ceils. The anti- proliferative rate in K562/FIV （3.26%） was significantly lower than that in K562/SHIP cells （26.42%）; the colony formation capacity of K562 decreased after transfected with SHIP （IL-3----- -K562/SHIP group 60.3 ± 6.6 vs IL-3-K562/FIV group 91.7 ± 4.2, P 〈 0.05）. An increase in the cells with morphologic features of apoptosis was also proved by TUNEL in SHIP-expressing cells. Western blot analysis showed SHIP down-regulated expression of pro-caspase-3. The level of phosphorylated Akt was decreased after stimulated by IL-3 for 1 h, and almost disappeared after stimulated for 24 h. The effect of IL-3 on K562 cell proliferation was inhibited by the transfected wild-type SHIP gene. The phosphorylated Akt in K562 cells were inhibited by the transfected SHIP gene at different time points. [Conclusion] SHIP is a negative regulator of IL-3-mediate PKB activation and K562 cell survival.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2009年第3期269-274,共6页
Journal of Sun Yat-Sen University:Medical Sciences
基金
国家自然科学基金(30240011)
河北省自然科学基金(2007000858)
