SHIP基因诱导白血病细胞株K562凋亡及其机制  

The mechanism for SHIP gene to induce the apoptosis of human leukemia cell line K562

作　　者：杨琳[1] 罗建民[1] 刘小军[1] 温树鹏[1] 杜行严[1] 姚丽[1] 杨敬慈[1] 

机构地区：[1]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

出　　处：《生理学报》2009年第2期146-154,共9页Acta Physiologica Sinica

基　　金：supported by the National Natural Science Foundation of China (No. 30240011);the Key Technologies Program of the Scientific and Technical Bureau of Hebei Province; China (No. 2007000858)

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、Hoechst33342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。The src homology 2 （SH2）-domain containing inositol-5-phosphatase （SHIP） is another recently identified lipid phos- phatase 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 NFrd3 were assayed by Western blot and ELISA, respectively. The results are as follows： （1） Human leukemia cell line K562 was SHIP-negative; （2） Transfec- tion 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 acti- vated 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-rd3 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 contribute

关 键 词：SHIP 慢病毒表达系统 细胞增殖 细胞凋亡 

分 类 号：R733.7[医药卫生—肿瘤] R730.231[医药卫生—临床医学]