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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。
Src homology 2 domain-containing inositol-5-phosphatase (SHIP) is another inositol phosphatase discovered after PTEN, which plays a key negative regulatory role on hematopoietic cell proliferation. At present, there are few reports on the relationship between SHIP gene and human tumor inhibition at home and abroad. In this study, K562 cells were stably infected with the lentivirus vector carrying the wild-type SHIP gene. The mRNA and protein levels of SHIP mRNA in the transfected cells were detected by real-time fluorescent PCR and Western blot. The proliferation of K562 cells was detected by MTT assay. Related kinase activity, TUNEL and Hoechst33342 to detect the apoptosis of K562 cells, and to explore the mechanism of SHIP gene inducing apoptosis of K562 and to analyze the significance of SHIP gene in the pathogenesis of leukemia. The results were as follows: (1) SHIP protein expression was negative in K562 cells; (2) SHIP mRNA and protein were expressed in K562 cells transfected with lentivirus vector carrying wild type SHIP gene; (3) Compared with control group, SHIP gene resulted in the inhibition of K562 cell growth and obvious apoptotic signs; (4) The Akt phosphorylation of K562 cells transfected with SHIP gene was decreased; the expression of NF-κB and bcl-xL was decreased; while the apoptosis-inducing gene bad , P27 expression and caspase-9, caspase-3 activity was significantly increased. These results suggest that SHIP can induce the apoptosis of leukemic cells by inhibiting the phosphorylation of Akt in PI3K / Akt pathway, promoting the downstream expression of bad, p27, activating caspase-9 and caspase-3 and inhibiting bcl-xL. In addition, κB downregulation is also an important mechanism of SHIP inhibiting leukemic cell proliferation and promoting apoptosis.