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目的研究第10号染色体缺失的磷酸酶及张力蛋白同源基因(PTEN)突变对人鼻咽癌细胞系CNE-1中蛋白激酶B(Akt)的磷酸化的影响。方法 CNE-1细胞用含100 m L/L胎牛血清RPMI1640培养基培养,并分别转染野生型PTEN(wt PTEN)质粒、突变型PTEN C124S质粒和突变型PTEN G129E质粒。各组细胞加刺激前用无血清RPMI1640培养基饥饿过夜,用0.15 IU/m L胰岛素或0.3μg/m L重组人表皮生长因子(rh EGF)刺激,Western blot法检测Akt的磷酸化水平。结果胰岛素和rh EGF刺激均可导致CNE-1细胞Akt信号激活;wt PTEN可抑制胰岛素或rh EGF刺激引起的Akt信号激活;PTEN C124S突变体可激活胰岛素刺激引起的Akt信号,但不可激活rh EGF刺激引起的Akt信号;PTEN G129E突变体抑制胰岛素刺激引起的Akt信号激活。结论 wt PTEN可抑制胰岛素或rh EGF刺激引起的Akt信号激活,但PTEN C124S和G129E突变体不能一致性激活Akt信号表达。这表明PTEN基因突变可能与Akt信号表达无关。
Objective To investigate the effect of PTEN mutation on the phosphorylation of protein kinase B (Akt) in human nasopharyngeal carcinoma cell line CNE-1. Methods CNE-1 cells were cultured in RPMI 1640 medium containing 100 m L / L fetal bovine serum and transfected into wild-type PTEN (wt PTEN) plasmid, mutant PTEN C124S plasmid and mutant PTEN G129E plasmid respectively. The cells in each group were starved overnight with serum-free RPMI1640 medium supplemented with 0.15 IU / mL insulin or 0.3 μg / mL recombinant human epidermal growth factor (rh EGF) before stimulation. The phosphorylation level of Akt was detected by Western blot. Results Insulin and rh EGF stimulation induced Akt signaling in CNE-1 cells; wt PTEN inhibited Akt signaling induced by insulin or rh EGF stimulation; PTEN C124S mutant activated Akt signaling induced by insulin stimulation but not rh EGF Akt signaling induced by stimulation; PTEN G129E mutant inhibits activation of Akt signaling by insulin stimulation. Conclusion wt PTEN can inhibit the activation of Akt signal induced by insulin or rh EGF stimulation, but PTEN C124S and G129E mutants can not activate Akt signaling consistently. This indicates that PTEN gene mutation may not be related to Akt signaling.