野生型及Thr187突变型p27Kip1 蛋白对HepG2细胞周期的影响

目的p27kip1氨基端第187号位置的苏氨酸(Thr187)磷酸化位点是该蛋白分子中重要的磷酸化位点,探讨人工诱变该位点苏氨酸为丙氨酸(T187A)后对肝癌细胞株HepG2细胞周期以及细胞增殖的影响。方法应用脂质体转染法将含人野生型和突变型p27kip1基因质粒DNA转染HepG2细胞,免疫细胞荧光化学检测p27kip1蛋白的表达,并用流式细胞仪分析细胞周期的变化。结果野生型和突变p27kip1基因转染HepG2细胞后均能在细胞核表达,并且可以明显抑制细胞增殖。p27kip1基因转染的HepG2细胞发生G0期阻滞,且突变型G0期阻滞作用明显强于野生型(P<0.01)。结论转染的p27kip1能够在HepG2细胞过度表达并能抑制HepG2细胞的增殖,Thr187磷酸化位点的人工诱变可能有利于p27kip1蛋白促使细胞G0期阻滞。

Over-expression of p27Kip1 Threonine Residue 187 Mutant Type Gene Induces G0 Phase Arrest in HepG2 Cell Lines

Objective: To evaluate the effects of p27Kip1 transfection on the cell cycle, we transfected HepG2 lines with a high activity of p27Kip1 threonine 187-to-alanine(T187A) mutant gene as well as with wild type gene, then to investigate the role of phosphorylation of Thr187 of p27kip1 protein in its subcellular localization and cell cycle and proliferation. Methods: We transfected p27Kip1 T187A mutant and wild type gene into HepG2 cell lines by using Lipofectamine(LF2000) separately. The exogenous p27kip1 protein expression and subcellular localization were monitored with immunoflurescence microscopy. FACS and growth curves were used to analyse its biological effects on cell cycle and proliferation. Results: Over-expression of p27kip1 protein inhibited cell growth and increased cell number at the G1 phase. Moreover, transfection of the T187A mutant gene was more effective in inhibiting cell growth. Conclusion: The over-expression of p27kip1 maybe inhibit the proliferation of HepG2, deletion of p27kip1 on Thr187 may be beneficial to tumor cell G0 arrest, and these results suggest that the transfection of the p27Kip1 T187A mutant gene can be a modality of cancer gene therapy target.