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目的:p27kip1氨基端第10号位置的丝氨酸(Ser10)磷酸化位点是该蛋白分子中最重要的磷酸化位点,探讨人工诱变该位点丝氨酸为丙氨酸(S10A)后对肝癌细胞株HepG2细胞周期以及细胞增生的影响.同时比较野生型p27kip1和Ser10突变型p27kip1基因转染对肝癌细胞株HepG2细胞周期和增生的影响. 方法:应用脂质体转染法将含人野生型和突变型p27kip1质粒DNA瞬时转染HepG2细胞,免疫细胞化学检测p27kip1蛋白的表达和细胞内分布,流式细胞计数仪分析细胞周期变化. 结果:野生型和突变p27kip1蛋白转基因后HepG2细胞均可以G0期阻滞,且突变型的阻滞作用强于野生型(P<0.05),细胞生长受到抑制.无血清培养96 h同步化于G0期,野生型和突变型p27kip1均分布于细胞核;而在20mL/L血清继续培养8 h后野生型向细胞质转运而主要分布于细胞质, 突变型仍然滞留于细胞核. 结论:p27kip1的过度表达可明显抑制HepG2细胞的增生, p27kip1Ser10磷酸可能介导其细胞核外转运的重要分子机制.
Objective: The serine (Ser10) phosphorylation site at amino-terminal 10 of p27kip1 is the most important phosphorylation site in this protein molecule. To investigate the effect of mutagenesis of serine to alanine (S10A) Strain HepG2 cell cycle and cell proliferation.Meanwhile, we compared the effect of wild-type p27kip1 and Ser10 mutant p27kip1 gene on the cell cycle and proliferation of HepG2 cell line.Methods: The mutant p27kip1 plasmid DNA was transiently transfected into HepG2 cells, the expression and intracellular distribution of p27kip1 protein were detected by immunocytochemistry, and the changes of cell cycle were analyzed by flow cytometry.Results: HepG2 cells transfected with wild-type and mutant p27kip1 protein could express G0 (P <0.05), and the cell growth was inhibited.The serum-free medium was synchronized to G0 stage after 96 h culture, wild-type and mutant p27kip1 were distributed in the nucleus, while in 20 mL / L serum for 8 h after the wild-type transfer to the cytoplasm and mainly distributed in the cytoplasm, the mutant still remain in the nucleus.Conclusion: overexpression of p27kip1 significantly inhibited HepG2 cells Hyperplasia, p27kip1Ser10 phosphate may mediate an important molecular mechanism of transport outside the nucleus.