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目的探讨核仁磷酸蛋白(nucleophosmin,NPM)突变基因对小鼠NIH3T3成纤维细胞系体外增殖和凋亡的影响以及其分子机制。方法通过脂质体介导将真核表达载体pEGFP-C1-NPMc+转染NIH3T3细胞,G418筛选稳定表达NPM突变蛋白细胞株,RT-PCR和Western blot检测NPM突变基因和蛋白的表达。MTT、克隆形成实验检测细胞体外增殖能力;FCM检测细胞周期分布及凋亡水平的改变,并通过对周期调控因子p21及凋亡相关分子Caspase-3活性检测,初步探讨NPM突变参与细胞恶性增殖的分子机制。结果建立了稳定表达NPM突变基因的NIH3T3细胞株。NPM-mA实验组细胞较对照组细胞增殖能力及平板克隆形成率明显增高(P<0.05);甲基纤维素集落形成实验显示各组细胞在甲基纤维素上均不能形成集落;与对照组比较,NPM-mA实验组细胞中G1期细胞比例(42.27±0.86)%显著减低(P<0.01),S期细胞比例(43.08±0.74)%显著增加(P<0.01),同时伴有p21 mRNA水平下降,稳定表达NPM突变基因的NIH3T3细胞凋亡率(1.00±0.13)%明显降低(P<0.01),Caspase-3活性(0.784±0.080)下降(P<0.01)。结论NPM突变基因可促进NIH3T3细胞体外增殖,抑制细胞凋亡发生。
Objective To investigate the effect of nucleophosmin (NPM) gene mutation on the proliferation and apoptosis of mouse NIH3T3 fibroblast cell line in vitro and its molecular mechanism. Methods The NIH3T3 cells were transfected with the eukaryotic expression vector pEGFP-C1-NPMc + by lipofectamine. The NPM mutant protein was stably expressed by G418. The expression of NPM gene and protein was detected by RT-PCR and Western blot. MTT assay and clonogenic assay were used to detect the proliferation of cells in vitro. FCM was used to detect cell cycle distribution and apoptosis. The expression of p21 and caspase-3 were detected by flow cytometry. Molecular mechanism. Results NIH3T3 cell line stably expressing NPM gene was established. Compared with the control group, the cell proliferation and the rate of plate clone formation in NPM-mA group were significantly increased (P <0.05). The results of methylcellulose colony formation assay showed that all the cells could not form colony on methylcellulose, The percentage of cells in G1 phase in NPM-mA group was significantly lower (42.27 ± 0.86)% (P <0.01), the percentage of cells in S phase was significantly higher (43.08 ± 0.74)% (P <0.01) (P <0.01), and the activity of Caspase-3 (0.784 ± 0.080) decreased (P <0.01) in NIH3T3 cells stably expressing NPM mutant gene. Conclusion NPM mutant gene can promote the proliferation of NIH3T3 cells in vitro and inhibit the apoptosis of NIH3T3 cells.