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目的探讨肿瘤抑制基因PTEN对人原代白血病细胞及K562细胞系的增殖、凋亡及细胞周期的影响。方法将携带有野生型PTEN、绿色荧光蛋白的腺病毒(Ad-PTEN-GFP)及空载体腺病毒(Ad-GFP)转染原代白血病细胞和K562细胞系。采用MTT法检测细胞生长曲线;流式细胞仪检测细胞凋亡率和细胞周期分布;光镜和电镜下观察细胞形态;TUNEL等方法检测细胞增殖及凋亡;培养细胞集落并比较不同组间集落形成的数量;实时荧光定量PCR(FQ-PCR)检测PTENmRNA水平变化,Westernblotting检测PTEN蛋白水平变化。结果以感染复数为200转染后,与Ad-GFP相比,Ad-PTEN-GFP转染人白血病K562细胞系后,细胞增殖受抑,凋亡率增加,最大生长抑制率为35.2%,最大凋亡率为30.0%。细胞周期显示G1期阻滞,G0/G1期细胞比例增加[(54.9±2.51)%vs(78.5±4.13)%],G2/M期比例降低[(30.2±1.91)%vs(13.6±1.02)%](均P<0.05)。结论过表达PTEN可以抑制白血病细胞增殖,促进细胞凋亡,并导致细胞周期阻滞在G0/G1期。
Objective To investigate the effect of tumor suppressor gene PTEN on the proliferation, apoptosis and cell cycle of human primary leukemia cells and K562 cell line. Methods The primary leukemia cells and K562 cell lines were transfected with wild type PTEN, green fluorescent protein (Ad-PTEN-GFP) and empty vector adenovirus (Ad-GFP). The cell growth curve was detected by MTT assay; the apoptosis rate and cell cycle distribution were detected by flow cytometry; the cell morphology was observed under light microscope and electron microscope; the cell proliferation and apoptosis were detected by TUNEL and other methods; The quantity of PTEN mRNA was detected by real-time fluorescence quantitative PCR (FQ-PCR). The change of PTEN protein level was detected by Western blotting. Results After transfection with Ad-GFP, Ad-PTEN-GFP transfected human K562 cell line inhibited cell proliferation and increased the apoptosis rate compared with Ad-GFP. The maximum growth inhibition rate was 35.2% The apoptosis rate was 30.0%. The percentage of cells in G2 / M phase decreased ([(30.2 ± 1.91)% vs (13.6 ± 1.02) vs (54.9 ± 2.51) vs (78.5 ± 4.13)%] %] (All P <0.05). Conclusion Overexpression of PTEN can inhibit leukemic cell proliferation, promote apoptosis and lead to cell cycle arrest in G0 / G1 phase.