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目的探讨凝血酶体外促进肺癌细胞Glc-82周期进程和增殖及其作用机制。方法采用CCK8法检测凝血酶(thrombin)对Glc-82细胞增殖活性的影响,确定其量效和时效关系;利用碘化丙锭(propidium iodide,PI)单染和AnnexinⅤ-FITC/PI双染在流式细胞仪上检测细胞周期和凋亡;以荧光实时定量PCR检测10号染色体上与张力蛋白同源缺失的磷酸酶基因(phosphatase and tensin homologue deleted on chromatosome 10,PTEN)mRNA水平的变化,免疫印迹法检测PTEN蛋白表达和AKT磷酸化水平的变化。结果凝血酶(0.5和1.0 U/ml)可以有效促进细胞的增殖(P<0.01)。凝血酶(0.5 U/ml)促进Glc-82细胞由G1期向S期转化,可以抑制PTEN的表达,提高AKT磷酸化水平,但对细胞凋亡没有影响。结论凝血酶(0.5 U/ml)可以促进肺癌细胞Glc-82周期进程和细胞增殖,可能系通过PTEN/PI3K/AKT信号分子途径发挥作用。
Objective To investigate the role of thrombin in promoting the progression and proliferation of Glc-82 lung cancer cells in vitro and its mechanism. Methods The effect of thrombin on the proliferation of Glc-82 cells was detected by CCK8 method. The dose-effect and time-effect relationship were determined. The single staining of propidium iodide (PI) and AnnexinV-FITC/PI double staining were used. The cell cycle and apoptosis were detected by flow cytometry; the level of phosphatase and tensin homologue deleted on chromatosome 10 (PTEN) mRNA on chromosome 10 was detected by fluorescent real-time quantitative PCR. The changes of PTEN protein expression and phosphorylation of AKT were detected by blotting. Results Thrombin (0.5 and 1.0 U/ml) could effectively promote cell proliferation (P<0.01). Thrombin (0.5 U/ml) promoted the transition from G1 phase to S phase of Glc-82 cells, which could inhibit the expression of PTEN and increase the phosphorylation level of AKT, but had no effect on apoptosis. Conclusion Thrombin (0.5 U/ml) can promote the Glc-82 cycle progression and cell proliferation in lung cancer cells, possibly through the PTEN/PI3K/AKT signaling pathway.