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目的 :研究二甲双胍对体外诱导的人骨肉瘤干细胞形态、凋亡及分化能力的生物学影响,并初步探讨其与磷酸腺苷激活的蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)活性及胚胎干细胞标志物Nanog、Oct4表达情况间的相关性。方法 :采用悬浮球生长实验将人骨肉瘤MG63细胞诱导为骨肉瘤干细胞,在倒置显微镜下呈现典型成球过程,并观察二甲双胍处理前、后骨肉瘤干细胞的形态及分化能力差异;采用流式细胞术分析二甲双胍对骨肉瘤干细胞凋亡诱导的情况及作用前、后胚胎干细胞标志物Nanog及Oct4的阳性率;用蛋白印迹法对比骨肉瘤细胞成球前、后的肝激酶B1(liver kinase B1,LKB1)/AMPK变化,并检测二甲双胍作用前、后骨肉瘤干细胞中AMPK/雷帕霉素靶蛋白(the mammalian target of rapamycin,m TOR)/p70核糖体蛋白S6激酶(p70 ribosomal protein S6 kinase,p70S6K)信号通路的表达情况。结果 :在倒置显微镜下发现,加入二甲双胍后骨肉瘤干细胞的球体形态及分化能力均受到一定程度的破坏。流式细胞术分析显示,二甲双胍具有微弱的诱导骨肉瘤干细胞凋亡的能力。二甲双胍组的Nanog及Oct4阳性率分别为45.2%和74.7%,均明显低于对照组的Nanog(73.5%)及Oct4(95.3%)阳性率。蛋白印迹法检测结果显示,骨肉瘤细胞成球后AMPK的活性降低;二甲双胍组中磷酸化AMPK(p-AMPK)相对表达量为0.637 3±0.143 1,明显高于对照组(0.272 5±0.125 1,P<0.05);p-m TOR及p-p70S6K的蛋白相对表达量分别为0.401 8±0.018 1和0.310 0±0.105 5,均低于对照组p-m TOR(0.630 1±0.221 8,P<0.05)及p-p70S6K(0.628 2±0.014 6,P<0.05)。结论:二甲双胍可破坏骨肉瘤干细胞的形态,诱导其发生凋亡,并对其分化能力具有一定抑制作用,其机制可能与Nanog、Oct4表达的下调和AMPK的激活有关。
OBJECTIVE: To study the biological effects of metformin on the morphology, apoptosis and differentiation of human osteosarcoma stem cells in vitro and to investigate the relationship between metformin and the activity of adenosine monophosphate-activated protein kinase (AMPK) and embryonic stem cell Correlation between Nanog and Oct4 expression. Methods: Human osteosarcoma MG63 cells were induced into osteosarcoma stem cells by suspension ball growth experiment, and typical morphological changes of osteosarcoma stem cells were observed under inverted microscope. The morphological and differentiation of osteosarcoma stem cells were observed before and after metformin treatment. Flow cytometry Metformin on osteosarcoma stem cell apoptosis induced by the situation and the role of pre-and post-embryonic stem cell markers Nanog and Oct4 positive rate; Western blotting osteosarcoma cells into the ball before and after the liver kinase B1 (LKB1 ) / AMPK changes and the expression of AMPK / mTOR / p70 ribosomal protein S6 kinase (p70S6K) in osteosarcoma stem cells before and after metformin treatment. Signal path expression. Results: Under inverted microscope, it was found that the morphology and differentiation of osteosarcoma stem cells after metformin were damaged to some extent. Flow cytometry analysis showed metformin has a weak ability to induce osteosarcoma stem cell apoptosis. The positive rates of Nanog and Oct4 in metformin group were 45.2% and 74.7% respectively, which were significantly lower than those in control group (73.5%) and Oct4 (95.3%). The results of Western blotting showed that the activity of AMPK in osteosarcoma cells decreased after intratracheal injection. The relative expression of phosphorylated AMPK (p-AMPK) in metformin group was 0.637 3 ± 0.143 1, which was significantly higher than that in control group (0.272 5 ± 0.125 1 , P <0.05). The relative expressions of pm TOR and p-p70S6K were 0.401 8 ± 0.018 1 and 0.310 0 ± 0.105 5, respectively, which were all lower than the pm TOR of control group (0.630 1 ± 0.221 8, P <0.05) and p-p70S6K (0.628 2 ± 0.014 6, P <0.05). CONCLUSION: Metformin can disrupt the morphology of osteosarcoma stem cells and induce their apoptosis. The mechanism may be related to the down-regulation of Nanog and Oct4 expression and the activation of AMPK.