人骨肉瘤细胞MG-63抗失巢凋亡的机制研究

Possible mechanism of anoikis resistance in human osteosarcoma MG-63 cells

目的:观察人骨肉瘤细胞(MG-63)是否存在抗失巢凋亡(anoikis)的特性,并进一步研究其分子机制。方法:将MG-63细胞分别在普通6孔板和事先用聚甲基丙烯酸2-羧乙基酯(poly-HEMA)处理过的6孔板中培育24 h、48 h、72 h和7 d,人正常成骨细胞hFOB 1.19和人正常肾上皮细胞293T作对照,在光镜、电镜下观察细胞的形态以及细胞间连接,用流式细胞术检测细胞的凋亡率。通过RT-PCR方法检测不同时期、不同生长状态下,细胞中β-catenin和磷脂酰肌醇激酶(PI3K)的转录水平,并用Western blotting方法检测细胞内β-catenin、PI3K、Bcl-2和caspase-3、8、9的蛋白表达。结果:MG-63细胞在悬浮状态下培养,细胞相互之间聚集成比较致密的团块,没有发生明显的细胞凋亡(凋亡比例最高为30.29%)。在贴壁生长和悬浮生长情况下,caspase-3和caspase-9的活化水平均无显著差异,而在悬浮状态下caspase-8在48 h时活化最多,然后活性减低,β-catenin和磷脂酰肌醇激酶的转录水平比对照组高,但蛋白表达水平对照组与实验组无显著差异。在悬浮状态下,Bcl-2的蛋白表达水平呈时间依赖性增长,在72 h达到最高。结论:MG-63细胞具有抗失巢凋亡的特性。细胞从细胞外基质中脱离后,早期可能激活caspase-8,从而启动并执行anoikis;Bcl-2的活化可能在后期抑制anoikis的发生。

AIM：To investigate the possible mechanism for the regulation of anchorage-independent survival by observing the resistance of human osteosarcoma cell line MG-63 to anoikis.METHODS：Human osteosarcoma cell line MG-63 was cultured in six-well plates under the conditions of pretreating with or without poly-2-hydroxyethyl methacrylate（poly-HEMA） for 24 h,48 h,72 h and 7 days.Human fetal osteoblasts cell hFOB 1.19 and human kidney epithelial cell 293T were used as controls.Morphological changes of the cells treated with ploy-HEMA were observed under microscope by aggregation assay.The cell junctions were evaluated under transmission electronic microscope.Apoptosis rate in response to anoikis was determined by flow cytometry.The transcriptional levels of β-catenin and phosphoinositide 3-kinase（PI3K） were analyzed by RT-PCR.The protein levels of β-catenin,PI3K,Bcl-2 and activation of caspase-3,8,9 were examined by Western blotting.RESULTS：Human kidney epithelial cell 293T and human osteoblast cell hFOB1.19 significantly underwent anoikis when adherence was denied.Human osteosarcoma MG-63 cells were distinctly anoikis-resistant when detached.Caspase-8 in suspension cultured MG-63 cells was activated by cell-matrix detachment.Translational level of Bcl-2 significantly increased in a time-dependent manner.CONCLUSION：The MG-63 cells show resistance to anoikis when detachment of adherent cells from extracellular matrix occurs.Over-expression of Bcl-2 participates in the process of anoikis by blocking the activation of caspase-8,resulting in the resistance of MG-63 cells to anoikis.