摘要
目的通过调节糖原合酶激酶-3β(GSK-3β)来观察其对肝癌细胞迁移能力的影响,研究GSK-3β在肝癌细胞迁移过程中发挥的作用。方法分别应用GSK-3特异性抑制剂LiCl和SB216763抑制GSK-3β的活性,或转染GSK-3βWT、GSK-3βS9A、GID5-6质粒影响GSK-3β的活性,通过划痕实验、小室迁移实验、微管组织中心体(MTOC)实验等观察处理前后肝癌细胞系SMMC-7721和Hep3B迁移能力的改变,并用免疫细胞化学法观察肝癌细胞迁移后磷酸化GSK-3β(pGSK-3β)的分布。结果划痕实验发现,用LiCl和SB216763处理后,SMMC-7721细胞的相对迁移率分别下降36.44%和41.78%,Hep3B细胞的相对迁移率都下降26.66%。转染GSK-3βWT后,GSK-3β和pGSK-3β表达上调,SMMC-7721细胞相对迁移率降为61.27%;转染GSK-3βS9A后,GSK-3β表达上调,pGSK-3β变化不明显,SMMC-7721细胞相对迁移率降为38.61%;转染GID5-6后,GSK-3β变化不明显,pGSK-3β表达增多,SMMC-7721细胞相对迁移率降为36.49%。免疫细胞化学可见,在正常培养的条件下肝癌细胞发生迁移3h后,细胞内pGSK-3β呈现出在细胞前沿边缘附集的现象;而加入LiCl后,pGSK-3β的分布则未呈现这种边缘附集的现象。MTOC实验发现,未加入抑制剂时Hep3B细胞MTOC阳性率在60%以上,而应用GSK-3β抑制剂后MTOC阳性率明显下降。结论GSK-3β在肝癌细胞的迁移过程中起到重要作用,应用针对GSK-3β的特异性抑制药物能够影响肝癌细胞的极性化,降低肝癌细胞的迁移能力。
Objective To study the effects of GSK-3β on the migration ability of hepatocellular carcinoma cells. Methods Inhibition tests with specific inhibitors of GSK -3 （ LiCl and SB216763） and transfection with different plasmids（ GSK-3β WT,GSK-3β S9A,and GID5 -6） were used to alter the activity of GSK -3β. The migration ability of SMMC-7221 and Hep3B cells was observed using wound healing assay,transwell assay and the microtubule-organizing center （ MTOC） test before and after treatment. Immunocytochemistry was used to investigate the distribution of phospho-GSK-3β. Results Wound healing assay showed that LiCl and SB216763 decreased the migration of SMMC-7721 cells by 36. 44% and 41. 78% ,respectively; and decreased the migration of Hep3B cells by both 26. 66%. Transfection with GSK-3β WT increased the expression of GSK-3β and pGSK-3β,and the migration rate of SMMC-7721 cells decreased to 61. 27%. Transfection with GSK-3β S9A increased GSK-3β expression and had no noticeable influence on pGSK -3β expression,and the migration rate of SMMC-7721 cells decreased to 38. 61%. Transfection with GID5-6 had no noticeable influence on GSK-3β and increased pGSK-3β expression,and the migration rate decreased to 36. 49%. Immunocytochemical staining showed that LiCl blocked pGSK-3β accumulation on cell edge as that in normally cultured cells. MTOC test showed that the MTOC positive rate was above 60% in Hep3B cells before adding inhibitors of GSK-3β,and it decreased after exposure to the inhibitors. Conclusion GSK-3β plays an important role in migration of hepatocellular carcinoma cells. Inhibitors of GSK-3β can affect the polarization and decrease the migration ability of hepatocellular carcinoma cells.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2010年第1期28-32,共5页
Academic Journal of Second Military Medical University
