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目的探讨糖原合酶激酶-3β(GSK-3β)对卵巢癌细胞增殖的影响及其意义。方法以蛋白印迹法检测GSK-3β和磷酸化GSK-3β(pGSK-3β)在卵巢癌细胞株SKOV3和ES-2中的表达水平；采用细胞计数法描记细胞生长曲线,以检测GSK-3β抑制剂LiCl和SB216763对SKOV3和ES-2细胞生长的影响；将SKOV3细胞分成4组,将增加GSK-3β活性的质粒GSK-3βS9A及其空载体对照质粒pCS2、降低GSK-3β活性的质粒GID5-6及其对照质粒GID5-6LP,分别用电转法与绿色荧光蛋白(GFP)质粒共同转染入细胞中,采用5-溴脱氧尿嘧啶(BrdU)掺入实验检测GSK-3β活性对卵巢癌细胞增殖的影响；利用G418筛选瞬时转染的SKOV3细胞得到稳定转染株后,采用克隆形成实验,观察改变GSK-3β的活性对卵巢癌细胞增殖的长期影响。结果SKOV3和ES-2细胞均表达GSK-3β和pGSK-3β,SKOV3细胞中pGSK-3β的表达水平比ES-2细胞低,而两者GSK-3β的表达水平相近。GSK-3β抑制剂LiCl和SB216763可抑制SKOV3和ES-2细胞的生长。与对照pCS2相比,转染GSK- 3βS9A质粒可提高GSK-3β的活性,增加SKOV3细胞BrdU的掺入率；与对照GID5-6LP相比,转染GID5-6质粒可降低GSK-3β的活性,减少SKOV3细胞BrdU的掺入率。分别与各自的对照质粒相比,稳定转染GSK-3βS9A可形成较多的细胞克隆,而稳定转染GID5-6则形成的细胞克隆较少。结论GSK-3β具有促进卵巢癌细胞增殖的作用,抑制GSK-3β的活性可望成为卵巢癌治疗的新靶点。
Objective To investigate the effect of glycogen synthase kinase-3β (GSK-3β) on the proliferation of ovarian cancer cells and its significance. The method of detecting ovarian cancer cell lines SKOV3 and expression levels of ES-2 in GSK-3β and phosphorylated GSK-3β (pGSK-3β) a protein blot; by cell counting method tracings cell growth curve, to detect the GSK-3β inhibition Effects of SB216763 agent LiCl and ES-2 and SKOV3 cell growth; SKOV3 cells were divided into four groups, the plasmid will increase GSK-3βS9A GSK-3β and empty vector control plasmid the pCS2 activity, reducing the activity of GSK-3β plasmid GID5- 6 and its control plasmid GID5-6LP were transfected into the cells by electroporation and green fluorescent protein (GFP) plasmids, respectively. BrdU incorporation assay was used to detect the effect of GSK-3β on ovarian cancer After G418 was screened for transient transfection of SKOV3 cells to obtain stable transfectants, clone formation assay was used to observe the long-term effect of changing the activity of GSK-3β on the proliferation of ovarian cancer cells. Results Both GSK-3β and pGSK-3β were expressed in SKOV3 and ES-2 cells. The expression of pGSK-3β in SKOV3 cells was lower than that in ES-2 cells, and the expression level of GSK-3β was similar in both SKOV3 and ES-2 cells. GSK-3β inhibitors LiCl and SB216763 can inhibit the growth of SKOV3 and ES-2 cells. Compared with the control the pCS2, GSK- 3βS9A transfected plasmid to increase the activity of GSK-3β, increased incorporation of BrdU SKOV3 cells; Compared with the control GID5-6LP, GID5-6 plasmid transfection may reduce the activity of GSK-3β , Reduce the incorporation rate of BrdU in SKOV3 cells. Stable transfection of GSK-3βS9A resulted in more cell clones compared to the respective control plasmids, whereas stable transfection of GID5-6 resulted in fewer cell clones. Conclusions GSK-3β can promote the proliferation of ovarian cancer cells. Inhibiting the activity of GSK-3β is expected to become a new target for the treatment of ovarian cancer.