目的 探讨磷脂酰肌醇3激酶( PI3K)/蛋白激酶B(Akt)/核因子κB(NF-κB)信号通路在卵泡刺激素(FSH)促进卵巢癌细胞增殖和侵袭中的作用.方法 培养卵巢癌细胞株SKOV3、3AO细胞,取对数生长期细胞用于以下实验,实验分为4组,即对照组、FSH组、LY294002(PI3K抑制剂)组、FSH+ LY294002组.采用四甲基偶氮唑蓝(MTT)比色法检测不同浓度(分别为10、20、40、80、160 U/L) FSH处理不同时间(分别为12、24、48、72h)后SKOV3、3AO细胞的增殖情况,倒置显微镜观察各组SKOV3、3AO细胞的形态,体外侵袭实验检测各组SKOV3、3AO细胞的侵袭能力,蛋白印迹法检测各组SKOV3、3AO细胞中FSH受体(FSHT)、Akt1/2、磷酸化Akt (p-Akt)、NF-κB p65蛋白的表达.结果 (1)SKOV3、3AO细胞增殖率的比较:SKOV3、3AO细胞经不同浓度FSH处理不同时间后,其增殖率明显高于相应对照细胞(P＜0.05).40U/L的FSH处理SKOV3细胞48 h、3AO细胞24h时,其细胞增殖率最高,分别为(166.9±1.1)％、(173.9±1.6)％.(2)各组细胞的形态:对照组SKOV3细胞为短梭形、3AO细胞为长梭形,胞核均呈圆形或椭圆形,胞质透亮；FSH组细胞变长或呈多角形,胞核较前稍增大,细胞密度较对照组增大；LY294002组细胞体积变小,胞质浓缩,部分细胞变圆漂浮；FSH+ LY294002组细胞形态较对照组无明显改变,细胞密度小于FSH组.(3)各组细胞的侵袭能力:对照组、FSH组、LY294002组及FSH+ LY294002组SKOV3细胞的穿膜细胞数分别为(26±6)、(118±19)、(18±5)、(38±7)个；3AO细胞分别为(19±4)、(134±20)、(12±3)、(58±11)个.FSH组两种细胞的穿膜细胞数均明显多于对照组(P＜0.05),LY294002组及FSH+ LY294002组两种细胞的穿膜细胞数均明显少于FSH组(P＜0.05).(4)各组细胞中相应蛋白的表达:FSH组、LY294002组、FSH+ LY294002组SKOV3、3AO细胞FSHR、Akt1/2蛋白的表达水平分别与对照组比较,差异均无统计学意义(P＞0.05).FSH组SKOV3、3AO细胞p-Akt蛋白的表达水平及胞核与胞质中NF-κB p65蛋白表达的比值高于对照组,差异有统计学意义(P＜0.05)；LY294002组、FSH+LY294002组SKOV3、3AO细胞p-Akt蛋白的表达水平及胞核与胞质中NF-κB p65蛋白表达的比值低于FSH组,差异有统计学意义(P＜0.05).结论 FSH可能是通过PI3K/Akt信号通路调节卵巢癌SKOV3、3AO细胞中NF-κB的活性,实现对卵巢癌细胞的促增殖和侵袭作用.%Objective To explore the effects of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)uclear factor-kappa B (NF-κB) signal pathway on the process of follicle-stimulating hormone (FSH) facilitating cell proliferation and invasion in human epithelial ovarian cancer. Methods Ovarian cancer cell lines SKOV3 and 3AO were cultured to exponential phase,then assigned to control group,FSH group,LY294002 group and FSH + LY294002 group,respectively.Cells were treated with different concentration of FSH and LY294002,respectively.The effects of FSH on cell proliferation were observed by methylthiazolyl tetrazolium (MTT).Morphological changes were observed by phase contrast microscope.The ability of cell invasion was investigated by transwell invasion assay.The expression of FSH receptor (FSHR),Akt1/2,phosphorylated-Akt (p-Akt) and NF-κB p65 protein were detected by western blot.Results ( 1 ) FSH could promote the proliferation of SKOV3 and 3AO cells.When the cells were treated with 40 U/L FSH for 48 hours (SKOV3) and 24 hours (3AO),compared with those in control groups,they reached the highest proliferation rate (P ＜ 0.05 ),respectively.(2) The morphology of SKOV3 and 3AO cells in four groups:in control group,SKOV3 cells were short spindle and 3AO cells were long spindle,the nuclei of them were both roundness or oval,the cytoplasm were bright.In FSH group,the cells changed to slightly longer or polygonal,they were full in shape,meanwhile,the cell intensity were higher than control group.In LY294002 group,some cells changed from spindle to round,and began to shrink.The cell intensity diminished.The morphology of FSH + LY294002 group was similar with control group,but the cell intensity was lower than that in FSH group.(3)The number of SKOV3 cell that passed through the membrane in control group,FSH group,LY294002 group and FSH + LY294002 group was (26 ± 6),( 118 ± 19),( 18 ± 5) and ( 38 ± 7 ),respectively.The number of 3AO cell was ( 19 ± 4 ),( 134 ± 20),(12 ±3) and (58 ± 11 ),respectively.The results showed that the number of cells in FSH group was significantly higher than that in control group ( P ＜ 0.05 ),while the number of cell in FSH + LY294002 group was significantly fewer than that in FSH group (P ＜ 0.05 ).(4) There was no significant difference in the expression of FSHR and Akt1/2 between FSH group and control group (P ＞ 0.05 ),but FSH increased the expression of p-Akt and the ratio of NF-κB p65 in the nucleus versus cytoplasm in SKOV3 and 3AO cells,there were significant differences compared with control group ( P ＜ 0.05 ).LY294002 reversed the effects of FSH on increasing the expression of p-Akt and the ratio of NF-κB p65 in the nucleus versus cytoplasm,there were significant differences among LY294002 group,FSH + LY294002 group and FSH group (P ＜ 0.05 ).Conclusion The effects of FSH on proliferation and invasion of ovarian cancer cell lines SKOV3 and 3AO may be realized by regulating the activity of NF-κB in PI3K/Akt signal pathway.
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