叶酸缺乏对L02细胞增殖、凋亡及纺锤体组装检查点相关蛋白表达的影响

Effects of folate deficiency on proliferation,apoptosis and expression of spindle assembly checkpoint-related proteins in L02 cells

目的:探讨叶酸缺乏对L02细胞增殖、凋亡以及纺锤体组装检查点相关蛋白基因表达的影响及其机制。方法:用不同浓度的叶酸(200 nmol/L、20 nmol/L、0 nmol/L)培养L02细胞2周后,用倒置显微镜观察L02细胞形态;流式细胞术检测细胞周期与凋亡;CCK8测定细胞增殖情况;qRT-PCR检测Mad2、Bub1、BubR1 mRNA的表达;Western blotting检测相关蛋白Mad2、Bub1、BubR1的表达。结果:与正常对照组相比,叶酸缺乏使L02细胞出现细胞体积变大、形态不规则现象;叶酸缺乏可降低L02细胞活性(P<0.01),诱导细胞发生S期阻滞(P<0.05),促进细胞凋亡(P<0.05),上调Mad2、Bub1、BubR1 mRNA及蛋白水平(P<0.01)。结论:在L02细胞培养中,叶酸浓度为200 nmol/L时导致叶酸缺乏,SAC功能受损,细胞增殖异常,严重缺乏时导致细胞发生凋亡。

Objective:To investigate the effect of folate deficiency on the proliferation,apoptosis,expression of spindle assembly checkpoint-related proteins,and its mechanism in L02 cells.Methods:L02 cells were cultured with different concentrations of folate(2000,200,20,0 nmol/L)for 2 weeks.Then the morphology of L02 cells was observed by inverted microscope,the cell cycle and apoptosis were detected by flow cytometry,cell proliferation was measured by CCK8,the expressions of Mad2,Bub1,BubR1 mRNA were detected by fluorescence quantitative PCR,and the expressions of Mad2,Bub1,BubR1 protein were analyzed by Western blotting.Results:Compared with the normal control group,folate deficiency made L02 cells to become larger and irregular in shape,reduced the cell viability(P<0.01),induced S-phase block(P<0.05),promoted apoptosis(P<0.05),and upregulated the mRNA and protein expressions of Mad2,Bub1,BubR1 in L02 cells(P<0.01).Conclusions:In the culture L02 cells,it induces folate deficiency at 200 nmol/L folate,which causes impaired spindle assembly checkpoint function,abnormal cell proliferation,and even apoptosis in severe deficiency.