邻苯二甲酸单乙基己基酯对神经干细胞增殖和迁移的作用

摘要

目的：探讨邻苯二甲酸单乙基己基酯（MEHP）对原代神经干细胞（NSC）和NE-4C小鼠细胞增殖和迁移的影响。方法原代NSC来源于孕15 d（E15）远交群SD胎大鼠脑皮质；NE-4C细胞为小鼠NSC。MEHP 0，1，10，100和1000μmol·L-1处理72 h后，CCK-8法检测细胞活力；5-乙炔基-2′脱氧尿嘧啶核苷（EdU）法检测细胞增殖能力；Transwell小室检测细胞迁移；Western蛋白印迹法检测糖皮质激素受体（GR）、Y性别决定区框2（Sox2）、信号传导与转录激活因子3（Stat3）等基因及蛋白的表达。结果 CCK-8结果显示，与正常对照组相比，MEHP1000μmol·L-1作用72 h后，NE-4C和NSC细胞活力明显减弱，分别为正常对照组的70.3%和40.0%。EdU结果显示，与正常对照组相比，MEHP 100μmol·L-1组细胞增殖率降低，分别为正常对照组的74.8%和12.0%（P<0.05）。Transwell实验发现，MEHP 100μmol·L-1处理72 h后， NE-4C细胞的迁移率降低至（63.4±2.0）%（P<0.05）。MEHP 100μmol·L-1组NE-4C中与增殖和迁移相关的基因GR，Stat3和Sox2的表达下调，分别为正常对照组的49.8%，26.0%和14.0%（P<0.05）；在NSC中的相应基因也下调，分别为正常对照组的10.0%，14.0%和15.3%；在NE-4C中与增殖及迁移相关的蛋白GR，GRβ， p-Stat3和Sox2的表达下调，相对表达量分别为0.92±0.17，0.87±0.35，0.62±0.24和0.81±0.22（P<0.05）；在NSC中相应蛋白表达也下调，相对表达量分别为0.82±0.20，0.56±0.12，0.53±0.20和0.84±0.36（P<0.05）。结论大剂量MEHP可抑制NSC的增殖和迁移能力，其机制可能是通过GR介导的Stat3及Sox2的作用实现。%OBJECTIVE To investigate the effect of mono-2-ethylhexyl phthalate(MEHP) on proliferation of primary neural stem cells(NSCs)of rats and NE-4C cells of mice and on the migration of NE-4C cells and the mechanism. METHODS NE-4C or NSCs were treated with MEHP 1,10,100 and 1000 μmol · L-1 for 72 h,respectively. The cytotoxicity was estimated with the cell counting kit-8 (CCK-8). Cell proliferation was analyzed by EdU assay. The mRNA expression levels of the glucocorticoid receptor(GR),signal transducer and activator of transcription 3(Stat3)and sex determining region Y (SRY)-box 2(Sox2) were detected by qRT-PCR. The protein expression levels of total GR,GRβ, Sox2,Stat3 and p-Stat3 were measured by Western blotting. RESULTS Cell viability of NE-4C cells and NSCs at MEHP 1000μmol·L-1 was significantly decreased,which was 70.3%and 40.0%of the control group, respectively. EdU assay showed that MEHP 100 μmol · L-1 decreased NE-4C cells and NSCs by 74.8%and 12.0%(P<0.05)compared with control. The effect of MEHP on the cell migration of NE-4C was evidenced by the fact that the migration was obviously reduced to (63.4±2.0)%(P<0.05)after treatment with MEHP 100μmol · L-1 for 72 h. The mRNA expression levels associated with proliferation and migration in NE-4C of GR,Stat3 and Sox2 in MEHP 100 μmol · L-1 group were down-regulated to 49.8%,26.0% and 14.0%of control(P<0.05). At MEHP 100μmol · L-1,mRNA of GR, Stat3 and Sox2 in NSCs declined to 10.0%,14.0% and 15.3% of normal control. Western blotting results revealed that protein expressions of GR,GRβ,Sox2 and p-Stat3 were remarkably inhibited by MEHP 100 μmol · L-1 in that the relative expression of NE-4C was 0.92 ± 0.17,0.87 ± 0.35,0.81 ± 0.22 and 0.62 ± 0.24(P<0.05). The corresponding protein expression in NSCs was 0.82 ± 0.20,0.56 ± 0.12,0.84 ± 0.36 and 0.53 ± 0.20(P<0.05)when the cells were treated with MEHP 100μmol · L-1 for 72 h. CONCLUSION MEHP can inhibit the proliferation and migration of NE-4C cells and NSCs possibly by decreasing Stat3 and Sox2 that are mediated by GRβ.