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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(Ed U)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%。Ed U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 MEHP on the proliferation and migration of primary neural stem cells (NSC) and NE-4C mice. Methods The primary NSC was derived from the cortex of the embryonic 15 d (E15) distant-felled cohort of SD fetus; the NE-4C cells were mouse NSC. Cell viability was detected by CCK-8 assay after MEHP treatment at 0, 1, 10, 100 and 1000 μmol·L -1 for 72 h. Cell proliferation was assayed by 5-ethynyl-2’-deoxyuridine (UU) Transwell chamber was used to detect cell migration. Western blotting was used to detect the expression of genes such as glucocorticoid receptor (GR), Y sex determination box 2 (Sox2), signal transducers and activators of transcription 3 (Stat3). Results The results of CCK-8 showed that compared with the normal control group, the viability of NE-4C and NSC significantly decreased after treated with 1000μmol·L -1 MEHP for 72 h, which were 70.3% and 40.0% of the normal control group, respectively. Ed U results showed that, compared with the normal control group, the proliferation rate of MEHP 100μmol·L -1 group decreased by 74.8% and 12.0% (P <0.05) of the normal control group respectively. Transwell experiments showed that the migration rate of NE-4C cells decreased to (63.4 ± 2.0)% (P <0.05) after treated with 100 μmol·L -1 MEHP for 72 h. The expressions of GR, Stat3 and Sox2 in the MEHP 100μmol·L -1 NE-4C were 49.8%, 26.0% and 14.0% lower than those in the normal control group respectively (P <0.05). The corresponding genes in NSC were also down-regulated to 10.0%, 14.0% and 15.3% of the normal control group respectively. The expressions of GR, GRβ, p-Stat3 and Sox2 in NE-4C were down-regulated, The expression levels were down-regulated in NSC (0.92 ± 0.17,0.87 ± 0.35,0.62 ± 0.24 and 0.81 ± 0.22, respectively), with relative expression levels of 0.82 ± 0.20, 0.56 ± 0.12 and 0.53 ± 0.20 And 0.84 ± 0.36 (P <0.05). Conclusion High-dose MEHP can inhibit the proliferation and migration of NSC, and its mechanism may be through GR-mediated effects of Stat3 and Sox2.