|摘要||目的 探讨不同链长脂肪酸对胎盘滋养细胞氧化应激和炎症反应的影响.方法 体外培养胎盘滋养细胞,将细胞分为5组,每组加入不同链长的脂肪酸孵育细胞,即分别为无游离脂肪酸( FFA-free,F-FFA)组、短链脂肪酸(SC-FFA)组、中链脂肪酸(MC-FFA)组、长链脂肪酸(LC-FFA)组、极长链脂肪酸(VLC-FFA)组.每组再分别以DMEM培养基、还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶抑制剂(NADPH-Ⅰ)和p38丝裂原活化蛋白激酶(p38MAPK)抑制剂(p38MAPK-Ⅰ)孵育细胞.采用实时荧光定量PCR技术和蛋白印迹法检测各组细胞p38MAPK和环氧合酶2(COX-2)mRNA和蛋白的表达变化.结果 (1)LC-FFA组+DMEM、VLC-FFA组+DMEM、LC-FFA组+NADPH-1、LC-FFA组+p38MAPK-Ⅰ、VLC-FFA组+NADPH-Ⅰ 、VLC-FFA组+p38MAPK-Ⅰ胎盘滋养细胞p38MAPK mRNA的表达量分别为4.56±0.28、22.65±2.40、0.87 ±0.06、1.02 ±0.15、19.87±1.93、10.22±0.75,蛋白的表达量分别为0.79 ±0.02、0.93 ±0.10、0.43 ±0.06、0.44±0.19、0.79±0.10、0.81± 0.14. LC-FFA组+DMEM、VLC-FFA组+DMEM细胞p38MAPK mRNA和蛋白的表达量升高(P＜0.05).与LC-FFA组+DMEM比较,LC-FFA组+NADPH-Ⅰ、LC-FFA组+p38MAPK-Ⅰ细胞p38MAPK mRNA和蛋白的表达量明显降低(P＜0.05).与VLC-FFA组+DMEM比较,VLC-FFA组+NADPH-Ⅰ细胞p38MAPK mRNA和蛋白的表达量差异无统计学意义(P＞0.05)；VLC-FFA组+p38MAPK-Ⅰ细胞p38 MAPK mRNA的表达量明显降低(P＜0.05),而蛋白的表达量无差异(P＞0.05).(2) LC-FFA组+DMEM、VLC-FFA组+DMEM、LC-FFA组+NADPH-Ⅰ、LC-FFA组+p38MAPK-Ⅰ、VLC-FFA组+NADPH-Ⅰ、VLC-FFA组+p38MAPK-Ⅰ细胞COX-2 mRNA的表达量分别为3.97 ±0.03、39.08±0.63、0.99 ±0.13、0.98 ±0.18、20.93±3.70、13.46±2.31,蛋白的表达量分别为1.32±0.20、1.33±0.25、0.59 ±0.13、0.58±0.30、0.88 ±0.18、0.91 ±0.24.与其他组比较,COX-2 mRNA和蛋白的表达量在LC-FFA组+DMEM、VLC-FFA组+DMEM细胞明显升高(P＜0.05).与LC-FFA组+DMEM比较,LC-FFA组+NADPH-Ⅰ、LC-FFA组+p38MAPK-Ⅰ细胞COX-2 mRNA和蛋白的表达量明显降低(P＜0.05).与VLC-FFA组+DMEM比较,VLC-FFA组+NADPH-Ⅰ、VLC-FFA组+p38MAPK-Ⅰ细胞COX-2 mRNA和蛋白的表达量均降低(P＜0.05).(3)相关性分析显示,在LC-FFA组中p38MAPK与 COX-2在mRNA和蛋白水平上均呈正相关(r=0.657、0.916,P＜0.05).在F-FFA、SC-FFA、MC-FFA、VLC-FFA组中,p38 MAPK与COX-2在mRNA水平上无相关性(P＞0.05),而在蛋白水平上呈正相关(P＜0.05).结论 LC-FFA和VLC-FFA刺激下的胎盘滋养细胞中存在氧化应激和炎症反应,该过程有p38MAPK信号通路参与.
Objective To investigate the oxidative stress and inflammation in trophoblast cells stimulated by different chain length fatty acids.Methods Serum-free trophoblast cells cultured in vitro were divided into five groups,which were incubated with DMEM medium without free fatty acid (F-FFA),short chain fatty acids (SC-FFA),medium chain fatty acids (MC-FFA),long chain fatty acids (LC-FFA),very long chain fatty acids (VLC-FFA).Then cells in each group were stimulated by DMEM medium,reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (apocynin) and p38 mitogen-activated protein kinases (p38MAPK) inhibitor (SB203580) and were subdivided as each FFA plus-DMEM group, plus-NADPH-Ⅰ and plus-p38MAPK-Ⅰ groups.Expressions of mRNA and protein of p38MAPK and cyclooxygenase 2 (COX-2) in trophoblast cells were detected by real-time PCR and western blot.Results (1) The mRNA expression of p38MAPK in LC-FFA + DMEM,VLC-FFA + DMEM,LC-FFA + NADPH-Ⅰ,LC-FFA + p38MAPK-Ⅰ,VLC-FFA + NADPH-Ⅰ,VLC-FFA + p38MAPK-Ⅰ group were 4.56 ±0.28,22.65 ±2.40,0.87 ±0.06,1.02 ±0.15,19.87 ± 1.93,10.22 ±0.75 separately,and the protein expressions were 0.79 ± 0.02,0.93 ± 0.10,0.43 ± 0.06,0.44 ± 0.19,0.79 ± 0.10,0.81 ±0.14.Compared with other groups,the mRNA and protein expressions of p38MAPK in LC-FFA + DMEM,VLC-FFA + DMEM group were increased ( P ＜ 0.05 ).Compared with LC-FFA + DMEM group,mRNA and protein expressions of p38MAPK in LC-FFA + NADPH-Ⅰ and LC-FFA + p38MAPK-Ⅰ group were significantly decreased (P ＜ 0.05 ).Compared with VLC-FFA + DMEM group,mRNA and protein expressions of p38MAPK had no difference in VLC-FFA + NADPH-Ⅰ group (P ＞ 0.05 ),mRNA expression of p38MAPK in VLC-FFA + p38MAPK-Ⅰ group was significantly decreased (P ＜ 0.05 ),but there was no difference in protein expression ( P ＞ 0.05).(2) The mRNA expression of COX-2 in LC-FFA + DMEM,VLC-FFA +DMEM,LC-FFA + NADPH-Ⅰ,LC-FFA + p38MAPK-Ⅰ,VLC-FFA + NADPH-Ⅰ,VLC-FFA + p38MAPK-Ⅰ group were 3.97 ±0.03,39.08 ±0.63,0.99 ±0.13,0.98 ±0.18,20.93 ±3.70,13.46 ± 2.31 separately,and the protein expressions were 1.32 ± 0.20,1.33 ± 0.25,0.59 ± 0.13,0.58 ± 0.30,0.88 ± 0.18,0.91 ± 0.24.Compared with other groups,mRNA and protein expressions of COX-2 in LC-FFA + DMEM and VLC-FFA + DMEM group were significantly increased ( P ＜ 0.05 ).Compared with LC-FFA + DMEM group,mRNA and protein expressions of COX-2 in LC-FFA + NADPH-Ⅰ and LC-FFA +p38MAPK-Ⅰ group were decreased ( P ＜ 0.05 ).Compared with VLC-FFA + DMEM group,mRNA and protein expressions of COX-2 in VLC-FFA + NADPH-Ⅰ and VLC-FFA + p38MAPK-Ⅰ group were all decreased ( P ＜ 0.05 ).( 3 ) The correlation analysis showed that there were significantly positive correlations between the mRNA and protein expressions of p38MAPK and COX-2 in LC-FFA group ( P ＜ 0.05 ).There were significantly positive correlations in protein expression ( P ＜ 0.05 ),but no conrelation in the mRNA expression between p38MAPK and COX-2 in the F-FFA,SC-FFA,MC-FFA,VLC-FFA groups (P ＞ 0.05).Conclusions The oxidative stress and inflammation may exist in trophoblast cells which were stimulated by LC-FFA and VLC-FFA.p38MAPK signal transduction pathway may contributed in this process.|