α1,2-岩藻糖转移酶基因转导对人卵巢癌细胞RMG-I p38MAPK信号通路介导的凋亡的影响

The Effect of α1,2-Fucosyl Transferase Gene Transfection on p38MAPK Signaling Pathway-mediated Apoptosis of Ovarian Carcinoma RMG-I Cells

以α1,2-岩藻糖转移酶基因转染前后卵巢癌细胞RMG-I、RMG-I-H为细胞模型,用细胞免疫荧光方法检测转染前后细胞p38MAPK和p-p38MAPK的细胞内定位,RT-PCR和Western blot方法从mRNA和蛋白质两个水平检测转染前后细胞p38MAPK表达的变化;以兔抗人IgG抗体处理组为对照,分别利用RT-PCR和Western blot方法检测Lewisy单克隆抗体处理前后RMG-I-H细胞p38MAPK mRNA和蛋白质表达水平的变化;以0.1%DMSO为对照,用流式细胞仪(FCM)检测p38MAPK特异性抑制剂SB203580处理后RMG-I-H凋亡比率的变化,并利用RT-PCR和Western blot方法检测caspase-3的mRNA和蛋白质水平的变化;用RT-PCR方法检测卡铂和SB203580处理后p38MAPK及caspase-3表达的变化.结果表明,RMG-I与RMG-I-H的p38MAPK蛋白主要定位在细胞质,p-p38MAPK蛋白定位在细胞核,转染后p38MAPK的mRNA水平明显高于转染前(P<0.05);Lewisy单克隆抗体处理后RMG-I-H细胞p38MAPK mRNA水平降低(P<0.05),不同浓度SB203580(0.1mmol/L,1mmol/L,10mmol/L)作用下RMG-I-H细胞的凋亡率分别为(15.927±0.861)%、(18.187±0.481)%及(33.565±0.912)%,明显高于空白组和对照组(P<0.05),SB203580处理后caspase-3的mRNA和蛋白质水平均明显高于空白组和对照组(P<0.05),卡铂处理后p38MAPK及caspase-3 mRNA水平均增加,SB203580处理后,caspase-3mRNA水平增加.说明Lewisy抗原介导的卵巢癌细胞凋亡与p38MAPK信号通路有关,可能通过p38MAPK信号通路抑制卵巢癌细胞凋亡,引起耐药性.

To study the effect of α1,2-fucosyltransferase gene transfection on p38MAPK signaling pathwaymediated apoptosis in ovarian carcinoma RMG-I cells.The localization of p38MAPK and p-p38MAPK was detected by immunofluorescence in RMG-I and RMG-I-H cells.The expression of p38MAPK and p-p38MAPK was analyzed by RT-PCR and Western blot,respectively.For inhibition assay,anti-Lewis y antibody was used to assess the change of p38MAPK of mRNA level in RMG-I-H cells by RT-PCR and Western blot.Using 0.1% DMSO as control,the apoptosis rate was detected by flow cytometry（FCM） in SB203580 treated RMG-I-H cells.Simultaneously,the expression of p38MAPK and caspase-3 was analyzed by RT-PCR and Western blot.Further more,the expression of p38MAPK and caspase-3 by RT-PCR after Carboplatin and/or SB203580 treatment were studied.Results showed that immunofluorescence staining of p38MAPK and p-p38MAPK in RMG-I and RMG-I-H cells showed cytoplasmic localization and nuclear localization,respectively,and the level of p-p38MAPK mRNA in RMG-I-H cells is significantly higher than that in RMG-I cells（P 0.05）,while the expression of p-p38MAPK mRNA decreased after anti-Lewis y antibody treatment（P 0.05）.FCM showed that the apoptosis rate increased in SB203580 treated RMG-I-H cells（P 0.05）.The mRNA level of p38MAPK and caspase-3 increased by treatment with Carboplatin.The mRNA level of caspase-3 also elevated by treatment with SB203580.In conclusion,high expression of Lewis y inhibits apoptosis in ovarian cancer cells,probably due to involvement of Lewis y in regulating p38MAPK signaling pathway,thereby causing drug resistance.