国家自然科学基金(30170980, 30571958, 30872757), 辽宁省教育厅攻关项目(20121268), 辽宁省自然基金(20052107), 教育部博士点基金(20070159023), 辽宁省教育厅重点实验室项目(2008S247)和盛京自由研究者计划(200807)资助项目
This work was supported by grants from The National Natural Science Foundation of China(30170980, 30571958, 30872757), Educational Department Science Foundation of Liaoning Province(20121268), Liaoning Natural Science Foundation(20052107), Educational Department Doctor Startup Fund(20070159023), Educational Department Key Laboratory of Liaoning Province(2008S247) and Shengjing Freedom Researchers Plan (200807)
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Lewis y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); Lewis y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(0.1 mmol/L,1 mmol/L,10 mmol/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Lewis y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 pathway- mediated 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.
丛建萍,林 蓓,刘娟娟,刘 晴,李飞飞,刘水策,高 嵩,张淑兰.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[J].生物化学与生物物理进展,2010,37(2):175-183
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