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CGI-58抵抗5-FU诱导的巨噬细胞凋亡 被引量：1

CGI-58 resists 5-FU induced macrophage apoptosis in vitro
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摘要 目的观察CGI-58对5-FU诱导的巨噬细胞凋亡的影响,并探讨其作用及机制。方法用5-FU处理巨噬细胞(PLKO)与CGI-58敲低(knockdown)的巨噬细胞(CGI-58 KD)后分别用CCK-8法检测其细胞增殖能力;流式细胞术检测其细胞周期与凋亡;Western blot检测其凋亡蛋白(cleaved Caspase-3)的表达水平;Seahorse Assays检测其线粒体功能。结果 CCK-8检测发现CGI-58 KD+5-FU组细胞活力降低更明显(P<0.01);流式细胞术结果显示经5-FU处理后,CGI-58 KD组的细胞凋亡率明显高于PLKO组(P<0.01),Western blot检测发现CGI-58 KD+5-FU组cleaved Caspase-3的表达水平明显高于PLKO+5-FU组(P<0.01);Seahorse Assays检测发现CGI-58 KD组其基础耗氧率OCR明显降低(P<0.01),ATP生成减少(P<0.05),ROS表达增加(P<0.01);抗氧化剂NAC处理可拮抗巨噬细胞CGI-58缺失诱导的ROS增加(P<0.05)、5-FU杀伤作用(P<0.05)及cleaved Caspase-3表达水平。结论 CGI-58缺失可增强5-FU对巨噬细胞的杀伤作用并损伤巨噬细胞的线粒体功能,其作用机制可能是通过增加ROS的产生来实现的。 Objective To determine the effect of CGI-58 on 5-fluorouracil （5-FU） induced maerophage apoptosis, and investigate the underlying mechanism. Methods CCK-8 assay was used to detect the effect of 5-FU on the proliferation of primary macrophage （PLKO） and CGI-58 knockdown macrophage （CGI-58 KD）. Flow cytometry was used to test the cell cycle and apoptosis of PLKO and CGI-58 KD which were induced by 5-FU. Western blotting was employed to detect apoptosis protein （cleaved Caspase-3 ） and expression levels of 5-FU induced PLKO and CGI-58 KD. Seahorse assay was applied to test the mitochondrial function of PLKO and CGI-58 KD. Results CCK-8 assay found that the cell vitality of CGI-58 KD ＋ 5-FU group was reduced significantly （ P 〈 O. O1 ）. Flow cytometry results showed that after 5-FU induction, the apoptotic rate of CGI-58 KD group was significantly higher than that of PLKO group （P 〈 0. 01 ）. Western blotting showed that the expression level of cleaved Caspase-3 in CGI-58 KD ＋ 5-FU group was obviously higher than that in PLKO ＋ 5-FU group （ P 〈 0. O1 ）. Seahorse assay detected that the oxygen consumption rate of CGI-58 KD group was significantly decreased （ P 〈 O. 01 ）, ATP generation was reduced （ P 〈 O. 05 ）, and ROS expression was increased （P 〈 0. 01 ）. Antioxidant N-acetyl-L-cysteine could antagonize ROS increase induced by CGI-58 deletion （ P 〈 O. 05 ）, 5-FU killing effect （ P 〈 O. 05 ） and expression of cleaved Caspase- 3. Conclusion CGI-58 deletion can strengthen the killing effect of 5-FU on macrophage and damage the mitochondrial function of macrophage through increasing ROS.
作者 郝丽君 陈玉娟 张璇 梁后杰 缪洪明 阮志华
机构地区 第三军医大学西南医院肿瘤科
出处 《第三军医大学学报》 CAS CSCD 北大核心 2016年第4期367-373,共7页 Journal of Third Military Medical University
基金 国家自然科学基金青年科学基金(81302136) 国家自然科学基金面上项目(81172115)~~
关键词 CGI-58 5-FU 巨噬细胞 细胞凋亡 CGI-58 5-FU macrophage cell apoptosis
分类号 R329.24 [医药卫生—人体解剖和组织胚胎学] R965 [医药卫生—药理学]
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参考文献28

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第三军医大学学报
2016年 第4期

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