文章摘要
赖露颖,张庆国,李 乐,赵 伟,雷洪伊,姜 珊,张 婧,徐世元.RNA干扰沉默MKK7对SH-SY5Y细胞凋亡的影响[J].南京医科大学学报,2015,(5):656~651
RNA干扰沉默MKK7对SH-SY5Y细胞凋亡的影响
Effects of the RNA interference silenceing MKK7 on SH-SY5Y cells apoptosis
投稿时间:2014-09-27  
DOI:10.7655/NYDXBNS20150511
中文关键词: SH-SY5Y细胞  RNA干扰  MKK7  凋亡
英文关键词: SH-SY5Y cells  RNA interference  MKK7  screening  apoptosis
基金项目:国家自然科学基金资助(81271390)
作者单位
赖露颖 南方医科大学珠江医院麻醉科,广东 广州 510282 
张庆国 南方医科大学珠江医院麻醉科,广东 广州 510282 
李 乐 南方医科大学珠江医院麻醉科,广东 广州 510282 
赵 伟 南方医科大学珠江医院麻醉科,广东 广州 510282 
雷洪伊 南方医科大学珠江医院麻醉科,广东 广州 510282 
姜 珊 南方医科大学珠江医院麻醉科,广东 广州 510282 
张 婧 南方医科大学珠江医院麻醉科,广东 广州 510282 
徐世元 南方医科大学珠江医院麻醉科,广东 广州 510282 
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中文摘要:
      目的:探讨RNA干扰沉默丝裂原激活的蛋白激酶激酶(mitogen-activated protein kinase kinase,MKK7)基因对SH-SY5Y细胞凋亡的影响。方法:设计并合成针对MKK7的小干扰RNA (small interference RNA,siRNA)3条(MKK7siRNA-1?MKK7siRNA-2?MKK7siRNA-3)及与MKK7基因无同源性的带有红色荧光的阴性对照siRNA(negative control siRNA),在lipofectamine 2000介导下转染SH-SY5Y细胞,荧光显微镜下观察转染效果,RT-PCR观察MKK7 mRNA表达及Western blot 检测MKK7蛋白的表达,进而筛选出转染效果好的MKK7siRNA,CCK-8检测各组细胞活力,Hochest 33258检测各组细胞凋亡染色,流式细胞仪检测细胞凋亡率,Western blot检测p-JNK?凋亡蛋白cleaved caspase-3的变化。结果:① MKK7siRNA-3组的MKK7 mRNA表达最低,MKK7蛋白表达最低;②与空白对照组(control group)比较,MKK7siRNA-3组的细胞活力无统计学差异,p-JNK?cleaved caspase-3水平降低,凋亡率降低。结论:RNA干扰沉默MKK7可通过下调JNK磷酸化进而抑制SH-SY5Y细胞的凋亡。
英文摘要:
      Objective:To discuss the effects of RNA interference silencing MKK7 on SH SY5Y cells apoptosis. Methods:Three articles MKK7 siRNA(MKK7siRNA-1,MKK7siRNA-2 and MKK7siRNA-3)were designed and synthesized,the negative control siRNA with red fluorescence,which had no homology to the MKK7 gene,was transfected into SH SY5Y cells with the lipofectamine 2000 and the transfection effect was tested using fluorescence microscope,the MKK7 was detected by RT-PCR and Western blot, then were screened MKK7siRNAs. After the MKK7siRNAs were transfected into SH-SY5Y cells,the cell vitality was detested using CCK-8,and the apoptosis rate was tested by Hochest 33258 dyeing and flow cytometry instrument,the expression of p-JNK and the cleaved caspase-3 protein was detected by western blot. Results:①MKK7siRNA-3 has lowest MKK7 mRNA and protein expression;② compared with the control group,MKK7siRNA-3 group has no statistical difference in cell viability,lower p-JNK and cleaved caspase-3 expression and apoptosis rate. Conclusion: The RNA interference silencing MKK7 can down-regulate the JNK phosphorylation ,which forther inhibit the SH SY5Y cell apoptosis.
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