摘要
【目的】通过苹果多酚(apple polyphenols,APs)处理RAW264.7小鼠巨噬细胞,以探讨苹果多酚的生物学作用.【方法】通过MTT试验确定苹果多酚的处理质量浓度,保证细胞的存活率;将RAW264.7巨噬细胞用10μg/mL的苹果多酚处理4h后,提取其总RNA,反转录后通过基因芯片技术检测,比较试验组与对照组TNF信号通路中基因表达量的差异,并进行生物信息学分析和实时荧光定量PCR验证.【结果】苹果多酚质量浓度为10μg/mL时,能够显著(P<0.05)促进RAW264.7巨噬细胞的增殖能力,而质量浓度为5μg/mL时,对巨噬细胞增殖能力的影响并不显著(P>0.05);基因芯片数据共筛选出差异倍数2倍以上的基因为3 149个(≥2-fold,P≤0.5),其中1 540个基因在RAW264.7巨噬细胞中表达上调,1 609个基因表达下调.基因体本轮(GO)分析结果显示:这些差异基因主要与核苷酸结合、DNA修复、细胞周期、细胞骨架与细胞核等相关,涉及的信号通路包括类固醇生物合成、真核细胞核糖体合成、TNF信号通路、抗生素生物合成、多巴胺能神经突触、烟酸和烟酰胺代谢等.但为了进一步研究TNF信号通路,利用qPCR验证该通路上(Map3k8、Mlkl、Pik3ca、Cflar、Atf2、Tnf)6个差异基因的表达,其中,Map3k8、Mlkl、Tnf 3个差异基因被显著(P<0.05)下调,而Pik3ca、Cflar、Atf2 3个基因则下调不显著(P>0.05).【结论】研究苹果多酚对TNF信号通路中差异表达基因的影响,对探索疾病发生发展的机制具有重要意义.
【Objective】To explore the biological effects of apple polyphenols on RAW264.7 murine macrophages.【Method】Optimal concentration of apple polyphenols was determined by MTT to guarantee the viability of keratinocytes.The total RNA of keratinocytes was extracted after treated with apple polyphenols for 4 h,the cDNA were acquired by reverse transcription and then detected by gene chip to investigate the difference of gene expression in TNF signaling pathway,and then validated through bioinformatics analysis as well as the quantitative real-time PCR test.【Result】The growth of RAW264.7 murine macro-phage was obviously promoted after the treatment with apple polyphenols at the concentration of 10μg/mL(P<0.05),however,it was not obvious with concentration of 5μg/mL(P>0.05).A total of 3149 expressed genes were screened by gene expression profile chip(Fold Change>2,P<0.05),including 1540 up regulation and 1609 down regulation.GO functional annotation results showed that these differential genes were mainly related to protein and nucleotide binding,DNA repair,cytoplasm,cytoskeleton and the nucleus,etc.The related pathways included steroid biosynthesis,ribosome synthesis in eukaryotes,biosynthesis of antibiotic,dopaminergic synapse,nicotinate acid and nicotinamide metabolism,etc.In order to study the TNF signaling pathway,qPCR was applied to verify 6 differential genes.As a result,Map3k8,Mlkl and Tnf were significantly down-regulated(P<0.05),while Pik3ca,Cflar,Atf2 were not significantly down-regulated(P>0.05).【Conclusion】It is of great significance to explore the mechanism of disease development by studying the impact of apple polyphenols on differentially expressed genes in TNF signaling pathway.
作者
徐倩
刘磊
郭玉蓉
XU Qian;LIU Lei;GUO Yu-rong(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;College of Food Engineering and Nutritional Science,Shanxi Normal University,Xi'an 710062,China)
出处
《甘肃农业大学学报》
CAS
CSCD
北大核心
2018年第6期10-18,共9页
Journal of Gansu Agricultural University
基金
国家现代农业产业技术体系建设专项资金项目(CARS-28)
