CAJ | 학술논문

文章从内蒙古野生蓝莓(Vaccinium uliginosum Lim)中提取花青素,观察其对口腔癌细胞株KB的增殖及凋亡的作用,探讨其作用机制与p53基因甲基化的相关性。利用含0.1%盐酸的甲醇提取花青素,用高效液相色谱-质谱(High performance liquid chromatography-mass spectrometry, HPLC-MS )鉴定花青素的成分。利用四甲基偶氮唑蓝(Methylthiazolyl-tetrazolium, MTT)比色法、流式细胞术、免疫荧光法、免疫细胞化学法和Western blot法分析蓝莓花青素对 KB 细胞增殖、细胞周期、细胞凋亡和 p53蛋白表达的影响；利用甲基化特异性 PCR 法(Methylation-specific PCR, MSP)分析蓝莓花青素诱导细胞凋亡与p53基因甲基化的关系。结果显示,内蒙古自治区的野生蓝莓中至少存在14种花青素成分；蓝莓花青素呈剂量依赖的方式抑制KB细胞增殖,诱导细胞周期阻滞在 G2/M 期,而且能诱导细胞凋亡；蓝莓花青素处理后 Caspase-9蛋白和细胞色素 C 的表达明显增加；Western blot结果表明蓝莓花青素可以诱导KB细胞中p53蛋白表达上调；MSP结果表明随蓝莓花青素浓度增加,未甲基化的p53的比例增加,说明p53的甲基化状态有所下调。
문장종내몽고야생람매(Vaccinium uliginosum Lim)중제취화청소,관찰기대구강암세포주KB적증식급조망적작용,탐토기작용궤제여p53기인갑기화적상관성。이용함0.1%염산적갑순제취화청소,용고효액상색보-질보(High performance liquid chromatography-mass spectrometry, HPLC-MS )감정화청소적성분。이용사갑기우담서람(Methylthiazolyl-tetrazolium, MTT)비색법、류식세포술、면역형광법、면역세포화학법화Western blot법분석람매화청소대 KB 세포증식、세포주기、세포조망화 p53단백표체적영향；이용갑기화특이성 PCR 법(Methylation-specific PCR, MSP)분석람매화청소유도세포조망여p53기인갑기화적관계。결과현시,내몽고자치구적야생람매중지소존재14충화청소성분；람매화청소정제량의뢰적방식억제KB세포증식,유도세포주기조체재 G2/M 기,이차능유도세포조망；람매화청소처리후 Caspase-9단백화세포색소 C 적표체명현증가；Western blot결과표명람매화청소가이유도KB세포중p53단백표체상조；MSP결과표명수람매화청소농도증가,미갑기화적p53적비례증가,설명p53적갑기화상태유소하조。
Blueberries are an excellent source of dietary polyphenols such as anthocyanins and phenolic acids. In this study, we investigated the ability of anthocyanins from the wild blueberries of Inner Mongolia to suppress the growth of the oral cancer cell line KB. The blueberry anthocyanins were extracted with methanol-containing 0.1%(v/v) hydrochloric acid. Fourteen unique anthocyanins were identified using high-performance liquid chromatography-mass spectrometry (HPLC-MS). The anticancer bioactivity of the extracts on KB cells was analyzed using methylthiazolyl-tetrazolium (MTT), flow cytometry (FCM) and immunocytochemistry. It was shown that the blueberry anthocyanins suppressed the prolifera-tion of KB cells in a dose-dependent manner, as well as induced G2/M cell cycle arrest and apoptosis of oral cancer KB cells. Immunocytochemistry analysis showed that the expression of caspase-9 and cytochrome c were obviously increased after the anthocyanins treatment. Western blot analysis also indicated that the expression of p53 was increased. Methyla-tion-specific PCR (MSP) showed that the amount of unmethylated p53 increased, indicating that the anthocyanins can down-regulate the methylation of p53.