【摘要】 背景与目的:信号转导和转录活化因子5(signaltransducersandactivatorsoftranscription5,STAT5)组成性激活在慢性粒细胞白血病(chronicmyeloidleukemia,CML)细胞恶性转化中起重要作用。本文研究STAT5诱骗核苷酸(decoyoligonucleotides,decoyODNs)对STAT5下游靶基因bcl鄄x转录激活的调控作用。方法:设计并合成decoyODNs、错配核苷酸(M鄄ODNs)和FAM标记decoyODNs。FAM鄄decoyODNs作为对照,在脂质体介导下转染白血病细胞K562,流式细胞仪(FCM)和倒置荧光显微镜检测FAM鄄decoyODNs的摄入情况。构建人bcl鄄x启动子的荧光素酶报告质粒pGL3b鄄bclxP,将其与decoyODNs或M鄄ODNs共转染K562细胞,检测转染后K562细胞中荧光素酶的表达。K562细胞分别转染decoyODNs和M鄄ODNs后,用RT鄄PCR检测bcl鄄xL表达,FCM检测细胞凋亡。结果:FAM鄄decoyODNs在终浓度0.04～4μmol/L范围内的摄入量呈剂量依赖性。24h时FAM鄄decoyODNs4μmol/L处理组转染效率高达99.1%,并在倒置荧光显微镜下也观察到细胞内有绿色荧光物质。decoyODNs处理组荧光素酶活性降低[(181.48±204.46)RLU/μgprotein],与对照组[(675.26±62.91)RLU/μgprotein]相比有显著性差异(P<0.05);而M鄄ODNs处理组细胞荧光素酶的活性[(632.07±98.95)RLU/μgprotein]与对照?更多还原

【Abstract】 BACKGROUND & OBJECTIVE: Constitutive activation of signal transducer s and activators of transcription 5 (STAT5) plays an important role in malignant transformation of chronic myeloid leukemia (CML) cells. This study was to explo re regulatory effect of STAT5 decoy oligonucleotides (ODNs) on trans-activation of its downstream target bcl-x gene in K562 cells. METHODS: STAT5 decoy ODNs, mi smatched ODNs (M-ODNs), and FAM-decoy ODNs were designed and synthesized. FAM-de coy ODNs were used as control, and transfected into K562 cells by cationic lipos omes, analyzed by flow cytometry (FCM) and fluorescent inversive microscopy. The bcl-x promoter fragment acquired by polymerase chain reaction (PCR) was inserte d into pGL3-basic to construct luciferase report plasmid pGL3b-bclxp, which was co-transfected with decoy ODNs or M-ODNs into K562 cells. The activity of lucife rase was detected. After transfection of decoy ODNs, and M-ODNs, expression of b cl-xL mRNA in K562 cells was detected by reverse transcription-PCR (RT-PCR),cell apoptosis was detected by FCM. RESULTS: FAM-decoy ODNs were incorporated into K 562 cells in a dose-dependent manner. The incorporation efficiency reached 99.1% at the concentration of 4 μmol/L 24 h after transfection, and green fluorescen ce could be observed in cells under fluorescent inversive microscope. The lucife rase activity was significantly lower in STAT5 decoy ODNs group than in control group <(181.48±204.46) RLU/μg protein vs. (675.26±62.91) RLU/μg protein, P < 0.05>, but that of M-ODNs group <(632.07±98.95) RLU/μg protein> has no signif icant difference with that of control group (P > 0.05). mRNA level of bcl-xL was decreased by STAT5 decoy ODNs, but not by M-ODNs. Moreover, Sub G1 peak was det ected in STAT5 decoy ODNs group by FCM. CONCLUSION: Transfection of STAT5 decoy ODNs can down-regulate the trans-activation of bcl-x in K562 cells.更多还原