【作者】 史梅；

【导师】 冯文莉；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2004， 硕士

【摘要】 目的：慢性粒细胞白血病（CML）具有特征性的Ph染色体，t(9;22)(q34;q11)形成BCR/ABL融合基因，编码产生具有强烈酪氨酸激酶活性的BCR/ABL融合蛋白，由此异常激活Ras/MAPK、PI3K和STAT5等信号转导途径，引起血细胞的恶性转化。其中，STAT5途径在CML的发病中具有十分重要的作用，BCR/ABL融合蛋白可以直接激活STAT5途径，启动bcl-xL、c-myc、cyclin D1等基因表达。而用显性失活技术抑制STAT5途径的激活可以导致CML细胞的增殖和转化的抑制，提示STAT5可能是CML治疗的新靶点。转录因子诱骗策略是近年来发展起来的一种研究转录因子和基因调控的有效手段，通过转入大量含有与转录因子结合的一致性序列的双链寡核苷酸分子，竞争结合转录因子，而调控转录因子下游的基因表达。本课题采用转录因子诱骗策略抑制CML细胞株K562 细胞中STAT5信号转导途径，观察STAT5诱骗核苷酸（Decoy ODNs）对K562细胞生长、增殖、凋亡等生物学性状的影响及对下游靶基因bcl-xL、cyclin D1和c-myc的作用，以进一步了解K562细胞中STAT5信号转导途径的通路和作用，探讨用诱骗核苷酸抑制STAT5途径作为慢粒治疗靶点的可能性。方法：设计合成针对STAT5转录因子的21bp硫代修饰的诱骗核苷<WP=6>酸，在脂质体介导下转染K562细胞，观察Decoy ODNs对K562细胞的生长的影响；MTT法观察对K562细胞增殖的作用；FCM检测对K562细胞细胞周期的影响；Annexin-V/PI染色和电镜观察的方法检测细胞凋亡；RT-PCR和Western blotting检测STAT5下游靶基因bcl-xL、cyclinD1和c-myc的mRNA和蛋白水平的改变情况，以进一步了解其作用机制。结果：1. Decoy ODNs 对K562细胞的生长抑制呈浓度依赖性，浓度越高，抑制作用越明显，5μΜ浓度抑制细胞基本不明显，15μΜ和25μΜ抑制效果明显，随着时间延长25μΜ可引起K562细胞的死亡。MTT实验也证实了Decoy ODNs对K562细胞的抑制，5μΜ浓度对K562细胞抑制率为15％，15μΜ为51％，25μΜ为82％，而相同浓度的作为对照的错配寡核苷酸（Mutant ODNs）的抑制率则较低。2. Decoy ODNs作用K562细胞24h后的细胞周期显示，S期细胞由48.71±5.42％减低为31.94±2.35％(P<0.05)，G0/G1期细胞由40.83±5.20％增加为61.39±2.18％(p<0.05)。而Mutant ODNs对细胞周期没有明显影响。3. Annexin-V/PI双染实验检测到Decoy ODNs作用24h后，凋亡细胞即有14.57±1.08％。Mutant ODNs引起的凋亡细胞只有4±2.02％。电镜检测方法观察到Decoy ODNs作用引起凋亡细胞体积缩小，细胞表面微绒毛结构明显减少，胞浆电子密度增大，染色质浓集，可见核固缩、核碎裂、可见凋亡小体等形态学改变。 <WP=7>4. RT-PCR实验证实Decoy ODNs作用24h后引起bcl-xL、cyclin D1和c-myc mRNA表达下调，bcl-xL下降了54.04％，cyclin D1下降了45.30％，c-myc下降了53.60％，。Western blotting实验也从蛋白水平检测到48h后bcl-xL、cyclin D1和c-myc的表达下调，bcl-xL下降了36.58％、cyclin D1下降了31.04％、c-myc下降了49.71％。结论：一定浓度的STAT5诱骗核苷酸可抑制K562细胞的生长增殖，抑制效果与Decoy ODNs浓度呈剂量依赖性；Decoy ODNs影响了K562细胞周期进程和诱导细胞发生凋亡；Decoy ODNs作用还引起了bcl-xL、cyclin D1和c-myc mRNA和蛋白水平的表达下调，而这些指标的变化也可能正是Decoy ODNs抑制K562细胞增殖和诱导凋亡的机制。更多还原

【Abstract】 Objective: CML is characterized by the reciprocal chromosomal translocation 9:22, which generates the Philadephia chromosome. This translocation generates a novel fusion gene, BCR/ABL, that encodes BCR/ABL fusion protein , with a hyperactive tyrosine kinase. Several studies have identified signaling pathways activated by BCR/ABL, including Ras/MAPK, PI3K, STAT5, and so on. Especialy, STAT5 is critical for CML transformation. Transcription factor decoy is a new techniques for gene therapy and in the study of transcriptional regulation. Nucleic acid molecules with high affinity for target transcription factor can be introduced into cells as decoy cis-elements to bind these facors and alter gene expression. When delivered into the nucleus of a target cell, the decoy ODNs can bind to free transcription factor protein and block its interaction with the promoter region of the target genes under its transcriptional regulation. Here, we try to use transcription factor decoy to inhibite STAT5 signaling pathway of K562, then observe the effect of Decoy ODNs in the proliferation, apoptosis and modulation of STAT5-mediated gene expression. This may help us to understand the mechanism of STAT5 in <WP=9>K562 more clearly and explore the STAT5 Decoy ODNs for therapy CML.Methods: Fist, the STAT5 Decoy ODNs, a double-stranded phosphorothioate 21mer, that exhibits a high sequence-specific binding affinity to the transcription factor STAT5 was synthesized. Cationic liposomes were used to transfect double-stranded Decoy ODNs into K562 cells. We make cell count experiments to determine the effects of the STAT5 Decoy ODNs on K562 cell growth. The MTT assay evaluated the effects of STAT5 Decy ODNs on the proliferative rate of K562 cells. The cell cycle assay and Annexin-V staining method to calculated apoptic cells by FCM. Then, we use RT-PCR method to observe the effects of STAT5 Decoy ODNs on the expression of bcl-xL、cyclin D1 and c-myc mRNA. Western blotting technique were used to determine the effect of stat5 Decoy ODNs on the expression of bcl-xL、cyclin D1 and c-myc proteins.Result: 1. The stat5 Decoy ODNs could inhibit the proliferation of K562 cells, and the inhibite increased with the dose increasing. 5μΜ Decoy ODNs could not inhibite the proliferation of K562 cells well, but 15μΜ and 25μΜ could . The result of MTT assay is in accordance with this. The proliferative inhibition rate of 5μΜ, 15μΜ and 25μΜ Decoy ODNs were 15%, 51% and 82%. But the proliferative inhibition of Mutant ODNs is very limited.2. The cell cycle assays of Decoy ODNs on K562 cells indicated that <WP=10>the percentage of S phase cells decreased from48.71±5.42％ to 31.94±2.35%（P<0.05）, while the percentage of M0/M1 phase cells increased from 40.83±5.20％ to 61.39±2.18％（P<0.05）. The untreated K562 cells and treated with Mutant ODNs did not effect their cell cycles significantly.3. After treated with Decoy ODNs 24h, FCM-Annexin-V/PI duel staining method determined the percentage of early apoptotic cells is 14.57±1.08%, while the cells and treated Mutant ODNs is 4±2.02%. Electron microscope also determined the microcosmine changes of apoptotic cells.4. RT-PCR tests indicated that STAT5 Decoy ODNs had decreased the mRNA expression of bcl-xL, cyclin D1 and c-myc. Western blotting tests also determined decrease of protein expression of bcl-xL, cyclin D1 and c-myc.Conclusion: First, STAT5 Decoy ODNs could inhibite the proliferation of K562, and the inhibite rate is dose-dependent. Second, Decoy ODNs altered the cell cycle progress and induced K562 apoptosis. Last, Decoy ODNs decreased the expression of of bcl-xL, cyclin D1 and c-myc both the mRNA and the protein, and it is maybe the cause of Decoy ODNs effecting the cell cycle and apoptosis.更多还原