STAT3基因沉默抑制人白血病细胞体外生长的实验研究
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江苏常州卫生创新人才基金


Effect of STAT3 gene silencing on growth of chronic myelogenous leukemia K562 cells
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    摘要:

    目的: 构建人STAT3 shRNA (short hairpin RNA)慢病毒载体,研究慢病毒介导的STAT3 SiRNA(small interference RNA)对人慢性粒细胞白血病K562细胞体外生长特性的影响-方法:针对STAT3基因RNAi有效靶点构建STAT3 shRNA慢病毒载体,293T细胞内包装产生STAT3 SiRNA慢病毒,感染K562细胞后RT-PCR和Western-blot方法验证STAT3 基因的敲减效率;细胞生长曲线和MTT法观察STAT3敲减后K562细胞体外生长和增殖情况;流式细胞术和Annexin V-FITC/PI法检测细胞周期改变和早期凋亡-结果:成功得到STAT3 SiRNA慢病毒,RT-PCR和Western-blot实验证实,针对其中两个靶点的STAT3 SiRNA对STAT3基因的敲减效率均在70%以上(P < 0.05)-STAT3敲减后,K562细胞生长变慢,细胞增殖活性降低40%(P < 0.05);细胞阻滞于G1→S期,并出现早期凋亡-结论:STAT3 SiRNA慢病毒可有效抑制K562细胞内STAT3基因表达,使细胞生长增殖受抑,细胞周期阻滞,细胞出现早期凋亡-

    Abstract:

    Objective: To construct the STAT3 shRNA (short hairpin RNA) lentivirus and investigate the effects of STAT3 SiRNA(small interference RNA)on the biological property of human chronic myelogenous leukemia (CML) K562 cells. Methods:The STAT3 shRNA lentiviral vector was constructed according to the effective target sequences for RNAi of STAT3 gene. The STAT3 SiRNA lentivirus were produced by transfecting into packaging cell line 293T. After transfected into K562 cells, the knockdown of STAT3 gene were determined by RT-PCR and Western-blot assay. The survival and proliferation of K562 cells were detected by both cell growth curve and MTT assay. The FCM was used to detected cell cycles alternation and the early apoptosis by Annexin V-PI assay. Results:PCR and sequencing analysis verified that the construction of the lentiviral vector of STAT3 shRNA was successful and the expression of STAT3 gene was knockdowned around 70 percent by two target sequences for STAT3 RNAi in K562 cells. Proliferation of K562 cells became slower and the proliferation activity of K562 cell was decreased to 40% post-transfection 5 days by lentiviral-mediated STAT3 SiRNA(P < 0.05). The proportion of cells in G0/G1 phase were increased while the cells in S phase decreased, which indicated a delay at G1 to S checkpoint in cell cycles. More than half the K562 cells underwent apoptosis after transfected with STAT3 SiRNA lentivirus compared to the control cells[(51.80±5.60)% vs (1.13±0.12)%],respectively(P < 0.01). Conclusion:Lentivirus-based STAT3 SiRNA can effectively mediate STAT3 gene silencing in K562 cells. SiRNA targeting STAT3 gene can inhibit K562 cell growth and proliferation, block cell cycle at G1 to S phase and induce cell apoptosis.

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马玲娣,周 民,蒋丽佳,岑 玲,范 静,夏 蕾. STAT3基因沉默抑制人白血病细胞体外生长的实验研究[J].南京医科大学学报(自然科学版),2011,(2):149-155

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  • 收稿日期:2010-08-06
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