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bcl-2 siRNA的设计、合成及其对HL-60 bcl-2基因表达的干扰作用 被引量:5

Design and synthesis of efficient bcl-2 siRNA and it's interference role to HL-60 cells in leukemia
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摘要 目的应用RNA干扰技术研究bcl-2siRNA对白血病细胞HL-60bcl-2基因表达的干扰作用。方法扫描bcl-2mRNA的全序列,记录从AUG启动子的AA及下游19个核苷酸肽作为潜在作用靶点。GC含量控制在30% ̄65%之间。由BLAST分析排除与其他基因有高度同源性的序列。由体外转录合成法合成双链bcl-2siRNA,将bcl-2siRNA转染入HL-60细胞。收集转染48h后的细胞,流式细胞术检测蛋白表达情况,筛选出最佳作用序列进一步作各项指标检测。从mRNA水平、蛋白质水平等观察bcl-2siRNA对细胞HL-60bcl-2基因表达的干扰作用。结果收集转染48h后的细胞,经细胞免疫荧光、RT-PCR、MTT及Hoechst33258荧光染色等指标检测,可见转染bcl-2siRNA阳性组的细胞bcl-2基因表达降低及HL-60细胞凋亡等现象。而转染GAPDHsiRNA组的细胞对bcl-2表达无影响。结论采用生物信息学的方法设计出有效siRNA靶位点;bcl-2siRNA可通过降低bcl-2mRNA水平特异性抑制bcl-2蛋白质的表达。 Objective To study the interference role of efficient bcl-2 siRNA to HL-60 cells in leukemia. Methods Beginning with the AUG start coclon, scan the length of bcl-2 mRNA for AA sequences. Record the AA and downstream 19 nucleoticles and compare the sequence to the appropriate genome database to eliminate any sequences with significant homology, to other genes. In this study, we selected three target sequences for testing and controlled the GC content between 30 % - 65 %, because early experiments suggest that target sequences with a low GC content may be more susceptible to siRNA-incluced degradation, bcl-2 siRNA was synthesized in vitro transcription with silencer siRNA construction kit. Synthesized siRNA was transfected into HL-60 cells with lipid siPORT transfection Kit. Forty-eight hours after transfection, we collected cells and detected the bcl-2 protein expression by flow cytometer. We selected the mostly efficient siRNA to do subsequently experiment according to the flow cytometerys result. We used RT-PCR and immunofluorescence to determine the expression level of bcl-2 mRNA and protein. Results bcl-2 siRNA1 specifically reduced bcl-2 expression on the level of mRNA and protein in HL-60 cells and induced cell apoptosis. There was no difference on the effect of other groups compared with the control groups. And GAPDH siRNA also only reduced the GAPDH gene expression while had no influence to bcl-2 gene. Conclusion We adopted bioinformatic technique to design effective target siRNA; Effective bcl-2 siRNA could specifically degrade bcl-2 protein expression by degraded bcl-2 mRNA level.
出处 《白血病.淋巴瘤》 CAS 2006年第5期323-326,共4页 Journal of Leukemia & Lymphoma
基金 国家自然科学基金资助青年项目(30300426)
关键词 BCL-2 SIRNA HL-60 BCL-2 基因表达 bcl-2 siRNA HL-60 cells Gene expression
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