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目的 :探讨bcl 2不同靶点的反义寡核苷酸 (ASODN)对白血病细胞株K5 6 2细胞凋亡的影响。方法 :用RNA二级结构预测程序预测bcl 2mRNA的二级结构 ;免疫荧光标记观察细胞bcl 2蛋白水平 ;细胞记数观察细胞的生存情况 ;用流式细胞仪观察细胞凋亡。结果 :在 5个初选的反义序列中 ,靶向bcl 2mRNA蛋白编码区和翻译起始区的ASODN能明显地下调bcl 2蛋白的表达 ,并且两个不同靶点的ASODN能有效地抑制K5 6 2细胞的生长活性、促进细胞凋亡 ;靶向bcl 2mRNA蛋白编码区的ASODN降低细胞bcl 2蛋白、促进细胞凋亡的作用较靶向翻译起始区的ASODN强。随机的无义寡核苷酸对K5 6 2细胞的生长活性、bcl 2蛋白水平及细胞凋亡率均无影响。结论 :分别在bcl 2mRNA翻译起始区和蛋白编码区发现了两个有效的反义作用靶点 ,针对这两个靶点的ASODN能特异性促进K5 6 2细胞的凋亡
AIM: To investigate the effect of antisense oligonucleotide (bcl-2) antisense oligonucleotide (ASODN) on the apoptosis of leukemia cell line K562. Methods: The secondary structure of bcl 2 mRNA was predicted by RNA secondary structure prediction program. The bcl 2 protein level was observed by immunofluorescence staining. The cell survival was observed by cell counting. Apoptosis was observed by flow cytometry. Results: Among the 5 primary antisense sequences, ASODN targeting the coding region and translation initiation region of bcl 2 mRNA significantly down-regulated the expression of bcl 2 protein, and ASODN at two different targets effectively inhibited K5 ASODNs targeting the bcl 2 mRNA protein coding region decreased the expression of bcl-2 protein and promoted cell apoptosis compared with ASODNs targeting the translation initiation region. Random nonsense oligonucleotides had no effect on the growth activity, bcl 2 protein level and apoptosis rate of K5 6 2 cells. CONCLUSION: Two effective antisense targets were found in the translation initiation region and the protein coding region of bcl 2 mRNA, respectively. ASODN targeting these two targets specifically promoted apoptosis of K5 6 2 cells