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目的 构建抗bcr/ablmRNA的小干扰RNA(smallinterferenceRNA ,siRNA)表达载体 ,转染K5 6 2细胞 ,检测诱导细胞凋亡的变化。方法参照siRNA模板设计原则 ,设计并合成两条siRNA模板序列 ,将其插入质粒pSilencer1.0 U6中得到重组子pBCR6 ,通过限制性酶切和测序鉴定 ,大量制备、纯化 ;以X tremeGENEQ2介导瞬时转染K5 6 2细胞 ,设置空载体作为对照。在转染后不同时间 ,利用原位缺口末端标记法 (TUNEL)、膜联蛋白Ⅴ +碘化丙锭染色法 (AnnexinⅤ /PI)通过流式细胞仪检测K5 6 2细胞凋亡的变化。结果针对bcr/ablmRNA融合区域设计的siRNA模板序列 ,经筛选合成寡核苷酸链后退火形成双链 ,再插入pSilencer1.0 U6 ,经酶切和测序鉴定提示构建成功 ;大量制备、纯化后进行转染 ,在转染后 4 8,72hTUNEL法检测和AnnexinⅤ /PI染色法均显示抗bcr/ablmRNA的siRNA表达载体可有效诱导K5 6 2细胞凋亡 ,且随转染时间的延长凋亡率增高 [转染pBCR6 72h的K5 6 2细胞凋亡率为 (47.80± 1.6 3) % ],与对照组 [(6 .6 7± 0 .37) % ]比较差异有显著性 (P <0 .0 0 0 1)。结论抗bcr/ablmRNA的siRNA表达载体构建成功 ,初步结果显示它可以有效地诱导K5 6 2细胞发生凋亡 ,预期siRNA有望成为慢性髓系白血病分子靶向治疗的一个新工具。
Objective To construct a small interfering RNA (siRNA) expression vector against bcr / ablmRNA and transfect it into K562 cells to detect the changes of induced apoptosis. Methods According to the principle of siRNA template design, two siRNA template sequences were designed and synthesized. The recombinant plasmid pBCR6 was inserted into plasmid pSilencer1.0 U6 and identified by restriction enzyme digestion and sequencing. A large number of siRNA templates were prepared and purified. X tremeGENEQ2 mediated transient K562 cells were transfected and empty vector was set as control. At different times after transfection, the apoptosis of K562 cells was detected by flow cytometry with TUNEL, AnnexinⅤ / PI staining. Results The sequence of the siRNA template designed for the fusion region of bcr / ablmRNA was annealed to form double strands after screening and synthesis, and inserted into pSilencer1.0 U6. The results of enzyme digestion and sequencing showed that the constructed siRNA template was successfully constructed and purified. After transfection, the expression of siRNA against bcr / ablmRNA was detected by TUNEL assay and Annexin V / PI staining at 48 and 72h after transfection, respectively. The apoptosis of K562 cells was also induced and the apoptosis rate was increased with the prolongation of transfection [The apoptosis rate of K562 cells transfected with pBCR6 for 72h was (47.80 ± 1.6 3)%], which was significantly different from that of the control group [(6.67 ± 0.37)%] (P <0. 0 0 0 1). Conclusion The siRNA expression vector against bcr / ablmRNA was successfully constructed. Preliminary results showed that it could induce apoptosis in K562 cells effectively. It is expected that siRNA will be a new tool for molecular targeted therapy of chronic myeloid leukemia.