【摘要】 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。更多还原

【Abstract】 ObjectivesTo construct a siRNA expression vector pBCR6 that produces siRNA against bcr/abl mRNA and detect apoptosis rate of K562 cells after pBCR6 transfection.MethodsTemplate sequence for siRNA was designed, synthesized and inserted into an expression vector pSilencer1.0 U6. Restriction analysis and sequencing were performed to verify the pBCR6 vector. Then pBCR6 was transfected into K562 cells by X tremeGene Q2. pSilencer1.0 U6 was used as the control. At different time point after transfection, apoptosis rate was determined by Tunel and Annexin Ⅴ+ PI with FCM.ResultpBCR6 was verified by restriction analysis and sequencing. The apoptosis rate of K562 cells markedly increased at 48 and 72 hour after transfected with pBCR6, and increased in a time dependent manner [the apoptosis rate of transfected K562 cells was (47.80±1.63)% at 72 hrs, wheras the control group was (6.67±0.37)%, P<0.0001] No prominent change in apoptosis rate was found in the control. ConclusionThe siRNA expression vector against bcr/abl mRNA was successfully constructed. The pilot study showed that pBCR6 could effectively induce K562 cells apoptosis. siRNA may be a new tool for molecular target therapy for chronic myelogenous leukemia.更多还原