Chen Bo-bin, Fan Hua-hua, Lin Guo-wei, Yuan Zheng-hong, Lu Hua-zhong, Gao Li, Liu Yan
Department of Hematology, Huashan Hospital, Fudan University, Shanghai 200040, China.
Zhonghua Xue Ye Xue Za Zhi. 2004 Dec;25(12):717-9.
To construct a siRNA expression vector pBCR6 that produces siRNA against bcr/abl mRNA and detect apoptosis rate of K562 cells after pBCR6 transfection.
Template 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.
pBCR6 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, whereas the control group was (6.67 +/- 0.37)%, P < 0.0001] No prominent change in apoptosis rate was found in the control.
The 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.
构建针对bcr/abl mRNA产生小干扰RNA（siRNA）的表达载体pBCR6，并检测pBCR6转染K562细胞后的凋亡率。
设计、合成针对siRNA的模板序列并插入表达载体pSilencer1.0-U6。进行酶切分析和测序以验证pBCR6载体。然后用X-tremeGene Q2将pBCR6转染至K562细胞，以pSilencer1.0-U6作为对照。转染后不同时间点，采用Tunel法和Annexin V+PI结合流式细胞术测定凋亡率。
通过酶切分析和测序验证了pBCR6。pBCR6转染K562细胞后48小时和72小时凋亡率显著增加，并呈时间依赖性增加[转染K562细胞72小时时凋亡率为（47.80±1.63）%，而对照组为（6.67±0.37）%，P<0.0001]，对照组凋亡率无明显变化。
成功构建了针对bcr/abl mRNA的siRNA表达载体。初步研究表明，pBCR6可有效诱导K562细胞凋亡。siRNA可能是慢性粒细胞白血病分子靶向治疗的新工具。
