目的: 研究慢病毒介导RNAi致bcr/abl基因长期沉默对K562白血病细胞各种生物学特性的影响。方法：构建含bcr/abl RNAi序列的pNLB/AEGFP慢病毒重组质粒载体并包装病毒，感染K562细胞，挑取稳定转化的克隆（B/AK562）。Realtime PCR及Western blotting验证干扰效应；锥虫蓝染色、集落形成实验检测细胞增殖能力变化；联苯胺染色观察细胞分化；ELISA法检测酪氨酸激酶活性；AOEB染色观察细胞凋亡；比色法检测Caspase3、Caspase9活性；以上检测均以K562细胞和转染空质粒EGFPK562作对照。结果：成功构建bcr/abl基因RNAi稳定转染的B/AK562细胞, Realtime PCR及Western blotting证实B/AK562细胞中bcr/abl mRNA及P210bcr/abl蛋白含量明显下调。bcr/abl基因稳定下调后，K562细胞倍增时间明显延长（37.1 vs 20.4、23.3 h）、集落形成能力减弱（P<0.01），K562细胞向红系分化，酪氨酸激酶活性下降（P<0.01），自发凋亡率显著提高（P<0.01），细胞中Caspase3（P<0.05）及Caspase9（P<0.01）活性明显提高。结论：慢病毒介导的RNAi能实现bcr/abl基因长期沉默，从而抑制K562细胞恶性增殖，诱导其分化及凋亡。

Objective: To study the effects of lentivirusmediated bcr/abl RNAi on the biological characteristics of human leukemia cell line K562. Methods:Bcr/abl RNAi lentivirus vector pNLB/AEGFP was constructed and was used to transfect K562 cells, the stable tansfectants（B/AK562）were selected. RNAi efficiency was assessed by Realtime PCR and Western blotting. Cell proliferation was detected by trypan blue staining and colony formation assay, cell differentiation was investigated by benzidine staining, PTK activity was determined by ELISA, apoptosis was observed by AOEB staining, and caspase3 and caspase9 activation were measured by chromometry. K562 cells and mock transfected EGFPK562 cells were used as controls.Results: pNLB/AEGFP stably transfected K562 cells (B/AK562) was successfully constructed. Realtime PCR and Western blotting analysis confirmed that lentivirusmediated bcr/abl RNAi downregulated bcr/abl mRNA and P210bcr/abl protein expression in K562 cells. The doubling time of B/AK562 cells was obviously longer than those of K562 cells and EGFPK562 cells (37.1 vs 20.4, 23.3 h). Furthermore, B/AK562 cells showed decreased colony formation ability, strengthened differentiation toward erythrocytes, decreased activation of PTK, increased apoptosis and enhanced caspase3 and caspase9 activation (P<0.05 or P<0.01). Conclusion: Lentivirusmediated bcr/abl RNAi can result in long time silencing of bcr/abl gene, inhibit malignant proliferation and induce differentiation and apoptosis in K562 cells.

国家自然科学基金资助项目(No. 30873101)