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目的:采用双链RNA能激活细胞内蛋白激酶PKR引起细胞凋亡的策略,研究重组慢性粒细胞白血病独特的bcr/ablb3a2型外源反义RNA诱导白血病K562细胞的凋亡及其作用机制。方法:用含bcr/abl融合基因序列40bp的反转录病毒载体RV-40AS作用于白血病K562细胞,以光学显微镜、电子显微镜、FCM法和DNA梯状条带法检测细胞凋亡情况,化学比色法检测caspase-8和caspase-9活性变化,RT-PCR法检测Bax、Bcl-2 mRNA表达变化,并以Western印迹法检测双链RNA依赖性蛋白激酶(double-stranded RNA-dependent protein kinase,PKR)、磷酸化PKR(p-PKR)、Bax和Bcl-2的蛋白变化。结果:RV-40AS作用K562细胞组在光学显微镜下出现胞体皱缩、染色质浓集以及折光性减弱,电子显微镜下可见细胞质电子密度增大、核固缩和出现凋亡小体;FCM法检测到凋亡细胞占(22.70±1.42)%[未处理组(3.24±0.66)%],DNA电泳出现典型的梯状条带;caspase-8和caspase-9活性升高,PKR蛋白无明显变化,但p-PKR水平升高;Bax的mRNA和蛋白表达均升高,而Bcl-2 mRNA和蛋白表达水平没有明显变化。对照组和PKR抑制剂处理组均未发生上述改变。结论:bcr/abl反义RNA反转录病毒载体RV-40AS可特异性激活PKR,从而诱导白血病K562细胞发生凋亡,其可能的机制是PKR活化激活了凋亡反应的上游分子caspase-8,并通过上调Bax表达水平、降低Bcl-2/Bax的比值,导致线粒体膜稳定性被破坏,促使凋亡小体形成和caspase-9活化,最终激活其他凋亡分子使细胞发生凋亡。
OBJECTIVE: To investigate the apoptosis of leukemia K562 cells induced by bcr / ablb3a2 exogenous antisense RNA, which is unique to recombinant chronic myeloid leukemia, using double-stranded RNA to activate PKR-induced apoptosis in human leukemia cells. Methods: A retroviral vector containing 40bp of bcr / abl fusion gene RV-40AS was used to act on leukemia K562 cells. The apoptosis of K562 cells was detected by light microscopy, electron microscopy, FCM and DNA laddering. The changes of caspase-8 and caspase-9 activities were detected by colorimetric assay. The expressions of Bax and Bcl-2 mRNA were detected by RT-PCR. The double-stranded RNA-dependent protein kinase PKR), phosphorylated PKR (p-PKR), Bax and Bcl-2. RESULTS: The effect of RV-40AS on K562 cells was observed under optical microscope. The chromatin condensation and refraction were observed under electron microscope. The electron density of cytoplasm, nuclear condensation and apoptotic bodies were observed under electron microscope. FCM assay The apoptotic cells accounted for (22.70 ± 1.42)% [untreated group (3.24 ± 0.66)%], DNA ladder typical ladder electrophoresis; caspase-8 and caspase-9 activity increased, PKR protein did not change significantly, But the level of p-PKR increased; Bax mRNA and protein expression increased, while Bcl-2 mRNA and protein expression levels did not change significantly. None of the above changes occurred in the control and PKR inhibitor groups. CONCLUSION: The bcr / abl antisense RNA retroviral vector RV-40AS can specifically activate PKR and induce the apoptosis of leukemia K562 cells. The possible mechanism is that PKR activation activates the upstream caspase-8, And by up-regulating the expression of Bax and decreasing the ratio of Bcl-2 / Bax, the stability of mitochondrial membrane was destroyed, which led to the formation of apoptotic bodies and the activation of caspase-9. Finally, other apoptotic molecules were activated to induce cell apoptosis.