Bcr-abl特异性系列核酶体外切割活性比较及诱导K562细胞凋亡的实验研究

Study on the in vitro cleavage abilities of ribozymes specific to different sites of bcr abl fusion gene and their induction of apoptosis in K562 cells

目的 构建单、双及三单位核酶 ,鉴定其体外切割活性及对K5 6 2生物学特性的影响 ,探讨核酶用于基因治疗的前景。方法 首先针对bcr abl融合位点设计并合成 3个单核酶 ,而后通过基因重组构建单、双及三核酶载体 ,以体外切割试验鉴定并比较其活性。然后以脂质体将bcr abl三核酶逆转录病毒载体转入K5 6 2细胞 ,观察其对K5 6 2细胞周期、凋亡、超微结构的影响。结果 构建成功bcr abl特异性系列核酶共 6个重组载体 ,切割试验证实系列核酶载体中三核酶体外切割活性最强 ,为 70 8% ,双核酶次之 ,分别为 6 0 7%和 30 3% ,不同位点的单核酶切割活性分别为 5 4 6 %、2 5 3%和3 6 %。bcr abl特异性多核酶通过诱导凋亡抑制K5 6 2细胞增殖。结论 bcr abl特异性核酶在慢性粒细胞白血病基因治疗中具有潜在的应用前景 ,为多核酶体外净化骨髓联合自体骨髓移植治疗提供了理论和实验依据。

Objective To investigate the in vitro cleavage abilities of ribozymes specific to different sites of bcr abl fusion gene and their effects on K562 cell Methods First, three single ribozymes specific to the fusion point were designed After recombination, 6 vectors including single , double and triple ribozymes were constructed and their in vitro cleavage abilities were compared Then the triple unit ribozyme retroviral vector was transfected into K562 cell to test its effect on cell cycle, apoptosis and cell structure Results These ribozymes can cleave the template in vitro with different efficiency The triple unit ribozyme, with an efficiency of 70 8%, was the most efficient one The cleavage efficiency of single unit RZ1, RZ2 and RZ3 was 54 6%, 25 3% and 3 6%, respectively Those of double unit RZ12 and RZ23 were 60 7% and 30 3% respectively The triple unit ribozyme could inhibit the K562 cell growth by inducing apoptosis Conclusion It is a new way to treat CML by ribozymes specific to bcr abl fusion gene, which made it available to purge bone marrow by bcr abl specific ribozymes