多单位核酶对慢性粒细胞白血病细胞的增殖抑制及凋亡诱导效应研究

The effect of multi-unit ribozymes on the growth inhibition and apoptosis induction of CML K562 cells

目的 探讨多单位核酶体外净化慢性粒细胞白血病 (慢粒 )骨髓的可能性 ,研究多单位核酶的体外切割活性及对慢粒细胞恶性表型的逆转作用。方法 针对慢粒发病中起重要作用的bcr abl融合基因 ,在融合位点两侧 4 4碱基范围内设计、合成 3个核酶 ,其中 2个切割位点位于bcr基因 ,1个位于abl基因。通过基因重组构建多单位核酶体外转录载体及逆转录病毒表达载体 ,鉴定其体外切割活性。将多单位核酶逆转录病毒表达载体转染K5 6 2细胞 ,应用MTT、3 H TdR掺入、RT PCR、斑点杂交、流式细胞仪、透射及扫描电镜等方法 ,检测多单位核酶对慢粒细胞增殖活性及细胞超微结构、细胞周期、凋亡等的影响。结果 多单位核酶的体外切割活性为 70 .8% ,逆转录病毒载体转染慢粒K5 6 2细胞系后 ,细胞DNA合成及细胞增殖受到明显抑制 ,转染后 96h抑制率达 5 0 %左右。多单位核酶转染细胞后能够有效切割细胞RNA ,使转染细胞RNA减少 10 0 0倍左右。流式细胞仪检测显示多单位核酶转染 72h后 ,18.4 %的细胞发生凋亡 ,大多数细胞被阻滞于G期 ,分裂期 (S期 )细胞数减少约 4 1.9%。透射、扫描电镜见转染细胞出现核固缩、凋亡小体等细胞凋亡的特异性表现。结论 多单位核酶不仅具有较高的体外切割活性 ,而且其逆转录病毒载体能够有效转染慢?

Objective To investigate the possibility of multi unit ribozymes to purge bone marrow of chronic myelocytic leukemia (CML), its in vitro cleavage ability and the reversal effect on CML cell′s malignant phenotype. Methods As bcr abl fusion gene plays an important role in CML pathology, three single unit ribozymes were designed and synthesized in 44 base pairs near the fusion point, two enzyme cleavage sites on bcr gene and one on abl gene. Multi unit ribozymes in vitro transcription and retroviral vector through gene recombination were constructed. Then, its in vitro cleavage ability was tested and the retroviral vector was transfected into K562 cell. Through MTT assay, the incorporation rate of 3 H TdR, RT PCR, Southern and Northern blot hybridization, flow cytometry, transmission and scanning electron microscopy were used to study the effect of multi unit ribozymes on CML cell proliferation, cell structure, cell cycle and the induction of apoptosis. Results Multi unit ribozymes had in vitro cleavage efficiency of 70.8%. After the transfection of multi unit ribozymes retroviral vector into K562 cell, cell proliferation and DNA synthesis were greatly reduced with an inhibition rate of about 50% after 96 hours of transfection. Multi unit ribozymes could cleave K562 cell′s RNA with a reduction rate about one 1 000 th of the original. By flow cytometry (FCM), 18.4% cells underwent apoptosis after 72 hours transfection with most of the cells blocked in the G phase. Here, the ratio in S phase was lowered by 41.9%. Under transmission and scanning electron microscope, compaction of nuclear chromation and apoptosis bodies were observed in the transfected cells. Conclusion Multi unit ribozymes possess high cleavage ability in vitro. The ribozymes, whose retroviral vector being transfected into CML cell, are able to express a lasting ability to cleave the fusion gene, induce apoptosis, reduce cell proliferation, revert the malignant phenotype. It is possible to make use of multi unit ribozymes to purge CML bone marrow. Therefore, multi unit ribozymes may very well be valuable in the gene therapy of CML.