重组反义c-myc腺病毒抑制K562细胞生长、诱导凋亡的作用

Recombinant antisense c-myc adenovirus inhibited proliferation and induced apoptosis of K562 cells

目的研究重组反义c-myc腺病毒(Ad-AS-c-myc)对人慢性粒细胞K562细胞系抑制生长,诱导凋亡作用及分子机制,探讨Ad-AS-c-myc用于慢性粒细胞白血病基因治疗的可能性。方法采用重组腺病毒Lac-Z(Ad-LacZ)及Ad-AS-c-myc联合多聚季胺转染K562细胞,X-gal染色判断转染效率。采用形态学、MTT试验、RT-PCR、DNA凝胶电泳、流式细胞仪及免疫细胞化学等方法进行研究。结果多聚阳离子可提高腺病毒载体对K562细胞的转染,Ad-LacZ+多聚季胺转染率达86%。Ad-AS-c-myc能抑制K562细胞c-myc基因在转录水平的表达,K562细胞生长受抑(抑制率38%);Ad-AS-c-myc可使K562细胞G1、G2期阻滞,增殖相关基因PCNA表达降低,Apo2.7蛋白阳性细胞率增高,DNA电泳出现DNA梯形条带。结论Ad-AS-c-myc对K562细胞具有抑制生长及诱导凋亡作用,其生物学作用与K562细胞c-myc及PCNA的表达降低有关。Ad-AS-c-myc在慢性粒细胞白血病基因治疗中具有潜在的临床价值。

Objective：To evaluate the effect of adenovirus antisense c-myc （Ad-AS-c-myc） on proliferation and apoptosis of human chronic myelocytic leukemia K562 cells and the mechanism. To explore the potential of Ad-AS-c-myc in gene therapy for human chronic myelocytic leukemia. Methods：Cultured K562 cells were transfected with Ad-LacZ and Ad-AS-c-myc combined with polybrene. The transfer efficiency was analyzed by X gal staining. Morphology examination, MTT assay, flow cytometric analysis,RT-PCR,DNA gel electrophoresis,and immunocytochemical techniques were used. Results： Infection with retroviruses was facilitated greatly by polybrene. The transfer efficiency of A＆-LacZq＋polybrene was 86 . Ad-AS-c-myc strongly inhibited c- myc transcription in the transfected K562 cells. Ad-AS-c-myc strongly inhibited cell growth in K562 cells（38% ）, arrested cells at G2/M stage, reduced proliferation-related gene expression such as PCNA,increased the number of Apo 2.7 positive cells,and induced typical DNA ＇ladder＇ in K562 cells. Ad-AS-c-myc could lead to G1 and G2 arrest of K562 cells. Ad-AS-c-myc also decreased the expression of PCNA and increased the positive rate of Apo 2.7 in K562 cells. Conclusions： The expression of Ad-AS-c-myc can inhibit proliferation and induce apoptosis of K562 cells in vitro . The biological effects of Ad-AS-c-myc may be closely associated with downregulation of c-myc and PCNA genes in K562 cells. Ad-AS-c-myc has clinical potential in gene therapy for chronic myelocytic leukemia.