脂质体转染细胞周期素A反义脱氧寡核苷酸对HL-60细胞增殖及凋亡的调控作用

目的探讨脂质体转染细胞周期素A(cyclin A)反义脱氧寡核苷酸(ASON)对HL-60细胞增殖及凋亡的影响.方法采用脂质体转染技术将cyclin A ASON与HL-60细胞共同培养,用MTT法绘制细胞生长曲线,用电镜、细胞原位凋亡检测(POD)等方法观察细胞凋亡,用流式细胞术(FACS)及RT-PCR法检测cyclin A、bcl-2表达,并通过裸鼠成瘤实验验证cyclin A在白血病发生中的作用.结果脂质体转染cyclin A ASON组和cyclin A ASON组的HL-60细胞生物抑制率分别为68.9%和24.8%(P<0.01).脂质体转染cyclin A ASON组cyclin A及bcl-2表达水平(1.1%和21.9%)较对照组(38.8%和65.0%)明显减低(P值均<0.01),并能检测出DNA断裂,观察到凋亡细胞.裸鼠实验中发现脂质体转染cyclin A ASON组与对照组相比,其成瘤率低,成瘤时间延长,同一时间成瘤的瘤体体积明显缩小.结论脂质体转染cyclin A ASON能在体外及动物体内明显抑制白血病细胞的生长,且有诱导细胞凋亡的作用,可能会成为基因治疗的一个新靶点.

Effect of liposomal transfection of cyclin A antisense oligodeoxynucleotide (ASON) on HL-60 cell proliferation and apoptosis

OBJECTIVE: To explore the effect of liposomal transfection of cyclin A antisense oligodeoxynucleotide (ASON) on HL-60 cell proliferation and apoptosis. METHODS: By liposomal transfection, cyclin A ASON was co-cultured with HL-60 cells, the cell growth curve was determined by MTT assay and cell apoptosis electron-microscopy in situ cell apoptosis detection kit (POD), the protein and mRNA of cyclin A and bcl-2 were measured by FACS and RT-PCR, the role of cyclin A ASON in the development of leukemia was tested by the tumor formation in nude mice. RESULTS: (1) In the cyclin A ASON liposomal transfection group (group A), the proliferation of HL-60 cell was significantly inhibited as compared to those in cyclin A ASON group (group B) (68.9% vs 24.8%) (P < 0.01). (2) The expressions of cyclin A and bcl-2 of group A were significantly lower than those in the control group (1.1% vs 38.8%, P < 0.01; 21.9% vs 65.0%, P < 0.01, respectively), and the DNA ladder and apoptosis body was displayed. (3) In group A, the rate of tumor formation in nude mice was lower, the time for tumor formation was longer and the volume of tumor was smaller than those in control group. CONCLUSION: Liposomal transfection of cyclin A ASON can inhibit in vitro proliferation of leukemia cells and induce in vivo apoptosis of the tumor cell, which might provide a new target for gene therapy.