自分泌血管内皮细胞生长因子对HL-60细胞生物学活性的影响

Effect of autocrine vascular endothelial growth factor on the biological activity of acute myeloid leukemia cell line HL-60

目的研究自分泌血管内皮细胞生长因子(VEGF)对髓系白血病细胞株HL-60的增殖及凋亡影响。方法采用脂质体转染VEGF165 cDNA入HL-60细胞,通过RT-PCR及ELISA方法鉴定转染克隆HL-60/VEGF165细胞中VEGF mRNA及蛋白的表达,MTT、集落形成实验比较HL-60/VEGF165及带有pcDNA3.1-- neo空载体的HL-60/neo细胞增殖活性,流式细胞仪观察转染细胞凋亡。结果HL-60/ VEGF165细胞的VEGF mRNA表达量增加,细胞培养上清中VEGF蛋白浓度为(399.07±12.45)ng/L,高于HL-60/neo 细胞的(184.45±10.53)ng/L(P<0.01);HL-60/VEGF165细胞与其对照组HL-60/neo 细胞相比较,生长速度明显加快,集落形成能力明显增加,集落数分别为(157.00±17.00)/500细胞和(110.00±12.90)/500细胞(P<0.05),而细胞凋亡率减少,分别为(3.18±0.33)%和(6.61±0.50)%。结论自分泌VGEF在白血病细胞的增殖与凋亡中起重要作用。

Objective To explore the effect of autocrine vascular endothelial growth factor (VEGF) on the abnormal proliferation of HL-60 cells. Methods HL-60 cells were transfected via lipofictamine with the VEGF 165 cDNA sense vector (HL-60/ VEGF 165 ) and with the PcDNA3.1 - -vector (HL-60/neo) as the control. Reverse transcription-polymerase chain reaction (RT-PCR) was used for detecting VEGF mRNA in the transfected cells. VEGF concentrations in the cell culture supernatant were determined by enzyme-linked immunosorbent assay (ELISA), and cell proliferation was determined by MTT assay and colony-forming assay in vitro. Flow cytometric Annexin-Ⅴ-FITC/ PI dual-labeling technique was employed to observe the effect of VEGF 165 cDNA transfection on the apoptosis of HL-60 cells. Results The levels of VEGF mRNA expression by HL-60/VEGF 165 cells were higher than HL-60/neo, with the mean VEGF quantity in the supernatant of HL-60/VEGF 165 cell culture being 399.07±12.45 ng/L, which was 2-fold higher than that in the supernatant of HL-60/neo cells (184.45±10.53 ng/L) (P<0.01). The growth rate of HL-60/VEGF 165 cells was significantly accelerated as compared with that of the control cells, and the colony formation capacity of the former cells also increased significantly (P<0.05), with the average colony number of 157.00±17.00/500 cells vs110.00±12.90/500 cells of HL-60/neo cells. Less apoptotic cells were identified among HL-60/VEGF 165 cells than in HL-60/neo cells in the same culture condition. Conclusion Autocrine VEGF plays an important role in the proliferation and apoptosis of acute myeloid leukemia cells.