K562细胞血管内皮生长因子自分泌链作用的实验研究  

Experimental study on the role of VEGF autocrine loop in K562 leukemia cells

作　　者：许文林[1] 沈慧玲[1] 袁伟[1] 江云伟[1] 吴朝阳[1] 费霞[1] 唐华容[1] 

机构地区：[1]江苏大学附属人民医院血液科,镇江212002

出　　处：《中华血液学杂志》2007年第3期189-193,共5页Chinese Journal of Hematology

摘　　要：目的探讨抗VEGF抗体及抗VEGF发夹状核酶基因对K562细胞增殖、凋亡及相关基因表达的影响及其分子机制。方法将不同浓度的VEGF抗体作用于K562细胞，应用脂质体介导的方法将抗VEGF发夹状核酶基因真核表达载体pcDNA-RZ转染K562细胞，采用MTT法、甲基纤维素半固体培养法和细胞周期测定分析抗VEGF抗体及发夹状核酶基因对白血病细胞增殖的影响；DNA凝胶电泳和AnnexinⅤ标记法检测白血病细胞的凋亡程度；RT-PCR法测定K562细胞中相关基因表达的变化。结果抗VEGF抗体能抑制K562细胞生长，促进其凋亡，这一作用呈剂量依赖关系。0．165μg／ml VEGF抗体作用于K562细胞72h，DNA凝胶电泳出现梯状条带，当抗体浓度增加到0．825μg／ml时，梯状条带更为清晰；RT-PCR显示，VEGF抗体作用后，K562细胞MRP、TOPOⅡ基因的表达较对照组下调，而GST基因表达无明显改变。与K562及K562／PC细胞（转染空质粒的K562细胞）相比，转染抗VEGF核酶基因的K562／RZ细胞VEGF mRNA和蛋白的表达量明显降低；生长曲线提示K562／RZ细胞生长缓慢，甲基纤维素集落形成率较对照组明显下降，对照组集落形成率为（30．5±3．3）％，而K562／RZ组为（16．3±2．8）％，细胞周期动力学分析结果显示K562／RZ细胞G1期细胞增多，S期细胞显著减少；在小剂量As2O3作用下，K562／RZ细胞凋亡数量较对照明显增高（凋亡率对照组为13．4％，K562／RZ组为31．5％）。结论抗VEGF抗体阻断K562细胞VEGF自分泌环路或者抗VEGF发夹状核酶减少K562细胞中VEGF的合成，能抑制K562细胞的增殖，促进K562细胞的凋亡，引起部分与细胞凋亡和多药耐药相关的基因表达改变。Objective To investigate the effects of anti-VEGF antibody and anti-VEGF hairpin ribozyme gene on the proliferation, apoptosis and related gene expression of the leukemia K562 cells and the possible molecular mechanisms. Methods K562 cells were cultured in different concentrations of anti-VEGF antibody. The recombinant eukaryotic expression plasmid（pcDNA3-RZ） containing anti-VEGF hairpin ribozyme gene and the vector-alone were introduced into K562 cells by lipofectamine mediation. Cell proliferative capacity was determined by MTr, colony formation assay and cells cycles analysis. Cell apoptosis was assayed by DNA ladder and Annexin Ⅴ-FITC/PI flow cytometry. Results The anti-VEGF antibody was able to inhibit growth and induce apoptosis of K562 cells in a dose-dependent manner. Exposure to anti-VEGF antibody at 0. 165 μg/ml for 72 hours, the cells exhibited typical DNA ladders. The apoptosis rate peaked at antibody concentration of 0. 825 μg/ml. RT-PCR showed a decrease of MRP and TOPO Ⅱexpression but a relative constant expression of GST. The introduction of exogenous anti-VEGF ribozyme gene resulted in a decrease of the proliferative capacity and colony forming efficiency from （ 30.5 ± 3.3 ） % in control group to （ 16.3 ± 2.8 ） % in K562/RZ group, higher G1 and lower S ratio in cell cycle distribution in comparison with the control groups. Typical DNA fragmentation and higher Annexin Ⅴ^＋ ratio occurred in K562/RZ cells after treated with 0. 5 μmol/L of As203 for 3 days, the apoptosis rate increased from 13.4% in control group to 31.5% in As2O3 treated group. Conclusion Anti-VEGF antibody can inhibit growth, induce apoptosis and down-regulate the expression of MRP, TOPO Ⅱ genes of K562 cells in vitro. Transfection with anti-VEGF ribozyme gene can inhibit the proliferation of the cells by delaying the progression GI into S phase in cell cycles and induce cell apoptosis by down-regulating VEGF gene expression.

关 键 词：K562细胞 内皮细胞生长因子 抗体 发夹状核酶 细胞凋亡 

分 类 号：R686[医药卫生—骨科学]