血管内皮生长因子反义核酸对细胞株HL60及耐药细胞株HL60/VCR的抑制作用

Inhibiting Effect of Vascular Endothelial Growth Factor Antisense Oligonucleotides on HL60 Cell and HL60/VCR Multidrug Resistance Cell

目的探讨反义血管内皮生长因子(AS-VEGF)对HL60、耐药细胞株HL60/VCR的影响及其机制,包括基因水平的耐药相关基因(Bcl-2,Mcl-1,MDR1,MRP,GSTπ,TopoⅡα,TopoⅡβ)和蛋白水平的耐药相关蛋白P糖蛋白(P-gp)表达量变化。方法分子生物学方法构建AS-VEGF表达真核载体,通过脂质体转染技术将其转染至细胞株HL60和耐药细胞株HL60/VCR内,观察并绘制细胞生长曲线,ELISA法检测细胞培养上清VEGF表达量变化,RT-PCR法从mRNA水平检测细胞内耐药相关基因表达,蛋白质印迹方法从蛋白水平检测细胞表面P-gp表达。结果1.25mmol/L的AS-VEGF真核表达载体转染HL60和HL60/VCR细胞株后继续培养24和48h与未转染组比较均可明显抑制细胞生长,细胞生长减慢;转染组细胞48与24h相比较VEGF、MDR1、MRP、GSTπ和TopoⅡβ的表达量均明显减少,Bcl-2、Mcl-1和TopoⅡα表达量则无明显变化;转染组HL60/VCR细胞表面P-gp表达量48与24h相比较也明显减少。结论AS-VEGF可通过抑制白血病细胞生长,改变细胞内的微环境和细胞膜相关蛋白转运通道,降低细胞解毒能力和自我修复能力逆转白血病细胞多药耐药。

Objective To explore the effect of vascular endothelial growth factor antisense oligonucleotides （ AS - VEGF） on HL60 cell and HL60/VCR multidrug resistance cell and analyze the function of P - gp and the expression of related multidrug resistance genes including Bcl - 2, Mcl - 1, MDR1, MRP, GSTπ,Topo Ⅱα and Topo Ⅱβ. Methods A vector AS - VEGF which expressed in eukaryotic cell was established ,then transfected the vector into HL60 and HL60/VCR by lirnposome transfection technology, observed and drew the growth curve by Tapanlan raining, RT - PCR was used to detect the expression of Bcl -2, Mcl - 1, MDR1, MRP, GSTπ, Topo Ⅱ α and Topo Ⅱβ in mRNA level after transfected 24 h and 48 h. Western Blot was used to detect the expression of P - gp in proteinum level after transfected 24 h and 48 h. Results The growth of HL60 and HL60/VCR was inhibited by AS - VEGF（ 1.25 mmol/L）. Between HL60 and HL60/VCR, AS - VEGF decreased the expression of MDRI, MRP, GSTπ and Topo I113 but could not influence the expression of Bcl - 2, Mcl - 1 and Topo Ⅱα, and the expression of P - gp was also obviously decreased in 48 h compared with that in 24 h. Conclusions AS - VEGF can inhibite the growth of HL60 and HL60/VCR and reverse multidrug resistance by changing cell microenvironment and the cell membrane correlated protein transportaring channel, reduce the cell disintoxicating and the self - repair ability.