叶酸受体-α在卵巢癌耐药细胞株中的表达及其介导的多药耐药的靶向逆转

Expression of folic acid receptor-α in drug resistant ovary cancer cells and its targeted effect for reversion of multidrug resistance

目的:研究叶酸受体-α(folic acid receptor-α,FR-α)在卵巢癌多药耐药细胞株中的表达水平,并探讨FR-α靶向的MDR1小干扰RNA(siRNA)纳米粒对卵巢癌多药耐药细胞的逆转效果。方法:采用体外浓度梯度递增法建立卵巢癌紫杉醇耐药细胞株SKOV3-ts,应用激光共聚焦法检测FR-α的表达水平。复凝聚法将叶酸修饰壳聚糖载体包裹靶向MDR1的PGPU6/GFP/Neo/PshRNA真核表达质粒(PshRNA-MDR1),形成叶酸修饰壳聚糖PshRNA-MDR1纳米粒(FA-CS-PshRNA)。采用RT-PCR、Western blot检测叶酸修饰前后,纳米粒转染细胞后MDR1 mRNA及蛋白的表达,MTT法检测SKOV3-ts半数抑制浓度(IC50)的变化。结果:激光共聚焦法显示,耐药细胞株SKOV3-ts胞膜层有FR-α强表达,经叶酸修饰后,FA-CS-PshRNA纳米粒较CS-PshRNA能更有效降低靶细胞SK-OV3-ts的靶基因MDR1 mRNA和P-gp蛋白表达(P<0.01),并逆转SKOV3-ts细胞针对紫杉醇的IC50(0.3830±0.0096 vs 0.0353±0.0006,P<0.01)。结论:经FR-α介导,FA-CS-PshRNA能更有效敲除MDR1的表达,靶向逆转卵巢癌多药耐药。

Objective：To investigate the expression of FR-α in drug resistant ovary cancer cells,and to compare the reversion effect of multidrug resistance before and after folic acid modification.Methods：The expression level of FR-α in SKOV3-ts cells which cultured using paclitaxel by concentration gradient method in vitro was detected using laser scanning confocal microscope.The chitosan modified with folic acid was used to parcel the eukaryon expression vector pGPU6/GFP/Neo/PshRNA targeting MDR1（PshRNA-MDR1）to form into FA-CS-PshRNA nanoparticles which were transfected into SKOV3-ts cells.The expression level of MDR1 mRNA and P-gp in SKOV3-ts cells were detected by RT-PCR and Western blot.The 50% inhibiting concentration（IC50）of SKOV3-ts cells to paclitaxel was detected by MTT assay.Results：There was strong expression of FRα in SKOV3-ts cells detected by laser scanning confocal microscope.Compared with unmodified nanoparticles,the FA-CS-PshRNA nanoparticles can effectively degrade the expression level of MDR1 mRNA and P-gp protein（P0.01）,and can effectively reverse the IC50 to paclitaxel of SKOV3-ts cells（0.3830±0.0096 vs 0.0353±0.0006;P0.01）.Conclusions：Through FRα mediation,FA-CS-PshRNA nanoparticles can effectively decrease the expression of MDR1 mRNA and P-gp of SKOV3-ts cells and can reverse multidrug resistance of ovary cancer in target.