论文部分内容阅读
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 study the expression of folic acid receptor-α (FR-α) in multidrug-resistant ovarian cancer cell lines and to explore the role of FR-α targeting MDR1 small interfering RNA (siRNA) Reversal effect on multidrug resistance of ovarian cancer cells. Methods: Paclitaxel-resistant cell line SKOV3-ts was established by in vitro concentration gradient method. The expression of FR-α was detected by laser scanning confocal microscopy. In the complex coacervation, the folic acid-modified chitosan vector was coated with the PshRNA-MDR1 PGPU6 / GFP / Neo / PshRNA eukaryotic expression plasmid (PshRNA-MDR1) targeting MDR1 to form the folate modified chitosan PshRNA- . The expression of MDR1 mRNA and protein was detected by RT-PCR and Western blot before and after folic acid modification. The half inhibitory concentration (IC50) of SKOV3-ts was detected by MTT assay. Results: Confocal laser scanning microscopy showed that FR-α was strongly expressed in the cell membrane of SKOV3-ts cell line, and the FA-CS-PshRNA nanoparticles were more effective than CS-PshRNA in reducing the expression of SK-OV3 (P <0.01), and reversed the IC50 of SKOV3-ts cells against paclitaxel (0.3830 ± 0.0096 vs 0.0353 ± 0.0006, P <0.01). CONCLUSION: FA-CS-PshRNA can knockdown the expression of MDR1 more effectively by FR-α, and reverse the multidrug resistance of ovarian cancer.