CS-TPGS-b-(PCL-ran-PGA)纳米粒递送HIF-1α siRNA联合顺铂对鼻咽癌裸鼠移植瘤影响研究

目的 乏氧诱导因子1α(hypoxia inducible factor-1α,HIF-1α)增强抗肿瘤药物敏感性研究在国内外已有诸多报道.本研究拟探讨纳米聚合物壳聚糖-聚乙二醇1 000维生素E琥珀酸酯-聚乙内酯-聚乙醇酸d-α-tocopheryl polyethylene glycol 1 000 succinate-b-poly(ε-caprolactone-ran-glycolide),CS-TPGS-b-(PCL-ran-PGA),简称CNP]包载HIF-1α小分子干扰RNA(small interfering RNA,siRNA)联合顺铂(cisplatin,DPP)对鼻咽癌裸鼠移植瘤生长的影响.方法 皮下接种CNE-2细胞建立人鼻咽癌裸鼠移植瘤模型,应用随机数字表法分磷酸盐缓冲液组(Control)、单纯纳米粒组(CNP,200 μg/kg)、单纯顺铂组(DPP,100 μg/kg)、单纯纳米粒联合顺铂组(CNP 200 μg/kg+DPP 100 μg/kg)、载HIF-1α siRNA纳米粒组(CNP-siRNA,200 μg/kg)和载HIF-1α siRNA纳米粒联合顺铂组(CNP-siRNA 200μg/kg+DPP 100μg/kg)6组.每组5只,腹腔注射3d1次,共7次.干预过程中3d测量1次肿瘤体积,治疗22d后拉颈处死裸鼠,称取瘤质量,计算抑瘤率;利用蛋白质印迹与RT-PCR检测移植瘤中HIF-1α和多重耐药基因-1/P糖蛋白(multidurg resistance gene 1/P-glycoprotein,MDR-1/P-gp)基因蛋白表达水平,HE染色观察各组肿瘤组织病理形态改变.结果 与Control组相比,CNP组抑瘤率为18.32％,DPP组为37.59％,CNP-siRNA组为43.32％,CNP+ DPP组为47.64％,CNP-siRNA+ DPP组为70.21％.其中CNP-siRNA+DPP组抑瘤效果最为显著,与其他治疗组相比,差异有统计学意义,F=253.511,P＜0.001.在HIF-1α mRNA表达上,与Control组相比,CNP组mRNA表达量为0.89±0.04,DPP组为0.99±0.02,CNP-siRNA组为0.95±0.02,CNP+ DPP组为0.35±0.04,CNP-siRNA+DPP组为0.15±0.07.CNP-siRNA+ DPP组mRNA抑制效果最为显著,与其他组相比,差异有统计学意义,F=351.194,P＜0.001.蛋白质印迹结果亦显示,CNP-siRNA+ DPP组中HIF-1α蛋白条带宽度明显低于其余各实验组,MDR-1/P-gp表达水平表现出与HIF-1α基因蛋白表达相似的实验结果.HE结果表明,CNP-siRNA+ DPP组能够显著促进肿瘤细胞坏死及凋亡.结论利用CNP纳米聚合物递送HIF-1α siRNA联合DDP能够有效下调肿瘤细胞HIF-1α及MDR 1/P-gp基因蛋白表达,抑制肿瘤生长,促进肿瘤细胞坏死凋亡.新型纳米聚合物CNP作为递送siRNA的有效载体,在肿瘤基因靶向治疗中具有较高的应用前景.

Anti-tumor effect of siRNA targeting HIF-1α loaded CS-TPGS-b-(PCL-ran-PGA) complexed with cisplatin on nasopharygeal carcinoma in nude mice

OBJECTIVE Hypoxia inducible factor-1α (HIF-1α) enhanced tumor chemosensitivity studies have been extensively reported at home and abroad.This research was to explore the effects of chitosan modified d-α-tocopheryl polyethylene glycol 1000 succinate-b-poly (ε-caprolactone-ran-glycolide)TPGS-b-(PCL-ran-PGA)] nanoparticle (CNP) entrapped HIF-1α siRNA and complexed with cisplatin (DPP) on the growth of nasopharyngeal carcinoma xenografts in nude mice.METHODS Human nasopharyngeal carcinoma xenograft model was established by subcutaneous inoculation of CNE-2 cell in nude mice.The xenografts were randomly divided into six groups through random number tabled,PBS group (control),Blank chitosan modified TPGS-b-(PCL-ran-PGA) group (CNP,200 μg/kg),Cisplatin group (DPP,100 μg/kg),SiRNA targeting HIF-1α loaded chitosan modified TPGS-b-(PCL-ran-PGA) group (CNP-siRNA,200 μg/kg),blank chitosan modified TPGS-b-(PCL-ran-PGA) complexed with cisplatin group (CNP 200μg/kg+DPP 100μg/kg) and SiRNA targeting HIF-1α loaded chitosan modified TPGS-b-(PCL-ran-PGA) complexed with cisplatin group (CNP siRNA 200 μg/kg+DPP 100 μg/kg),each group of five.All groups received intraperitoneal injection every three days for seven times.Tumor volume was measured every three days during the course of treatment.After treatment,The tumor weight and inhibition ration were measured and calculated.The expression levels of HIF-1α and multidurg resistance gene 1 (MDR1)/P-glycoprotein (P-gp) in tumors were detected by Western blot and Real-time PCR.Histopathological changes were observed by hematoxylin and eosin stain.RESULTS In comparison with control group,the inhibition rate of blank CNP,siRNA loaded CNP,Cisplatin,blank CNP complexed with cisplatin or siRNA loaded CNPs complexed with cispla tin group were 18.32％,37.59％,47.64％,43.32％,70.21％ respectively.The inhibition rate were significantly decreased in siRNA loaded CNP complexed with cisplatin treatment group compared with the other groups(F=253.511,P＜0.001).In comparison with control group the expression of HIF-1α on mRNA in each group were as follows:CNP group(0.89±0.04),DPP group(0.99 ± 0.02),CNP siRNA group(0.95 ± 0.02),CNP+ DPP group(0.35 ± 0.04) and CNP-siRNA+ DPP group(0.15±0.07).Compared with the other five groups,The expression level of HIF-1α were sig nificantly decreased in CNP siRNA+ DPP group (F=351.194,P＜0.001) and the western blot results also showed that the band width of HIF-1α protein in the CNP-siRNA+DPP group was significantly lower than that in the other experimental groups.Similar results were observed in the expression of MDR1 and P-gp.Tumor cells necrosis and apoptosis were also effectively observed in siRNA loaded CNPs combine with cisplatin treatment group.CONCLUSIONS These findings show that using siRNA loaded chitosan-modified TPGS-b (PCL-ran-PGA) (CNP) to reduce HIF-1α mediated P-gp expression can effectively inhibite the growth of tumor and significantly promote the tumor cells necrosis and apoptosis.CNP function as an effective carrier for siRNA targeting HIF-1a delivery may have a promised prospect in the field of gene targeted therapy.