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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 000succinate-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,200μg/kg)、单纯顺铂组(DPP,100μg/kg)、单纯纳米粒联合顺铂组(CNP 200μg/kg+DPP 100μg/kg)、载HIF-1αsiRNA纳米粒组(CNP-siRNA,200μg/kg)和载HIF-1αsiRNA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-1αmRNA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-1αsiRNA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。
Objective Hypoxia inducible factor-1α (HIF-1α) enhances the antitumor drug sensitivity studies at home and abroad have been reported. This study was to investigate the effects of nano-polymer chitosan-PEG-1000 vitamin E succinate-polyglycolide- ran-glycolide) and CS-TPGS-b- (PCL-ran-PGA) Effect of tumor growth in nude mice. Methods CNE-2 cells were inoculated subcutaneously into human nasopharyngeal carcinoma xenografts in nude mice model. Random number table was used to observe the effects of phosphate buffer group (CNP, 200μg / kg), pure cisplatin group DPP 100μg / kg), simple nanoparticles combined with cisplatin (CNP 200μg / kg + DPP 100μg / kg), HIF-1αsiRNA nanoparticles group (CNP-siRNA, 200μg / kg) Cisplatin group (CNP-siRNA 200μg / kg + DPP 100μg / kg) 6 groups. Each group of 5, abdominal injection 3d1 times, a total of 7 times. The tumor volume was measured in 3d and the nude mice were sacrificed after 22 days of treatment. The tumor weight was weighed and the tumor inhibition rate was calculated. The expression of HIF-1α and multi-drug resistance gene-1 / P-glycoprotein (MDR-1 / P-gp) gene protein expression levels were observed by HE staining of the tumor tissue histological changes. Results Compared with Control group, the tumor inhibition rate of CNP group was 18.32%, that of DPP group was 37.59%, that of CNP-siRNA group was 43.32%, that of CNP + DPP group was 47.64%, and that of CNP-siRNA + DPP group was 70.21%. Among them, CNP-siRNA + DPP had the most significant antitumor effect, with statistical significance compared with other treatment groups (F = 253.511, P <0.001). The expression of HIF-1αmRNA in CNP group was 0.89 ± 0.04, in the DPP group was 0.99 ± 0.02, in CNP-siRNA group was 0.95 ± 0.02, in CNP + DPP group was 0.35 ± 0.04, The siRNA + DPP group was 0.15 ± 0.07. The inhibitory effect of CNP-siRNA + DPP mRNA was the most significant, with statistical significance compared with other groups (F = 351.194, P <0.001). Western blotting also showed that the band width of HIF-1α protein in CNP-siRNA + DPP group was significantly lower than that in other experimental groups, and the expression of MDR-1 / P-gp showed similar results with HIF-1α gene protein . HE results showed that CNP-siRNA + DPP group can significantly promote tumor cell necrosis and apoptosis. Conclusions The delivery of HIF-1α siRNA combined with DDP via CNP nanopolymer can effectively down-regulate the expression of HIF-1α and MDR-1 / P-gp in tumor cells, inhibit tumor growth and promote apoptosis of tumor cells. As an effective carrier for delivery of siRNA, novel CNP polymer CNP has high application prospect in tumor gene targeting therapy.