论文部分内容阅读
目的:研究适配体SP94修饰的阳离子基因载体H_3R_5,合成新型肝靶向纳米复合物SP94-H_3R_5/miR195,增加对肝癌细胞的靶向性,提高基因的转染效率。方法:经半胱氨酸修饰的SP94与H_3R_5末端的半胱氨酸发生氧化反应,组装得到SP94-H_3R_5,利用~1 H-NMR鉴定SP94-H_3R_5载体的结构,通过电位粒度仪测定纳米复合物的电位和粒径,利用琼脂糖凝胶电泳考察载体对miR195的压缩能力。以体外培养的人肝癌SK-Hep-1为研究对象,CCK8法检测SP94-H_3R_5和H_3R_5对细胞增殖的抑制作用,采用激光共聚焦显微镜考察肝癌细胞对纳米复合物的摄取,以pEGFP为报告基因考察基因转染效率,Western blot实验检测SK-Hep-1细胞VEGF的蛋白表达。结果:SP94-H_3R_5具有生物相容性,可以压缩miR195形成稳定的纳米复合物,SP94-H_3R_5/miR195与H_3R_5/miR195相比可以更多地被SK-Hep-1摄取(P<0.01),SP94-H_3R_5转染效率高于非靶向载体H_3R_5,对VEGF的阻滞作用也更高(P<0.01)。结论:SP94-H_3R_5兼具纳米材料的被动靶向作用和适配体的主动靶向作用,有潜力成为肝癌治疗中的新型载体。
OBJECTIVE: To study the effect of SP94-H_3R_5 / miR195, a new type of liver-targeting nanocomposite, on the gene carrier H_3R_5 modified by aptamer SP94, to increase the targeting of hepatoma cells and increase the transfection efficiency. METHODS: SP94-H_3R_5 was assembled by cysteine-modified SP94 and cysteine at the end of H_3R_5, and the structure of SP94-H_3R_5 was identified by ~ 1 H-NMR. The nanocomposite The potential and particle size, using agarose gel electrophoresis carrier miR195 compression capacity. The human hepatocellular carcinoma SK-Hep-1 cultured in vitro was used as the research object. The inhibitory effect of SP94-H_3R_5 and H_3R_5 on the cell proliferation was detected by CCK8 assay. The uptake of the nanocomplex by the liver cancer cells was examined by confocal laser scanning microscopy. The efficiency of gene transfection was investigated. The protein expression of VEGF in SK-Hep-1 cells was detected by Western blot. RESULTS: SP94-H_3R_5 was biocompatible and could compress miR195 to form stable nanocomposites. Compared with H_3R_5 / miR195, SP94-H_3R_5 / miR195 could be more upregulated by SK-Hep-1 (P <0.01) The transfection efficiency of -H_3R_5 was higher than that of non-target vector H_3R_5, and the blocking effect on VEGF was also higher (P <0.01). Conclusion: SP94-H_3R_5 has the potential of both passive targeting of nanomaterials and active targeting of aptamers and has the potential to become a new carrier in the treatment of liver cancer.