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目的制备PCEC阳离子纳米粒并复合靶向FAK的shRNA的重组质粒,观察其感染人乳腺癌435S细胞所引起的FAK下调对体外肿瘤细胞凋亡的影响。方法用乳液溶剂扩散法制备PCEC阳离子纳米粒;将PCEC阳离子纳米粒与靶向FAK的shRNA的重组质粒静电复合并感染人乳腺癌435S细胞;用Western-bolt检测感染细胞中FAK的下调;通过MTT、PI荧光染色及DNA Ladder分别检测感染细胞的增殖与凋亡情况。结果乳液溶剂扩散法制备出100~200 nm的PCEC阳离子纳米粒;选取100 nm的纳米粒以40:1的质量比与靶向FAK的shRNA的重组质粒复合,感染人乳腺癌435S细胞后,经Western-blot测得FAK蛋白水平明显下调,MTT检测结果为细胞增殖受到明显抑制,经PI染色后细胞、细胞核出现明显的凋亡形态学变化,琼脂糖凝胶电泳显示其基因组DNA有明显的梯状条带(DNA ladder)。结论PCEC阳离子纳米粒复合靶向FAK的shRNA的重组质粒具有抑制435S细胞增殖和诱导凋亡的作用。
Objective To prepare PCEC cationic nanoparticles and recombine the recombinant plasmids targeting FAK shRNA to observe the effect of FAK down-regulation on the apoptosis of tumor cells induced by human breast cancer 435S cells. Methods PCEC cationic nanoparticles were prepared by emulsion solvent diffusion method. The recombinant plasmids of PCEC cationic nanoparticles and shRNA targeting FAK were electrostatically complexed and infected into human breast cancer cell line 435S. The down-regulation of FAK in infected cells was detected by Western-blot. , PI staining and DNA Ladder were used to detect the proliferation and apoptosis of infected cells respectively. RESULTS: 100-200 nm PCEC cationic nanoparticles were prepared by emulsion solvent diffusion method. Nanoparticles of 100 nm were combined with recombinant plasmids targeting shRNA targeting FAK at a mass ratio of 40: 1. After infecting human breast cancer cells 435S, The level of FAK protein was down-regulated by Western-blot. MTT assay showed that the cell proliferation was significantly inhibited. The morphological changes of apoptotic cells and nuclei were obvious after PI staining. The agarose gel electrophoresis showed that the genomic DNA had obvious ladder DNA ladder. Conclusions The recombinant plasmid targeting PCK cationic nanoparticle complexed with FAK shRNA can inhibit the proliferation and induce apoptosis of 435S cells.