聚氰基丙烯酸正丁酯纳米粒载P16a基因的制备及在喉癌细胞中的表达

发布时间：2018-10-26 08:34

【摘要】：目的制备携带P16a基因的聚氰基丙烯酸正丁酯纳米粒并在喉癌细胞中进行表达。方法选用乳化聚合法制备PBCA-NPs,以激光粒度分析仪及透射电子显微镜分析纳米粒的形态和粒径。用阳离子表面活性剂十六烷基三乙基溴化铵(CTAB)对纳米粒进行表面修饰。构建真核表达载体pIRES2-EGFP-P16a经过酶切鉴定及测序后与PBCA-NPs连接,转染喉癌细胞Hep-2,荧光显微镜检测转染效率,蛋白印迹法检验P16a基因的表达,流式细胞术检测细胞凋亡率。结果所制PBCA-NPs粒径均匀、Zeta电位较高,较为理想;重组质粒pIRES2-EGFP-P16a经过酶切鉴定及测序后,表明真核表达载体构建正确;PBCA-NPs可以介导pIRES2-EGFP-P16a高效转染喉癌细胞,蛋白印迹检测表明转染后喉癌细胞能够表达外源P16a基因,在其介导下P16a能有效抑制喉癌细胞的增殖并能诱导细胞凋亡。结论聚氰基丙烯酸正丁酯纳米粒可以作为一种良好的基因载体,为喉癌的基因治疗提供了新思路。
[Abstract]:Objective to prepare polybutyl cyanoacrylate nanoparticles carrying P16a gene and express them in laryngeal cancer cells. Methods PBCA-NPs, was prepared by emulsion polymerization method. The morphology and particle size of nanoparticles were analyzed by laser particle size analyzer and transmission electron microscope. Cationic surfactant cetyltriethylammonium bromide (CTAB) was used to modify the surface of nanoparticles. The eukaryotic expression vector pIRES2-EGFP-P16a was ligated with PBCA-NPs after restriction endonuclease digestion and sequencing. The transfection efficiency was detected by Hep-2, fluorescence microscope and the expression of P16a gene was detected by Western blot. Apoptosis rate was detected by flow cytometry. Results the size of PBCA-NPs was uniform and the potential of Zeta was high. The recombinant plasmid pIRES2-EGFP-P16a was identified by restriction endonuclease digestion and sequenced, which indicated that the eukaryotic expression vector was constructed correctly. PBCA-NPs can efficiently transfect pIRES2-EGFP-P16a into laryngeal carcinoma cells. Western blot analysis shows that the transfected laryngeal carcinoma cells can express exogenous P16a gene, and P16a can effectively inhibit the proliferation of laryngeal cancer cells and induce apoptosis. Conclusion Poly (n-butyl cyanoacrylate) nanoparticles can be used as a good gene vector for gene therapy of laryngeal cancer.
【作者单位】：福建医科大学附属厦门第一医院耳鼻咽喉头颈外科;厦门大学生命科学院;
【基金】：厦门市卫生局科研立项专项资金(序号wsk09)资助
【分类号】：R739.65

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