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

Construction of polybutycyanocrylate nanoparticles(PBCA-NPs) loaded P16a gene and expression in Hep-2 cell

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。

[Objective] To prepare polybutycyanocrylate nanoparticles（PBCA-NPs）loaded P16a gene as the gene delivery system,and to express the P16a protein in Hep-2 cell line.[Methods] PBCA-NPs were prepared by the emulsion polymerization method.Surface of PBCA-NPs was surveyed by transmission electron micrographs（TEM）,the grain distribution and zeta potentials of PBCA-NPs were determined with the laser grain analyzer.The PBCA-NPs surface was modified by the cationic surfactant cetyltrimethylammonium bromide（CTAB）.Construct the eukaryotic expression vector pIRES2-EGFP-P16a and the recombined plasmid were extracted and purified.After verification by double enzyme digestion and sequencing,the constructed eukaryotic expression plasmid was transfected to Hep-2 line.Transfect ion efficiency was observed by fluorescence microscope and the expression of P16a gene was detected by Western blot.Apoptosis was detected by flow cytomery.[Results] Nps with even size and smooth surface were successfully obtained,holding the higher zeta electric potential.The new constructed vector was confirmed by restricted enzyme and sequencing.pIRES2-EGFP-P16 can be mediated by PBCA-NPs with high transfection efficiency.Exogenous P16a gene can be expressed in transfected Hep-2 cell line detected by Western blot.P16a mediated by Nps can effectively inhibit the proliferation of Hep-2 cell,and induce Hep-2 cell apoptosis in vitro.[Conclusions] PBCA-NPs could be a good vector and provided a new way for gene therapy of laryngocarcinoma.