聚氰基丙烯酸正丁酯纳米粒载BDNF基因的制备及在活体大鼠脑内表达的实验研究  

Construction of polybutylcyanoacrylate nanoparticles loaded brain derived neurotrophic factor gene and their expressions in rat brain tissues

作　　者：赖雪[1] 钟武[1] 胡迎春[1] 李昊 熊雨 陈礼刚 

机构地区：[1]四川医科大学附属第一医院急诊医学部,泸州646000 [2]四川医科大学附属第一医院神经外科,泸州646000

出　　处：《中华神经医学杂志》2015年第10期973-978,共6页Chinese Journal of Neuromedicine

基　　金：四川省卫生和计划生育委员会课题（2012-a-37）

摘　　要：目的制备携带脑源性神经营养因子基因（BDNF）的聚氰基丙烯酸正丁酯（PBCA）纳米粒并在活体颅脑损伤大鼠脑内表达，观察PBCA纳米粒是否具有提高BDNF基因转染率的能力。方法选用乳化聚合法制备PBCA纳米粒，以透射电子显微镜分析纳米粒的形态和粒径。构建真核表达载体P^BGFP-BDNF，经过酶切鉴定及测序后利用PBCA纳米粒进行包裹。选用48只雄性SD大鼠按随机数字表法分为空白组、PBCA组、P^BGFP-BDNF组、PBCA-P^BGFP-BDNF组，每组12只，按照自由落体致伤原理制备颅脑损伤大鼠模型并分别注入生理盐水、PBCA纳米粒、质粒P^BGFP-BDNF、PBCA-P^BGFP-BDNF各1mL。7d后取大鼠右侧大脑创伤周围脑组织，经荧光显微镜，RT-PCR及Westernblotting检测BDNFmRNA及蛋白在大鼠脑组织中的表达情况。结果所制PBCA纳米粒粒径均匀、电动电位较高，负载率为（62．23±2．15）％。采用PBCA纳米粒包裹BDNF并转染大鼠后可见BDNF在模型大鼠脑内表达。RT-PCR结果提示，4组大鼠BDNF mRNA表达量差异有统计学意义（F=112．668，P=0．000），与前3组比较，PBCA-P^BGFP-BDNF组BDNFmRNA表达量明显升高，差异有统计学意义（P〈0．05）。Westernblotting结果提示，4组大鼠BDNF差异有统计学意义（F=66．629，P=0．000）．P^BGFP-BDNF组中BDNF蛋白的表达明显高于空白组和PBCA组，而PBCA-P^BGFP-BDNF组中BDNF的表达明显高于前3组，差异均有统计学意义（P〈0．05）。结论PBCA纳米粒是一种良好的基因载体．可携带基因进入组织高效表达。Objective To prepare the polybutylcyanoacrylate nanoparticles （PBCA-NPs） loaded brain derived neurotrophic factor （BDNF） gene as the gene delivery system and explore their expressions in rat brain tissues so as to observe the influence of PBCA-NPs in BDNF expression. Methods PBCA-NPs were prepared by emulsion polymerization method. Surface of PBCA-NPs was surveyed by transmission electron micrograph （TEM） and zeta potentials of PBCA-NPs were determined with laser grain analyzer. The PBCA-NPs surface was modified by cationic surfactant cetyltrimethylammonium bromide （CTAB）. The eukaryotic expression vectors P^BGFP-BDNF were constructed; after verification by double enzyme digestion and sequencing, P^BGFP-BDNF was packaged by PBCA-NPs. Forty-eight male SD rats were randomly divided into blank-control group, PBCA group, P^BGFP-BDNF group and PBCA-P^BGFP-BDNF group （n=12）, and Feeney＇s method was used to induce craniocerebral injury models in these rats, and then, one mL normal saline, PBCA-NPs, P^BGFP-BDNFplasmids and PBCA-P^BGFP-BDNF plasmids were given to the above groups. Seven d after that, peripheral brain tissues of right injury brain tissues were chosen; expressions ofBDNF gene were detected by pathological examination, real time-PCR and Western blotting. Results Nps with even size and smooth surface were successfully obtained, holding the high zeta electric potential （[62.23±2.15] %）. The new constructed vectors were confirmed by restricted enzyme and sequencing. Real time-PCR indicated significant difference of BDNF mRNA expressions among the four groups （F=112.668, P=0.000）; as compared with that in the other three groups, the BDNF mRNA expression in the PBCA-P^BGFP-BDNFgroup was significantly higher （P〈0.05）. Western blotting indicated significant difference of BDNF protein expressions among the four groups （F=66.629, P=-0.000）; as compared with that in the blank group and PBCA group, the BDNF protein expression in the P^BGFP-BDNF group was significantly higher （

关 键 词：PBCA纳米粒 神经营养因子 颅脑外伤 

分 类 号：R651.15[医药卫生—外科学]