CAJ | 학술논문

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PEGFP-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、PEGFP-BDF组、PBCA-PEGFP-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PEGFP-BDNF、PBCA-PEGP-BDNF各1 mL.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-PEGFP-BDNF组BDNFmRNA表达量明显升高,差异有统计学意义(P＜0.05).Western blotting结果提示,4组大鼠BDNF差异有统计学意义(F=66.629,P=0.000),PEGFP-BDNF组中BDNF蛋白的表达明显高于空白组和PBCA组,而PBCA-PEGFP-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.
목적 제비휴대뇌원성신경영양인자기인(BDNF)적취청기병희산정정지(PBCA)납미립병재활체로뇌손상대서뇌내표체,관찰PBCA납미립시부구유제고BDNF기인전염솔적능력. 방법 선용유화취합법제비PBCA납미립,이투사전자현미경분석납미립적형태화립경.구건진핵표체재체PEGFP-BDNF,경과매절감정급측서후이용PBCA납미립진행포과.선용48지웅성SD대서안수궤수자표법분위공백조、PBCA조、PEGFP-BDF조、PBCA-PEGFP-BDNF조,매조12지,안조자유락체치상원리제비로뇌손상대서모형병분별주입생리염수、PBCA납미립、질립PEGFP-BDNF、PBCA-PEGP-BDNF각1 mL.7d후취대서우측대뇌창상주위뇌조직,경형광현미경,RT-PCR급Westernblotting검측BDNFmRNA급단백재대서뇌조직중적표체정황. 결과 소제PBCA납미립립경균균、전동전위교고,부재솔위(62.23±2.15)％.채용PBCA납미립포과BDNF병전염대서후가견BDNF재모형대서뇌내표체.RT-PCR결과제시,4조대서BDNF mRNA표체량차이유통계학의의(F=112.668,P=0.000),여전3조비교,PBCA-PEGFP-BDNF조BDNFmRNA표체량명현승고,차이유통계학의의(P＜0.05).Western blotting결과제시,4조대서BDNF차이유통계학의의(F=66.629,P=0.000),PEGFP-BDNF조중BDNF단백적표체명현고우공백조화PBCA조,이PBCA-PEGFP-BDNF조중BDNF적표체명현고우전3조,차이균유통계학의의(P＜0.05). 결론 PBCA납미립시일충량호적기인재체,가휴대기인진입조직고효표체.
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 PPEGFP-BDNF were constructed;after verification by double enzyme digestion and sequencing, pPEGFP-BDNF was packaged by PBCA-NPs.Forty-eight male SD rats were randomly divided into blank-control group, PBCA group, pPEGFP-BDNF group and PBCA-PEGFP-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, PEGFP-BDNF plasmids and PBCA-PEGFP-BDNF plasmids were given to the above groups.Seven d after that, peripheral brain tissues of right injury brain tissues were chosen;expressions of BDNF 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-PEGFP-BDNF group 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 PEGFP-BDNF group was significantly higher (P＜0.05), and that in the PBCA-PEGFP-BDNF group was significantly higher than the other three groups (P＜0.05).Conclusion PBCA-NPs could be a good vector and provide a new way for gene therapy of craniocerebral injury.