CAJ | 학술논문

目的：采用中心组合设计法优化载基因壳聚糖纳米粒的最佳转染制备区域。方法采用复凝聚法制备载质粒基因的壳聚糖纳米粒，选择壳聚糖浓度和质粒基因浓度作为实验考察因素，应用两因素五水平中心组合设计优化最佳转染制备区域，优化指标选择平均粒径和基因转染率。通过透射电镜观察纳米粒的形态；通过动态光散射和电泳光散射技术分别测量纳米粒的粒径和Zeta电位；通过凝胶电泳分析考察质粒在纳米粒制备过程中的稳定性；通过倒置荧光显微镜观察质粒基因在细胞内的表达；通过流式细胞技术测定纳米粒的转染效率。结果成功优化了载基因壳聚糖纳米粒的最佳转染制备区域。优选条件下制备的纳米粒大多呈球形，纳米粒平均粒径为217．6 nm，粒径多分散系数为0．241，表明粒径分布较窄。纳米粒zeta电位为＋22．4 mV，表明纳米粒表面带有正电荷，可以增加纳米粒混悬液的稳定性。凝胶电泳分析结果表明质粒基因在纳米粒制备过程中没有遭到破坏。纳米粒的细胞转染效率比较高，能够高效地将绿色荧光蛋白质粒基因递送到细胞内，并且基因表达产生绿色荧光蛋白。结论本研究建立的数学模型具有良好的预测性。在优化的制备区域内制备的载基因壳聚糖纳米粒的转染性能比较理想。
목적：채용중심조합설계법우화재기인각취당납미립적최가전염제비구역。방법채용복응취법제비재질립기인적각취당납미립，선택각취당농도화질립기인농도작위실험고찰인소，응용량인소오수평중심조합설계우화최가전염제비구역，우화지표선택평균립경화기인전염솔。통과투사전경관찰납미립적형태；통과동태광산사화전영광산사기술분별측량납미립적립경화Zeta전위；통과응효전영분석고찰질립재납미립제비과정중적은정성；통과도치형광현미경관찰질립기인재세포내적표체；통과류식세포기술측정납미립적전염효솔。결과성공우화료재기인각취당납미립적최가전염제비구역。우선조건하제비적납미립대다정구형，납미립평균립경위217．6 nm，립경다분산계수위0．241，표명립경분포교착。납미립zeta전위위＋22．4 mV，표명납미립표면대유정전하，가이증가납미립혼현액적은정성。응효전영분석결과표명질립기인재납미립제비과정중몰유조도파배。납미립적세포전염효솔비교고，능구고효지장록색형광단백질립기인체송도세포내，병차기인표체산생록색형광단백。결론본연구건립적수학모형구유량호적예측성。재우화적제비구역내제비적재기인각취당납미립적전염성능비교이상。
Objective This study aimed to optimize the preparation condition of DNA-chitosan nanoparticles with high transfec-tion efficency through a central composition design .Methods The DNA-chitosan nanoparticles were prepared by complex coacervation between pEGFP and chitosan .We selected the concentrations of chitosan and plasmid as two experimental factors , and a central compos-ite design with two factors and five levels was used to optimize the preparation condition of DNA-chitosan nanoparticles for high transfec-tion efficency .The concentrations of chitosan and plasmid were selected as the independent variables , respectively .The dependent varia-bles included average particle size and transfection efficiency .The morphology of DNA-chitosan nanoparticles was observed using a trans-mission electron microscope .The size and zeta potential of nanoparticles were measured by dynamic light scattering ( DLS) and electro-phoretic light scattering ( ELS ) , respectively .The stability of plasmids in the process of nanoparticles preparation was investigated through the agrose gel electrophoresis .The expression of plasmids delivered by nanoparticles was observed under an inverted fluorescence microscope .The transfection efficiency of DNA-chitosan nanoparticles was assayed by flow cytometry .Results The preparation condition of DNA-chitosan nanoparticles with high transfection efficency was optimized successfully .Under the optimum preparative conditions , the DNA-chitosan nanoparticles were almost spherical .The average size of nanoparticles was 217.6nm, and distributed in a narrow range with a polydispersity index of 0.241.The zeta potential was +22.4 mV, which suggested that a den-sity of positive charge exist onto the surface of nanoparticles and consequently enhanced the stability of nanoparticles suspension .The results of gel electrophoresis showed that plasmids were not destroyed in the process of nanoparticles preparation .The cell transfection of nanoparticles was very highly efficient .The nanoparticles could effectively deliver the pEGFP plasmids into cells to express the green fluorescent protein at a high level.Conclusion The established mathematic models have the good predictive function .Under the optimum preparative condi-tions, the DNA-chitosan nanoparticles have the high potential of cell transfection .