CAJ | 학술논문

背景：壳聚糖对软骨细胞具有良好的生物相容性和可降解性，但存在基因转染效率偏低的缺陷。目的：构建负载增强型绿色荧光蛋白基因的聚乙烯亚胺-壳聚糖/DNA 纳米粒，检测其理化性能，以及体外对关节软骨细胞的基因转染效率。方法：将聚乙烯亚胺共价连接于壳聚糖骨架上构建聚乙烯亚胺-壳聚糖复合物，再将聚乙烯亚胺-壳聚糖与负载增强型绿色荧光蛋白基因的质粒 DNA 以复凝聚法制成纳米粒，以扫描电镜检测纳米粒形态，Zeta 电位粒度分析仪测定其粒径、表面电位；凝胶电泳阻滞实验观察聚乙烯亚胺-壳聚糖和质粒DNA的结合力。以聚乙烯亚胺-壳聚糖/DNA纳米粒、裸质粒DNA、脂质体2000及壳聚糖/DNA纳米粒转染体外培养的兔关节软骨细胞，流式细胞仪及荧光显微镜检测基因转染率；激光共聚焦显微镜检测DNA的入核情况。结果与结论：聚乙烯亚胺-壳聚糖/DNA 纳米粒多呈球形，粒径为(154.6±18.6) nm，表面 Zeta 电位为(24.68±6.82) mV，可有效保护质粒DNA免受 DNaseⅠ的降解。体外转染实验证明聚乙烯亚胺-壳聚糖/DNA纳米粒能介导增强型绿色荧光蛋白基因转染关节软骨细胞并在细胞内表达绿色荧光蛋白，转染率达(23.80±1.74)%，转染率高于裸质粒DNA组及壳聚糖/DNA纳米粒组(P<0.05)，与脂质体2000组无显著差别(P=0.522)。表明聚乙烯亚胺-壳聚糖/DNA纳米粒能有效保护质粒DNA免受核酸酶降解，对关节软骨细胞有良好的基因转染能力。
배경：각취당대연골세포구유량호적생물상용성화가강해성，단존재기인전염효솔편저적결함。목적：구건부재증강형록색형광단백기인적취을희아알-각취당/DNA 납미립，검측기이화성능，이급체외대관절연골세포적기인전염효솔。방법：장취을희아알공개련접우각취당골가상구건취을희아알-각취당복합물，재장취을희아알-각취당여부재증강형록색형광단백기인적질립 DNA 이복응취법제성납미립，이소묘전경검측납미립형태，Zeta 전위립도분석의측정기립경、표면전위；응효전영조체실험관찰취을희아알-각취당화질립DNA적결합력。이취을희아알-각취당/DNA납미립、라질립DNA、지질체2000급각취당/DNA납미립전염체외배양적토관절연골세포，류식세포의급형광현미경검측기인전염솔；격광공취초현미경검측DNA적입핵정황。결과여결론：취을희아알-각취당/DNA 납미립다정구형，립경위(154.6±18.6) nm，표면 Zeta 전위위(24.68±6.82) mV，가유효보호질립DNA면수 DNaseⅠ적강해。체외전염실험증명취을희아알-각취당/DNA납미립능개도증강형록색형광단백기인전염관절연골세포병재세포내표체록색형광단백，전염솔체(23.80±1.74)%，전염솔고우라질립DNA조급각취당/DNA납미립조(P<0.05)，여지질체2000조무현저차별(P=0.522)。표명취을희아알-각취당/DNA납미립능유효보호질립DNA면수핵산매강해，대관절연골세포유량호적기인전염능력。
BACKGROUND:Chitosan is wel known as good biocompatibility and biodegradability;however, its extensive use in biomedical applications is restricted due to its poor transfection efficiency. OBJECTIVE:To prepare the polyethyleneimine-chitosan/DNA nanoparticles loading enhanced green fluorescent protein gene, and to detect their physicochemical properties and gene transfection efficiency towards chondrocytes in vitro. <br> METHODS:Low molecular weight polyethyleneimine was covalently linked to chitosan backbone to construct chitosan-graft-polyethyleneimine;then the chitosan-graft-polyethyleneimine was mixed with DNA nanoparticles, which loaded enhanced green fluorescent protein gene, by a complex coacervation method. The nanoparticle morphology was observed under a scanning electron microscopy. The sizes and zeta-potentials of the <br> nanoparticles were measured by a Marven-nano laser diffractometer. The binding capacity of plasmid DNA was evaluated by agarose gel electrophoresis analysis. The gene transfection experiments in vitro were performed towards rabbit’s chondrocytes. The gene transfection efficiency was measured with flow cytometry and under fluorescence microscope. How marked DNA entered into the nucleus of chondrocytes mediated by the nanoparticles was detected by laser scanning confocal microscopy. <br> RESULTS AND CONCLUSION:The prepared nanoparticles were mainly spherical, with an average size of (154.6±18.6) nm, and zeta-potential of (24.68±6.82) mV. The agarose gel electrophoresis analysis confirmed that the nanoparticles could effectively protect plasmid DNA from DNase Ⅰ-induced degradation. Gene transfection in vitro proved that the nanoparticles were efficient in transfecting rabbit’s chondrocytes and the expression of green fluorescent proteins was observed under fluorescent microscope, with a transfection efficiency of (23.80±1.74)%that was significantly higher than that of the naked plasmid DNA and chitosan/DNA nanoparticles (P<0.05). But no significant differences were observed between polyethyleneimine-chitosan/DNA nanoparticles and LipofectamineTM 2000. These findings indicate that the polyethyleneimine-chitosan/DNA nanoparticles can effectively protect plasmid DNA from nuclease degradation, and exhibit the favorable transfection ability towards articular chondrocytes.