摘要
目的：研究壳聚糖纳米颗粒在体外和体内实验中的转染能力。方法：用亚硝酸钠降解壳聚糖的方法制得低分子质量壳聚糖，用zeta电位仪测定粒径、多分散度、zeta电位，并使用乌式黏度计法测定其相对分子质量；通过静电吸附复合绿色荧光蛋白表达质粒pIRES-eGFP（报告基因），采用琼脂糖凝胶电泳分析载体与DNA结合能力；用体外和体内基因转染实验，评价纳米颗粒的转染能力。结果：制得的壳聚糖粒径200～600nm，多分散度最好的达到0．005，zeta电位0、89mV，相对分子质量7．7×10^7，粒径250nm。体外对3T3细胞的转染实验显示，该壳聚糖具有一定的转染效率；体内对Balbc57／BL6小鼠的股四头肌肌肉的转染实验显示，肌肉组织中有大量绿色荧光蛋白的表达，并且在炎症部位尤为明显。结论：本研究制备的壳聚糖，能够在体外和体内均实现有效转染，为基因治疗提供了一种潜在的载体。
Objective To study the gene transfection efficiency of the low molecular weight chitosan （LMWC） nanoperticles both in vitro and in vivo under optimizing the experimental condition. Methods The low molecular weight chitosan （LMWC） particles were prepared by oxidative degradation with NaNO2. Molecular weight was measured by testing the intrinsic viscosity. Its diameter,the diametric distribution range and zeta potential were measured by scanning electronic microscope. The LMWC particles were loaded with plasmid PIRES-eGFP through electrostatic forces and its loading ability and the protection ability to plasmid was studied. The transfection efficiency of chitosan-DNA nanoparticles was tested both in vitro and in vivo. Results The size of LMWC was optimized to 200-600 nm. The optimized condition of depolymerization of the chitosan lead us to the No. A chitosan. The surface charge was slightly positive with a zeta potential of ＋ 0. 89 mV. The molecular weight was around 7.7 × 10^7. The transfection efficiency of chitosan-DNA nanoparticles were two to five times higher than the background level in 3T3 cells in relative light units, and two times lower than that achieved by Lipofect AMINEE-DNA complexes. The study of the chitosan-DNA microspheres injected intramuscularly into the quadriceps muscle of thigh of a rat model showed a strong eGFP expression, especially in the inflammatory areas. However the toxicity of the LMWC chitosan was much less than the traditional gene vector, PEI and Lipofect AMINEE, according to previous report, which showed a superior biocompatibility. Conclusion The LMWC chitosan,ean be transfected both in vitro and in vivo ,may be a suitable polymeric carrier for gene therapy.
出处
《东南大学学报（医学版）》
CAS
2007年第1期4-6,共3页
Journal of Southeast University(Medical Science Edition)
基金
国家自然科学基金资助项目(30028012
30330530
30425009)
教育部基金资助项目(TRAPOY199028418
SRFDP20030284040)
关键词
壳聚糖
基因治疗
细胞转染
载体
chitosan
gene therapy
cell transfection
vector
