He Qin, Liu Ji, Sun Xun, Zhang Zhi-Rong
West China School of Pharmacy, Sichuan University, Chengdu 610041, Sichuan Province, China.
World J Gastroenterol. 2004 Mar 1;10(5):660-3. doi: 10.3748/wjg.v10.i5.660.
To prepare thymidine kinase gene (TK gene) nanoparticles and to investigate the expression of TK gene.
Poly(D,L-lactic-co-glycolic acid) (PLGA), a biodegradable and biocompatible polymer, was used to prepare recombinant plasmid P(EGFP-AFP) nanoparticles by a double-emulsion evaporation technique. Characteristics of the nanoparticles were investigated in this study, including morphology, entrapment efficiency, and tissue distribution. The expression of TK gene was also investigated by MTT assay, by which the viable cells were determined after the addition of ganciclovir (GCV). The enhanced green fluorescent protein (EGFP) expression in human hepatocellular carcinoma SMMC-7721 cells and normal parenchymal Chang liver cells were assessed by flow cytometry.
The prepared plasmid-nanoparticles had regular spherical surface and narrow particle size span with a mean diameter of 72+/-12 nm. The mean entrapment efficiency was 91.25%. A total of 80.14% DNA was found to be localized in the livers after 1-h injection with (32)P-DNA-PLGA nanoparticles in mouse caudal vein. The expression of DNA encapsulated in nanoparticles was much higher than that in naked DNA, and human hepatocellular carcinoma SMMC-7721 cells were more sensitive to GCV than human normal parenchymal Chang liver cells.
The enhanced transfection efficiency and stronger ability to protect plasmid DNA from being degraded by nucleases are due to nanoparticles encapsulation.
制备胸苷激酶基因（TK基因）纳米粒并研究TK基因的表达。
采用可生物降解且具有生物相容性的聚（D，L-乳酸-乙醇酸共聚物）（PLGA），通过复乳蒸发技术制备重组质粒P（EGFP-AFP）纳米粒。本研究对纳米粒的特性进行了研究，包括形态、包封率和组织分布。还通过MTT法研究TK基因的表达，在加入更昔洛韦（GCV）后测定活细胞。通过流式细胞术评估人肝癌SMMC-7721细胞和正常实质细胞Chang肝细胞中增强型绿色荧光蛋白（EGFP）的表达。
制备的质粒纳米粒表面呈规则球形，粒径跨度窄，平均直径为72±12nm。平均包封率为91.25%。在小鼠尾静脉注射（32）P-DNA-PLGA纳米粒1小时后，共发现80.14%的DNA定位于肝脏。纳米粒中包裹的DNA的表达远高于裸DNA，并且人肝癌SMMC-7721细胞比人正常实质细胞Chang肝细胞对GCV更敏感。
纳米粒包封导致转染效率提高以及保护质粒DNA不被核酸酶降解的能力增强。
