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目的研究纳米粒子PLGA携带特异性HIF-1α基因转染的可行性及其效率。方法构建HIF-1α基因的真核表达质粒pcDNA3.1(+)的重组体。应用复乳溶剂挥发法制备pcDNA3.1(+)-HIF-1α的PLGA纳米粒,用正交设计法对纳米粒子的制备工艺进行优化,检测其包埋率、体外释放情况及粒度。观察纳米粒的形态、大小和粒度,研究体外释药特性、抗核酸酶降解性能。用PLGA-pcDNA3.1(+)-HIF-1α纳米粒转染U251细胞,Western blot检测PLGA纳米粒子中HIF-1α蛋白表达。结果用正交设计法优化制备工艺成功构建pcDNA3.1(+)-HIF-1α的PLGA纳米粒,形态圆整,大小均匀,平均粒径102 nm,含药量0.83%,包封率可达72%,有对抗核酸酶降解能力,体外释药缓慢。PLGA-pcDNA3.1(+)-HIF-1α纳米粒可持续高效表达HIF-1α蛋白。结论 PLGA-pcDNA3.1(+)-HIF-1α纳米粒可用于特异性基因的转染。
Objective To study the feasibility and efficiency of transfection of PLGA-carrying HIF-1α gene by nanoparticle. Methods Recombinant plasmid pcDNA3.1 (+) of HIF-1α gene was constructed. PLGA nanoparticles of pcDNA3.1 (+) - HIF-1α were prepared by double emulsion evaporation method. The preparation process of nanoparticles was optimized by orthogonal design. The embedding rate, in vitro release and particle size of pcDNA3.1 (+) - HIF- The morphology, size and particle size of the nanoparticles were observed to study the in vitro release characteristics and the resistance to nuclease degradation. U251 cells were transfected with PLGA-pcDNA3.1 (+) - HIF-1α nanoparticles and Western blot was used to detect the expression of HIF-1α protein in PLGA nanoparticles. Results The optimized preparation process of orthogonal design was used to successfully construct pcDNA3.1 (+) - HIF-1α PLGA nanoparticles with the average size of 102 nm and the drug content of 0.83% 72%, resistant to nuclease degradation, slow release in vitro. PLGA-pcDNA3.1 (+) - HIF-1α nanoparticles can sustainably and efficiently express HIF-1α protein. Conclusion PLGA-pcDNA3.1 (+) - HIF-1α nanoparticles can be used for transfection of specific genes.