不同非病毒载体对原代肌腱细胞转染效率和毒性作用研究

Study on the transfection efficiencyand toxicity of primary tendon cells by different non-viral vectors

目的:研究并比较非病毒载体纳米球和脂质体对原代肌腱细胞的转染效率以及毒性作用。方法:制备不同N/P比例的PLGA纳米球,原代培养鸡、大鼠、小鼠肌腱细胞,将低、中、高不同剂量的pEGFP-N1质粒负载于纳米球和脂质体,对三种肌腱细胞进行转染。通过荧光拍照和流式细胞分析,检测质粒在24、48、72、96 h的转染效率,Western Blot法检测GFP表达水平,CCK8法检测转染24、48、72 h细胞活力。结果:对于鸡肌腱细胞,纳米球的转染效率显著高于脂质体,尤其是NP10的GFP表达最明显,且对细胞无毒性作用。对于大鼠肌腱细胞,NP10的转染效率最佳,其次是NP6和脂质体,但脂质体降低了细胞活性。对于小鼠肌腱细胞,纳米球和脂质体的转染效率均较低,且随着时间与剂量的增加,细胞毒性作用越来越明显。结论:NP10是鸡和大鼠肌腱细胞转染质粒的最佳试剂,但小鼠肌腱细胞最合适的转染试剂还需进一步探索。

Objective:To investigate and compare the transfection efficiency and toxicity of non-viral vector nanoparticles and liposomes on primary tendon cells.Methods:PLGA nanoparticles with different NP ratios were prepared,and the tendon cells of chicken,rat and mouse were cultured.The low,medium and high doses of pEGFP-N1 plasmid were loaded into nanoparticles and liposomes,and the three cells were transfected.The transfection efficiency of the plasmid at 24,48,72 and 96 hours was detected by fluorescence photography and flow cytometry.The expression level of GFP was detected by Western Blot,and cell activity was detected by CCK8 at 24,48 and 72 hours after transfection.Results:For chicken tendon cells,the transfection efficiency of nanoparticles was significantly higher than that of liposomes,especially the GFP expression of NP10 was the most obvious,and it was not toxic to cells.For rat tendon cells,NP10 had the best transfection efficiency,followed by NP6 and liposomes,but liposomes reduced cell activity.For mouse tendon cells,the transfection efficiency of nanoparticles and liposomes was low,and the toxic effect on cells became more and more obvious with the increase of time and dose.Conclusion:NP10 is the best transfection reagent for plasmid transfection in chicken and rat tendon cells,but the most suitable transfection reagent for mouse tendon cells needs further exploration.