基于流体动力学的mA20质粒及其突变体转染对实验性小鼠内毒素血症的治疗作用

Effect of hydrodynamics-based delivery of mA20 and its mutant plasmid on experimental endotoxemia in mice

目的研究mA20及其突变体mA20-ZnF4-7质粒对脂多糖(lipopolysaccharide,LPS)攻击所致实验性内毒素血症小鼠的治疗作用。方法用LPS攻击小白鼠制备内毒血症动物模型。按10μg质粒/1.6ml生理盐水比例溶于生理盐水中,采用基于流体动力学基因转染方法对实验组小鼠进行质粒注射。以ELISA检测各组血浆和组织TNF-α、IL-1β表达。12h观察组织病理变化。结果①mA20质粒治疗组、mA20-ZnF4-7质粒治疗组的TNF-α在各时间点的表达分别为:6h:(201.797±4.789)、(235.710±6.108)pg/ml,12h:(236.580±7.497)、(255.130±7.827)pg/ml,24h:(154.551±16.460)、(223.246±11.316)pg/ml;两组间无显著性差异(P>0.05);与单纯内毒素组、空质粒组的TNF-α表达比较有显著性降低(P<0.05)。②mA20质粒治疗组、mA20-ZnF4-7质粒治疗组的IL-1β在各时间点的表达分别为:6h:(87.103±3.840)、(101.586±2.486)pg/ml,12h:(83.655±4.973)、(94.460±3.110)pg/ml,24h:(77.678±6.555),(87.103±4.826)pg/ml;该两组间无显著性差异(P>0.05);与单纯内毒素组、空质粒组的IL-1β表达比较有显著性降低(P<0.05);各时间点之间比较无明显差别(P>0.05)。③病理学观察结果显示:mA20治疗组、mA20-ZnF4-7治疗组的肝、肺损伤均较单纯内毒素组、空质粒组轻;与生理盐水对照组相比,肝、肺损伤无明显差别(P>0.05)。结论“基于血流动力学的基因转染方法”能有效地把目的质粒DNA转入肝、肺;对实验性类毒素血症小鼠体外转染mA20-ZnF4-7和mA20有相似的治疗作用,这为临床应用提供一定的实验基础。

Objective To study the therapic effect of mA20 and its mutant mA20-ZnF4-7 plasmid on endotoxemia induced by lipopolysaccharide （LPS） in mice. Methods We prepared the endotoxemia animal model by assaulting mice with LPS （2.5 mg/kg）. Using the method of hydrodynamics-based gene transfection, we intravenously injected mice with 10 g of plasmid DNA in 1.6 ml saline through the tail vein. The contents of TNF-α, IL-1 β in blood plasma were detected by ELISA, and inflammatory reaction was also observed pathologically. Results The TNF-α expressions of mA20 plasmid treated group and mA20-ZnF4-7 plasmid treated group in each time point were （201. 797 ±4.789） and （235.710 ± 6. 108 ） pg/ml in 6 h, （236.580 ± 7. 497 ） and （255. 130 ±7.827） pg/ml in 12 h, （154. 551 ± 16.460） and （223. 246 ± 11. 316） pg/ml in 24 h, which were significantly lower than those of LPS assaulting group and empty plasmid group. The IL-1 β expressions of mA20 plasmid treated group and mA20-ZnF4-Tplasmid treated group in each time point were （87. 103 ± 3.84） and （ 101. 586 ± 2.486） pg/ml in 6 h, （83. 655 ± 4.973） and （94. 460 ± 3.110） pg/ml in 12 h, （77.678 ± 6.555 ） and （87. 103 ± 4.826 ） pg/ml in 24 h, which also significantly lower than those of LPS assaulting group and empty plasmid group. The inflammatory damage in mA20 plasmid treated group and mA20-ZnF4-7 plasmid treated group was lighter than those of LPS assaulting group and empty plasmid group, but of no significant difference in comparison with normal saline group. Conclusion The method of hydrodynamics-based gene transfection ean efficiently deliver plasmid DNA into liver and hmg. In vitro transfection of mA20-ZnF4-7 plasmid showed the similar therapeutic effect on experimental endotoxemia in mice as that of mA20 plasmid, which provides some experimental base for its clinical application.±