亲环素A小干扰RNA对载脂蛋白E基因敲除小鼠动脉粥样硬化斑块形成的影响

The impact of cyclophilin A small interfering RNA on the formation of atherosclerotic plaque in apolipoprotein E-deficient mice

目的：观察慢病毒介导的亲环素A（cyclophilin A，CyPA）小干扰RNA对载脂蛋白E基因敲除（ ApoE^-/-）小鼠动脉粥样斑块的影响。方法采用ApoE^-/-小鼠颈动脉硅胶圈置入法快速建立动脉粥样斑块模型，然后将小鼠分为CyPA小干扰RNA慢病毒载体治疗组（ CyPA-RNAi-LV组）和绿色荧光蛋白-慢病毒载体对照组（NC-GFP-LV组），采用局部注射法将10μl（1.0×108 TU/ml）慢病毒载体局部作用硅胶圈置入处10 min，干预ApoE^-/-小鼠颈动脉斑块。采用苏木素-伊红染色法观察各组小鼠动脉粥样斑块大小。采用实时聚合酶链反应（ RT-PCR）法检测各组小鼠动脉粥样硬化斑块中CyPA mRNA的表达水平，Western blot法检测其蛋白水平。结果转染6周后，CyPA-RNAi-LV组小鼠动脉粥样硬化斑块进展慢于NC-GFP-LV组，斑块面积分别为（9085±671）μm^2（18021±1901）μm^2两组比较差异有统计学意义（P＜0.05），斑块中脂质含量较NC-GFP-LV组少[（17.8±4.5）％比（28.9±6.3）％， P＜0.05]，胶原含量较NC-GFP-LV组多[（35.1±5.2）％比（24.2±4.8）％， P＜0.05]，内膜/中膜比值NC-GFP-LV组较高（0.65±0.12比0.36±0.11，P＜0.05），管腔狭窄较NC-GFP-LV组轻（内膜/管腔比值，0.18±0.02比0.33±0.03， P＜0.05）。 CyPA-RNAi-LV组小鼠促炎因子和基质金属蛋白酶表达水平均较NC-GFP-LV组低（ P均＜0.05）。结论慢病毒介导的CyPA小干扰RNA能抑制ApoE^-/-小鼠动脉粥样硬化斑块的进展，减轻斑块局部炎症反应。

Objective The aim of this study was to determine whether inhibition of cyclophilin A by lentivirus-mediated RNA interference （ RNAi ） could inhibit progression of atherosclerotic plaques and increase collagen production .Methods Atherosclerostic plaque model was induced by rapid perivascular carotid silicone collar placement in ApoE -/-mice.The recombinant CyPA-RNAi-Lentivirus （ CyPA-RNAi-LV） or negative control-green fluorescent protein-Lentivirus （ NC-GFP-LV） were constructed and transfected into right carotid plaques , respectively.Using the local injection method , ApoE^-/- mice carotid artery plaque were intervened 10 min in the silicone collar placement with 10 μl （ 1.0 ×108 TU/ml ） lentivirus vector.The areas and CyPA expression of plaques were analyzed by morphological observation , real-time polymerase chain reaction （ RT-PCR ） and Western blot respectively .Results CyPA-RNAi-LV not only prevented plaques progression （（9 085 ±671） μm2 to （18 021 ±1 901） μm2）, but also decreased plaque lipid content （（28.9 ±6.3）%to （17.8 ±4.5）%）, increased plaque collagen content （（24.2 ±4.8）%to （35.1 ±5.2）%） at 6 weeks after lentivirus transfection.The intima/media ratio （0.36 ±0.11 vs.0.65 ±0.12, P〈0.05） and degree of lumen stenosis （intima/lumen ratios, 0.18 ±0.02 vs.0.33 ±0.03, P〈0.05 ） were also significantly reduced by CyPA-RNAi-LV.Moreover , RT-PCR analysis revealed downregulated expressions of proinflammatory cytokines and matrix metalloproteinases （ MMP-9 -17.5%） in the CyPA-RNAi-LV group.Conclusion Lentivirus-mediated CyPA silencing by siRNA could inhibit plaques progression and reduce local inflammation through the anti-inflammatory effects in this model .