载LOX-1-siRNA靶向超声微泡的制备及其对大鼠动脉粥样硬化斑块的生物学作用

发布时间：2018-12-16 22:25

【摘要】：目的构建载血凝素样氧化低密度脂蛋白受体1(lectin-like oxidized low density lipoprotein receptor-1,LOX-1)-si RNA靶向超声微泡,研究其对大鼠动脉粥样硬化斑块的生物学作用。方法薄膜-水化-超声乳化法构建载LOX1-si RNA纳米级超声微泡(nanobubbles,NBs-si RNA)。从Wistar大鼠体内分离、培养动脉平滑肌细胞(vascular smooth muscle cells,VSMCs)。通过凝胶迁移阻滞实验与si RNA细胞内化实验验证超声微泡可运载si RNA进入靶细胞内;q RT-PCR及Western blot检测LOX1的m RNA和蛋白表达水平;油红O染色观察细胞内脂质聚集情况。构建Wistar大鼠动脉硬化模型,实验组经鼠尾静脉注射含有LOX-1-si RNA的NBs-si RNA微泡100μg,并使用超声仪照射。治疗15 d后,取标本HE染色后与对照组对比动脉硬化斑块病变程度;免疫组化法检测斑块组织中LOX-1表达情况。结果凝胶迁移阻滞实验结果显示,证实NBs与si RNA结合成功。激光共聚焦显微镜观察证实NBs可通过超声照射介导si RNA内化入VSMCs之中。q RT-PCR及Western blot实验结果显示实验组VSMCs LOX-1 m RNA及蛋白表达水平降低(P0.01)。油红O染色显示实验组细胞泡沫化较ox LDL组减弱。组织标本HE染色表明实验组大鼠动脉斑块病变中内膜增厚、中膜萎缩及炎细胞浸润情况较对照组减轻;免疫组化实验结果显示,实验组动脉斑块中LOX-1表达水平较各对照组降低(P0.01)。结论成功制备能够有效运载LOX-1-si RNA的超声微泡,体内外实验证实该微泡可以在超声辐照下内化入靶细胞内部,下调LOX-1表达,减少泡沫细胞形成,降低动脉硬化斑块病变的严重程度。
[Abstract]:Objective to study the biological effects of lectin-like oxidized low density lipoprotein receptor-1,LOX-1-si RNA targeted ultrasound microbubbles on atherosclerotic plaques in rats. Methods LOX1-si RNA loaded nano-scale ultrasonic microbubbles (nanobubbles,NBs-si RNA).) were prepared by thin-film hydration-phacoemulsification method. Arterial smooth muscle cells (vascular smooth muscle cells,VSMCs) were isolated from Wistar rats. Gel migration arrest assay and si RNA cell internalization test showed that ultrasound microbubbles could transport si RNA into target cells; Q RT-PCR and Western blot were used to detect the expression level of m RNA and protein of LOX1; oil red O staining was used to observe lipid accumulation in the cells. The arteriosclerosis model of Wistar rats was established. The experimental group was injected with 100 渭 g NBs-si RNA microbubbles containing LOX-1-si RNA via tail vein and irradiated with ultrasound. After 15 days of treatment, the degree of atherosclerotic plaque lesion was detected by HE staining and the expression of LOX-1 in plaque tissue was detected by immunohistochemical method. Results the results of gel migration block test showed that NBs was successfully combined with si RNA. Laser confocal microscopy showed that NBs could mediate si RNA internalization into VSMCs by ultrasound irradiation. Q RT-PCR and Western blot experiments showed that the expression of VSMCs LOX-1 m RNA and protein in the experimental group was decreased (P0.01). Oil red O staining showed that cell foaming was weaker in experimental group than in ox LDL group. The results of HE staining showed that the intima thickening, medial atrophy and inflammatory cell infiltration were lighter in the experimental group than in the control group. Immunohistochemical results showed that the expression of LOX-1 was lower in the experimental group than that in the control group (P0.01). Conclusion Ultrasonic microbubbles capable of carrying LOX-1-si RNA effectively were successfully prepared. The results of in vitro and in vivo experiments showed that the microbubbles could be internalized into the target cells under ultrasound irradiation, down-regulating the expression of LOX-1 and reducing the formation of foam cells. Reduce the severity of atherosclerotic plaque lesions.
【作者单位】：南方医科大学第三附属医院心内科超声室;南方医科大学珠江医院超声诊断科;
【基金】：广东省自然科学基金(10151051501000016)~~
【分类号】：R543.5

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