浏览全部资源
扫码关注微信
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
Preparation of ultrasound-targeted LOX-1-siRNA-loaded microbubbles and their effect on atherosclerotic plaque in rats
- 第三军医大学学报 2017年39卷第24期 页码：2359-2365
- 作者机构：
1.510600 广州，南方医科大学第三附属医院心内科超声室
2.510220 广州，南方医科大学珠江医院超声诊断科
- 作者简介：
[陈霞，E-mail：chenxiaxx1@163.comCHEN Xia, E-mail:chenxiaxx1@163.com
- 基金信息：广东省自然科学基金(10151051501000016)
DOI：10.16016/j.1000-5404.201708032
中图分类号： R543.5; R965; R981收稿：2017-08-06，
修回：2017-10-16，
纸质出版：2017-12-30
- 稿件说明：
移动端阅览
陈霞, 肖煦. 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[J]. 第三军医大学学报, 2017,39(24):2359-2365.
Xia CHEN, Xu XIAO. Preparation of ultrasound-targeted LOX-1-siRNA-loaded microbubbles and their effect on atherosclerotic plaque in rats[J]. Journal of Third Military Medical University, 2017, 39(24): 2359-2365.
陈霞, 肖煦. 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[J]. 第三军医大学学报, 2017,39(24):2359-2365. DOI： 10.16016/j.1000-5404.201708032.
Xia CHEN, Xu XIAO. Preparation of ultrasound-targeted LOX-1-siRNA-loaded microbubbles and their effect on atherosclerotic plaque in rats[J]. Journal of Third Military Medical University, 2017, 39(24): 2359-2365. DOI： 10.16016/j.1000-5404.201708032.
摘要
目的
2
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)-siRNA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
2
薄膜-水化-超声乳化法构建载LOX1-siRNA纳米级超声微泡(nanobubbles
NBs-siRNA)。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siRNA细胞内化实验验证超声微泡可运载siRNA进入靶细胞内；qRT-PCR及Western blot检测LOX1的mRNA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-siRNA的NBs-siRNA微泡100 μg，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
2
凝胶迁移阻滞实验结果显示，证实NBs与siRNA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siRNA内化入VSMCs之中。qRT-PCR及Western blot实验结果显示实验组VSMCs LOX-1 mRNA及蛋白表达水平降低(
P
＜0.01)。油红O染色显示实验组细胞泡沫化较oxLDL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(
P
＜0.01)。
结论
2
成功制备能够有效运载LOX-1-siRNA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
2
Toconstructlectin-like oxidized low density lipoprotein receptor-1 (LOX-1)-siRNA loaded ultrasound microbubbles
and investigate their biological function to the atherosclerosis plaques in mice.
Methods
2
The LOX1-siRNA loaded ultrasound nanobubbles(NBs-siRNA) were constructed by membrane-hydration-ultrasonic emulsification. Vascular smooth muscle cells (VSMCs) were isolatedfrom the Wistar rats and then cultured
in vitro
. Gel shift assay and cell internalization assay were used to confirm that the NBs can deliver siRNA into the targeted VSMCs cells. The expression of LOX-1 was detected by qRT-PCR and Western blot assay. Oil red O staining was used to analyze the intracellular lipid aggregation. After the atherosclerosis model was constructed in Wistar rats
100 μg NBs-siRNA was injected into the mice via the tail v
ein
and the atherosclerosis site was exposure to the ultrasound wave. The severity of atherosclerosis was analyzed by HE staining
and the LOX-1 expression in the atherosclerosis was detected by immunohistochemistry (IHC).
Results
2
The connection of NBs and siRNA was proved by gel shift assay
and siRNA can be delivered into the VSMCs cells by NBs under irradiation of ultrasound as observed by laser confocal scanning microscopy. qRT-PCR and Western blot assay indicated that the expression of LOX-1 was downregulated at transcription and translation levels. Oil red O staining displayed that the intracellular lipid aggregation was alleviated in the experimental group. HE staining showed attenuated tunica intimal thickening
tunica media atrophy
and inflammatory cell infiltration in the plagues of the experimental group. IHC assay demonstrated that the expression of LOX-1 was downregulated in the plaques of the experimental group.
Conclusion
2
The ultrasound microbubbles are successfully constructed and can deliver LOX-1-siRNA into targeted cells under the irradiation of ultrasound
and downregulate the expression of LOX-1 both
in vitro
and
in vivo
. Thus
less foam cells are formed and the severity of atherosclerosis is alleviated.
关键词
Keywords
references
MOORE K J, SHEEDY F J, FISHER E A. Macrophages in atherosclerosis: A dynamic balance[J]. Nat Rev Immunol, 2013, 13(10): 709-721. DOI: 10.1038/nri3520.
ZERNECKE A, WEBER C. Chemokines in atherosclerosis: Proceedings resumed[J]. Arterioscler Thromb Vasc Biol, 2014, 34(4): 742. DOI: 10.1161/ATVBAHA.113.301655.
TRPKOVIC A, RESANOVIC I, STANIMIROVIC J, et al. Oxidized low-density lipoprotein as a biomarker of cardiovascular diseases[J]. Crit Rev Clin Lab Sci, 2015, 52(2): 70. DOI: 10.3109/10408363.2014.992063.
李倩, 芮耀诚. LOX-1—心血管疾病防治的新靶标[J].药学实践杂志, 2014, (5): 321-323. DOI: 10.3969/j.issn.1006-0111.2014.05.001.
LI Q, RUI Y C. LOX-1, a new target in cardiovascular disease [J]. J Pharm Prac, 2014, (5): 321-323. DOI: 10.3969/j.issn.1006-0111.2014.05.001.
熊晓昉, 蒋雯, 祝芬, 等.小蘖碱对oxLDL诱导平滑肌细胞泡沫化及LOX-1、NF-κB表达变化的影响[J].微循环学杂志, 2014, 24(2): 31-34. DOI: 10.3969/j.issn.1005-1740.2014.02.008.
XIONG X F, JIANG W, ZHU F, et al. Effect of berberine on oxLDL induced foam cell formation and LOX-1, NF-κB expression in vascular smooth muscle cells[J]. Chin J Microcirc, 2014, 24(2): 31-34. DOI: 10.3969/j.issn.1005-1740.2014.02.008.
李硕阳, 尹庭辉, 李景果, 等.载sirna微泡实现肿瘤治疗与疗效评估一体化的可行性分析[J].南方医科大学学报, 2015, 35(6): 874-878. DOI: 10.3969/j.issn.1673-4254.2015.06.019.
LI S Y, YIN T H, LI J G, et al. Feasibility of integrating tumor therapy with therapeutic effect evaluation using siRNA-loaded microbubbles[J]. J Southern Med Univ, 2015, 35(6): 874-878. DOI: 10.3969/j.issn.1673-4254.2015.06.019.
郭延松, 吴宗贵, 杨军柯, 等.三种大鼠动脉粥样硬化模型复制方法的比较[J].中国动脉硬化杂志, 2003, 11(5): 465-469.
GUO Y S, WU Z G, YANG J K, et al. Comparison on the three duplication methods of atherosclerosis model in rats[J]. Chin J Arteriosler, 2003, 11(5): 465-469. DOI: 10.3969/j.issn.1007-3949.2003.05.021.
刘莉, 陆远, 王媛, 等. Egfr、her2、cxcr4在非小细胞肺癌中的表达及临床意义[J].现代生物医学进展, 2014, 14(6): 1069-1073. DOI: 10.13241/j.cnki.pmb.2014.06.018
LIU L, LU Y, WANG Y, et al. Expression and clinical significance of EGFR, HER2 and CXCR4 in non-small cell lung cancer[J]. Prog Mod Biomed, 2014, 14(6): 1069-1073. DOI: 10.13241/j.cnki.pmb.2014.06.018.
刘军锋, 贾克刚, 刘运德.血凝素样氧化低密度脂蛋白受体-1与冠心病损伤机制的研究现状[J].中华临床医师杂志:电子版, 2013, 7(9): 147-149. DOI: 10.3877/cma.j.issn.1674-0785.2013.09.051.
LIU J F, JIA K G, LIU Y D. The research progress of lectin like oxidized low-density lipoprotein receptor -1 and the mechanism of coronary heart disease injury[J]. Chin J Clinicians (Electr Ed), 2013, 7(9): 147-149. DOI: 10.3877/cma.j.issn.1674-0785.2013.09.051.
CHEN B, LI J, ZHU H. AMP-activated protein kinase attenuates oxLDL uptake in macrophages through PP2A/NF-κB/LOX-1 pathway[J]. Vascul Pharmacol, 2016, 85: 1-10. DOI: 10.1016/j.vph.2015.08.012
HOSSAIN E, OTA A, KARNAN S, et al. Arsenic augments the uptake of oxidized ldl by upregulating the expression of lectin-like oxidized ldl receptor in mouse aortic endothelial cells[J]. Toxicol Appl Pharmacol, 2013, 273(3):651-658.DOI: 10.1016/j.taap.2013.10.012
林清飞, 王华军, 林静, 等.虾青素对糖尿病大鼠血管内皮功能的保护作用及机制[J].中华高血压杂志, 2015, 23(6): 530-536. DOI: 10.16439/j.cnki.1673-7245.2015.06.016.
LIN Q F, WANG H J, LIN J, et al.The protective effect and related mechanisms of astaxanthin on endothelial function in diabeticrats[J]. Chin J Hypertens, 2015, 23(6): 530-536. DOI: 10.16439/j.cnki.1673-7245.2015.06.016.
BIOCCA S, IACOVELLI F, MATARAZZO S, et al. Molecular mechanism of statin-mediated lox-1 inhibition[J]. Cell Cycle, 2015, 14(10): 1583-1595. DOI: 10.1080/15384101.2015.1026486.
张聪, 王玉凤, 李隆敏, 等.靶向超声微泡在肝癌治疗领域的研究进展[J].现代肿瘤医学, 2017, 25(1): 154-157. DOI: 10.3969/j.issn.1672-4992.2017.01.42
ZHANG C, WANG Y F, LI L M, et al. Advances of ultrasound targeted microbubbles in the field of liver cancer treatment[J]. J Mod Oncol, 2017, 25(1): 154-157. DOI: 10.3969/j.issn.1672-4992.2017.01.42.
WILLIFORD J M, WU J, REN Y, et al. Recent advances in nanoparticle-mediated siRNA delivery[J]. Ann Rev Biomed Eng, 2014, 16: 347-370. DOI: 10.1146/annurev-bioeng-071813-105119.
LU Y, WANG Y, ZHANG M, et al. Her2-sirna delivered by egfr-specific single chain antibody inhibits nsclc cell proliferation and tumor growth[J]. Oncotarget, 2016, 7(17): 23594-23607. DOI: 10.18632/oncotarget.8053.
0
浏览量
0
下载量
0
CSCD
- Meta-XML下载
- BibTeX下载
- Endnote下载
- RefMan 下载
- NoteExpress下载
- NoteFirst下载
关联资源
相关文章
相关作者
相关机构
- 技术支持由北京北大方正电子有限公司提供 京ICP备09064830号-19
京公网安备11010802024621 - 本系统建议在Chrome、 IE9+ 以上版本浏览器阅读本站内容，360浏览器请切换至极速模式
- Cookies帮助我们提供服务并提供个性化体验。使用本网站，即表示您同意我们使用Cookies
