【摘要】：目的構(gòu)建載血凝素樣氧化低密度脂蛋白受體1(lectin-like oxidized low density lipoprotein receptor-1,LOX-1)-si RNA靶向超聲微泡,研究其對(duì)大鼠動(dòng)脈粥樣硬化斑塊的生物學(xué)作用。方法薄膜-水化-超聲乳化法構(gòu)建載LOX1-si RNA納米級(jí)超聲微泡(nanobubbles,NBs-si RNA)。從Wistar大鼠體內(nèi)分離、培養(yǎng)動(dòng)脈平滑肌細(xì)胞(vascular smooth muscle cells,VSMCs)。通過凝膠遷移阻滯實(shí)驗(yàn)與si RNA細(xì)胞內(nèi)化實(shí)驗(yàn)驗(yàn)證超聲微泡可運(yùn)載si RNA進(jìn)入靶細(xì)胞內(nèi);q RT-PCR及Western blot檢測(cè)LOX1的m RNA和蛋白表達(dá)水平;油紅O染色觀察細(xì)胞內(nèi)脂質(zhì)聚集情況。構(gòu)建Wistar大鼠動(dòng)脈硬化模型,實(shí)驗(yàn)組經(jīng)鼠尾靜脈注射含有LOX-1-si RNA的NBs-si RNA微泡100μg,并使用超聲儀照射。治療15 d后,取標(biāo)本HE染色后與對(duì)照組對(duì)比動(dòng)脈硬化斑塊病變程度;免疫組化法檢測(cè)斑塊組織中LOX-1表達(dá)情況。結(jié)果凝膠遷移阻滯實(shí)驗(yàn)結(jié)果顯示,證實(shí)NBs與si RNA結(jié)合成功。激光共聚焦顯微鏡觀察證實(shí)NBs可通過超聲照射介導(dǎo)si RNA內(nèi)化入VSMCs之中。q RT-PCR及Western blot實(shí)驗(yàn)結(jié)果顯示實(shí)驗(yàn)組VSMCs LOX-1 m RNA及蛋白表達(dá)水平降低(P0.01)。油紅O染色顯示實(shí)驗(yàn)組細(xì)胞泡沫化較ox LDL組減弱。組織標(biāo)本HE染色表明實(shí)驗(yàn)組大鼠動(dòng)脈斑塊病變中內(nèi)膜增厚、中膜萎縮及炎細(xì)胞浸潤(rùn)情況較對(duì)照組減輕;免疫組化實(shí)驗(yàn)結(jié)果顯示,實(shí)驗(yàn)組動(dòng)脈斑塊中LOX-1表達(dá)水平較各對(duì)照組降低(P0.01)。結(jié)論成功制備能夠有效運(yùn)載LOX-1-si RNA的超聲微泡,體內(nèi)外實(shí)驗(yàn)證實(shí)該微泡可以在超聲輻照下內(nèi)化入靶細(xì)胞內(nèi)部,下調(diào)LOX-1表達(dá),減少泡沫細(xì)胞形成,降低動(dòng)脈硬化斑塊病變的嚴(yán)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.
【作者單位】： 南方醫(yī)科大學(xué)第三附屬醫(yī)院心內(nèi)科超聲室;南方醫(yī)科大學(xué)珠江醫(yī)院超聲診斷科;
【基金】：廣東省自然科學(xué)基金(10151051501000016)~~
【分類號(hào)】：R543.5

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