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海洋生物毒素是一类由海洋生物产生的天然活性物质,具有化学结构新颖,生物活性高,作用于钠、钾、钙等离子通道等特点,有望开发为先导化合物,为新型海洋药物的研发提供新策略,但其强烈毒性和高致死率也对人类健康产生巨大威胁[1]。目前,药物毒性评价多采用细胞模型和动物模型,由于细胞模型不能重现体内真实环境,动物模型存在伦理和物种差异等问题,近年来,在微流控技术基础上发展起来的器官芯片模型,可以模拟体内组织器官的结构和功能,再现体内微环境,在药物毒性评价与检测领域展现出极大潜力。本文构建了一种可模拟心血管结构与功能的心血管芯片模型,用于四种典型的深海毒素,即大田软海绵酸(OA)、芋螺毒素(CTX)、河豚毒素(TTX)和环亚胺毒素(GYM)的致伤评价,并初步探索了雷公藤甲素对毒素致伤的保护作用,旨在为深海毒素的防护提供参考。
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以1 μl/min的流速动态培养细胞,HUVEC细胞在所构建的心血管器官芯片模型中生长状态良好,存活率高。经WGA-FITC免疫荧光染色,可清楚观察到结构完整的血管内皮糖萼,符合体外心血管模型的要求,如图2所示。
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采用CCK-8法考察不同浓度的雷公藤甲素对HUVEC细胞活力的影响,结果如图3所示,在测试浓度范围内,雷公藤甲素对HUVEC细胞不存在明显的活力抑制,选择浓度为1 μmol/L的雷公藤甲素进行后续实验。
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采用CCK-8法测定雷公藤甲素预保护前后,低、中、高三个浓度的毒素对HUVEC细胞活力的影响。与对照组相比,OA在中、高浓度下对细胞活力具有较强的抑制作用(P<0.01),其IC50为54.12 nmol/L。GYM在高浓度对细胞活力有较大影响(P<0.05),但是在加入1 μmol/L雷公藤甲素预保护后,OA与GYM对细胞的毒性显著降低,细胞活力与对照组相比无显著性差异,说明雷公藤甲素对OA和GYM损伤HUVEC细胞具有一定的保护作用。而CTX、TTX在雷公藤甲素预保护前后,对HUVEC细胞活力均无显著抑制作用,结果如图4、5所示。
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在所建立的心血管芯片平台上研究雷公藤甲素对四种毒素产生细胞内皮糖萼损伤的保护作用,免疫荧光染色结果如图6和图7所示。OA作用后,HUVEC细胞出现皱缩、变圆、体积变小等形态变化,且随着实验浓度的增大,所呈现的上述细胞形态变化加剧,培养液中可见大量细胞碎片和悬浮的死细胞,说明OA对HUVEC细胞毒性较强,不仅对细胞糖萼有较严重的损伤,还影响了细胞与芯片PDMS基底的贴附水平,心血管芯片结构和功能受损。CTX、TTX和GYM作用后,HUVEC细胞受到不同程度的损伤,内皮细胞糖萼的WGA-FITC染色荧光强度信号与对照组相比均明显降低,具有统计学意义(P<0.01)。经雷公藤甲素预保护后,低浓度OA所产生的细胞损伤得以减轻,糖萼损伤率为28.20%,但是中浓度和高浓度作用下的细胞依旧损伤严重。除OA外,其余三种毒素作用后的糖萼WGA-FITC染色荧光强度均有明显升高,糖萼损伤率见表1。
表 1 心血管芯片上的糖萼损伤率
毒素 给药浓度(μmol/L) 糖萼损伤率(%) 预保护前 预保护后 CTX 0.5 36.30 14.90 5 48.04 13.47 20 52.28 26.52 TTX 0.04 29.55 6.52 0.4 40.30 6.69 4 41.97 14.60 GYM 4 23.53 0.22 10 25.89 3.65 40 40.41 6.01
Evaluation of toxin-induced injury and protective effect of triptolide based on a cardiovascular chip model
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摘要:
目的 构建一种心血管芯片模型,评估四种海洋毒素即大田软海绵酸(OA)、芋螺毒素(CTX)、河豚毒素(TTX)和环亚胺毒素(GYM)对血管糖萼组织的损伤,初步探索雷公藤甲素对毒素致伤的保护作用。 方法 将人脐静脉内皮细胞(HUVEC)接种于三通道结构的微流控芯片,并对所构建的心血管芯片模型进行表征。采用CCK-8法和免疫荧光染色法分析低、中、高三个浓度的海洋毒素对细胞活力和糖萼组织损伤情况,以及雷公藤甲素对毒素致伤的保护作用。 结果 所构建心血管器官芯片中的细胞生长状态良好,具有结构完整的糖萼组织,与对照组相比,OA中、高浓度以及GYM高浓度均对细胞活力具有一定抑制(P<0.05),CTX和TTX在所测浓度下对细胞没有明显活力抑制,但是四种毒素均引起细胞表面糖萼组织的严重损伤(P<0.01),且随着浓度增加,糖萼损伤率升高。经雷公藤甲素预保护后,四种毒素对HUVEC的毒性显著降低,糖萼组织损伤率下降。 结论 四种毒素对HUVEC细胞活力以及表面的糖萼组织具有损伤,且呈一定的剂量关系,而雷公藤甲素对毒素损伤后的HUVEC细胞具有保护作用。 Abstract:Objective To construct a cardiovascular chip model for evaluating the damage of vascular glycocalyx induced by four marine toxins: okadaic acid (OA), conotoxin (CTX), tetrodotoxin (TTX) and gymnodimine (GYM), and explore the protective effect of triptolide on toxin-induced injury. Methods Human umbilical vein endothelial cells(HUVEC) were inoculated into a three-channel microfluidic chip. CCK-8 method and immunofluorescence staining were used to analyze the damage of cell viability and glycocalyx tissue induced by low, middle and high concentrations of marine toxin, as well as the protective effect of triptolide on toxin-induced injury. Results The cells in the cardiovascular chip grew well and had structurally intact glycocalyx. Compared with the control group, the activity of HUVEC cells were inhibited in group of the medium and high concentration of OA and high concentration of GYM (P<0.05). The activity of cells had not been inhibited by CTX and TTX significantly , but all the four toxins caused serious damage to the glycocalyx tissue (P<0.01). After pre-protection with triptolide, the toxicity of the four toxins to HUVEC cells and the damage rate of glycocalyx decreased significantly. Conclusion The four marine biotoxins could damage the activity and glycocalyx of HUVEC cells in a dose-dependent manner, while triptolide has a protective effect on HUVEC cells injured by toxin. -
Key words:
- cardiovascular chip /
- marine biotoxins /
- glycocalyx damage /
- triptolide
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表 1 心血管芯片上的糖萼损伤率
毒素 给药浓度(μmol/L) 糖萼损伤率(%) 预保护前 预保护后 CTX 0.5 36.30 14.90 5 48.04 13.47 20 52.28 26.52 TTX 0.04 29.55 6.52 0.4 40.30 6.69 4 41.97 14.60 GYM 4 23.53 0.22 10 25.89 3.65 40 40.41 6.01 -
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