CN114507598B - 一种基于心血管芯片模型的深海生物毒素的致伤评价方法与应用 - Google Patents
一种基于心血管芯片模型的深海生物毒素的致伤评价方法与应用 Download PDFInfo
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Abstract
本发明公开了一种基于心血管芯片模型的深海毒素致伤评价方法，首先建立了心血管芯片模型，模拟心血管结构与功能，包括血管表面的糖萼组织；分别考察了四种深海重要生物毒素，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)在心血管芯片模型上的毒性效应，每个毒素设置低、中、高三个浓度梯度，结果发现，OA、CTX、TTX和GYM对脐静脉内皮细胞(HUVEC)增殖具有一定的影响，对芯片模型的糖萼损伤呈现出一定的量‑毒效应，随着浓度增加，糖萼损伤愈发严重。考察了雷公藤甲素对以上四种毒素致伤的预保护作用，在心血管芯片模型上，雷公藤甲素预保护后显著降低四种毒素对HUVEC的毒性，有望为深海生物毒素的致伤防护提供新的策略和候选药物。
Description
技术领域
本发明涉及医药技术领域，具体涉及一种基于心血管芯片模型的深海生物毒素的致伤评价方法，以及在药物筛选中的应用。
背景技术
深海生物毒素是海洋生物中天然存在的、具有强烈毒性的化学物质。在生物体自生的化学防御和进攻机制方面具有重要的作用。典型深海毒素包括大田软海绵酸(OA)、河豚毒素(TTX)、芋螺毒素(CTX)和裸甲藻亚胺毒素(GYM)。其中，大田软海绵酸(OA)是一种主要存在于藻类植物中的小分子腹泻类毒素，OA会抑制蛋白质磷酸化过程，中毒患者会出现肠胃失调，还具有潜在的慢性毒性，包括损伤DNA、影响胚胎发育等。河豚毒素(TTX)是一种非蛋白高毒性的天然海洋活性物质，也是一种特殊的选择性钠离子通道阻断剂。芋螺毒素(CTX)是由芋螺毒液管和毒囊内壁的毒腺所分泌的混合毒素，能特异性地作用于乙酰胆碱受体及其他神经递质受体亚型，以及多种离子通道。环亚胺毒素(GYM)由于其缓慢的降解速度，存在长期的生态系统潜在风险。
深海生物毒素可特异作用于神经与肌肉细胞，兴奋细胞膜上的关键靶位，即神经受体或离子通道，从而影响与受体相关的细胞调控活动，具有广泛的神经系统活性、心血管系统活性和细胞活性，由于毒素对受体的高度选择性和亲和性，造成其较大毒性。对于深海毒素最好的防护是物理防护，如穿上防护衣或物理隔离，不接触有毒生物和染毒食品，一旦致伤中毒，目前救护的方法不多，主要是对症治疗，如用激素等，特异性的针对毒素的救治药物或者防护策略几乎是空白。深海生物毒素致伤中毒的评价手段匮乏，导致无法采取针对性的防护。
心血管器官芯片是近年来发展起来的一类器官芯片，相较于常规细胞模型，心血管器官芯片更仿生、可提供更丰富的生理及毒理信息，同时比实验动物模型速度快、通量高、成本低。通过芯片设计、参数优化、流体控制以及模拟血液开发，使多种人源细胞联合工作并接近器官真实生理结构，模拟真实人体情况再现生物毒素的毒性作用过程，是基于器官芯片技术对生物毒素进行毒性评价方法开发的关键。心血管芯片可以更好地模拟人体对毒素真实反应，显著减少毒性评估的成本和时间，在毒性测试动物替代技术研究领域具有很大的潜力。
中国专利申请CN202010884867.3，申请公布号：CN112143642A，公开了一种用于体外培养的血管化肿瘤微流控器官芯片及其制备方法，芯片包括：PMMA模块和玻璃片，PMMA模块设有贯通其厚度方向的三个通孔；玻璃片的上表面与PMMA模块的下表面的四周通过粘结层键合为一体，使三个通孔分别形成用于容置肿瘤小球的第一腔室、用于储存培养液的第二腔室、第三腔室；且PMMA模块下表面的中间区域与玻璃片构成中空的组织腔室；具有液面高度差的培养液所产生的静态压力差可促进三维毛细血管网络生成，通过三维毛细血管网络与肿瘤小球共培养能实现对毛细血管的募集，构建成为三维血管化肿瘤微组织。本发明克服了目前微流控器官芯片制作的耗时与高成本缺点，工艺简单，尤其是体外血管化肿瘤疾病的研究提供了应用价值。
中国专利CN201711368061.3，授权公布号：CN108004144B，公开了一种含管道网络的器官芯片单元，包括：至少一层含输入管道网络的芯片，输入管道网络用于物质的输入、分流和扩散；至少一层含输出管道网络的芯片，输出管道网络用于物质的扩散、汇流和输出；至少一层腔室芯片，腔室芯片中盛放实质器官组织；至少两层微孔膜，实质器官组织与输入、输出管道网络之间由微孔膜间隔；至少两层屏障细胞，屏障细胞处于输入、输出管道网络中，粘附在微孔膜上；两层封板，用以实现封装。使用该器官芯片单元，能够模拟物质经过血管/淋巴管/支气管/腺体分泌网络等各种管道网络的分流后进入组织并在组织内代谢后逐渐汇流的过程，在保证低成本、迅速的芯片优势的同时，应用于药物筛选、病例研究和生理分析时更加准确。
发明内容
本发明着眼于模拟人体真实生理环境和反应，构建了心血管器官芯片模型，探索基于心血管芯片模型的深海重要生物毒素致伤评价方法，包括大田软海绵酸(OA),河豚毒素(TTX)，芋螺毒素CTX和环亚胺毒素(GYM)；在此基础上，本发明进行了相关的药物筛选。
进一步地，本发明发现了雷公藤甲素对于这四种毒素的致伤有一定的保护作用，这为海洋毒素致伤中毒的防护和解救提供了候选化合物，有望开发成新的防护药物。
本发明的第一方面，提供一种基于心血管芯片模型的深海生物毒素的致伤评价方法。
一种基于心血管芯片模型的深海生物毒素的致伤评价方法，所述的心血管芯片模型由三通道组成，其中一侧通道为血管通道，接种脐静脉内皮细胞；
配置纤维蛋白原母液和凝血酶母液，将纤维蛋白原母液和凝血酶母液按一定浓度混合，加入中间通道，恒温孵育至纤维蛋白原凝胶化；
另一通道为细胞培养液通道；
基质胶包被血管通道表面，将脐静脉内皮细胞的细胞悬液加入血管通道；
血管通道进口插入软管，并与蠕动泵相连，参照体内血管流体剪切应力注入细胞培养基以模拟体内血管动态微环境；
通过血管通道加入一定浓度的深海生物毒素溶液，观察芯片上糖萼损伤情况。
所述的深海生物毒素为大田软海绵酸、环亚胺毒素、芋螺毒素和河豚毒素等。
在低、中、高浓度梯度下考察深海生物毒素对脐静脉内皮细胞的毒性效应，通过细胞形态观察和CCK-8法评价四种毒素对细胞形态和增殖的影响。
具体步骤为：纤维蛋白原和凝血酶溶解于PBS，配置纤维蛋白原母液和凝血酶母液；将纤维蛋白原母液和凝血酶母液混合(优选的，纤维蛋白原最终浓度为6mg/ml，凝血酶最终浓度为2U/ml)，加入中间通道，恒温孵育15min至纤维蛋白原凝胶化；
在血管通道加入基质胶(优选的，1:50，v/v)包被血管通道表面40min，PBS冲洗通道内多余基质胶；
将脐静脉内皮细胞(优选的，细胞终浓度为1×106cells/ml)加入血管通道，每24h更换通道内细胞培养液；
所述的细胞培养液，优选的，配方为DMEM/F-12(Dulbecco改良的Eagle培养基/营养混合物F-12)，胎牛血清，青霉素/链霉素混合液，三者的比例为90：10：1(v/v/v)。
本发明的第二方面，提供一种基于心血管芯片模型的深海生物毒素的致伤评价方法在药物筛选中的应用。
所述的药物为针对深海生物毒素致伤的救治药物或者防护药物，如雷公藤甲素等。
通过血管通道加入雷公藤甲素溶液，1h后加入毒素溶液，观察雷公藤甲素预保护后毒素对芯片内糖萼组织损伤情况，评价雷公藤甲素的保护作用；
本发明首次基于心血管微流控芯片模型分析毒素的致伤情况以及雷公藤甲素的保护作用。四种深海生物毒素对脐静脉内皮细胞的生长具有抑制作用，呈一定的量-毒效应。考察了雷公藤甲素对四种毒素致伤的预保护作用，结果发现，雷公藤甲素可以降低四种毒素对脐静脉内皮细胞的毒性作用。
本发明的第三方面，提供了雷公藤甲素对深海重要生物毒素致伤的预保护作用。
考察雷公藤甲素对糖萼组织的保护作用时，先在血管通道加入雷公藤甲素溶液保护一定时间，在血管通道加入一定浓度的毒素溶液，观察芯片上糖萼损伤情况。与对照组相比，经雷公藤甲素预保护后GYM对细胞的毒性显著降低，孔板实验中，在未加入雷公藤甲素的组别中，高浓度对HUVEC存在较为明显的生长抑制作用，但是在加入保护剂雷公藤甲素后，由CCK-8实验得出细胞活力与空白组相当，说明雷公藤甲素对高浓度GYM致伤的HUVEC具有一定的保护作用。与对照组相比，OA经雷公藤甲素预保护后对细胞的毒性有所降低，孔板实验中，在未加入雷公藤甲素的组别中，中、高两个浓度的OA对HUVEC存在较为明显的生长抑制作用，但是在加入保护剂雷公藤甲素后，由CCK-8实验得出细胞活力与空白组相当，说明雷公藤甲素对中、高两个浓度的OA致伤的HUVEC具有一定的保护作用。CTX、TTX在浓度范围内均对HUVEC细胞本来不存在毒性，雷公藤甲素预保护后，对HUVEC细胞生长无抑制作用。
附图说明
图1：本发明心血管器官芯片构建模拟图；其中左通道1、中间通道2、右通道3、液体注入口4-6、液体出口7-8；
图2：四种毒素对HUVEC的毒性评价结果图；其中(A)大田软海绵酸；(B)芋螺毒素；(C)河豚毒素；(D)环亚胺毒素；
图3：雷公藤甲素对HUVEC毒性评价结果图；
图4：四种毒素在雷公藤甲素预保护后对HUVEC的毒性评价结果图；其中(A)大田软海绵酸；(B)芋螺毒素；(C)河豚毒素；(D)环亚胺毒素；
图5：雷公藤甲素对心血管芯片上四种毒素损伤糖萼的保护作用结果；其中(A)大田软海绵酸；(B)芋螺毒素；(C)河豚毒素；(D)环亚胺毒素。
具体实施方式
下面结合实施例对本发明提供的具体实施方式作详细说明。
现结合实施例和附图，对本发明作进一步描述，但本发明的实施并不仅限于此。
实施例1：微流控芯片、心血管芯片模型和深海生物毒素的致伤评价方法的建立
一种微流控芯片，其结构参阅图1所示，所述微流控芯片包括左通道1、中间通道2、右通道3，左通道1的液体注入口4用于注入脐静脉内皮细胞，中间通道2的液体注入口5用于注入纤维蛋白原和凝血酶混合液，右通道3的液体注入口6用于注入细胞培养液，为了便于换液，在左通道1和右通道3的另一侧对应设置液体出口7和液体出口8，更换细胞培养液时，缓慢谨慎向液体注入口4和液体注入口6注入细胞培养液，液体出口7和液体出口8吸取细胞培养液。
本发明提供的微流控芯片，由上下两层不可逆键合而成，两层均为聚二甲基硅氧烷聚合物，上层含有通道，下层是空白层。上下两层进行等离子体处理后进行键合，键合后的芯片进行灭菌处理。
所述的细胞培养液配方为DMEM/F-12(Dulbecco改良的Eagle培养基/营养混合物F-12)，胎牛血清，青霉素/链霉素混合液，三者的比例为90：10：1(v/v/v)。
一种基于心血管芯片模型的深海重要生物毒素的致伤评价方法与雷公藤甲素的保护作用研究采用上述微流控芯片，按照以下步骤进行：
(1)芯片内细胞接种与培养
配置纤维蛋白原母液和凝血酶母液，将纤维蛋白原母液和凝血酶母液按一定浓度(优选的，纤维蛋白原最终浓度为6mg/ml，凝血酶最终浓度为2U/ml)，混合，加入中间通道，恒温孵育至纤维蛋白原凝胶化，基质胶包被血管通道表面，将脐静脉内皮细胞的细胞悬液加入血管通道，细胞贴壁生长，血管通道与蠕动泵相连，模拟体内血管动态微环境。
(2)深海生物毒素的致伤评价方法与雷公藤甲素的保护作用研究
分别在孔板和芯片上考察不同浓度的毒素对细胞的毒性及糖萼组织损伤情况，与此同时考察雷公藤甲素的保护作用。
实施例2：四种生物毒素的细胞毒性实验
取对数生长期的HUVEC细胞接种于96孔板，考察以下四种毒素在低、中、高三个浓度的毒性效应，分别包括：大田软海绵酸的实验浓度为10、50、100nM，环亚胺毒素的实验浓度为1、4、10μM，芋螺毒素的实验浓度为0.5、5、20μM，河豚毒素的实验浓度为0.04、0.4、4μM，通过细胞形态观察和CCK-8法检测四种毒素对其形态结构和细胞增殖的影响，结果如图2所示。大田软海绵酸在高浓度下对HUVEC具有较大的毒性，HUVEC细胞活性与毒素浓度呈正相关。环亚胺毒素10μM以上，对HUVEC细胞活性有较大的影响。芋螺毒素和河豚毒素在所测浓度下对HUVEC细胞没有明显生长抑制作用。
实施例3：四种生物毒素的致伤评价方法
采用实验室自行设计制作的微流控芯片，如图1所示。芯片处理后，采用上述细胞接种和培养方式建立心血管芯片模型。血管通道内分别加入同实施例2中的不同浓度的毒素，用FITC标记小麦胚芽凝集素(WGA-FITC)考察毒素对血管糖萼组织损伤情况，具体步骤如下：芯片通道用PBS缓慢冲洗三次，将预冷的4％多聚甲醛缓慢注入到芯片微通道内，固定20min。使用PBS缓慢冲洗通道内细胞三次，去除残留的多聚甲醛。通道内加入山羊血清工作液，封闭30min。避光操作，在通道内加入适量的WGA-FITC，室温避光放置1h。用PBS通道冲洗三次，在荧光倒置显微镜下进行观察，结果发现河豚毒素给药后，糖萼的WGA-FITC染色荧光强度信号与对照组相比，在高浓度剂量组毒素对细胞存在毒性作用；实验发现环亚胺毒素给药，中浓度和高浓度组中糖萼的WGA-FITC染色荧光强度信号与对照组相比，显著降低；实验发现芋螺毒素给药后，中浓度和高浓度组中糖萼的WGA-FITC染色荧光强度信号与对照组相比，有所降低，与孔板实验相比，TTX和CTX对细胞虽无明显毒性，但是对心血管上的糖萼组织还是存在一定程度上的抑制作用，可见这两种毒素虽然本身对细胞不存在明显的毒性作用，但是通过其他途径与细胞上的结构组织相互作用，而影响细胞的功能表达。
实施例4：雷公藤甲素的细胞毒性实验
取对数生长期的HUVEC细胞接种于96孔板，考察不同浓度的雷公藤甲素(0.5、1、3、10、20、50μM)对脐静脉内皮细胞的增殖的影响，结果如图3所示。在测试浓度范围内，雷公藤甲素对HUVEC细胞不存在明显的生长抑制作用。根据相关实验选择浓度为1μM的雷公藤甲素进行后续预保护实验。
实施例5：雷公藤甲素对毒素致伤的保护作用
取对数生长期的HUVEC细胞接种于96孔板，向实验组各孔中分别加入含有1μM的雷公藤甲素，培养箱中预保护1h。向实验组各孔中分别加入不同浓度的毒素。24h后，通过CCK-8法检测四种毒素对细胞增殖的影响。在药物作用时间结束时，向各组每孔中加入10％CCK-8，在培养箱中培养1h，以空白组调零，用酶标仪检测各样样品的OD值，计算细胞存活率，结果如图4所示。
实验选取浓度为1μM雷公藤甲素对HUVEC细胞进行预保护。与对照组相比，经雷公藤甲素预保护后GYM对细胞的毒性显著降低，孔板实验中，在未加入雷公藤甲素的组别中，高浓度对HUVEC存在较为明显的生长抑制作用，但是在加入保护剂雷公藤甲素后，由CCK-8实验得出细胞活力与空白组相当，说明雷公藤甲素对高浓度GYM致伤的HUVEC具有一定的保护作用。与对照组相比，OA经雷公藤甲素预保护后对细胞的毒性有所降低，孔板实验中，在未加入雷公藤甲素的组别中，中、高两个浓度的OA对HUVEC存在较为明显的生长抑制作用，但是在加入保护剂雷公藤甲素后，由CCK-8实验得出细胞活力与空白组相当，说明雷公藤甲素对中、高两个浓度的OA致伤的HUVEC具有一定的保护作用。CTX、TTX在浓度范围内均对HUVEC细胞本来不存在毒性，雷公藤甲素预保护后，对HUVEC细胞生长无抑制作用。
实施例6：雷公藤甲素对心血管芯片上毒素糖萼损伤的保护作用
采用实验室自行设计制作的微流控芯片，如图1所示。芯片处理后，采用上述细胞接种和培养方式建立心血管芯片模型。血管通道内加入浓度为1μM雷公藤甲素对HUVEC细胞进行预保护1h，分别加入不同浓度的毒素，用FITC标记小麦胚芽凝集素(WGA-FITC)考察雷公藤甲素对毒素损伤血管糖萼组织保护作用，具体步骤如实施例3，结果如图5所示。
实验发现经雷公藤甲素保护、河豚毒素给药后，糖萼的WGA-FITC染色荧光强度信号与对照组相比，雷公藤甲素对低浓度和中浓度组毒素损伤糖萼有一定的保护作用；实验发现经雷公藤甲素保护、大田软海绵酸给药后，糖萼的WGA-FITC染色荧光强度信号与对照组相比，雷公藤甲素的保护作用不明显；实验发现经雷公藤甲素保护、环亚胺毒素给药，糖萼的WGA-FITC染色荧光强度信号与对照组相比，雷公藤甲素对低浓度组毒素损伤糖萼有保护作用；实验发现经雷公藤甲素保护、芋螺毒素给药后，糖萼的WGA-FITC染色荧光强度信号与对照组相比，雷公藤甲素的保护作用较弱。
以上已对本发明创造的较佳实施例进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
Claims (7)
1.一种基于心血管芯片模型的深海生物毒素的致伤评价方法，所述的心血管芯片模型由三通道组成，其中一个通道为血管通道，接种脐静脉内皮细胞；中间通道为纤维蛋白原母液和凝血酶母液按一定浓度混合，恒温孵育至纤维蛋白原凝胶化；另一通道为细胞培养液通道；
且血管通道进口插入软管，并与蠕动泵相连，参照体内血管流体剪切应力注入细胞培养液以模拟体内血管动态微环境；
通过血管通道加入一定浓度的深海生物毒素溶液，观察对细胞的毒性及血管糖萼组织损伤情况，对深海生物毒素的致伤进行评价；
对深海生物毒素的致伤评价是在低、中、高浓度梯度下考察深海生物毒素对脐静脉内皮细胞的毒性效应，通过细胞形态观察和CCK-8法评价四种毒素对细胞形态和增殖的影响；
所述的心血管芯片模型包括左通道、中间通道、右通道，左通道的液体注入口用于注入脐静脉内皮细胞，中间通道的液体注入口用于注入纤维蛋白原和凝血酶混合液，右通道的液体注入口用于注入细胞培养液；为了便于换液，在左通道和右通道的另一侧对应设置液体出口。
2.根据权利要求1所述的一种基于心血管芯片模型的深海生物毒素的致伤评价方法，其特征在于，所述的深海生物毒素为大田软海绵酸、环亚胺毒素、芋螺毒素和河豚毒素。
3.根据权利要求1或2所述的一种基于心血管芯片模型的深海生物毒素的致伤评价方法，其特征在于，将纤维蛋白原和凝血酶溶解于PBS，配置纤维蛋白原母液和凝血酶母液；最终浓度为6mg/ml的纤维蛋白原母液和最终浓度为2U/ml的凝血酶母液混合，加入中间通道，恒温孵育至纤维蛋白原凝胶化。
4.根据权利要求1或2所述的一种基于心血管芯片模型的深海生物毒素的致伤评价方法，其特征在于，基质胶包被血管通道表面，将脐静脉内皮细胞的细胞悬液加入血管通道。
5.根据权利要求4所述的一种基于心血管芯片模型的深海生物毒素的致伤评价方法，其特征在于，在血管通道加入基质胶1:50，v/v包被血管通道表面40min，PBS冲洗通道内多余基质胶；将脐静脉内皮细胞，细胞终浓度为1×106cells/ml加入血管通道，每24h更换通道内培养基。
6.一种如权利要求1至5任一项所述的基于心血管芯片模型的深海生物毒素的致伤评价方法在药物筛选中的应用。
7.根据权利要求6所述的应用，所述的药物为针对深海生物毒素致伤的救治药物或者防护药物。
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