CN114164165A - 一种微流控芯片在构建疱疹性脑炎模型中的应用 - Google Patents
一种微流控芯片在构建疱疹性脑炎模型中的应用 Download PDFInfo
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Abstract
本发明公开了一种微流控芯片在构建疱疹性脑炎模型中的应用。本发明所述微流控芯片包括开放式培养区、培养室和基质室；在开放式培养区内加入脑微血管内皮细胞，在培养室内加入小胶质细胞，在基质室内加入基质液和按一定比例混合的星形胶质细胞和神经元细胞悬液，构建获得疱疹性脑炎模型。本发明利用微流控芯片技术，以具有良好生物相容性，透光性的PDMS为芯片材料，设计的装置可直接记录和观察病毒颗粒作用下人神经血管单元组成细胞的损伤，并且可实时动态监测小胶质细胞、单个核细胞的迁移行为。
Description
技术领域
本发明属于病毒性脑炎模型构建技术领域，具体涉及一种微流控芯片在构建疱疹性脑炎模型中的应用。
背景技术
单纯疱疹性脑炎是由单纯疱疹病毒引起的中枢神经系统最常见的病毒感染性疾病。其常累及大脑颞叶、额叶及边缘系统，引起脑组织出血性坏死和变态反应性脑损害。单纯疱疹病毒是一种嗜神经DNA病毒，分为Ⅰ、Ⅱ型，近90％是由Ⅰ型病毒引起，余为Ⅱ型所致，病毒先引起2～3周的口腔和呼吸道原发感染，然后沿三叉神经分支经轴突逆行至三叉神经节，在此潜伏。机体免疫力下降时，诱发病毒激活，病毒由嗅球和嗅束直接侵入脑叶，或口腔感染后病毒经三叉神经入脑而引起脑炎。疱疹性脑炎呈散发性，在非流行性病毒脑炎中系最常见的一种，据统计约占病毒性脑炎的10％～20％。目前临床用药以抗病毒药物和糖皮质激素为主，难以实现对疱疹性脑炎免疫损伤的完全阻断，重症型患者往往预后较差。建立疹性脑炎体外模型，对于寻找新的治疗干预手段，具有重要意义。
现阶段，仍缺少近体内的疱疹性脑炎体外模型。体外实验仍以二维细胞培养为主。难以反应人体神经血管单元的复杂组成和外周血免疫细胞在疾病进程中的作用。器官芯片技术作为一门迅速发展起来的科学技术，已经在生物医学领域展现了其独特的优势，更因其同细胞尺寸匹配、环境同生理环境相近、在时间和空间维度上能够提供更为精确的操控，易于通过灵活设计实现多种细胞功能研究等特点而成为新一代细胞研究的重要平台。因其高通量，低消耗，以及对流体的精准操控，更易于构建接近于体内真实环境的神经血管单元细胞组成。而将其应用于疱疹性脑炎体外模型构建，以反映疱疹性脑炎复杂的多细胞共同参与效应以及免疫系统在其中的作用，尚属于该研究领域的空白区域。
发明内容
本发明的目的在于提供一种微流控芯片在构建疱疹性脑炎模型中的应用。基于该器官芯片的疱疹性脑炎模型不仅可以模拟病毒颗粒对神经血管单元的损伤，对多种细胞功能障碍进行实时监测，还能够对外周血免疫细胞的组织浸润实现实时追踪观察。
本发明提供了一种微流控芯片在构建疱疹性脑炎模型中的应用，所述微流控芯片包括开放式培养区、培养室和基质室，所述基质室与开放式培养区之间设有多孔薄膜；在开放式培养区内加入脑微血管内皮细胞，在培养室内加入小胶质细胞，在基质室内加入基质液和按一定比例混合的星形胶质细胞和神经元细胞悬液，构建获得疱疹性脑炎模型。
进一步地，所述星形胶质细胞与神经元细胞的数量比为10:1～1:1。
进一步地，所述微流控芯片包括上层芯片和下层芯片两层，所述上层芯片设置于所述下层芯片上方，所述上层芯片设置于所述下层芯片上方，所述上层芯片设有通孔a、通孔b、通孔c、通孔d和通孔状的开放式培养区；所述下层芯片设有基质室，所述基质室设置于所述开放式培养区下方，所述基质室与所述开放式培养区之间设有多孔薄膜，所述基质室两侧各设有一个培养室，所述基质室另外两侧均分别设有基质入口a和基质入口b，所述基质入口a的位置与所述通孔a的位置在竖直方向上相对应设置，所述基质入口b的位置与所述通孔b的位置在竖直方向上相对应设置；所述培养室为条状，所述培养室两端分别设有培养室入口和培养室出口，所述培养室入口位置与所述通孔c的位置在竖直方向上相对应设置，所述培养室出口位置与所述通孔d的位置在竖直方向上相对应设置。
进一步地，所述基质室设有一条条状通道a，所述条状通道a两侧各设置一条与通道a平行的条状通道b，所述通道b与所述培养室平行，所述通道b上设有数个与通道b垂直的通道c，所述通道c使所述通道a、所述通道b、所述培养室三者连通，数个所述通道c与所述通道b组合形成栅栏结构；所述通道a两端各设有一个基质入口a，所述通道b两端各设有一个基质入口b。
进一步地，每条所述通道b上的所述通道c的数量为7-10个。
进一步地，所述培养室为类n状，两个培养室组成类x状，每个培养室两端分别设有所述培养室入口和所述培养室出口。
进一步地，所述上层芯片和所述下层芯片为聚二甲基硅氧烷简称PDMS材质，所述多孔薄膜为聚苯二甲酸乙二醇酯简称PET材质。
进一步地，所述培养室的深度为100-1000μm，所述基质室的深度为50-200μm。
进一步地，所述疱疹性脑炎模型的应用方法，包括如下步骤：
(1)芯片基质灌注
采用matrigel工作液，将按一定比例混合的星形胶质细胞和神经元细胞悬液与工作液混合。用移液器将其加入基质入口a、基质入口b，每孔10-50μl；加1-5ml PBS缓冲液于培养皿中，将固定芯片的培养皿放入培养箱中孵育30-45min凝胶；
(2)脑微血管内皮细胞和小胶质细胞接种及培养
脑微血管内皮细胞制成悬液，取100μl脑微血管内皮细胞悬液加入开放式培养区内。小胶质细胞制成悬液，取50μl小胶质细胞悬液分别加入到两个小培养室入口。将芯片放平移入37℃培养箱中培养、每隔18-24h换液一次；
(3)芯片神经血管单元功能监测
采用血管内皮细胞钙粘蛋白VE-cadherin和Claudin-5检测芯片神经血管单元的完整性，采用FITC标记的右旋糖苷FITC-Dextran监测芯片神经血管单元血脑屏障的通透性。
(4)加入病毒颗粒
待多种细胞在芯片上共培养5-8天后，在培养室入口加入不同滴度的单纯疱疹病毒颗粒。病毒暴露2-4h后，培养基清洗。将芯片放平移入37℃培养箱中培养、每日换液一次；培养2-5天。
(5)加入外周血单个核细胞
在开放式培养区中灌流加入外周血单个核细胞，模拟体内循环血液中的免疫细胞。30-45min后，除去细胞悬液，培养基清洗。37℃培养箱中培养，每隔24h拍照记录单个核细胞位置，记录细胞运动及形态改变。进行细胞行为的跟踪观察，每隔1-2天进行拍照，实时记录细胞黏附，运动、屏障功能破坏和细胞位置的改变。
原理：本发明所述微流控芯片在构建疱疹性脑炎模型中的应用中，神经血管单元由脑微血管内皮细胞，星形胶质细胞，神经细胞，小胶质细胞共同构成。在培养室入口池加入单纯疱疹病毒颗粒后，在开放式培养区中灌流加入外周血单个核细胞，模拟体内循环血液中的外周血免疫细胞，共同构成疱疹性脑炎脑损伤病理微环境。
本发明所述微流控芯片在构建疱疹性脑炎模型中的应用中，所述基质成分为Matrigel，4℃以下时下它是呈粘稠状的液体，当pH＝7，温度达到常温的情况下，5min，即可呈现果冻状的凝胶。
有益效果：
本发明所述微流控芯片在构建疱疹性脑炎模型中的应用中，所述疱疹性脑炎模型不仅可以模拟病毒颗粒对神经血管单元的损伤，对多种细胞功能障碍进行实时监测，还能够对外周血免疫细胞的组织浸润实现实时追踪观察。
本发明所述疱疹性脑炎模型中，可采用生物学上常用的细胞检测手段对神经血管单元的组成细胞以及迁移运动到基质中的外周血免疫细胞进行检测，包括细胞死活标记染色、细胞免疫荧光染色、PCR检测、蛋白质检测。
本发明利用微流控芯片技术，以具有良好生物相容性，透光性的PDMS为芯片材料，设计的装置可横向直接记录和观察病毒颗粒作用下人神经血管单元组成细胞损伤和单个核细胞迁移行为的芯片，功能完备，并且在芯片上可以独立完成各项信号检测，如细胞蛋白表达，细胞因子分泌，细胞增殖，凋亡检测等。
附图说明
图1为本发明所述微流控芯片的结构示意图，其中(a)为微流控芯片的整体结构示意图，(b)为上层芯片，下层芯片分层示意图。
图2为本发明所述微流控芯片的结构及局部放大图，其中a为微流控芯片的整体结构示意图，b为局部放大图。
图中，1.上层芯片，2.通孔a，3.通孔b，4.通孔c，5.开放式培养区，6.通孔d，7.多孔薄膜，8.基质室，9.培养室，10.下层芯片，81.基质入口a，82.基质入口b，83.通道a，84.通道b，85.通道c，91.培养室入口，92.培养室出口。
图3为病毒作用下神经血管单元不同细胞类型的形态变化图。
图4为病毒作用后外周血单个核细胞在神经血管单元中的组织浸润图；其中，箭头处为浸润的单个核细胞。
具体实施方式
下面的实施例将对本发明予以进一步的说明，但并不因此而限制本发明。
实施例1
如图1-2所示，本实施例提供一种微流控芯片，所述微流控芯片包括上层芯片1和下层芯片10两层，所述上层芯片1设置于所述下层芯片10上方，所述上层芯片1设有通孔a2、通孔b3、通孔c4、通孔d6和通孔状的开放式培养区5；所述下层芯片10设有基质室8，所述基质室8设置于开放式培养区5下方，所述基质室8与开放式培养区5之间设有多孔薄膜7，所述基质室8两侧各设有一个培养室9，所述基质室8另外两侧均分别设有基质入口a81和基质入口b82，所述基质入口a81的位置与通孔a2的位置在竖直方向上相对应设置，所述基质入口b82的位置与通孔b3的位置在竖直方向上相对应设置；所述培养室9为条状，所述培养室9两端分别设有培养室入口91和培养室出口92，所述培养室入口91位置与通孔c4的位置在竖直方向上相对应设置，所述培养室出口92位置与通孔d6的位置在竖直方向上相对应设置。
所述基质室8设有一条条状通道a83，所述通道a83两侧各设置一条与通道a83平行的条状通道b84，所述通道b84与所述培养室9平行，所述通道b84上设有数个与通道b84垂直的通道c85，所述通道c85使所述通道a83、所述通道b84、所述培养室9三者连通，数个所述通道c85与所述通道b84组合形成栅栏结构；所述通道a83两端各设有一个基质入口a81，所述通道b84两端各设有一个基质入口b82。
每条所述通道b84上的所述通道c85的数量为7-10个。
所述培养室9为类n状，两个培养室9组成类x状，每个培养室9两端分别设有培养室入口91和培养室出口92。
所述上层芯片1和所述下层芯片10为聚二甲基硅氧烷材质，所述多孔薄膜7为聚苯二甲酸乙二醇酯材质。
所述培养室9的深度为100-1000μm，所述基质室8的深度为50-200μm。
实施例2
本实施例在实施例1中所述开放式培养区5内加入脑微血管血管内皮细胞，所述培养室9内加入小胶质细胞，所述基质室8内加入基质液和按一定比例混合的星形胶质细胞和神经元细胞悬液，所述星形胶质细胞与神经元细胞的数量比为10:1～1:1。
实施例3
一种基于微流控芯片的疱疹性脑炎模型构建方法。具体过程如下：
采用matrigel工作液，将按一定比例混合的星形胶质细胞和神经元细胞悬液与之混合。用移液器将其加入基质入口a81和基质入口b82，每孔10-50μl；加1-5ml PBS缓冲液于培养皿中，将固定芯片的培养皿放入培养箱中孵育30min凝胶；脑微血管内皮细胞制成悬液，取100μl悬液加入开放式培养区5。小胶质细胞制成悬液，取50μl悬液分别加入两培养室入口91。将芯片放平移入37℃培养箱中培养、每隔24h换液一次。共培养5天后，在培养室入口91加入不同滴度的单纯疱疹病毒颗粒。病毒暴露2h后，培养基清洗。将芯片放平移入37℃培养箱中培养、每日换液一次；培养2天。在培养室9中灌流加入外周血单个核细胞，30min后，除去细胞悬液，培养基清洗。37℃培养箱中培养，每隔24h拍照记录单个核细胞位置，记录细胞运动及形态改变。进行细胞行为的跟踪观察，实时记录细胞黏附，运动、屏障功能破坏和细胞位置的改变。加入单个核细胞3天后，记录神经血管单元各类型细胞形态，其结果如图3所示。可见多种细胞出现细胞变形，皱缩，血管内皮细胞脱落。单个核细胞浸润入星形胶质细胞和神经元三维培养区，其结果如图4所示。
以上所述，仅是本申请的几个实施例，并非对本申请做任何形式的限制，虽然本申请以较佳实施例揭示如上，然而并非用以限制本申请，任何熟悉本专业的技术人员，在不脱离本申请技术方案的范围内，利用上述揭示的技术内容做出些许的变动或修饰均等同于等效实施案例，均属于技术方案范围内。
Claims (9)
1.一种微流控芯片在构建疱疹性脑炎模型中的应用，其特征在于，所述微流控芯片包括开放式培养区(5)、培养室(9)和基质室(8)，所述基质室(8)与开放式培养区(5)之间设有多孔薄膜(7)；在开放式培养区(5)内加入脑微血管内皮细胞，在培养室(9)内加入小胶质细胞，在基质室(8)内加入基质液和按一定比例混合的星形胶质细胞和神经元细胞悬液，构建获得疱疹性脑炎模型。
2.根据权利要求1所述的应用，其特征在于，所述星形胶质细胞与神经元细胞的数量比为10:1～1:1。
3.根据权利要求1所述的应用，其特征在于，所述微流控芯片包括上层芯片(1)和下层芯片(10)两层，所述上层芯片(1)设有通孔a(2)、通孔b(3)、通孔c(4)、通孔d(6)和通孔状的开放式培养区(5)；所述下层芯片(10)设有基质室(8)，所述基质室(8)设置于开放式培养区(5)下方，所述基质室(8)与开放式培养区(5)之间设有多孔薄膜(7)，所述基质室(8)两侧各设有一个培养室(9)，所述基质室(8)另外两侧均分别设有基质入口a(81)和基质入口b(82)，所述基质入口a(81)的位置与通孔a(2)的位置在竖直方向上相对应设置，所述基质入口b(82)的位置与通孔b(3)的位置在竖直方向上相对应设置；所述培养室(9)为条状，所述培养室(9)两端分别设有培养室入口(91)和培养室出口(92)，所述培养室入口(91)位置与通孔c(4)的位置在竖直方向上相对应设置，所述培养室出口(92)位置与通孔d(6)的位置在竖直方向上相对应设置。
4.根据权利要求3所述的应用，其特征在于，所述基质室(8)设有一条条状通道a(83)，所述通道a(83)两侧各设置一条与通道a(83)平行的条状通道b(84)，所述通道b(84)与所述培养室(9)平行，所述通道b(84)上设有数个与通道b(84)垂直的通道c(85)，所述通道c(85)使所述通道a(83)、所述通道b(84)、所述培养室(9)三者连通，数个所述通道c(85)与所述通道b(84)组合形成栅栏结构；所述通道a(83)两端各设有一个基质入口a(81)，所述通道b(84)两端各设有一个基质入口b(82)。
5.根据权利要求4所述的应用，其特征在于，每条所述通道b(84)上的所述通道c(85)的数量为7-10个。
6.根据权利要求3所述的应用，其特征在于，所述培养室(9)为类n状，两个培养室(9)组成类x状，每个培养室(9)两端分别设有培养室入口(91)和培养室出口(92)。
7.根据权利要求3所述的应用，其特征在于，所述上层芯片(1)和所述下层芯片(10)为聚二甲基硅氧烷材质，所述多孔薄膜(7)为聚苯二甲酸乙二醇酯材质。
8.根据权利要求3所述的应用，其特征在于，所述培养室(9)的深度为100-1000μm，所述基质室(8)的深度为50-200μm。
9.根据权利要求1所述的应用，其特征在于，所述疱疹性脑炎模型的应用方法，包括如下步骤：
(1)芯片基质灌注
采用matrigel工作液，将星形胶质细胞和神经元细胞悬液与工作液混合，加入基质入口a(81)、基质入口b(82)；加PBS缓冲液于培养皿中，将固定芯片的培养皿放入培养箱中孵育凝胶；
(2)脑微血管内皮细胞和小胶质细胞接种及培养
脑微血管内皮细胞制成悬液，取脑微血管内皮细胞悬液加入开放式培养区(5)内；小胶质细胞制成悬液，取小胶质细胞悬液分别加入到两个培养室入口(91)；将芯片放平移入培养箱中培养；
(3)芯片神经血管单元功能监测
采用血管内皮细胞钙粘蛋白VE-cadherin和Claudin-5检测芯片神经血管单元的完整性，采用FITC标记的右旋糖苷监测芯片神经血管单元血脑屏障的通透性；
(4)加入病毒颗粒
待多种细胞在芯片上共培养5-8天后，在培养室入口(91)加入不同滴度的单纯疱疹病毒颗粒；病毒暴露2-4h后，培养基清洗；将芯片放平移入培养箱中培养；
(5)加入外周血单个核细胞
在开放式培养区(5)中灌流加入外周血单个核细胞，模拟体内循环血液中的免疫细胞，记录细胞黏附，运动，屏障功能破坏和细胞位置的改变。
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