CN116254180A - 一种脑缺血再灌注损伤芯片及其在开发新药中的应用 - Google Patents
一种脑缺血再灌注损伤芯片及其在开发新药中的应用 Download PDFInfo
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Abstract
本发明公开了一种脑缺血再灌注损伤芯片,自下而上依次包括层叠设置的芯片基板、下层芯片、透气薄膜和上层芯片,其中,下层芯片上设置有多个相互连通的耗氧腔室,上层芯片上对应耗氧腔室的位置设置有细胞培养腔室,用于培养神经细胞;透气薄膜位于上层芯片与下层芯片之间,且为透气但不透液的薄膜;上层芯片和下层芯片上还设有对应的注射孔,注射孔与耗氧腔室连通,用于向耗氧腔室内注入耗氧试剂以模拟脑缺氧。本发明还公开了所述脑缺血再灌注损伤芯片的应用。本发明的脑缺血再灌注损伤芯片,实现了不依赖缺氧小室、三气培养箱等复杂设备就可以实现缺血再灌注损伤模型的构建,且可以进行原位的实时氧浓度监测和脑中风药物的研发。
Description
技术领域
本发明涉及器官芯片技术和药物药效评价技术领域,具体涉及一种脑缺血再灌注损伤芯片及其在开发新药中的应用。
背景技术
脑卒中,又名脑中风或脑血管意外,是一组以脑部缺血或出血性损伤症状为主要临床表现的疾病。中国卒中协会发布《中国卒中流行报告》,我国每12秒就有一人发生卒中,每21秒就有一人死于卒中,每年新发脑血管病患者约270万,且逐年呈上升趋势。据统计,全球25岁之后发生卒中的风险约为25%,不同地域和国家存在差异,中国风险最高,为39.3%,其次为中欧31.7%和东欧31.6%。脑卒中又分为缺血性脑卒中和出血性脑卒中两种类型,缺血性脑卒中占所有卒中的85%,具有高发病率、高病死率、高致残率和高复发率的特点。
目前,及时恢复缺血区域的血液灌流是临床上减小脑缺血梗死体积、减少病死率和致残率的最有效策略之一。然而,临床和实验研究均发现缺血区恢复血液灌流后缺血损伤进一步加重,这种血流再通后引起的继发性损伤称为脑缺血再灌注损伤。脑缺血再灌注损伤是缺血性脑卒中发病的重要病理生理环节,其急性期(<24h)主要的变化是神经细胞、胶质细胞及内皮细胞呈明显缺血改变,由中心坏死区和周围缺血半暗带组成。而在亚急性期(1-7天)脑组织明显水肿并伴随大量神经细胞消失,其特点是中心坏死区逐渐扩大,半暗带则逐渐减小。迄今为止,在治疗学上尚无理想的治疗脑缺血再灌注损伤的药物。所以脑缺血再灌注损伤的研究具有重要意义。
体外脑缺血再灌注损伤模型在研究再灌注损伤机制、缺血相关疾病和药物筛选及评价中发挥着重要作用。体外模拟是通过氧糖剥夺/复氧复糖(OGD/R)进行。目前,OGD/R体外模型必须借助三气培养箱、缺氧小室等仪器设备才能实现,且这些设备构建的缺氧是环境中缺氧,并不能反应培养基中氧水平的情况。
微流控芯片被认为是当代极为重要的新兴科学技术平台。是一种以在微米尺度空间对流体进行操控为主要特征的科学技术。主流形式的微流控芯片指的是把化学和生物等领域中所涉及的样品制备、反应、分离、检测,细胞培养、裂解等基本操作单元集成或基本集成到一块几平方厘米(甚至更小)的芯片上,由微通道形成网络,以可控流体贯穿整个系统,用以实现常规化学,生物,材料,光学等不同实验室的各种功能的一种技术。基于芯片设计灵活、消耗试剂少等特点,本研究应用微加工技术,构建脑缺血再灌注损伤芯片,并利用该芯片进行天然产物的药效学研究。
发明内容
本发明要解决的技术问题是提供一种脑缺血再灌注损伤芯片,基于芯片结构的灵活多变,利用PDMS薄膜透气不透水,在薄膜下腔室中加入耗氧试剂,再用PVDC膜将芯片缠绕以隔绝外界空气的影响,实现了芯片细胞腔室氧糖剥夺的技术,克服了依赖缺氧小室或者三气培养箱等设备的缺陷。
为了解决上述技术问题,本发明提供了一种脑缺血再灌注损伤芯片,自下而上依次包括层叠设置的芯片基板、下层芯片、透气薄膜和上层芯片,其中,所述下层芯片上设置有多个相互连通的耗氧腔室,所述上层芯片上对应所述耗氧腔室的位置设置有细胞培养腔室,用于培养神经细胞;所述透气薄膜位于上层芯片与下层芯片之间,所述透气薄膜为透气但不透液的薄膜;所述上层芯片和下层芯片上还设有对应的注射孔,所述注射孔与所述耗氧腔室连通,用于向所述耗氧腔室内注入耗氧试剂以模拟脑缺氧。
进一步地,所述芯片基板的材质可为玻璃、聚二甲基硅氧烷(PDMS)或聚甲基丙烯酸甲酯(PMMA)等透明材料,所述上层芯片、下层芯片的材质均为PDMS等透气的生物相容性材料;所述透气薄膜优选为厚度为100μm的PDMS薄膜。
进一步地,所述芯片基板的表面进行了常压等离子体处理,再依次与下层芯片、透气薄膜和上层芯片通过不可逆封接组装在一起。
本发明中,可根据实验需要灵活调整芯片尺寸和细胞培养腔室大小和数量。所述耗氧腔室的数量可为1~96个,例如可1个、2个、3个、4个、8个、12个、24个、36个、48个、96个等。所述芯片的边框尺寸可为(10~100)×(10~100)mm,例如10×10mm、20×20mm、30×30mm、50×50mm等;所述耗氧腔室的形状不限,优选为圆形,其直径可为2~20mm,例如2mm、4mm、5mm、8mm、10mm、20mm等。
所述耗氧腔室为多个,且相互连通。在一种实施方式中,耗氧腔室呈胡芦串结构。相邻两耗氧腔室的互通尺寸(即连接处的尺寸)可为(0.1~1.5)×(0.2~2.0)mm,优选为1.2×0.5mm。
注入孔的数量优选为两个,分别设于耗氧腔室的两端。其直径优选为1.5mm。
进一步地,所述神经细胞选自人原代细胞、动物原代细胞、人源细胞系、动物源细胞系和干细胞诱导分化的人源细胞,但不仅限于上述细胞来源。
进一步地,所述细胞的培养方式为基质胶中三维培养、球状培养、类器官培养或贴壁的二维培养,但不仅限于上述培养方式。
为了在芯片上形成缺氧环境,需要在细胞培养腔室、耗氧腔室之间封接上一层PDMS薄膜,PDMS薄膜只允许气体通过而阻止液体通过。当在下层注入耗氧试剂,就可以将上层细胞培养腔室中的氧气迅速消耗从而达到缺氧。PDMS薄膜的制作过程如下:先将光学玻璃进行硅烷化,目的是使其疏水;然后将PDMS倒到玻璃上,根据所需膜的厚度,精确调整好涂膜器的刻度,然后进行涂膜,之后放到90℃热板上固化,制作100μm厚的PDMS薄膜。之后进行等离子体处理后进行封接。
本发明还提供了所述的脑缺血再灌注损伤芯片在脑缺血再灌注损伤模型或氧糖剥夺/复氧复糖模型构建中的应用。
进一步地,构建脑缺血再灌注损伤模型的方法为:将芯片清洗干净后,进行高温高压灭菌处理;烘干后取出芯片,细胞培养腔室采用Matrix基质胶进行包被,置于培养箱包被过夜后,向胞培养腔室中接种SHSY5Y细胞,培养24h;接着,通过注射孔向耗氧腔室中注入NaSO3溶液;然后用PVDC膜围绕芯片缠绕,进行氧糖剥夺模拟脑缺血,氧糖剥夺的同时插入氧探针实时记录细胞培养腔室中的氧浓度;氧糖剥夺2h后,将PVDC膜去除,吸弃NaSO3溶液,将培养基更换为含葡萄糖的培养基,即构建得到脑缺血再灌注损伤模型。
本发明还提高了所述的脑缺血再灌注损伤芯片在脑中风药物开发中的应用。
三叶苷(trilobatin,TLB)是多穗石柯主要活性成分之一,目前,关于三叶苷的药理活性的报道不多,主要包括抗炎、抗病毒、抗氧化等。然而,三叶苷是否抗脑缺血再灌注损伤及其可能的作用机制还不是很清楚。因此,可通过本发明芯片探究三叶苷对脑缺血再灌注损伤的作用,旨在为三叶苷用于治疗缺血性脑卒中提供基础药理学依据。
同样的,利用本发明的脑缺血再灌注损伤芯片,可评价化合物C21对脑缺血再灌注损伤的保护作用。
本发明的有益效果:
1.本发明的脑缺血再灌注损伤芯片,简单、灵活、制作和加工的复杂度比其他方法均低,氧糖剥夺的实现不再依赖于缺氧小室、三气培养箱等复杂的设备。
2.本发明的脑缺血再灌注损伤芯片,可以原位检测氧气的实时浓度,根据亚硫酸钠的不同添加浓度可以获得不同的缺氧浓度。
3.本发明的脑缺血再灌注芯片可以在缺氧操作过程中置于显微镜下观察细胞状态,这是缺氧小室、三气培养箱等设备无法实现的。
附图说明
图1为脑缺血再灌注损伤芯片的设计图;
图2为PDMS薄膜的制作图;
图3为脑缺血再灌注芯片的实物图和原位实时监测氧浓度;
图4为脑缺血再灌注损伤芯片模型构建成功结果图;
图5为三叶苷降低脑缺血再灌注损伤芯片中SHSY5Y细胞ROS水平;
图6为C21对脑缺血再灌注损伤芯片中SHSY5Y细胞活力的影响;
图7为C21降低脑缺血再灌注损伤芯片中SHSY5Y细胞ROS水平;
图8为C21在不同氧糖剥夺条件下对HIF-1α基因水平的影响;
图9为C21在不同氧糖剥夺条件下对Nrf2基因水平的影响;
其中:1、第一基板;2、耗氧腔室;3、第一通孔;4、第三基板;5、第二通孔;6、细胞培养腔室。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。本文所使用的术语“及/或”包括一个或多个相关的所列项目的任意的和所有的组合。
实施例1:脑缺血再灌注损伤芯片构建及验证
临床和实验研究均发现缺血区恢复血液灌流后缺血损伤进一步加重,这种血流再通后引起的继发性损伤称为脑缺血再灌注损伤。脑缺血再灌注损伤是缺血性脑卒中发病的重要病理生理环节。脑缺血再灌注损伤机制复杂,可能涉及到自由基形成、细胞内钙超载、氧化应激等多方面。在治疗学方面,目前没有理想的治疗缺血性中风的药物。在研究平台方面,体外模拟脑缺血再灌注损伤模型是通过OGD/R进行。目前,OGD/R体外模型必须借助三气培养箱、缺氧小室等仪器设备才能实现,这些设备构建的缺氧是环境中缺氧,并不能反应培养基中氧水平的情况,且不能进行实时的显微镜观察。
1.芯片的设计
本实施例设计的脑缺血再灌注损伤芯片方法简单、灵活、制作和加工的复杂度不高。如图1所示,该芯片包括自下而上依次层叠设置的第一基板1、第二基板、第三基板4和第四基板。
第一基板1可以是玻璃做底衬,也可以是聚二甲基硅氧烷(PDMS)、聚甲基丙烯酸甲酯(PMMA)材料。
第二基板设置有葫芦串结构的耗氧腔室2,每个耗氧腔室2的直径是5mm,高度是2mm。耗氧腔室2的大小可以根据需要灵活变小或者变大;另外,第二基板上还设有两个第一通孔3,分别位于耗氧腔室2的两端,并与耗氧腔室2连通,用于向耗氧腔室2中加入耗氧物质。
第三基板4是PDMS薄膜,该薄膜的特点是透气不透水。制备过程如图2所示,预先将玻璃板进行硅烷化处理。图2A是未硅烷化的玻璃,图2B是硅烷化后的玻璃,具有明显的疏水特性,硅烷化的目的是可以将PDMS薄膜轻松从玻璃底板上揭起。PDMS混合物(基质:固化剂=10:1)经四维旋转混匀器混匀,再用真空泵抽去气泡,然后将PDMS混合物取适量置于硅烷化的玻璃上,用KTQ-III可调式涂膜器均匀涂布。制得的100μm PDMS薄膜如图2C所示。
第四基板上设置有第二通孔5和细胞培养腔室6,且第二通孔5和细胞培养腔室6分别对应于第一通孔3和耗氧腔室2设置。每个细胞培养腔室6的直径是5mm,高是4mm,腔室大小可以根据需要灵活变小或者变大。
2.芯片的组装
将第一基板清洗干净,自然风干后使用常压等离子体进行处理,依次和耗氧试剂腔室、PDMS薄膜、细胞培养腔室进行不可逆封接。在组装好的芯片中注入有色墨水后,芯片不漏液,封接成功,实物图如图3A、图3B所示。
3.脑缺血再灌注损伤模型的构建
将制备好的芯片清洗干净后进行高温高压灭菌处理,烘箱烘干4h后取出芯片,细胞培养腔室用Matrix基质胶进行包被,置于培养箱包被过夜后,向腔室中接种SHSY5Y细胞,细胞在芯片中生长24h后,接着在下腔室中通过通孔注入25%的NaSO3溶液,然后用PVDC膜围绕芯片缠绕两圈,进行氧糖剥夺模拟脑缺血,氧糖剥夺的同时插入氧探针实时记录上腔室中氧浓度,如图3C所示,20分钟后,细胞腔室中的氧浓度从20%迅速下降到5%左右,之后一直维持在2%左右。氧糖剥夺2h后,将PVDC膜去除,吸弃NaSO3溶液,将培养基更换为含葡萄糖的培养基,模拟脑缺血再灌注。如图4所示,正常组SHSY5Y细胞形态正常,荧光染色没有观察到ROS,而OGD/R模型组细胞皱缩,数量减少,部分细胞漂浮,荧光染色观察到ROS。结果说明了脑缺血再灌注损伤模型芯片构建和验证成功。
实施例2:利用脑缺血再灌注损伤芯片研究天然产物三叶苷的保护作用
脑缺血再灌注损伤机制复杂,与自由基的形成、细胞内钙超载、兴奋性氨基酸毒性等有关。ROS是指在生物体内与氧代谢有关的含氧自由基和易形成自由基的过氧化物的总称。ROS可作用于不饱和脂肪酸从而发生脂质过氧化,也可诱导DNA、RNA、多糖和氨基酸等大分子物质发生交联,继而失去或者原来的活性和功能。脑缺血再灌注损伤后,观察到ROS明显的升高,引起细胞氧化损伤。细胞内抗氧化应激损伤的防御机制主要是诱导抗氧化酶的生成,核转录因子E-2相关因子-2(nuclear factor-erythroid 2-related factor2,Nrf2)被诱导后进入胞核识别并结合抗氧化反应元件促进保护基因的转录。诱导多种Ⅱ相解毒酶生成对抗氧化损伤。
在芯片上氧糖剥夺2h,复氧复糖24h后显微镜下观察细胞状态,并进行ROS染色。结果如图5所示。
从图5中可以看出,与正常组相比,芯片上OGD/R后SHSY5Y细胞形态发生改变,且ROS增多,部分细胞死亡,用三叶苷处理后,细胞损伤情况明显得到改善,结果说明三叶苷可以通过抗氧化作用从而保护SHSY5Y细胞OGD/R损伤。
实施例3:利用脑缺血再灌注损伤芯片研究化合物C21的保护作用
C21对脑缺血再灌注损伤芯片中SHSY5Y细胞活力的影响结果如图6。从图中可以看出,氧糖剥夺后无论3h还是4.5h,模型组吸光度显著上升,考虑细胞周期尚未完成,可能是氧糖剥夺后,脱氢酶应激升高所致。氧糖剥夺3h,复氧复糖24h,无论加C21与否,均对细胞活力没有显著影响,氧糖剥夺4.5h,复氧复糖24h,细胞活力显著下降,加入C21可对细胞产生一定的保护作用。氧糖剥夺4.5h,复氧复糖48h,细胞活力进一步下降,加入C21依然会对细胞产生一定的保护作用。
C21降低脑缺血再灌注损伤芯片中SHSY5Y细胞ROS水平,结果如图7所示。从图7中可以看到,氧糖剥夺无论是3h后还是4.5h后,对照组与模型组中细胞ROS情况没有显著差异。在复氧复糖24h后,模型组中的ROS显著升高,C21可以抑制模型ROS的产生。复氧复糖48h后,细胞开始脱落,氧糖剥夺时间越长,复氧复糖后,脱落的细胞越多。
C21在不同氧糖剥夺条件下对HIF-1α基因水平的影响,结果如图8所示。从图8中可以看到,氧糖剥夺4.5h后,模型组的HIF-1a显著升高,但复氧复糖后,HIF-1a显著下降。对于氧糖剥夺3h组,复氧复糖24h,C21的加入可以使HIF-1a的表达进一步降低,但复氧复糖48后,C21的加入可以使HIF-1a的表达显著升高。对于氧糖剥夺4.5h组,复氧复糖24h,C21的加入没有显著影响,但复氧复糖48h后,C21的加入可以使HIF-1a的表达显著下降。
C21在不同氧糖剥夺条件下对Nrf2基因水平的影响,结果如图9所示。从图9中可以看出,Nrf2作为内源性抗氧化通路的开关,与HIF-1a的表达趋势相反,氧糖剥夺3h与4.5h,C21对Nrf2的表达的影响趋势相反,对于氧糖剥夺时间较短,损伤较轻的细胞,抗氧化内源作用的贡献高于外源C21添加产生的作用。对于氧糖剥夺时间较长的情况,与对照组相比,C21处理后明显增加Nrf2的水平。
综上,说明C21可以通过抗氧化作用从而保护SHSY5Y细胞OGD/R损伤。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (10)
1.一种脑缺血再灌注损伤芯片,其特征在于,自下而上依次包括层叠设置的芯片基板、下层芯片、透气薄膜和上层芯片,其中,所述下层芯片上设置有多个相互连通的耗氧腔室,所述上层芯片上对应所述耗氧腔室的位置设置有细胞培养腔室,用于培养神经细胞;所述透气薄膜位于上层芯片与下层芯片之间,所述透气薄膜为透气但不透液的薄膜;所述上层芯片和下层芯片上还设有对应的注射孔,所述注射孔与所述耗氧腔室连通,用于向所述耗氧腔室内注入耗氧试剂以模拟脑缺氧。
2.如权利要求1所述的一种脑缺血再灌注损伤芯片,其特征在于,所述芯片基板的材质为玻璃、PDMS或PMMA,所述上层芯片、下层芯片的材质均为PDMS,所述透气薄膜为PDMS薄膜。
3.如权利要求1所述的一种脑缺血再灌注损伤芯片,其特征在于,所述芯片基板的表面进行了等离子体处理,再依次与下层芯片、透气薄膜和上层芯片通过不可逆封接组装在一起。
4.如权利要求1所述的一种脑缺血再灌注损伤芯片,其特征在于,所述耗氧腔室的数量为1~96个。
5.如权利要求1所述的一种脑缺血再灌注损伤芯片,其特征在于,所述芯片的尺寸为(10~100)×(10~100)mm;所述耗氧腔室为圆形,其直径为2~20mm;所述耗氧腔室为多个,相邻两耗氧腔室的连接处的尺寸为(0.1~1.5)×(0.2~2.0)mm。
6.如权利要求1所述的一种脑缺血再灌注损伤芯片,其特征在于,所述神经细胞选自人原代细胞、动物原代细胞、人源细胞系、动物源细胞系和干细胞诱导分化的人源细胞。
7.如权利要求1所述的一种脑缺血再灌注损伤芯片,其特征在于,所述细胞的培养方式为基质胶中三维培养、球状培养、类器官培养或贴壁的二维培养。
8.权利要求1-7所述的脑缺血再灌注损伤芯片在脑缺血再灌注损伤模型或氧糖剥夺/复氧复糖模型构建中的应用。
9.如权利要求8所述的应用,其特征在于,构建脑缺血再灌注损伤模型的方法为:将芯片清洗干净后,进行高温高压灭菌处理;烘干后取出芯片,细胞培养腔室采用Matrix基质胶进行包被,置于培养箱包被过夜后,向胞培养腔室中接种SHSY5Y细胞,培养24h;
通过注射孔向耗氧腔室中注入NaSO3溶液;然后用PVDC膜围绕芯片缠绕,进行氧糖剥夺模拟脑缺血,氧糖剥夺的同时插入氧探针实时记录细胞培养腔室中的氧浓度;
氧糖剥夺2h后,将PVDC膜去除,吸弃NaSO3溶液,将培养基更换为含葡萄糖的培养基,即构建得到脑缺血再灌注损伤模型。
10.权利要求1-7所述的脑缺血再灌注损伤芯片在脑中风药物开发中的应用。
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