CN114181887A - 一种基于细菌纤维素复合凝胶材料构建体外肠道模型的方法 - Google Patents
一种基于细菌纤维素复合凝胶材料构建体外肠道模型的方法 Download PDFInfo
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Abstract
本发明公开了一种基于细菌纤维素复合凝胶材料构建体外肠道模型的方法。细菌纤维素、明胶和糖溶液经过共混,冷冻干燥得到复合凝胶,高温热处理,制备出具有高稳定特性的细菌纤维素复合凝胶;以细菌纤维素复合凝胶的薄片为肠道细胞的载体材料,经过体外培养,获得具有肠道组织相关生理功能的体外肠道模型。肠道细胞体外培养使用的载体材料绿色天然,无毒无害,可耐受长时间的体外细胞培养,制备工艺简单,经济环保。所构建的体外肠道模型在药物筛选、营养吸收评估、肠道菌群互作以及组织工程领域等方面具有广泛的应用。
Description
技术领域
本发明属于生物技术领域,具体涉及一种基于细菌纤维素复合凝胶材料构建体外肠道模型的方法。
背景技术
肠道在营养吸收、药物利用、与肠道菌群互作等方面发挥着至关重要的作用。体外肠道模型的构建可为医药学、食品营养学、生物学等领域的基础研究和应用研究提供便捷准确的体外分析途径。目前,主要采用的是基于Caco-2等肠道癌细胞的2D体外肠道模型和基于原代肠道细胞的3D类器官体外肠道模型。前者操作简便,但癌细胞本质使该体外肠道模型的生物功能与正常肠道组织有较大差别;后者表达的生物性状与正常肠道组织接近,但封闭的囊形结构使得应用阶段的操作变得复杂。因此,开发新型的体外肠道模型,使之既为2D的体外肠道模型,具有便捷的可操作性,又具备正常肠道组织的分化特性,已成为一个亟需解决的现状。
发明内容
有鉴于此,本发明的目的在于提供一种基于细菌纤维素复合凝胶材料构建体外肠道模型的方法,将细菌纤维素/明胶复合材料经过高温热处理,制备出具有高稳定特性的细菌纤维素/明胶复合凝胶,体外培养肠道细胞形成完整的肠道细胞层,获得具有正常肠道组织相关生理功能的体外肠道模型。
为实现上述发明目的,本发明提供如下技术方案:
本发明一方面提供了一种基于细菌纤维素复合凝胶材料构建体外肠道模型的方法,其包括如下步骤:细菌纤维素、明胶和糖经过溶解、共混、冷冻干燥和热处理,制备出复合凝胶;以复合凝胶为载体材料,体外培养肠道细胞形成完整的肠道细胞层,获得具有肠道组织相关生理功能的体外肠道模型。
作为本发明的优选方案,所述的细菌纤维素、明胶和糖经过溶解、共混、冷冻干燥和热处理,制备出复合凝胶,具体为:细菌纤维素在水中分散均匀;将明胶加入到细菌纤维素分散均匀的水溶液中,充分溶解;最后,将糖加入到细菌纤维素和明胶的混合溶液中,充分溶解,得到复合凝胶的制备溶液;将制备溶液转移至容器中,低温冷冻;冷冻后的样品经过真空冷冻干燥,制备得到细菌纤维素的复合凝胶;冷冻干燥的复合凝胶在高温环境中处理,得到最终的复合凝胶。
优选的,细菌纤维素在水分散可以采用超声分散、均质分散、搅拌分散等辅助方法,冷冻温度可选择常规的-20℃、-80℃、-196℃;高温环境中处理的温度为120~200℃,处理时间为0.5~4小时。
优选的,所述的糖为具有多羟基结构的水溶性糖类。更为优选的,所述的糖为单糖、二糖或多糖,单糖包括葡萄糖、果糖、半乳糖等中的一种或多种,二糖包括蔗糖、麦芽糖、乳糖等中的一种或多种,多糖包括可以水解后能生成多个单糖的淀粉、纤维素或低聚糖等。所述的低聚糖包括葡聚糖、低聚果糖、魔芋寡糖、菊粉等。
作为本发明的优选方案,细菌纤维素、明胶和糖溶解在溶剂水中分散和共混,细菌纤维素在水溶液中的浓度为1g/L~20g/L,明胶在水溶液中的浓度为1g/L~20g/L,糖在水溶液中的浓度为0.1g/L~6g/L。
作为本发明的优选方案,所使用的细菌纤维素包括来自各种微生物合成的细菌纤维素,去可以为未改性的细菌纤维素,或经过物理法、化学法和/或生物法处理的改性细菌纤维素。
本发明方法制备的用于构建体外肠道模型的细菌纤维素复合凝胶材料有利于肠道细胞的生长和正常分化。该细菌纤维素复合凝胶材料具有与肠道细胞相应来源肠道组织相接近的机械性能,复合凝胶材料杨氏模量在0.1~6MPa的范围内。优选的,用于构建体外肠道模型的细菌纤维素复合凝胶材料的孔隙与所选肠道细胞来源一致的物种特定肠道部位隐窝尺寸保持一致,合凝胶材料的孔隙直径为50~120μm,深度为50~290μm。优选的,用于构建体外肠道模型的细菌纤维素复合凝胶材料在液体中具有长期稳定性,细胞培养阶段能稳定维持其整体的形貌结构。
作为本发明的优选方案,所述体外培养肠道细胞包括肠道癌细胞(如Caco-2等)、肠道原代细胞(包括小肠部位和大肠部位的细胞),体外培养肠道细胞在细菌纤维素/明胶复合凝胶材料上可快速生长和分化。
本发明还提供了上述方法构建的体外肠道模型在评估药物、营养成分和其他成分的吸收或生物利用度中的应用。
本发明还提供了上述方法构建的体外肠道模型在评估肠道菌群与肠道上皮互作中的应用。
本发明还提供了上述方法构建的体外肠道模型在评估肠道其他功能中的应用。
本发明还提供了上述方法构建的体外肠道模型在肠道组织工程中的应用。
本发明的有益效果在于:本发明公开了一种基于细菌纤维素复合凝胶材料构建体外肠道模型的方法,主要利用细菌纤维素复合凝胶材料作为肠道细胞的载体材料,基于细菌纤维素复合凝胶材料与肠道组织在机械性能和隐窝结构上的匹配性以及细菌纤维素复合凝胶材料的结构稳定性,促进肠道细胞在细菌纤维素复合凝胶材料上的正常生长和分化。本发明构建的体外肠道模型具有模仿正常肠道组织的能力,可广泛用于评估药物、营养成分和其他成分的吸收或生物利用度,也可用于肠道菌群的功能研究,还可用于组织工程等领域。
本发明构建的体外肠道模型的优点在于:
本发明涉及的细菌纤维素复合凝胶材料具有成本低廉、绿色天然、生物相容性好的特点,其结构和机械性能可模拟正常肠道组织,促进肠道细胞的正常生长和分化,真实模拟体内肠道上皮组织。
为了达到一定的机械性能和水溶液中的稳定性,现有技术采用交联剂来制备载体材料,然而交联剂一般具有细胞毒性,需要除去得很干净才能用于细胞的培养。本发明在复合凝胶材料的制备过程中避免了具有生物毒性的交联剂的使用,通过对制备方法的创新,使用的原料都是天然的,交联方式也无毒无害,得到的复合凝胶材料仍具有足够的机械强度和水溶液里的稳定性。
本发明载体材料构建的体外肠道模型为开放式的二维肠道模型,与真实肠道组织保持一致,便于模型在各个领域的应用。
说明书附图
图1为本发明构建的细菌纤维素复合凝胶材料在低倍数下的扫描电镜照片。
图2为本发明构建的细菌纤维素复合凝胶材料在高倍数下的扫描电镜照片。
图3为Caco-2细胞在本发明构建的细菌纤维素复合凝胶材料上培养后的扫描电镜照片。
图4为Caco-2细胞在本发明构建的细菌纤维素复合凝胶材料上培养后的激光共聚焦显微镜照片。
图5为十二指肠细胞在本发明构建的细菌纤维素复合凝胶材料上培养后的扫描电镜照片。
图6为十二指肠细胞在本发明构建的细菌纤维素复合凝胶材料上培养后的激光共聚焦显微镜照片。
图7为本发明构建的十二指肠体外模型(实验样品)与传统Transwell体外肠道模型(对照)的细胞增殖能力比较(MTT)。
图8为本发明构建的十二指肠体外模型(实验样品)与传统Transwell体外肠道模型(对照)的碱性磷酸酶表达能力比较。
图9为本发明构建的十二指肠体外模型(实验样品)与传统Transwell体外肠道模型(对照)的基因分化能力比较。
图10为细菌纤维素复合凝胶材料与文献报道的人和猪的部分肠道组织的杨氏模量比较。
图11为结肠细胞在本发明构建的细菌纤维素复合凝胶材料上培养后的扫描电镜照片。
图12为结肠细胞在本发明构建的细菌纤维素复合凝胶材料上培养后的激光共聚焦显微镜照片。
图13为本发明构建的结肠体外模型(实验样品)与传统Transwell体外肠道模型(对照)的细胞增殖能力比较(MTT)。
图14为本发明构建的结肠体外模型(实验样品)与传统Transwell体外肠道模型(对照)的碱性磷酸酶表达能力比较。
图15为本发明构建的结肠体外模型(实验样品)与传统Transwell体外肠道模型(对照)的基因分化能力比较。
图16为本发明构建的结肠体外模型评估益生菌对致病菌大肠杆菌促进肠道炎症因子分泌的影响。
图17为本发明构建的结肠体外模型评估益生菌对肠道抗炎因子分泌的影响。
具体实施方式
下面将对本发明的优选实施例进行详细的描述,但又不限于下述的实施例。实施例中未注明具体条件的实验方法,通常按照常规条件或与本发明所述的领域相关的普通技术人员和科研人员通常所理解的条件进行。
实施例1、
1g细菌纤维素、1g明胶和0.1g葡萄糖共溶于100mL水中,冷冻干燥,在140℃下热处理3h,制备得到细菌纤维素复合凝胶。细菌纤维素复合凝胶的表面孔直径和深度在湿态下约为70μm,小鼠小肠的隐窝直径约为60μm,深度约130μm,细菌纤维素复合凝胶的孔尺寸与小肠组织的隐窝尺寸相当。
细菌纤维素/明胶复合凝胶的结构如图1和图2所示。将Caco-2细胞悬液以8×104cells/mL的密度接种到细菌纤维素/明胶复合凝胶上,在37℃下5%CO2的气氛中恒温培养。培养基为高糖培养基(DMEM),其中加入10%胎牛血清,1%非必须氨基酸和1%双抗(青霉素和链霉素)。经过5天的培养,在细菌纤维素/明胶复合凝胶上形成的细胞层形貌如图3所示。图4显示在细菌纤维素/明胶复合凝胶上Caco-2细胞生长良好。说明在细菌纤维素/明胶复合凝胶上,Caco-2细胞可以快速生长和分化,在短时间内形成常见的Caco-2细胞模型,而常规的基于Transwell的体外Caco-2细胞模型需要21天的培养。
实施例2、
四甲基哌啶氧化物(TEMPO)氧化的细菌纤维素1g,明胶1g和葡萄糖0.2g共溶于100mL水中,冷冻干燥,在140℃下热处理3h,制备得到细菌纤维素复合凝胶,其杨氏模量为0.10MPa。细菌纤维素复合凝胶的表面孔直径和深度在湿态下约为70μm,小鼠小肠的隐窝直径约为60μm,深度约130μm,细菌纤维素复合凝胶的孔尺寸与小肠组织的隐窝尺寸相当。
十二指肠原代隐窝细胞的分离按常规方法分离:取小鼠原代肠段,清洗干净后,剪成1mm左右的碎片,移入15ml离心管,加入10mL的EDTA(5mM)解离液,置于摇床上解离50min(其间换两次新鲜的解离液);用PBS洗涤3次,开始边摇边收集隐窝(先用100μm滤膜收集,再用70μm滤膜过滤)。收集完后放离心机1450r/min离心5min,换空白培养基再次离心,去上清,根据细胞量加入适量细胞完全培养基重悬,得到隐窝悬液。
接种十二指肠原代隐窝细胞前,先用1mg/mL基质胶溶液处理细菌纤维素/明胶复合凝胶2h,吸掉多余的液体。将隐窝悬液以1000个隐窝/mL的密度接种到细菌纤维素/明胶复合凝胶上,在37℃下5%CO2的气氛中恒温培养。未形成完整细胞单层前每2天更换一次培养基,形成完整细胞单层后每天更换一次培养基。培养基为肠道细胞培养的常规AdvancedDMEM/F12培养基,在此基础上添加1%HEPES(1M)、1%Anti-Anti(100X)和1%GlutaMAX,并在每mL培养基中添加50μL R-spondin、5μL EGF、10μL Y2763210、10μL N-acetylcysteine、10μL N-2、20μL B27、1μL LDN-193189等细胞因子。
在细菌纤维素/明胶复合凝胶上形成的十二指肠细胞层形貌如图5和图6所示,在细菌纤维素/明胶复合凝胶上,形成了十二指肠单细胞层。如图7所示,与常规的基于Transwell的体外十二指肠细胞模型相比,在细菌纤维素/明胶复合凝胶上,十二指肠细胞的生长状态更好,在1天到5天的培养周期内,MTT数值具有极显著的提高。同时,细胞分化更好,如图8所示,在细菌纤维素/明胶复合凝胶上的十二指肠细胞表达的碱性磷酸酶较之对照同样具有极显著的提高(p<0.001)。图9表明,与正常的肠道组织相比,在转录物水平上,在细菌纤维素/明胶复合凝胶上十二指肠细胞的多种表达更接近肠道组织。说明基于细菌纤维素/明胶复合凝胶构建的小肠体外模型具有更好的生长能力和功能表达,更接近真实的小肠组织。
实施例3、
四甲基哌啶氧化物(TEMPO)氧化的细菌纤维素1g,明胶1g和葡萄糖0.2g共溶于100mL水中,冷冻干燥,在140℃下热处理3h,制备得到细菌纤维素/明胶复合凝胶。其杨氏模量为0.10MPa,与文献报道的人的结肠和猪的直肠的杨氏模量在同一数量级,且与猪的肠道组织较为接近,如图10所示。细菌纤维素复合凝胶的表面孔直径和深度在湿态下约为70μm,小鼠结肠的隐窝顶端直径约为80-120μm,底部直径约为48-68μm,长度约为192~290μm。细菌纤维素复合凝胶的孔尺寸涵盖在结肠组织的隐窝尺寸范围内。
结肠原代隐窝细胞的分离按常规方法分离:取原代肠段,清洗干净后,剪成1mm左右的碎片,移入15ml离心管,加入10mL的EDTA(5mM)解离液,置于摇床上解离20min(其间换两次新鲜的解离液),再在37℃下酶解30min;用PBS洗涤3次,开始边摇边收集隐窝(先用100μm滤膜收集,再用70μm滤膜过滤)。收集完后放离心机1450r/min离心5min,换空白培养基再次离心,去上清,根据细胞量加入适量细胞完全培养基重悬,得到隐窝悬液。
接种结肠原代隐窝细胞前,先用1mg/mL基质胶溶液处理细菌纤维素/明胶复合凝胶2h,吸掉多余的液体。将隐窝悬液以1000个隐窝/mL的密度接种到细菌纤维素/明胶复合凝胶上,在37℃下5%CO2的气氛中恒温培养。未形成完整细胞单层前每2天更换一次培养基,形成完整细胞单层后每天更换一次培养基。培养基为肠道细胞培养的常规AdvancedDMEM/F12培养基,在此基础上添加1%HEPES(1M)、1%Anti-Anti(100X)和1%GlutaMAX,并在每mL培养基中添加50μL R-spondin、5μL EGF、10μL Y2763210、10μL N-acetylcysteine、10μL N-2、20μL B27、1μL LDN-193189等细胞因子。
在细菌纤维素/明胶复合凝胶上形成的结肠细胞层形貌如图11和图12所示,在细菌纤维素/明胶复合凝胶上,形成了结肠单细胞层。如图13所示,与常规的基于Transwell的体外结肠细胞模型相比,在细菌纤维素/明胶复合凝胶上,结肠细胞的生长状态更好,在1天到5天的培养周期内,MTT数值具有极显著的提高。同时,细胞分化更好,如图14所示,在细菌纤维素/明胶复合凝胶上的结肠细胞表达的碱性磷酸酶较之对照同样具有极显著的提高。图15表明,与正常的肠道组织相比,在转录物水平上,在细菌纤维素/明胶复合凝胶上结肠细胞的多种表达更接近肠道组织。说明基于细菌纤维素/明胶复合凝胶构建的结肠体外模型具有更好的生长能力和功能表达,更接近真实的结肠组织。
实施例4、
基于实施例3构建的体外结肠模型用于益生菌益生功能的评估。如图16和图17所示,该体外结肠模型构建成熟之后,加入致病菌大肠杆菌(Escherichia coli),发现肠道细胞的促炎因子TNF-α分泌增加;副干酪乳杆菌(L.paracasei)和双歧杆菌(Bifidobacterium)在一定程度上减少了TNF-α分泌,罗伊氏乳杆菌(L.reuteri)则没有影响。三种益生菌的上清液均能在一定程度上下调大肠杆菌对结肠细胞造成的炎症反应。整体而言,益生菌及其上清液对致病菌的炎症效应有一定的作用,但没有显著性。与此同时,体外结肠模型中添加大肠杆菌导致细胞的抗炎水平下降,与对照组(med)存在显著性差异(p<0.05);体外结肠模型中添加益生菌则能提高结肠细胞的抗炎水平(L.paracasei没有明显效果);在添加大肠杆菌的体外结肠模型中,益生菌及其上清液的加入,显著提高了结肠的抗炎反应。该体外结肠模型评估了三种常规益生菌对致病菌大肠杆菌造成的肠道炎症的影响,发现益生菌及其上清液可以在一定程度上缓解结肠的炎症反应,显著提高结肠细胞的抗炎能力。说明本方法构建的体外肠道模型可用于评估益生菌的益生功能。
最后说明的是,以上优选实施例仅用以说明本发明的技术方案而非限制,尽管通过上述优选实施例已经对本发明进行了详细的描述,但本领域技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离本发明权利要求书所限定的范围。
Claims (10)
1.一种基于细菌纤维素复合凝胶材料构建体外肠道模型的方法,其特征在于包括如下步骤:细菌纤维素、明胶和糖经过溶解、共混、冷冻干燥和热处理,制备出复合凝胶;以复合凝胶为载体材料,体外培养肠道细胞形成完整的肠道细胞层,获得具有肠道组织相关生理功能的体外肠道模型。
2.根据权利要求1所述的基于细菌纤维素复合凝胶材料构建体外肠道模型的方法,其特征在于,所述的糖为具有多羟基结构的水溶性糖类。
3.根据权利要求1所述的基于细菌纤维素复合凝胶材料构建体外肠道模型的方法,其特征在于,所述的糖为单糖、二糖或多糖,单糖包括葡萄糖、果糖、半乳糖中的一种或多种,二糖包括蔗糖、麦芽糖、乳糖中的一种或多种,多糖包括可以水解后能生成多个单糖的淀粉、纤维素或低聚糖。
4.根据权利要求1所述的基于细菌纤维素复合凝胶材料构建体外肠道模型的方法,其特征在于细菌纤维素、明胶和糖溶解在水中共混,细菌纤维素在水溶液中的浓度为1g/L~20g/L,明胶在水溶液中的浓度为1g/L~20g/L,糖在水溶液中的浓度为0.1g/L~6g/L。
5.根据权利要求1所述的基于细菌纤维素复合凝胶材料构建体外肠道模型的方法,其特征在于:所使用的细菌纤维素为未改性的细菌纤维素,或经过物理法、化学法和/或生物法处理的改性细菌纤维素。
6.根据权利要求1所述的基于细菌纤维素复合凝胶材料构建体外肠道模型的方法,其特征在于所述的细菌纤维素、明胶和糖经过溶解、共混、冷冻干燥和热处理,制备出复合凝胶,具体为:细菌纤维素在水中分散均匀;将明胶加入到细菌纤维素分散均匀的水溶液中,充分溶解;最后,将糖加入到细菌纤维素和明胶的混合溶液中,充分溶解,得到复合凝胶的制备溶液;将制备溶液转移至容器中,低温冷冻;冷冻后的样品经过真空冷冻干燥,制备得到细菌纤维素的复合凝胶;冷冻干燥的复合凝胶在高温环境中处理,温度为120~200℃,处理时间为0.5~4小时。
7.根据权利要求1-6任一项所述的基于细菌纤维素复合凝胶材料构建体外肠道模型的方法,其特征在于:用于构建体外肠道模型的细菌纤维素复合凝胶材料的杨氏模量在0.1~6MPa的范围内。
8.根据权利要求1-6任一项所述的基于细菌纤维素复合凝胶材料构建体外肠道模型的方法,其特征在于:用于构建体外肠道模型的细菌纤维素复合凝胶材料的孔隙直径为50~120μm,深度为50~290μm。
9.由权利要求1-6任一项所述方法构建的体外肠道模型在评估药物、营养物质及其他成分的吸收或生物利用度中的应用。
10.由权利要求1-6任一项所述方法构建的体外肠道模型在评估肠道菌群与肠道的互作中的应用以及在组织工程中的应用。
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