CN112342183A - 一种仔猪胃肠道体外环境模型的建立方法以及应用 - Google Patents
一种仔猪胃肠道体外环境模型的建立方法以及应用 Download PDFInfo
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Abstract
本发明属于细胞模型构建的技术领域，具体涉及一种仔猪胃肠道体外环境模型的建立方法以及应用，包括如下步骤：选取仔猪的小肠上皮细胞系和胃黏膜上皮细胞系，进行细胞传代培养，待细胞传代3～4代至细胞活力较强，采用trypsin‑EDTA进行消化、计数并利用Transwell培养体系接种细胞，培养之后弃完全培养基，采用PBS洗涤，添加分化培养基，通过胃黏膜上皮细胞GES‑1分泌的因子影响IPEC‑1细胞的分化及形态，该模型中的IPEC‑1细胞对氮营养素的响应机制与仔猪空肠黏膜细胞对日粮氮水平的响应机制基本一致，为体外研究氮营养素响应机制建立了模型，更进一步推动生猪肠道营养及健康研究的发展。
Description
技术领域
本发明属于细胞模型构建的技术领域，具体涉及一种仔猪胃肠道体外环境模型的建立方法以及应用。
背景技术
细胞在体外的培养通常首先模拟体内环境，包括无菌、适宜温度、酸碱度和一定营养条件等，使得在体外的细胞能够生存、生长、繁殖并维持主要结构和功能，从而进行细胞信号转导、细胞代谢、细胞增殖以及细胞死亡等相关研究。然而，单个细胞存在细胞出现单一型化，与体内细胞环境仍有很大差异，单细胞培养不能很好的模拟体内细胞环境，存在科学研究的局限性。
目前，尚未发现体外氮营养素响应的研究，肠道是主要的消化器官，肠道健康直接影响动物健康及生长。而肠上皮细胞作为主要的功能性细胞，通过调节消化吸收、肠道屏障、宿主防御及炎症等功能，保持肠道稳态及健康。然而，胃的分泌的相关因子影响肠道生理环境及功能。
肠上皮细胞IPEC-1细胞常用于肠道氧化应激、免疫应激等肠道健康相关研究，然而，常出现与体内研究结果不一致的现象。
发明内容
针对上述问题，本发明的目的在于提供一种仔猪胃肠道体外环境模型的建立方法以及应用，利用Transwell培养体系，建立了猪小肠上皮细胞IPEC-1细胞系与胃粘膜上皮细胞GES-1共培养的系统，成功建立了仔猪胃肠道环境的模型。
本发明的技术内容如下：
本发明的一种仔猪胃肠道体外环境模型的建立方法，包括如下步骤：
选取仔猪的小肠上皮细胞系和胃黏膜上皮细胞系，进行细胞传代培养，待细胞传代3～4代至细胞活力较强，采用trypsin-EDTA进行消化、计数并利用Transwell培养体系接种细胞，在37℃，5％CO2条件下培养，之后弃完全培养基，采用PBS洗涤，添加分化培养基；
所述细胞系包括小肠上皮细胞IPEC-1细胞系和胃黏膜上皮细胞GES-1细胞系，分别进行复苏、生长至80％～90％之后进行细胞传代；
所述细胞传代于完全培养基中培养，所述完全培养基的组成包括基础培养基、5～10％胎牛血清以及0.05～0.10μg/ml表皮生长因子(Epidermal growth factor，EGF)；
所述分化培养基的组成包括基础培养基、0.5～1％ITS(Insulin-Transferrin-Selenium)、以及0.05～0.10μg/ml表皮生长因子(Epidermal growth factor，EGF)，分化15～20天，所述分化培养基能够满足细胞增殖需要、促进细胞的功能性分化、模拟肠上皮细胞的体内微环境；
所述基础培养基的组成包括甘氨酸、丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酸、谷氨酰胺、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、丙苯氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸以及其它营养成分(生物素、胆碱、泛素钙、烟酰胺、VB6、核黄素、硫胺素、VB12、肌醇、葡萄糖、HEPES、次黄嘌呤、亚油酸、硫辛酸、酚红、腐胺、丙酸钠、胸腺嘧啶、CaCl2、CuSO4、Fe(NO3)3、FeSO4、MgCl2、MgSO4、KCl、NaCl、Na2HPO4、NaH2PO4以及ZnSO4)；
所述Transwell培养体系接种细胞为采用6孔或96孔Transwell；
所述6孔Transwell下室接种2×105～3×105个/孔的GES-1细胞，上室接种2×105～3×105个/孔IPEC-1细胞。
所述96孔Transwell下室接种5×103～1×104个/孔的GES-1细胞，上室接种5×103～1×104个/孔IPEC-1细胞。
本发明所建立的仔猪胃肠道体外环境模型用于模拟仔猪胃肠道环境。
本发明所建立的仔猪胃肠道体外环境模型用于体外研究仔猪氮营养素响应机制。
本发明的有益效果如下：
本发明所建立的仔猪胃肠道环境体外模型，成功地模拟了仔猪肠道黏膜环境，在Transwell培养体系下，通过胃黏膜上皮细胞GES-1分泌的因子影响IPEC-1细胞的分化及形态，该模型中的IPEC-1细胞对氮营养素的响应机制与仔猪空肠黏膜细胞对日粮氮水平的响应机制基本一致，通过体外建立仔猪胃肠道环境的模型，研究氮营养素响应机制，比肠上皮细胞的单培养研究更具有科学性，为体外研究氮营养素响应机制建立了模型，更进一步推动生猪肠道营养及健康研究的发展。
附图说明
图1为Transwell单细胞培养和本发明模型的结构示意图；
图2为单细胞培养和本发明模型中的IPEC-1细胞形态；
图3单细胞培养和本发明模型中IPEC-1细胞增殖情况。
具体实施方式
以下通过具体的实施案例以及附图说明对本发明作进一步详细的描述，应理解这些实施例仅用于说明本发明而不用于限制本发明的保护范围，在阅读了本发明之后，本领域技术人员对本发明的各种等价形式的修改均落于本申请所附权利要求所限定。
若无特殊说明，本发明的所有原料和试剂均为常规市场的原料、试剂。
实施例1
一种仔猪胃肠道体外环境模型的建立：
选用仔猪小肠上皮细胞IPEC-1细胞系以及胃粘膜上皮细胞GES-1细胞系，分别复苏，待细胞长至80％～90％进行细胞传代，完全培养基为基础培养基、5％～10％的胎牛血清及0.05μg/ml的表皮生长因子(Epidermal growth factor，EGF)；
细胞传代3～4代至细胞活力较强，0.25％trypsin-EDTA进行消化、计数并利用Transwell培养体系接种细胞；
采用6孔Transwell下室接种2×105个/孔的GES-1细胞，上室接种2×105个/孔IPEC-1细胞；
采用96孔Transwell下室接种5×103个/孔的GES-1细胞，上室接种5×103个/孔IPEC-1细胞；
37℃，5％CO2条件下培养24h后，弃完全培养基，1×PBS洗3次，添加分化培养基(基础培养基、0.5％ITS(Insulin-Transferrin-Selenium)、0.05μg/ml EGF分化15天后，IPEC-1模型进行后续研究。
所述基础培养基的成分如表1所示，并且配制不同氮营养素水平的培养基NNL(179mg/L)、LNL(152mg/L)、VLNL(125mg/L)，用于细胞培养及氮营养素响应机制研究。
表1基础培养基组成(mg/L)
其它营养成分为每升培养基添加物质：生物素3.5μg/L、胆碱8.98mg/L、泛素钙2.24mg/L、叶酸2.65mg/L、烟酰胺2.02mg/L、VB62.03mg/L、核黄素0.219mg/L、硫胺素2.17mg/L、VB120.68 mg/L、肌醇12.6mg/L、葡萄糖3.15g/L、HEPES 3.58g/L、次黄嘌呤2.39mg/L、亚油酸42μg/L、硫辛酸105μg/L、酚红8.1mg/L、腐胺81μg/L、丙酸钠55mg/L、胸腺嘧啶365μg/L CaCl2117 mg/L、CuSO4 1.3μg/L、Fe(NO3)3 0.05mg/L、FeSO4 417μg/L、MgCl228.6mg/L、MgSO4 48.8mg/L；KCl，312mg/L、NaCl 7g/L、Na2HPO4，71.0mg/L、NaH2PO4 62.5mg/L、ZnSO4 432μg/L。
实施例2
一种仔猪胃肠道体外环境模型的建立：
选用仔猪小肠上皮细胞IPEC-1细胞系以及胃粘膜上皮细胞GES-1细胞系，分别复苏，待细胞长至80％～90％进行细胞传代，完全培养基为基础培养基、5％的胎牛血清及0.05μg/ml的表皮生长因子(Epidermal growth factor，EGF)；
细胞传代3～4代至细胞活力较强，0.25％trypsin-EDTA进行消化、计数并利用Transwell培养体系接种细胞；
采用6孔Transwell下室接种3×105个/孔的GES-1细胞，上室接种3×105个/孔IPEC-1细胞；
37℃，5％CO2条件下培养24h后，弃完全培养基，1×PBS洗3次，添加分化培养基(基础培养基、0.6％ITS(Insulin-Transferrin-Selenium)、0.08μg/ml EGF)分化16天后，IPEC-1模型进行后续研究。
实施例3
一种仔猪胃肠道体外环境模型的建立：
选用仔猪小肠上皮细胞IPEC-1细胞系以及胃粘膜上皮细胞GES-1细胞系，分别复苏，待细胞长至80％～90％进行细胞传代，完全培养基为基础培养基、8％的胎牛血清及0.10μg/ml的表皮生长因子(Epidermal growth factor，EGF)；
细胞传代3～4代至细胞活力较强，0.25％trypsin-EDTA进行消化、计数并利用Transwell培养体系接种细胞；
采用96孔Transwell下室接种1×104个/孔的GES-1细胞，上室接种1×104个/孔IPEC-1细胞；
37℃，5％CO2条件下培养24h后，弃完全培养基，1×PBS洗3次，添加分化培养基(基础培养基、0.5％-1％ITS(Insulin-Transferrin-Selenium)、0.08μg/ml EGF)分化18天后，IPEC-1模型进行后续研究。
实施例4
一种仔猪胃肠道体外环境模型的建立：
选用仔猪小肠上皮细胞IPEC-1细胞系以及胃粘膜上皮细胞GES-1细胞系，分别复苏，待细胞长至80％～90％进行细胞传代，完全培养基为基础培养基、10％的胎牛血清及0.10μg/ml的表皮生长因子(Epidermal growth factor，EGF)；
细胞传代3～4代至细胞活力较强，0.25％trypsin-EDTA进行消化、计数并利用Transwell培养体系接种细胞；
6孔Transwell下室接种3×105个/孔的GES-1细胞，上室接种3×105个/孔IPEC-1细胞；
37℃，5％CO2条件下培养24h后，弃完全培养基，1×PBS洗3次，添加分化培养基(基础培养基、1％ITS(Insulin-Transferrin-Selenium)、0.1μg/ml EGF)分化20天后，IPEC-1模型进行后续研究。
将得到的体外细胞模型用于动物试验设计：
选择108头(杜×长×大)三元杂21日龄断奶仔猪随机分为3组，每组6个重复，每个重复6头，对照组(NCP)饲喂基础日粮(NRC2012)，处理组日粮粗蛋白水平分别降低组3％(LCP)及6％(VLCP)，试验周期为2周，试验期间动物进行自由采食及饮水。
试验结束前12小时断料，不断水。试验结束当天，每栏挑选1头接近平均体重的仔猪，每组共6头仔猪。进行麻醉后，放血，将腹腔破开，立即取空肠组织，用预冷1×PBS冲洗，纵向剪开，滤纸吸去表面水分，用载玻片刮取黏膜组织并分装于1.5mL离心管中，立即至于液氮中，待采样结束后将样品转移至-80℃保存备用。
选用仔猪小肠上皮细胞IPEC-1细胞系以及胃粘膜上皮细胞GES-1细胞系按照实施例1中的方法建立体外模型，在所建立的细胞体外模型中，选取培养的IPEC-1细胞和GES-1细胞，用TRIzol试剂盒提取总RNA(Takara，Ostu，Japan)，提取的RNA溶解在RNase-free水中，将RNA(1μg)反转录为cDNA(Takara，Cat.#RR047A)；
利用primer premier 5.0(Applied Biosystems，Carlsbad，CA)进行设计引物，如表2所示，并进行Real-time PCR检测分析，反应体系如下(总体积20μL):2μL cDNA，10μLSYBR Green mix(Bio-Rad，1725265)，上下游引物各0.8μL(100nM)，6.4μL ddH2O。Real-time PCR(Bio-Rad System)条件为预变性95℃，3min；变性95℃，15s；退火30s；延伸72℃，30s；扩增39个循环，β-actin为Real-time PCR的内参基因。
表2 Real-time PCR引物信息Artificial Sequence
所设计的引物进行PCR检测结果的分析如下所述。
1.单细胞培养与模型中IPEC-1细胞形态学观察
图1中左图为采用Transwell体系培养IPEC-1单细胞，右图为采用本发明Transwell体系上室培养IPEC-1细胞、下室培养GES-1细胞的模型，对单细胞培养与模型中IPEC-1细胞进行形态学观察，如图2所示，单细胞培养中IPEC-1细胞较模型中IPEC-1细胞的体积小，其立体结构性较差，由此可见，模型中Transwell下室中的GES-1细胞分泌的因子透过Transwell聚碳酸酯膜，影响IPEC-1细胞分化，从而影响IPEC-1形态，因此，模型中的IPEC-1细胞更接近肠道上皮细胞形态，更适合应用于肠道相关研究。
2.单细胞培养与模型中IPEC-1细胞增殖的影响
利用96孔transwell进行单细胞培养与模型中IPEC-1细胞培养，利用MTT法，每24h测定IPEC-1细胞增殖情况。
由图3可知，氮营养素对单细胞培养IPEC-1细胞或模型中IPEC-1细胞的增殖无显著影响(P>0.05)。
3.仔猪肠道黏膜氨基酸转运及蛋白合成信号通路的影响
如表3所示，降低日粮粗蛋白水平可显著降低EAAT3及CAT1氨基酸转运载体的表达(P<0.05)，同时降低mTOR-MAPK、Class3PI3K表达(P<0.05)。而与NCP组相比，VLCP显著降低PI3K、TSC2、mTORC1及S6K1表达(P<0.05)。由此可见，降低日粮蛋白水平通过mTOR-MAPK、Class 3PI3K信号通路调控氨基酸转运，同时通过PI3K、TSC2、mTORC1信号通路调控下游S6K1基因表达，从而降低仔猪空肠黏膜蛋白合成。
表3日粮蛋白水平对仔猪空肠蛋白转运及合成信号通路的影响
4.单细胞培养IPEC-1细胞中氨基酸转运及蛋白合成信号通路的影响
由表4可知，与NNL培养基相比，降低氮水平减少IPEC-1细胞中PI3K、Class 3PI3K、TSC2及4E-BP1表达(P<0.05)，VLNL增加EAAT3及TGF-β表达(P<0.05)，LNL增加4F2hc，降低EIF4E表达(P<0.05)；
由此可见，降低氮水平通过PI3K、Class 3 PI3K、TSC2信号通路调控下游4E-BP1基因表达，从而降低仔猪IPEC-1细胞的蛋白合成。
表4培养基氮水平对单细胞培养IPEC-1细胞氨基酸转运及蛋白合成信号通路的影响
5.模型中IPEC-1细胞中氨基酸转运及蛋白合成信号通路的影响
由表5可知，与NNL培养基相比，降低氮水平降低IPEC-1细胞中氨基酸转运载体B0+AT、Y+LAT1表达，增加EAAT3转运载体表达(P<0.05)。降低氮水平降低IPEC-1细胞中mTOR-MAPK、Class 3 PI3K的转录表达(P<0.05)。VLNL降低PI3K、TSC2、S6K1及LC3B的表达的基因表达(P<0.05)；
由此可见，降低氮水平通过mTOR-MAPK、Class 3 PI3K信号通路调控IPEC-1细胞氨基酸转运，同时通过PI3K、TSC2信号通路调控下游S6K1基因表达，从而调节模型中IPEC-1细胞的蛋白合成。
表5氮水平对模型中IPEC-1细胞氨基酸转运及蛋白合成信号通路的影响
综上所述，胃粘膜上皮细胞GES-1分泌的因子影响IPEC-1细胞的分化及形态。同时，该模型中的IPEC-1细胞对氮营养素的响应机制与仔猪空肠黏膜细胞对日粮氮水平的响应机制基本一致，而单细胞培养的IPEC-1细胞对氮营养素的响应机制与仔猪空肠黏膜细胞的响应机制存在差异。因此，该模型成功模拟仔猪肠道粘膜环境，可应用于肠道氮营养素调控等机制研究。
序列表
<110> 广东省农业科学院动物科学研究所
<120> 一种仔猪胃肠道体外环境模型的建立方法以及应用
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Claims (8)
1.一种仔猪胃肠道体外环境模型的建立方法，其特征在于，包括如下步骤：选取仔猪的小肠上皮细胞系和胃黏膜上皮细胞系，进行细胞传代培养，待细胞传代3～4代至细胞活力较强，采用trypsin-EDTA进行消化、计数并利用Transwell培养体系接种细胞，在37℃，5％CO2条件下培养，之后弃完全培养基，采用PBS洗涤，添加分化培养基。
2.由权利要求1所述的体外环境模型的建立方法，其特征在于，所述细胞系包括小肠上皮细胞IPEC-1细胞系和胃黏膜上皮细胞GES-1细胞系，分别进行复苏、生长至80％～90％之后进行细胞传代。
3.由权利要求1所述的体外环境模型的建立方法，其特征在于，所述细胞传代于完全培养基中培养，所述完全培养基的组成包括基础培养基、5～10％胎牛血清以及0.05～0.10μg/ml表皮生长因子。
4.由权利要求1所述的体外环境模型的建立方法，其特征在于，所述分化培养基的组成包括基础培养基、0.5～1％ITS、以及0.05～0.10μg/ml表皮生长因子，分化15～20天。
5.由权利要求1所述的体外环境模型的建立方法，其特征在于，所述Transwell培养体系接种细胞为采用6孔或96孔Transwell；所述6孔Transwell下室接种2×105～3×105个/孔的GES-1细胞，上室接种2×105～3×105个/孔IPEC-1细胞。
6.由权利要求5所述的体外环境模型的建立方法，其特征在于，所述96孔Transwell下室接种5×103～1×104个/孔的GES-1细胞，上室接种5×103～1×104个/孔IPEC-1细胞。
7.一种权利要求1～6任一项所述方法所建立的仔猪胃肠道体外环境模型用于模拟仔猪胃肠道环境。
8.一种权利要求1～6任一项所述方法所建立的仔猪胃肠道体外环境模型用于体外研究仔猪氮营养素响应机制。
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| CN117503800A (zh) * | 2024-01-04 | 2024-02-06 | 北京益华生物科技有限公司 | 一种胃黏膜上皮细胞提取液及其制备方法与应用 |
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