CN113025561A - 一种母乳外泌体及其制备方法与应用 - Google Patents
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Abstract
本发明公开了一种母乳外泌体及其制备方法与应用，具体为母乳外泌体在制备肠道上皮屏障蛋白保护剂或者损伤修复剂中的应用。现有技术公开了母乳外泌体促进肠道细胞迁移与增殖，对抗肠道损伤，但是没有给出母乳外泌体对肠道上皮屏障蛋白的作用启示。本发明创造性的提出母乳外泌体在制备肠道上皮屏障蛋白保护剂中的应用，为母乳外泌体的新应用，扩展了母乳外泌体的应用范围。
Description
技术领域
本发明属于新生儿科技术领域，具体涉及母乳外泌体及其制备方法与应用。
背景技术
新生儿坏死性小肠结肠炎（NEC）为一种获得性疾病，是多种原因引起的肠黏膜损害，使之缺血、缺氧，导致小肠、结肠发生弥漫性或局部坏死的一种疾病。尽管新生儿医疗和全球新生儿重症监护室（NICUs）的建立，新生儿坏死性小肠结肠炎（NEC）仍是一种毁灭性疾病，特别是早产儿。外泌体是从细胞中释放的微小（100-200 nm）小泡，携带蛋白质、微RNA（miRNA）和信使RNA（mRNA），可以信号和调控细胞间行为，作为粒径纳米级的天然囊泡具有介导细胞间信号转导、抗原呈递和兔疫应答等功能。母乳中富含外泌体，可促进细胞增殖和迁移，减轻炎症，对新生儿相关疾病具有保护潜能，现有技术分析母乳性黄疸患儿肠道菌群特征,筛选与差异细菌有相互作用位点的母乳外泌体miRNA,说明miRNA可能通过调节乳儿肠道菌群影响新生儿母乳性黄疸的发生发展。利用16S rRNA高通量测序分析两组粪便样本肠道菌群特征,筛选出可能与新生儿胆红素代谢相关的差异性特殊菌种,然后用透射电镜和Western blotting两种方法鉴定从母乳中提取的外泌体囊泡,并通过miRBase数据库预测与特异性细菌基因组有相互作用位点的miRNA,利用荧光定量PCR验证预测的miRNA在母乳外泌体中表达的差异,探究母乳中差异表达的miRNA与肠道菌群的相关性。但是未见母乳外泌体对肠道上皮屏障蛋白的影响报道。
发明内容
本发明公开了母乳外泌体的新应用，公开了母乳外泌体对肠道上皮屏障蛋白的保护。现有技术探究母乳外泌体miRNA与肠道菌群的相关性，通过miRBase数据库预测与E.coli基因组有相互作用位点的miRNA，筛选出组间存在差异表达的分子，经预测，hsa-miR-16-5p与大肠杆菌ATP结合蛋白基因具有相互作用位点。采取预扩增法定量miRNA，结果发现miR-16-5p在两组间表达具有显著差异。但是该研究与母乳外泌体对肠道上皮屏障蛋白的作用无关。
本发明采用如下技术方案：
母乳外泌体在制备肠道上皮屏障蛋白保护剂中的应用。
母乳外泌体在制备结肠炎治疗剂中的应用。
本发明中，母乳为人母乳。母乳外泌体的制备方法为，将母乳离心处理后，取乳清用母乳外泌体提取试剂盒提取，得到母乳外泌体。优选的，离心处理、试剂盒提取都在室温下进行，离心处理为2000×g离心10分钟。
本发明中，保护剂为药物、食物、营养品或者保健品，以母乳外泌体为活性成分即可；治疗剂为药物、食物、营养品或者保健品，以母乳外泌体为活性成分即可。
本发明中，肠道上皮屏障蛋白为肠上皮细胞紧密连接蛋白，具体为咬合蛋白（Occludin）、闭合蛋白（Claudin1）及闭合小环蛋白（ZO-1，一种胞浆蛋白）。
本发明中，结肠炎为新生儿坏死性小肠结肠炎。
目前母乳缓解或者治疗新生儿坏死性小肠结肠炎的机理不明，现有技术公开了母乳外泌体促进肠道细胞迁移与增殖，对抗肠道损伤，但是没有给出母乳外泌体对肠道上皮屏障蛋白的作用启示。本发明创造性的提出母乳外泌体在制备肠道上皮屏障蛋白保护剂中的应用，为母乳外泌体的新应用，扩展了母乳外泌体的应用范围。
附图说明
图1为母乳外泌体的TEM图；
图2为母乳外泌体的粒径分布；
图3为母乳外泌体的蛋白表达电泳图；
图4为母乳外泌体的蛋白含量；
图5为mRNA的表达（Caco-2）；
图6为mRNA的表达（NCM460）；
图7为肠道上皮屏障蛋白电泳图（Caco-2）；
图8为肠道上皮屏障蛋白电泳图（NCM460）；
图9为不同制备方法得到的母乳外泌体mRNA的表达（Caco-2）；
图10为动物实验结果。
具体实施方式
在签署知情同意书后，招募健康的哺乳期母亲，她们在分娩后在苏州儿童医院住院，33名母亲分娩了足月儿（≥37周）、21名母亲分娩了早产儿（24-36周）。所有的母亲都生产过多的母乳，这些母乳通常被丢弃，这些多余的母乳样本是为研究目的而收集的。泌乳时间，在婴儿出生后的第3天采集初乳样本。对于出院的婴儿，在新生儿随访门诊采集其母亲的母乳，时间为出生后3-10天。母乳采集得到了母亲和苏州儿童医院伦理委员会的批准。
ZO-1、claudin 1和occludin的mRNA表达水平通过两步RT-qPCR进行检测。用TRIzol试剂(Thermo Fisher Scientific, Inc., US)从细胞中提取mRNA。用qScript cDNASuperMix（Quantabio，US）和S1000热循环器（Bio-Rad Laboratories, Inc., US）制备互补DNA（cDNA）。RT-qPCR采用Advanced qPCR Master Mix与CFX384实时系统(Bio-RadLaboratories, Inc.)进行，以甘油醛-3-磷酸脱氢酶（GAPDH）做内参；引物如下：
ZO-1: 正向引物, 5ʹ-TGCCATTACACGGTCCTCTG-3ʹ；反向引物, 5ʹ-GGTTCTGCCTCATCATTTCCTC-3ʹ
Claudin 1: 正向引物, 5ʹ-GAGCGAGTCATGGCCAAC-3ʹ；反向引物, 5ʹ-TCTCAATGTCCATTTTCGGTTT-3ʹ
Occludin: 正向引物, 5ʹ-AGTGTGATAATAGTGAGTGCTATCC-3ʹ；反向引物5ʹ-TGTCATACCTGTCCATCTTTCTTC-3ʹ。
蛋白质印迹（Western blot.）。细胞接种在6孔板上，密度为3.5×105，用RIPA缓冲液（150mM NaCl，1.0%Triton X-100，0.5%脱氧胆酸钠，0.1%十二烷基硫酸钠，1mM苯甲基磺酰氟和50mM Tris；pH 8.0）溶解。总蛋白浓度用常规BCA法测定，调整至1μg/μl；添加5倍加样缓冲液并在100℃下煮沸10分钟，之后将每个样品的蛋白质体积分离到10%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳。蛋白质从凝胶转移到PVDF膜(Millipore Corporation, US)上，然后室温封闭1小时（TBS，5%脱脂牛奶，0.05% TBS-T），进行一抗孵育(anti-ZO-1，1:1000,anti-occludin，1:1000, anti-claudin-1，1:1000, anti-β-actin，1:5000)，4℃过夜；然后在吐温-20（TBS-T）缓冲液中洗涤后，将膜在室温下放置1h进行常规二级抗体孵育。用Immobilon Western Substrate (Millipore Corporation, US)检测蛋白条带基质，并用生物图像分析系统(Syngene, US)分析，内源性参考蛋白GAPDH比较。
以上这些测试都是常规方法；提取母乳外泌体时，具体离心以及试剂盒操作都是常规方法。
实施例一
母乳收集后放入50ml无菌试管中，用冰袋冷藏保存，运输至实验室进行处理。室温下，以2000×g离心10min，然后常规取乳清，转移到另一管中，并使用外泌体提取试剂盒（Total
Exosome Isolation Kit ，Invitrogen，美国），按照试剂盒的方法提取外泌体并在−80°C下储存；剩余的母乳在−20°C下储存（不含外泌体的母乳）。
用透射电子显微镜观察上述母乳外泌体；纳米颗粒追踪分析（NTA）评估外泌体的大小分布；以CD81和CD63的蛋白印迹（western blot）作为外泌体阳性指标；按照制造商的方案，通过外泌体分离试剂盒（Invitrogen，美国）提取母乳外泌体的蛋白质；使用双肌酸蛋白质分析试剂盒（Thermo Scientific,美国）测定蛋白质浓度。这些测试都是常规方法。
图1为TEM结果，图2为粒径分布，图3为CD81和CD63的凝胶电泳结果，以GAPDH为内参，可以可看出足月儿（term）母亲、早产儿（preterm）母亲的母乳外泌体表达CD81和CD63，而结直肠癌细胞系（Caco-2）、正常结肠粘膜上皮细胞系（NCM460）作为参考；图4为足月儿（term）母亲、早产儿（preterm）母亲的母乳外泌体总蛋白表达情况，看出无差异。图3、图4中，足月儿（term）母亲、早产儿（preterm）母亲的母乳外泌体混合在一起，形成一组母乳外泌体；图3、图4中，足月儿（term）母亲、早产儿（preterm）母亲的母乳外泌体分别混合，形成两组母乳分泌体，再常规取样测试。
取结直肠癌细胞系（Caco-2）、正常结肠粘膜上皮细胞系（NCM460）进行分组孵育，证实母乳外泌体对肠道上皮屏障蛋白的保护作用，结直肠癌细胞系（Caco-2）、正常结肠粘膜上皮细胞系（NCM460）来自武汉普诺赛生命科技有限公司（中国）。Caco-2接种在常规DMEM/F12培养基中，NCM460接种在常规RPMI培养基中，每个培养基都添加10% exosome-free fetal serum supplemented（含有100 U/ml青霉素和100μg/ml链霉素）；在37℃、5%CO2环境中孵育。分为五组：（1）不添加任何试剂的对照组，（2）添加母乳外泌体，（3）用脂多糖（LPS，Invitrogen，美国）刺激12小时，（4）用母乳外泌体处理6小时，然后用脂多糖（LPS，Invitrogen，美国）刺激12小时，（5）用不含外泌体的母乳处理6小时，然后用脂多糖（LPS，Invitrogen，美国）刺激12小时。一共孵育24小时，LPS用量为10μg/mL。采用常规逆转录实时定量聚合酶链反应（RT-qPCR）、蛋白质印迹（Western blot）进行测试。
图5、图6分别为直肠癌细胞系（Caco-2）、正常结肠粘膜上皮细胞系（NCM460）的各组mPNA的表达结果；图7、图8分别为直肠癌细胞系（Caco-2）、正常结肠粘膜上皮细胞系（NCM460）的电泳结果。结果可以看出，肠道上皮屏障蛋白受到LPS的损伤，第3组和第5组中ZO-1、claudin-1和occludin的表达降低，第4组中肠道上皮屏障蛋白表达增加，说明母乳外泌体发挥了有益的作用，修复了肠道上皮屏障蛋白。
实施例二
将实施例一提取母乳外泌体时2000×g离心10min更换为10000×g离心10min，其余不变，得到母乳外泌体。
实施例三
将实施例一提取母乳外泌体时2000×g离心10min更换为1000×g离心10min，取乳清后，再1000×g离心10min；其余不变，得到母乳外泌体。
实施例四
将实施例一提取母乳外泌体时2000×g离心10min更换为2000×g离心30min，其余不变，得到母乳外泌体。
实施例五
以Caco-2的ZO-1作为评价标准，采用实施例一一样的细胞培养方法与测试方法，测试mRNA的表达，结果见图9，都按照实施例一（4）用母乳外泌体处理6小时，然后用脂多糖（LPS，Invitrogen，美国）刺激12小时的方法。
常规小鼠（C57BL/6）动物实验也可证实母乳外泌体对肠道上皮屏障蛋白的损伤模型有修复作用，见图10，模型建立后母乳外泌体具有修复作用。
母乳外泌体在母乳中含量较高，相较于其他体液更容易获取，且更易被接受，另外具备低免疫原性的天然优势，是一个极具应用前景的研究方向。然而现有技术针对母乳外泌体的应用都是基于细胞，未涉及保护蛋白，尤其没有针对肠道上皮屏障蛋白的技术方案。肠道上皮屏障蛋白与多种肠道疾病（肠道炎症、肠道肿瘤、肠道感染）的发生、发展以及全身缺血缺氧后出现肠损伤、肠黏膜屏障功能受损、肠道菌群、激素等有着密切关系，而且肠道上皮屏障蛋白受到多重信号通路的调节，影响着蛋白的结构与功能，而且肠道上皮屏障蛋白反过来影响细胞与外界的信息传递，是一个复杂的体系；本发明公开的技术方案为肠道上皮屏障蛋白损伤提供新的治疗方法。
Claims (10)
1.一种母乳外泌体，其特征在于，将母乳离心处理后，取乳清用母乳外泌体提取试剂盒提取，得到母乳外泌体。
2.根据权利要求1所述母乳外泌体，其特征在于，离心处理为2000×g离心10分钟。
3.根据权利要求3所述母乳外泌体，其特征在于，离心处理、试剂盒提取都在室温下进行。
4.权利要求1所述母乳外泌体在制备肠道上皮屏障蛋白保护剂或者损伤修复剂中的应用。
5.根据权利要求3所述的应用，其特征在于，母乳为人母乳。
6.根据权利要求3所述的应用，其特征在于，保护剂或者损伤修复剂为药物、食物、营养品或者保健品。
7.根据权利要求3所述的应用，其特征在于，肠道上皮屏障蛋白为肠上皮细胞紧密连接蛋白。
8.根据权利要求3所述的应用，其特征在于，所述损伤为肠道疾病损伤、缺血缺氧肠损伤、肠黏膜屏障功能受损、肠道菌群损伤或者激素损伤。
9.权利要求1所述母乳外泌体在制备结肠炎治疗剂中的应用。
10.根据权利要求9所述的应用，其特征在于，治疗剂为药物、食物、营养品或者保健品。
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