WO2022188892A1 - Breast milk exosome, and preparation method and use therefor - Google Patents
Breast milk exosome, and preparation method and use therefor Download PDFInfo
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- WO2022188892A1 WO2022188892A1 PCT/CN2022/087780 CN2022087780W WO2022188892A1 WO 2022188892 A1 WO2022188892 A1 WO 2022188892A1 CN 2022087780 W CN2022087780 W CN 2022087780W WO 2022188892 A1 WO2022188892 A1 WO 2022188892A1
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- breast milk
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0631—Mammary cells
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/20—Milk; Whey; Colostrum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the invention belongs to the technical field of neonatology, and particularly relates to breast milk exosomes and a preparation method and application thereof.
- Neonatal necrotizing enterocolitis is an acquired disease, which is a disease caused by a variety of causes of intestinal mucosal damage, resulting in ischemia and hypoxia, leading to diffuse or local necrosis of the small intestine and colon.
- NICUs neonatal intensive care units
- NEC neonatal necrotizing enterocolitis
- Exosomes are tiny (100-200 nm) vesicles released from cells that carry proteins, microRNAs (miRNAs), and messenger RNAs (mRNAs) that can signal and regulate intercellular behaviors, serving as natural vesicles of nanometer size Vesicles have the functions of mediating intercellular signal transduction, antigen presentation and rabbit immune response.
- Breast milk is rich in exosomes, which can promote cell proliferation and migration, reduce inflammation, and have protective potential for neonatal-related diseases.
- the existing technology analyzes the characteristics of intestinal flora in children with breast milk jaundice, and screens for interacting sites with differential bacteria. The results indicated that miRNAs may affect the occurrence and development of neonatal breast milk jaundice by regulating the intestinal flora of infants.
- 16S rRNA high-throughput sequencing was used to analyze the characteristics of intestinal flora in the two groups of fecal samples, and to screen out the differential special bacteria that may be related to neonatal bilirubin metabolism.
- the extracted exosome vesicles were used to predict the miRNAs that interact with specific bacterial genomes through the miRBase database.
- Fluorescence quantitative PCR was used to verify the differences in the expression of the predicted miRNAs in breast milk exosomes, and to explore the differential expression in breast milk. Correlation of miRNAs with gut microbiota. However, there is no report on the effect of breast milk exosomes on intestinal epithelial barrier proteins.
- the invention discloses a new application of breast milk exosomes, and discloses the protection of intestinal epithelial barrier proteins by breast milk exosomes.
- Existing technology explores the correlation between breast milk exosomal miRNAs and intestinal flora, predicts miRNAs that interact with the E. coli genome through the miRBase database, and screens out the differentially expressed molecules between groups.
- miR-16-5p has an interaction site with Escherichia coli ATP-binding protein gene. The pre-amplification method was used to quantify miRNA, and it was found that the expression of miR-16-5p was significantly different between the two groups. However, this study is not related to the effect of breast milk exosomes on intestinal epithelial barrier proteins.
- the present invention adopts the following technical scheme: the application of breast milk exosomes in the preparation of intestinal epithelial barrier protein protective agent.
- breast milk is human breast milk.
- the preparation method of breast milk exosomes is as follows: after breast milk is centrifuged, whey is extracted with a breast milk exosome extraction kit to obtain breast milk exosomes.
- the centrifugation treatment and kit extraction are performed at room temperature, and the centrifugation treatment is 2000 ⁇ g for 10 minutes.
- the protective agent is medicine, food, nutritional product or health care product, and breast milk exosomes can be used as the active ingredient;
- the therapeutic agent is medicine, food, nutritional product or health care product, and breast milk exosomes are used as the active ingredient Can.
- the intestinal epithelial barrier protein is an intestinal epithelial cell tight junction protein, specifically, occludin (Occludin), occludin (Claudin1) and occludin (ZO-1, a cytoplasmic protein).
- colitis is neonatal necrotizing enterocolitis.
- breast milk exosomes promote intestinal cell migration and proliferation and resist intestinal damage, but there is no evidence that breast milk exosomes have effects on intestinal epithelium. Implications for the role of barrier proteins.
- the invention creatively proposes the application of breast milk exosomes in the preparation of intestinal epithelial barrier protein protective agent, which is a new application of breast milk exosomes and expands the application scope of breast milk exosomes.
- Figure 1 is a TEM image of breast milk exosomes.
- Figure 2 shows the particle size distribution of breast milk exosomes.
- Figure 3 is a protein expression electropherogram of breast milk exosomes.
- Figure 4 shows the protein content of breast milk exosomes.
- Figure 6 is the expression of mRNA (NCM460).
- Figure 7 is an electrophoresis image of intestinal epithelial barrier protein (Caco-2).
- Figure 8 is an electrophoresis image of intestinal epithelial barrier protein (NCM460).
- Figure 9 shows the expression of human milk exosome mRNA (Caco-2) obtained by different preparation methods.
- Figure 10 shows the results of animal experiments.
- RNA expression levels of ZO-1, claudin 1 and occludin were detected by two-step RT-qPCR.
- TRIzol reagent Thermo Fisher Scientific, Inc., US
- qScript cDNA SuperMix Quantabio, US
- S1000 thermal cycler Bio-Rad Laboratories, Inc., US
- RT-qPCR was performed using Advanced qPCR Master Mix and CFX384 real-time system (Bio-Rad Laboratories, Inc.) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal reference; primers are as follows.
- ZO-1 forward primer, 5 ⁇ -TGCCATTACACGGTCCTCTG-3 ⁇ ; reverse primer, 5 ⁇ -GGTTCTGCCTCATCATTTCCTC-3 ⁇ .
- Claudin 1 forward primer, 5 ⁇ -GAGCGAGTCATGGCCAAC-3 ⁇ ; reverse primer, 5 ⁇ -TCTCAATGTCCATTTTCGGTTT-3 ⁇ .
- Occludin forward primer, 5 ⁇ -AGTGTGATAATAGTGAGTGCTATCC-3 ⁇ ; reverse primer 5 ⁇ -TGTCATACCTGTCCATCTTTCTTC-3 ⁇ .
- Proteins were transferred from the gel to PVDF membrane (Millipore Corporation, US), then blocked for 1 hour at room temperature (TBS, 5% nonfat milk, 0.05% TBS-T) and incubated with primary antibody (anti-ZO-1, 1:1000 , anti-occludin, 1:1000, anti-claudin-1, 1:1000, anti- ⁇ -actin, 1:5000), overnight at 4°C; then after washing in Tween-20 (TBS-T) buffer , the membrane was incubated at room temperature for 1 h with conventional secondary antibodies.
- the protein band matrix was detected with Immobilon Western Substrate (Millipore Corporation, US) and analyzed with the Bioimage Analysis System (Syngene, US), compared to the endogenous reference protein GAPDH.
- Example 1 After the breast milk was collected, put it into a 50ml sterile test tube, refrigerate it in an ice pack, and transport it to the laboratory for processing. Centrifuge at 2000 ⁇ g for 10 min at room temperature, then routinely take the whey, transfer it to another tube, and use the exosome extraction kit (Total Exosome Isolation). Kit, Invitrogen, USA), exosomes were extracted according to the kit method and stored at ⁇ 80°C; the remaining breast milk was stored at ⁇ 20°C (exosome-free breast milk).
- kit Total Exosome Isolation
- the breast milk exosomes described above were observed by transmission electron microscopy; the size distribution of exosomes was assessed by nanoparticle tracking assay (NTA); Western blotting of CD81 and CD63 was used as exosome positivity indicator; following the manufacturer's protocol, Proteins from breast milk exosomes were extracted by exosome isolation kit (Invitrogen, USA); protein concentration was determined using double creatine protein assay kit (Thermo Scientific, USA). These tests are conventional methods.
- Figure 1 is the TEM results
- Figure 2 is the particle size distribution
- Figure 3 is the gel electrophoresis results of CD81 and CD63.
- GAPDH GAPDH
- the breast milk of term mothers and preterm mothers The exosomes express CD81 and CD63, while a colorectal cancer cell line (Caco-2) and a normal colonic mucosal epithelial cell line (NCM460) are used as reference;
- Figure 4 is breast milk from mothers of term infants (term) and preterm infants (preterm) There was no difference in the total protein expression of exosomes.
- breast milk exosomes from mothers of term infants (term) and preterm infants (preterm) are mixed together to form a group of breast milk exosomes; in Figures 3 and 4, term infants (term ) breast milk exosomes from mothers and preterm mothers were mixed to form two groups of breast milk exosomes, which were then routinely sampled and tested.
- Colorectal cancer cell line (Caco-2) and normal colonic mucosal epithelial cell line (NCM460) were grouped and incubated to confirm the protective effect of breast milk exosomes on intestinal epithelial barrier proteins.
- Colorectal cancer cell line (Caco-2) , Normal colonic mucosal epithelial cell line (NCM460) was from Wuhan Proceeds Life Technology Co., Ltd. (China).
- Caco-2 was inoculated in conventional DMEM/F12 medium
- NCM460 was inoculated in conventional RPMI medium, each supplemented with 10% exosome-free fetal serum supplemented (containing 100 U/ml penicillin and 100 ⁇ g/ml streptomycin) ); incubate at 37°C, 5% CO 2 .
- Figures 5 and 6 show the expression results of mPNA in each group of rectal cancer cell line (Caco-2) and normal colonic mucosal epithelial cell line (NCM460), respectively;
- Figures 7 and 8 are respectively rectal cancer cell line (Caco-2) ,
- the results showed that the intestinal epithelial barrier proteins were damaged by LPS, the expressions of ZO-1, claudin-1 and occludin decreased in groups 3 and 5, and the expression of intestinal epithelial barrier proteins increased in group 4, indicating that breast milk Exosomes played a beneficial role in repairing intestinal epithelial barrier proteins.
- Example 2 When extracting breast milk exosomes in Example 1, the centrifugation at 2000 ⁇ g for 10 minutes was replaced with centrifugation at 10000 ⁇ g for 10 minutes, and the rest remained unchanged to obtain breast milk exosomes.
- Example 3 When extracting breast milk exosomes in Example 1, the centrifugation at 2000 ⁇ g for 10 minutes was replaced with centrifugation at 1000 ⁇ g for 10 minutes, and after taking the whey, centrifugation at 1000 ⁇ g for 10 minutes was performed; the rest remained unchanged to obtain breast milk exosomes.
- Example 4 The centrifugation at 2000 ⁇ g for 10 min in Example 1 was replaced by centrifugation at 2000 ⁇ g for 30 min, and the rest remained unchanged to obtain breast milk exosomes.
- Example 5 Using the ZO-1 of Caco-2 as the evaluation standard, the same cell culture method and testing method as in Example 1 were used to test the expression of mRNA. The results are shown in Figure 9, and breast milk was used according to Example 1 (4). The exosomes were treated for 6 hours and then stimulated with lipopolysaccharide (LPS, Invitrogen, USA) for 12 hours.
- LPS lipopolysaccharide
- Routine mouse (C57BL/6) animal experiments can also confirm that breast milk exosomes have a repairing effect on the damaged model of intestinal epithelial barrier proteins, as shown in Figure 10. After the model is established, breast milk exosomes have a repairing effect.
- breast milk exosomes have a high content in breast milk, are easier to obtain and more acceptable than other body fluids, and have the natural advantage of low immunogenicity, which is a very promising research direction.
- the applications of the prior art for breast milk exosomes are all based on cells and do not involve protective proteins, especially no technical solutions for intestinal epithelial barrier proteins.
- Intestinal epithelial barrier proteins and the occurrence and development of various intestinal diseases intestinal inflammation, intestinal tumor, intestinal infection), and intestinal injury after systemic ischemia and hypoxia, impaired intestinal mucosal barrier function, intestinal flora Intestinal epithelial barrier proteins are closely related to hormones, and intestinal epithelial barrier proteins are regulated by multiple signaling pathways, affecting the structure and function of the protein, and intestinal epithelial barrier proteins in turn affect the information transmission between cells and the outside world, which is a complex system;
- the technical solution disclosed in the present invention provides a new treatment method for intestinal epithelial barrier protein damage.
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Abstract
Provided are a breast milk exosome, and a preparation method and use therefor, the use being an application of a breast milk exosome in the preparation of an intestinal epithelial barrier protein protective agent or a damage repairing agent.
Description
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。The invention belongs to the technical field of neonatology, and particularly relates to breast milk exosomes and a preparation method and application thereof.
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。Neonatal necrotizing enterocolitis (NEC) is an acquired disease, which is a disease caused by a variety of causes of intestinal mucosal damage, resulting in ischemia and hypoxia, leading to diffuse or local necrosis of the small intestine and colon. Despite neonatal care and the establishment of neonatal intensive care units (NICUs) worldwide, neonatal necrotizing enterocolitis (NEC) remains a devastating disease, especially in preterm infants. Exosomes are tiny (100-200 nm) vesicles released from cells that carry proteins, microRNAs (miRNAs), and messenger RNAs (mRNAs) that can signal and regulate intercellular behaviors, serving as natural vesicles of nanometer size Vesicles have the functions of mediating intercellular signal transduction, antigen presentation and rabbit immune response. Breast milk is rich in exosomes, which can promote cell proliferation and migration, reduce inflammation, and have protective potential for neonatal-related diseases. The existing technology analyzes the characteristics of intestinal flora in children with breast milk jaundice, and screens for interacting sites with differential bacteria. The results indicated that miRNAs may affect the occurrence and development of neonatal breast milk jaundice by regulating the intestinal flora of infants. 16S rRNA high-throughput sequencing was used to analyze the characteristics of intestinal flora in the two groups of fecal samples, and to screen out the differential special bacteria that may be related to neonatal bilirubin metabolism. The extracted exosome vesicles were used to predict the miRNAs that interact with specific bacterial genomes through the miRBase database. Fluorescence quantitative PCR was used to verify the differences in the expression of the predicted miRNAs in breast milk exosomes, and to explore the differential expression in breast milk. Correlation of miRNAs with gut microbiota. However, there is no report on the effect of breast milk exosomes on intestinal epithelial barrier proteins.
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。The invention discloses a new application of breast milk exosomes, and discloses the protection of intestinal epithelial barrier proteins by breast milk exosomes. Existing technology explores the correlation between breast milk exosomal miRNAs and intestinal flora, predicts miRNAs that interact with the E. coli genome through the miRBase database, and screens out the differentially expressed molecules between groups. miR-16-5p has an interaction site with Escherichia coli ATP-binding protein gene. The pre-amplification method was used to quantify miRNA, and it was found that the expression of miR-16-5p was significantly different between the two groups. However, this study is not related to the effect of breast milk exosomes on intestinal epithelial barrier proteins.
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。The present invention adopts the following technical scheme: the application of breast milk exosomes in the preparation of intestinal epithelial barrier protein protective agent.
母乳外泌体在制备结肠炎治疗剂中的应用。Application of breast milk exosomes in the preparation of a therapeutic agent for colitis.
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。In the present invention, breast milk is human breast milk. The preparation method of breast milk exosomes is as follows: after breast milk is centrifuged, whey is extracted with a breast milk exosome extraction kit to obtain breast milk exosomes. Preferably, the centrifugation treatment and kit extraction are performed at room temperature, and the centrifugation treatment is 2000×g for 10 minutes.
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。In the present invention, the protective agent is medicine, food, nutritional product or health care product, and breast milk exosomes can be used as the active ingredient; the therapeutic agent is medicine, food, nutritional product or health care product, and breast milk exosomes are used as the active ingredient Can.
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）。In the present invention, the intestinal epithelial barrier protein is an intestinal epithelial cell tight junction protein, specifically, occludin (Occludin), occludin (Claudin1) and occludin (ZO-1, a cytoplasmic protein).
本发明中，结肠炎为新生儿坏死性小肠结肠炎。In the present invention, colitis is neonatal necrotizing enterocolitis.
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。At present, the mechanism of breast milk relieving or treating neonatal necrotizing enterocolitis is unclear. The prior art discloses that breast milk exosomes promote intestinal cell migration and proliferation and resist intestinal damage, but there is no evidence that breast milk exosomes have effects on intestinal epithelium. Implications for the role of barrier proteins. The invention creatively proposes the application of breast milk exosomes in the preparation of intestinal epithelial barrier protein protective agent, which is a new application of breast milk exosomes and expands the application scope of breast milk exosomes.
图1为母乳外泌体的TEM图。Figure 1 is a TEM image of breast milk exosomes.
图2为母乳外泌体的粒径分布。Figure 2 shows the particle size distribution of breast milk exosomes.
图3为母乳外泌体的蛋白表达电泳图。Figure 3 is a protein expression electropherogram of breast milk exosomes.
图4为母乳外泌体的蛋白含量。Figure 4 shows the protein content of breast milk exosomes.
图5为mRNA的表达（Caco-2）。Figure 5. Expression of mRNA (Caco-2).
图6为mRNA的表达（NCM460）。Figure 6 is the expression of mRNA (NCM460).
图7为肠道上皮屏障蛋白电泳图（Caco-2）。Figure 7 is an electrophoresis image of intestinal epithelial barrier protein (Caco-2).
图8为肠道上皮屏障蛋白电泳图（NCM460）。Figure 8 is an electrophoresis image of intestinal epithelial barrier protein (NCM460).
图9为不同制备方法得到的母乳外泌体mRNA的表达（Caco-2）。Figure 9 shows the expression of human milk exosome mRNA (Caco-2) obtained by different preparation methods.
图10为动物实验结果。Figure 10 shows the results of animal experiments.
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。Healthy breastfeeding mothers who were admitted to Suzhou Children's Hospital after giving birth were recruited after signing the informed consent form, 33 mothers delivered term infants (≥37 weeks) and 21 mothers delivered preterm infants (24-36 weeks). . All mothers produce excess breast milk, which is often discarded, and samples of this excess breast milk are collected for research purposes. Lactation time, colostrum samples were collected on the 3rd day after the baby was born. For discharged infants, the mother's breast milk was collected at the Neonatal Follow-up Clinic 3-10 days after birth. Breast milk collection was approved by the ethics committee of the mother and Suzhou Children's Hospital.
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 cDNA SuperMix（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-Rad Laboratories,
Inc.)进行，以甘油醛-3-磷酸脱氢酶（GAPDH）做内参；引物如下。The mRNA expression levels of ZO-1, claudin 1 and occludin were detected by two-step RT-qPCR. TRIzol reagent (Thermo Fisher Scientific,
Inc., US) to extract mRNA from cells. with qScript cDNA SuperMix (Quantabio, US) and S1000 thermal cycler (Bio-Rad Laboratories,
Inc., US) to prepare complementary DNA (cDNA). RT-qPCR was performed using Advanced qPCR Master Mix and CFX384 real-time system (Bio-Rad Laboratories,
Inc.) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal reference; primers are as follows.
ZO-1: 正向引物, 5ʹ-TGCCATTACACGGTCCTCTG-3ʹ；反向引物, 5ʹ-GGTTCTGCCTCATCATTTCCTC-3ʹ。ZO-1: forward primer, 5ʹ-TGCCATTACACGGTCCTCTG-3ʹ; reverse primer, 5ʹ-GGTTCTGCCTCATCATTTCCTC-3ʹ.
Claudin 1: 正向引物, 5ʹ-GAGCGAGTCATGGCCAAC-3ʹ；反向引物, 5ʹ-TCTCAATGTCCATTTTCGGTTT-3ʹ。Claudin 1: forward primer, 5ʹ-GAGCGAGTCATGGCCAAC-3ʹ; reverse primer, 5ʹ-TCTCAATGTCCATTTTCGGTTT-3ʹ.
Occludin: 正向引物, 5ʹ-AGTGTGATAATAGTGAGTGCTATCC-3ʹ；反向引物5ʹ-TGTCATACCTGTCCATCTTTCTTC-3ʹ。Occludin: forward primer, 5ʹ-AGTGTGATAATAGTGAGTGCTATCC-3ʹ; reverse primer 5ʹ-TGTCATACCTGTCCATCTTTCTTC-3ʹ.
蛋白质印迹（Western blot.）。细胞接种在6孔板上，密度为3.5×10
5，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。
Western blot. Cells were seeded on 6-well plates at a density of 3.5×10 5 with RIPA buffer (150 mM NaCl, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM benzyl Sulfonyl fluoride and 50 mM Tris; pH 8.0) were dissolved. Total protein concentration was determined by conventional BCA method and adjusted to 1 μg/μl; 5x loading buffer was added and boiled at 100°C for 10 min, after which the protein volume of each sample was separated into 10% sodium dodecyl sulfate-polyethylene Acrylamide gel electrophoresis. Proteins were transferred from the gel to PVDF membrane (Millipore Corporation, US), then blocked for 1 hour at room temperature (TBS, 5% nonfat milk, 0.05% TBS-T) and incubated with primary antibody (anti-ZO-1, 1:1000 , anti-occludin, 1:1000, anti-claudin-1, 1:1000, anti-β-actin, 1:5000), overnight at 4°C; then after washing in Tween-20 (TBS-T) buffer , the membrane was incubated at room temperature for 1 h with conventional secondary antibodies. The protein band matrix was detected with Immobilon Western Substrate (Millipore Corporation, US) and analyzed with the Bioimage Analysis System (Syngene, US), compared to the endogenous reference protein GAPDH.
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。The above tests are all routine methods; when extracting breast milk exosomes, specific centrifugation and kit operations are routine methods.
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）。Example 1: After the breast milk was collected, put it into a 50ml sterile test tube, refrigerate it in an ice pack, and transport it to the laboratory for processing. Centrifuge at 2000 × g for 10 min at room temperature, then routinely take the whey, transfer it to another tube, and use the exosome extraction kit (Total Exosome Isolation).
Kit, Invitrogen, USA), exosomes were extracted according to the kit method and stored at −80°C; the remaining breast milk was stored at −20°C (exosome-free breast milk).
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。The breast milk exosomes described above were observed by transmission electron microscopy; the size distribution of exosomes was assessed by nanoparticle tracking assay (NTA); Western blotting of CD81 and CD63 was used as exosome positivity indicator; following the manufacturer's protocol, Proteins from breast milk exosomes were extracted by exosome isolation kit (Invitrogen, USA); protein concentration was determined using double creatine protein assay kit (Thermo Scientific, USA). These tests are conventional methods.
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。Figure 1 is the TEM results, Figure 2 is the particle size distribution, and Figure 3 is the gel electrophoresis results of CD81 and CD63. Using GAPDH as the internal reference, it can be seen that the breast milk of term mothers and preterm mothers The exosomes express CD81 and CD63, while a colorectal cancer cell line (Caco-2) and a normal colonic mucosal epithelial cell line (NCM460) are used as reference; Figure 4 is breast milk from mothers of term infants (term) and preterm infants (preterm) There was no difference in the total protein expression of exosomes. In Figures 3 and 4, breast milk exosomes from mothers of term infants (term) and preterm infants (preterm) are mixed together to form a group of breast milk exosomes; in Figures 3 and 4, term infants (term ) breast milk exosomes from mothers and preterm mothers were mixed to form two groups of breast milk exosomes, which were then routinely sampled and tested.
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%CO
2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。
Colorectal cancer cell line (Caco-2) and normal colonic mucosal epithelial cell line (NCM460) were grouped and incubated to confirm the protective effect of breast milk exosomes on intestinal epithelial barrier proteins. Colorectal cancer cell line (Caco-2) , Normal colonic mucosal epithelial cell line (NCM460) was from Wuhan Proceeds Life Technology Co., Ltd. (China). Caco-2 was inoculated in conventional DMEM/F12 medium, NCM460 was inoculated in conventional RPMI medium, each supplemented with 10% exosome-free fetal serum supplemented (containing 100 U/ml penicillin and 100 μg/ml streptomycin) ); incubate at 37°C, 5% CO 2 . Divided into five groups: (1) control group without any reagent added, (2) supplemented with breast milk exosomes, (3) stimulated with lipopolysaccharide (LPS, Invitrogen, USA) for 12 hours, (4) with breast milk exosomes Treated for 6 hours, then stimulated with lipopolysaccharide (LPS, Invitrogen, USA) for 12 hours, (5) treated with exosome-free breast milk for 6 hours, then stimulated with lipopolysaccharide (LPS, Invitrogen, USA) for 12 hours. A total of 24 hours of incubation, the dosage of LPS was 10 μg/mL. Routine reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot were used for testing.
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。Figures 5 and 6 show the expression results of mPNA in each group of rectal cancer cell line (Caco-2) and normal colonic mucosal epithelial cell line (NCM460), respectively; Figures 7 and 8 are respectively rectal cancer cell line (Caco-2) , Electrophoresis results of normal colonic mucosal epithelial cell line (NCM460). The results showed that the intestinal epithelial barrier proteins were damaged by LPS, the expressions of ZO-1, claudin-1 and occludin decreased in groups 3 and 5, and the expression of intestinal epithelial barrier proteins increased in group 4, indicating that breast milk Exosomes played a beneficial role in repairing intestinal epithelial barrier proteins.
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。Example 2: When extracting breast milk exosomes in Example 1, the centrifugation at 2000×g for 10 minutes was replaced with centrifugation at 10000×g for 10 minutes, and the rest remained unchanged to obtain breast milk exosomes.
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。Example 3: When extracting breast milk exosomes in Example 1, the centrifugation at 2000×g for 10 minutes was replaced with centrifugation at 1000×g for 10 minutes, and after taking the whey, centrifugation at 1000×g for 10 minutes was performed; the rest remained unchanged to obtain breast milk exosomes.
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。Example 4: The centrifugation at 2000×g for 10 min in Example 1 was replaced by centrifugation at 2000×g for 30 min, and the rest remained unchanged to obtain breast milk exosomes.
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。Example 5: Using the ZO-1 of Caco-2 as the evaluation standard, the same cell culture method and testing method as in Example 1 were used to test the expression of mRNA. The results are shown in Figure 9, and breast milk was used according to Example 1 (4). The exosomes were treated for 6 hours and then stimulated with lipopolysaccharide (LPS, Invitrogen, USA) for 12 hours.
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。Routine mouse (C57BL/6) animal experiments can also confirm that breast milk exosomes have a repairing effect on the damaged model of intestinal epithelial barrier proteins, as shown in Figure 10. After the model is established, breast milk exosomes have a repairing effect.
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。Breast milk exosomes have a high content in breast milk, are easier to obtain and more acceptable than other body fluids, and have the natural advantage of low immunogenicity, which is a very promising research direction. However, the applications of the prior art for breast milk exosomes are all based on cells and do not involve protective proteins, especially no technical solutions for intestinal epithelial barrier proteins. Intestinal epithelial barrier proteins and the occurrence and development of various intestinal diseases (intestinal inflammation, intestinal tumor, intestinal infection), and intestinal injury after systemic ischemia and hypoxia, impaired intestinal mucosal barrier function, intestinal flora Intestinal epithelial barrier proteins are closely related to hormones, and intestinal epithelial barrier proteins are regulated by multiple signaling pathways, affecting the structure and function of the protein, and intestinal epithelial barrier proteins in turn affect the information transmission between cells and the outside world, which is a complex system; The technical solution disclosed in the present invention provides a new treatment method for intestinal epithelial barrier protein damage.
Claims (10)
- 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。A breast milk exosome is characterized in that, after centrifuging breast milk, extracting whey with a breast milk exosome extraction kit to obtain breast milk exosomes.
- 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。The breast milk exosome according to claim 1, wherein the centrifugation treatment is 2000×g centrifugation for 10 minutes.
- 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。The breast milk exosome according to claim 3, wherein the centrifugation treatment and the extraction by the kit are all performed at room temperature.
- 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。The application of the breast milk exosomes of claim 1 in the preparation of an intestinal epithelial barrier protein protective agent or a damage repairing agent.
- 根据权利要求3所述的应用，其特征在于，母乳为人母乳。The application according to claim 3, wherein the breast milk is human breast milk.
- 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。The application according to claim 3, wherein the protective agent or the damage repairing agent is a drug, food, nutritional product or health care product.
- 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。The application according to claim 3, wherein the intestinal epithelial barrier protein is an intestinal epithelial cell tight junction protein.
- 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。The application according to claim 3, wherein the damage is intestinal disease damage, ischemia-hypoxic intestinal damage, intestinal mucosal barrier function damage, intestinal flora damage or hormone damage.
- 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。The application of the breast milk exosome of claim 1 in the preparation of a colitis therapeutic agent.
- 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。The application according to claim 9, wherein the therapeutic agent is a drug, food, nutritional product or health care product.
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