WO2024001457A1 - 一种乳源外泌体及提取方法 - Google Patents
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- milk
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Classifications
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- 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
-
- 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
Definitions
- This application relates to the technical field of dairy products, especially milk-derived exosomes and extraction methods.
- Exosomes are double-membrane round or oval vesicles actively secreted by cells, with a diameter of 30-200nm and a density of 1.13-1.19g/mL.
- small vesicles of 50 nm were first discovered in the study of transferrin receptors in reticulocytes.
- Canadian scholar Rose Johnstone coined the term "Exosome” and believed that exosomes are just a tool for cells to "excret" to the external environment.
- Valadi et al. proposed that exosomes contain mRNA and miRNA and can deliver them to another cell. Since then, the function and mechanism of exosomes have been widely studied.
- Exosomes exist in various body fluid environments, such as serum, urine, saliva, breast milk, amniotic fluid, etc. Exosomes in biological fluids indicate the functional status of their originating cells and can be used in medicine as biodiagnostic markers for liver cancer, prostate cancer and other diseases; due to their membrane structure, exosomes can also be used as drug carriers to demonstrate efficient drug delivery. Properties such as exosome loading of labile curcumin may enhance therapeutic efficacy.
- Milk-derived exosomes are small vesicles secreted by mammary epithelial cells, and their main components are proteins, lipids and nucleic acids.
- the membrane surface of milk-derived exosomes has specific membrane proteins, such as the most abundant tetraspanins (CD9, CD82, CD81 and CD63), as well as costimulatory molecules (CD54) and adhesion molecules (CD11b).
- Lipids phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, and phosphatidylinositol are also commonly found in exosomes.
- Nucleic acids in milk-derived exosomes mainly include non-coding single-stranded RNA molecules (micro RNA, mi RNA), long non-coding RNA (long non-coding RNA, lnc RNA), circular RNA (circular RNA, circ RNA), mRNA and tRNA, etc.
- Milk-derived exosomes play important functions in physiological processes, such as mediating cell communication, promoting cell growth, and participating in immune responses.
- exosome isolation technology is centrifugation, but centrifugation can cause damage to the morphology of exosomes.
- this application provides a new extraction method of milk-derived exosomes, which has less impact on the morphology of exosomes.
- this application provides a method for extracting milk-derived exosomes, which follows the following steps:
- step (3) Centrifuge the middle layer supernatant obtained in step (2), take the supernatant, mix it with PBS buffer and exosome extraction reagent, and incubate;
- the centrifugation process in step (2) is centrifugation at 8500-10000 ⁇ g for 25-35 minutes at room temperature.
- the centrifugation time is 30 minutes.
- the centrifugation process in step (5) is centrifugation at 8500-10000 ⁇ g for 4-6 minutes at room temperature.
- the centrifugation time is 5 minutes.
- this application also provides a milk-derived exosome prepared by the above-mentioned extraction method.
- milk-derived exosomes prepared by the milk-derived exosome extraction method provided by this application have a more complete shape, require less sample volume, and after separation, Milk-derived exosomes contain more types of proteins, which is more conducive to analysis and research operations.
- Figure 2 is a transmission electron microscope image of milk-derived exosomes prepared in Example 2;
- Figure 3 is a transmission electron microscope image of milk-derived exosomes prepared in Comparative Example 1;
- Figure 4 is a transmission electron microscope image of milk-derived exosomes prepared in Comparative Example 2;
- Figure 6 is a transmission electron microscope image of milk-derived exosomes prepared in Comparative Example 4.
- Figure 7 is a Venn diagram of exosome protein types isolated from milk-derived exosomes prepared in Example 1 and Comparative Examples 1 and 3.
- This embodiment provides a method for extracting milk-derived exosomes.
- the milk raw material of the milk-derived exosomes comes from the fresh milk filling truck of Beijing Sanyuan Food Co., Ltd.
- This embodiment provides a method for extracting milk-derived exosomes.
- the milk raw material of the milk-derived exosomes comes from Ailiyou milk powder produced by Beijing Sanyuan Food Co., Ltd.
- Example 2 The extraction process in Example 2 is the same as that in Example 1.
- This comparative example provides a method for extracting milk-derived exosomes.
- the milk raw material of the milk-derived exosomes comes from the fresh milk filling truck of Beijing Sanyuan Food Co., Ltd.
- Centrifuge the middle layer supernatant at room temperature and 10,000 ⁇ g for 30 minutes take 200 ⁇ L of the supernatant, mix it with 200 ⁇ L PBS buffer, and then add 200 ⁇ L of high-efficiency exosome precipitation reagent produced by Intex Biotechnology (Beijing) Co., Ltd. , mix well, incubate the reaction at room temperature for 30 minutes, centrifuge at 10,000 ⁇ g for 10 minutes at room temperature, and collect the precipitate.
- Intex Biotechnology Beijing
- This comparative example provides a method for extracting milk-derived exosomes.
- the milk raw material of the milk-derived exosomes comes from Ailiyou milk powder produced by Beijing Sanyuan Food Co., Ltd.
- Comparative Example 2 The extraction process in Comparative Example 2 is the same as Comparative Example 1.
- This comparative example provides a method for extracting milk-derived exosomes.
- the milk raw material of the milk-derived exosomes comes from the fresh milk filling truck of Beijing Sanyuan Food Co., Ltd.
- the milk raw material of the milk-derived exosomes comes from the fresh milk filling truck of Beijing Sanyuan Food Co., Ltd.
- the diameter range of the milk-derived exosomes obtained in Examples 1-2 and Comparative Examples 1-3 is between 30-150 nm, and both have a cup-filled double-layer membrane structure.
- the morphology of milk-derived exosomes obtained by the method provided in Comparative Examples 2-3 is incomplete and the background is messy, while the morphology of the milk-derived exosomes obtained by the method in Comparative Example 1 and Example 1 is very complete, showing a complete " Cup and plate” structure.
- the morphology of milk-derived exosomes obtained by the method in Comparative Example 4 was completely broken.
- the processed milk-derived exosomes were detected using a nanoliter liquid chromatography tandem Q Exactive Orbitrap mass spectrometer, and a Venn diagram of protein types was drawn based on the detection results, as shown in Figure 7.
- This application relates to a method for extracting milk-derived exosomes.
- the milk-derived exosomes prepared by the milk-derived exosomes extraction method provided by this application have better morphology. It is more complete, requires less sample volume, and contains more types of proteins in milk-derived exosomes after separation, which is more conducive to analysis and research operations.
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- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Dermatology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Compounds Of Unknown Constitution (AREA)
- Extraction Or Liquid Replacement (AREA)
Abstract
提供了一种乳源外泌体的提取方法，依次按下述步骤进行：(1)将乳原料分装，离心，取清液；(2)将步骤(1)得到的清液离心，取中间层清液；(3)将步骤(2)得到的中间层清液离心，取清液，与PBS缓冲液和外泌体提取试剂混匀，孵育；(4)将步骤(3)的产物离心，保留沉淀，用PBS缓冲液重悬；(5)将步骤(4)的产物离心，取上清，即得。
Description
交叉引用
本申请要求在中国专利局提交的、申请号为202210755949.7、申请日为2022年06月30日、申请名称为“一种乳源外泌体及提取方法”的中国专利申请的优先权，该申请的全部内容通过引用结合在本申请中。
本申请涉及乳制品技术领域，特别是乳源外泌体及提取方法。
外泌体是细胞主动分泌产生的双层膜结构圆形或椭圆形囊泡，直径在30-200nm，密度为1.13-1.19g/mL。1983年，首次在网织红细胞中转铁蛋白受体的研究中发现50nm的小囊泡。1987年，加拿大学者Rose Johnstone创造“Exosome”一词并认为外泌体只是细胞向外界环境“排泄”的工具。2007年，Valadi等提出外泌体包含mRNA和miRNA并可以将其传递给另一个细胞，至此外泌体的功能及其机制开始得到广泛研究。外泌体存在于各种体液环境中，如血清、尿液、唾液、乳汁、羊水等。生物体液中的外泌体表明其起源细胞的功能状态，在医学上可作为肝癌、前列腺癌等疾病的生物诊断标记物；由于外泌体的膜结构还可以作为药物载体表现出高效药物传递的特性，例如外泌体负载不稳定的姜黄素可提高治疗效果。
乳源外泌体是由乳腺上皮细胞分泌的小囊泡，其主要组成成分是蛋白质、脂质以及核酸。乳源外泌体膜表面具有特异性膜蛋白，如最丰富的四跨膜蛋白(CD9、CD82、CD81和CD63)，以及共刺激分子(CD54)和粘附分子(CD11b)。脂类(磷脂酰胆碱、磷脂酰丝氨酸、磷脂酰乙醇胺、鞘磷脂和磷脂酰肌醇)也常见于外泌体中。乳源外泌体中核酸主要包括非编码单链RNA分子(micro RNA，mi RNA)、长链非编码RNA(long non-coding RNA，lnc RNA)、环状RNA(circular RNA，circ RNA)、m RNA和t RNA等。乳源外泌体在生理过程中发挥着重要功能，如介导细胞交流、促进细胞生长以及参与免疫反应等。
目前常用的外泌体分离技术是离心法，但是离心法会对外泌体的形态造成损伤。
因鉴于此，特提出此申请。
发明内容
为了解决现有的技术问题，本申请提供了一种新的乳源外泌体的提取方法，该提取方法对外泌体的形态影响较小。
为实现上述目的，本申请采用的技术方案如下：
第一方面，本申请提供了一种乳源外泌体的提取方法，依次按下述步骤进行：
(1)将乳原料分装，离心，取清液；
(2)将步骤(1)得到的清液离心，取中间层清液；
(3)将步骤(2)得到的中间层清液离心，取清液，与PBS缓冲液和外泌体提取试剂混匀，孵育；
(4)将步骤(3)的产物离心，保留沉淀，用PBS缓冲液重悬；
(5)将步骤(4)的产物离心，取
优选或可选地，步骤(1)中的离心工艺为室温下1500-2500×g离心5-15min。
优选或可选地，步骤(2)中的离心工艺为室温下8500-10000×g离心25-35min，优选的，离心时间为30min。
优选或可选地，步骤(3)中的离心工艺为室温下8500-10000×g离心25-35min，优选的，离心时间为30min。
优选或可选地，步骤(3)中，清液、PBS缓冲液和外泌体提取试剂的体积比为1:1:1。
优选或可选地，所述外泌体提取试剂为Thermo Fisher公司生产的总外泌体分离试剂。
优选或可选地，步骤(3)中，孵育时间为25-35min，优选的，孵育时间为30min。
优选或可选地，步骤(4)中离心工艺为室温下8500-10000×g离心9-11min，优选的，离心时间为10min。
优选或可选地，步骤(5)中离心工艺为室温下8500-10000×g离心4-6min，优选的，离心时间为5min。
第二方面，本申请还提供了一种乳源外泌体，采用上述的提取方法制备而成。
采用本申请提供的乳源外泌体提取方法制备的乳源外泌体，相较于其他方法制备的乳源外泌体而言，形态更完整，需要的样品体积更少，且在分离后包含的乳源外泌体的蛋白种类更多，更有利于分析研究作业。
图1为实施例1制备的乳源外泌体的投射电子显微镜图像；
图2为实施例2制备的乳源外泌体的投射电子显微镜图像；
图3为对比例1制备的乳源外泌体的投射电子显微镜图像；
图4为对比例2制备的乳源外泌体的投射电子显微镜图像；
图5为对比例3制备的乳源外泌体的投射电子显微镜图像；
图6为对比例4制备的乳源外泌体的投射电子显微镜图像；
图7为实施例1和对比例1和3制备的乳源外泌体分离外泌体蛋白种类韦恩图。
为了便于理解本申请，下面将结合说明书附图和较佳实验例对本申请作更全面、细致地描述，但本申请的保护范围并不限于以下具体的实施例。
除非另有定义，下文中所使用的所有专业术语与本领域技术人员通常理解的含义相同。本文中所使用的专业术语只是为了描述具体实施例的目的，并不是旨在限制本申请的保护范围。
除非另有特别说明，本申请中用到的各种原材料、试剂、仪器和设备等均可通过市场购买得到或者可通过现有方法制备得到。
实施例1
本实施例提供了一种乳源外泌体的提取方法。
所述乳源外泌体的乳原料来自于北京三元食品有限公司的鲜奶灌装车。
将乳原料分装在1mL离心管中，每管中均添加1mL样品，在室温，2000×g下离心10min，离心完成后取清液，在室温，10000×g下再次离心30min，取中间层清液。
将中间层清液在室温，10000×g下离心30min后，取200μL清液，并与200μL PBS缓冲液混匀，再添加200μL Thermo Fisher公司生产的总外泌体分离试剂，混匀，在室温下孵育反应30min后，在室温下，10000×g离心10min，取沉淀。
将沉淀用50μL PBS缓冲液重悬，在室温下，10000×g离心5min，取上清即为提取出的外泌体。
实施例2
本实施例提供了一种乳源外泌体的提取方法。
所述乳源外泌体的乳原料来自于北京三元食品有限公司生产的爱力优奶粉。
实施例2中的所述提取工艺与实施例1相同。
对比例1
该对比例提供了一种乳源外泌体的提取方法。
所述乳源外泌体的乳原料来自于北京三元食品有限公司的鲜奶灌装车。
将乳原料分装在1mL离心管中，每管中均添加1mL样品，在室温，2000×g下离心10min，离心完成后取清液，在室温，10000×g下再次离心30min，取中间层清液。
将中间层清液在室温，10000×g下离心30min后，取200μL清液，并与200μL PBS缓冲液混匀，再添加200μL英文特生物技术(北京)有限公司生产的高效外泌体沉淀试剂，混匀，在室温下孵育反应30min后，在室温下，10000×g离心10min，取沉淀。
将沉淀用50μL PBS缓冲液重悬，在室温下，10000×g离心5min，取上清即为提取出的外泌体。
对比例2
该对比例提供了一种乳源外泌体的提取方法。
所述乳源外泌体的乳原料来自于北京三元食品有限公司生产的爱力优奶粉。
对比例2中的所述提取工艺与对比例1相同。
对比例3
该对比例提供了一种乳源外泌体的提取方法。
所述乳源外泌体的乳原料来自于北京三元食品有限公司的鲜奶灌装车。
将50mL乳原料置于超速离心管内，在4℃，3000rpm下离心60min，取上清液，在4℃，16000rpm下离心20min，去除表层的脂肪，取清液，在4℃，30000rpm下离心60min，并收集松散的颗粒。
将收集到的松散的颗粒用PBS重悬后，用0.3mol/L的蔗糖溶液溶解，在4℃下，20000rpm离心120min，取沉淀用PBS重悬后通过0.22μ的膜进行过滤，收集过滤后的清液，即为外泌体。
对比例4
该对比例提供了一种乳源外泌体的提取方法。
所述乳源外泌体的乳原料来自于北京三元食品有限公司的鲜奶灌装车。
将50mL乳原料置于超速离心管内，在4℃，3000rpm下离心60min，取上清液，在4℃，16000rpm下离心20min，去除表层的脂肪，取清液。
将200μL清液，并与200μL PBS缓冲液混匀，再添加200μL英文特生物技术(北京)
有限公司生产的高效外泌体沉淀试剂，混匀，在室温下孵育反应30min后，在室温下，10000×g离心10min，取沉淀。
将沉淀用50μL PBS缓冲液重悬，在室温下，10000×g离心5min，取上清即为提取出的外泌体。
效果例1
通过投射电子显微镜(TEM)观察实施例1-2、对比例1-4中得到的乳源外泌体的形态，结果如图1-3所示。
由图1-6可见，实施例1-2和对比例1-3得到的乳源外泌体的直径范围均在30-150nm之间，且均具有杯装双层膜结构。对比例2-3提供的方法得到的乳源外泌体形态不完整且背景杂乱，而对比例1和实施例1中方法得到的乳源外泌体的形态则很完整，呈现出完整的“杯盘”结构。对比例4中方法得到的乳源外泌体形态则完全破碎。
效果例2
将实施例1，对比例1和3得到的乳源外泌体分别进行处理，具体的处理方法为：向乳源外泌体中加入100μL 0.1％的Waters公司生产的RapiGest SF试剂，混合均匀后，超声溶解30min，之后载入100μL DTT试剂，置于56℃下水浴反应1h。反应完成后，自然冷却至室温，加入0.1mol/L IAA 100μL，避光反应40min，再加入1mg/mL的胰蛋白酶50μL，进行酶切过夜，中间复加一次；酶解结束后，加入适量的0.5mol/L的HCl终止反应50min，使RapiGest SF试剂沉淀。使用HLB柱进行脱盐处理，除去RapiGest SF试剂在酸性条件下产生的沉淀，收集样品进行冻干处理，冻干后的样品使用0.1％甲酸水溶液复溶，10000×g离心10min，取上清备用。
将处理后的乳源外泌体采用纳升液相色谱串联Q Exactive Orbitrap质谱仪检测，并根据检测结果绘制蛋白种类韦恩图，如图7所示。
由图7可以看出，实施例1中制备的乳源外泌体可以分离得到315种牛乳外泌体蛋白，对比例1制备的乳源外泌体可以分离得到267种牛乳外泌体蛋白，对比例3制备的乳源外泌体可以分离得到307种牛乳外泌体蛋白。
综上所述，本申请提供的乳源外泌体的提取方法，通过对于工艺的优化和试剂的选择，相较于传统的高速离心法而言，制备的乳源外泌体在形态上更加完整，且需要的样品体积更少，且相较于同类试剂而言，制备的乳源外泌体在分离后得到的外泌体的蛋白
种类更多，因此，更适合后续外泌体蛋白组学的分析作业。
最后应说明的是：以上实施例仅用以说明本申请的技术方案，而非对其限制；尽管参照前述实施例对本申请进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本申请各实施例技术方案的精神和范围。
本申请涉及一种乳源外泌体的提取方法，采用本申请提供的乳源外泌体提取方法制备的乳源外泌体，相较于其他方法制备的乳源外泌体而言，形态更完整，需要的样品体积更少，且在分离后包含的乳源外泌体的蛋白种类更多，更有利于分析研究作业。
Claims (10)
- 一种乳源外泌体的提取方法，其特征在于，依次按下述步骤进行：(1)将乳原料分装，离心，取清液；(2)将步骤(1)得到的清液离心，取中间层清液；(3)将步骤(2)得到的中间层清液离心，取清液，与PBS缓冲液和外泌体提取试剂混匀，孵育；(4)将步骤(3)的产物离心，保留沉淀，用PBS缓冲液重悬；(5)将步骤(4)的产物离心，取上清，即得。
- 根据权利要求1所述的乳源外泌体的提取方法，其特征在于，步骤(1)中的离心工艺为室温下1500-2500×g离心5-15min。
- 根据权利要求1所述的乳源外泌体的提取方法，其特征在于，步骤(2)中的离心工艺为室温下8500-10000×g离心25-35min，优选的，离心时间为30min。
- 根据权利要求1所述的乳源外泌体的提取方法，其特征在于，步骤(3)中的离心工艺为室温下8500-10000×g离心25-35min，优选的，离心时间为30min。
- 根据权利要求1所述的乳源外泌体的提取方法，其特征在于，步骤(3)中，清液、PBS缓冲液和外泌体提取试剂的体积比为1:1:1。
- 根据权利要求5所述的乳源外泌体的提取方法，其特征在于，所述外泌体提取试剂为Thermo Fisher公司生产的总外泌体分离试剂。
- 根据权利要求1所述的乳源外泌体的提取方法，其特征在于，步骤(3)中，孵育时间为25-35min，优选的，孵育时间为30min。
- 根据权利要求1所述的乳源外泌体的提取方法，其特征在于，步骤(4)中离心工艺为室温下8500-10000×g离心9-11min，优选的，离心时间为10min。
- 根据权利要求1所述的乳源外泌体的提取方法，其特征在于，步骤(5)中离心工艺为室温下8500-10000×g离心4-6min，优选的，离心时间为5min。
- 一种乳源外泌体，其特征在于，采用权利要求1-9中任一所述的提取方法制备而成。
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| US10166259B1 (en) * | 2016-03-15 | 2019-01-01 | 3P Biotechnologies, Inc. | Isolation of exosomes from colostrum powder and exosomal drug formulations using the same |
| CN109468265A (zh) * | 2018-11-06 | 2019-03-15 | 广州市创唯曦旺生物科技有限公司 | 一种提取乳外泌体的方法 |
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