WO2023246012A1 - 一种分离提取胃癌组织来源细胞外囊泡多种亚群的方法 - Google Patents

一种分离提取胃癌组织来源细胞外囊泡多种亚群的方法 Download PDF

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WO2023246012A1
WO2023246012A1 PCT/CN2022/138912 CN2022138912W WO2023246012A1 WO 2023246012 A1 WO2023246012 A1 WO 2023246012A1 CN 2022138912 W CN2022138912 W CN 2022138912W WO 2023246012 A1 WO2023246012 A1 WO 2023246012A1
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gastric cancer
supernatant
extracellular vesicles
cancer tissue
minutes
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French (fr)
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张常华
霍明宇
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中山大学附属第七医院(深圳)
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    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

Definitions

  • the present invention relates to the field of biomedical technology, and in particular to a method for isolating and extracting multiple subpopulations of extracellular vesicles derived from gastric cancer tissue.
  • Extracellular vesicles are mostly 30nm-150nm in diameter and have a double-layer lipid membrane structure. Extracellular vesicles are secreted by a variety of cells in the body and contain a variety of proteins derived from mother cells. RNA and other biologically active substances can participate in processes such as cell communication, cell migration, angiogenesis, and tumor cell growth. They are widely present in various body fluids and tissue gaps, and stably carry some important signaling molecules. After extracellular vesicles enter the extracellular matrix, they can move in a directional manner and specifically bind to target cells. They activate intracellular signaling cascades by binding to receptors on the target cell surface. They can also pass through the vesicles of extracellular vesicles.
  • Extracellular vesicles have a wide range of biological functions, and the specific bioactive substances carried by EVs derived from different cells can play important regulatory roles in different environments. Moreover, extracellular vesicles can resist most degradation and damage due to their stable bilayer lipid membrane structure and have good carrying potential. In current research, most extracellular vesicles are secreted by cell lines. To explore the pathophysiological functions of extracellular vesicles on the body, it can be used in tumor vaccines, biomarkers, chemotherapy drug carriers, and targeted organisms. It has important guiding value in areas such as treatment.
  • tissue-derived extracellular vesicles can be directly isolated and extracted from tissue samples, and through subgroup classification, proteomics and transcriptomics of the extracted extracellular vesicles, Detection can discover more pathophysiological information contained in tissue extracellular vesicles, and can more truly reflect the biological functions and modes of action of tumor-derived extracellular vesicles.
  • the specific composition of the body is not yet clear.
  • the main methods for extracting tissue-derived extracellular vesicles include enzymatic digestion and leaching.
  • SEC size exclusion chromatography
  • ultrafiltration tubes a size exclusion chromatography
  • digestive enzymes the reagents used The application is complex and the operation steps are cumbersome, so it cannot be widely used.
  • extracellular vesicles derived from renal cancer and melanoma tissues are not a unified whole, and there are multiple subgroups.
  • Gastric cancer as one of the malignant tumors with a high incidence rate, also has There is no clear classification of subpopulations of extracellular vesicles derived from gastric cancer tissue, and there are no relevant extraction technical standards. Therefore, if multiple subpopulations of extracellular vesicles derived from gastric cancer tissue can be isolated and extracted, it can truly reflect the biological functions and mode of action of extracellular vesicles derived from gastric cancer tissue.
  • the present invention optimizes the enzymatic digestion process by preparing a digestive juice, and then combines it with differential centrifugation to separate and extract multiple subpopulations of extracellular vesicles derived from gastric cancer tissue, in order to solve the current problem of separation of subpopulations of extracellular vesicles derived from gastric cancer tissue. Problems with few extraction methods and excessive costs.
  • a method for isolating and extracting multiple subpopulations of extracellular vesicles derived from gastric cancer tissue characterized in that the method includes the following steps:
  • Step 1 Dip the gastric cancer tissue into the prepared digestive juice for enzymatic digestion and filtration to obtain the tissue digestion supernatant.
  • the digestive juice is a mixture of collagenase II and deoxyribonuclease I, or collagenase IV and deoxyribose.
  • the digestive solution may also be a combined solution of a mixed solution of ethylenediaminetetraacetic acid and dithiothreitol and collagenase IV;
  • Step 2 Take the digestion supernatant from step 1 and centrifuge it at 500g-1000g for 10-15 minutes at 4°C. Then take the centrifuged supernatant and centrifuge it at 3000g-5000g for 15-20 minutes at 4°C and collect the supernatant;
  • Step 3 Take the supernatant collected in step 2 and ultracentrifuge it again at 15000-17000g and 4°C for 15-30 minutes. Collect the supernatant and filter it and then ultracentrifuge it at 50000-120000g and 4°C for 60-100 minutes to obtain the precipitate. It is the first subgroup of extracellular vesicles derived from gastric cancer tissue;
  • Step 4 Collect the supernatant after centrifugation in step 3, ultracentrifuge at 150,000-200,000g for 60-100 minutes at 4°C, and obtain the precipitate again as subpopulation 2 of extracellular vesicles derived from gastric cancer tissue.
  • the digestive fluid is preferably a mixture of collagenase II and deoxyribonuclease I.
  • the supernatant of step 1 is preferably collected and centrifuged at 500g and 4°C for 10 minutes, and the centrifuged supernatant is then centrifuged at 3000g and 4°C for 20 minutes before collecting the supernatant.
  • the ultracentrifugation in step 3 preferably collects the supernatant and centrifuges it at 16,500g and 4°C for 30 minutes, then collects the supernatant, filters it and ultracentrifuges it at 100,000g and 4°C for 60 minutes to obtain a precipitate of extracellular vesicle subtypes. Group one.
  • the supernatant is preferably collected at 160,000g and ultracentrifuged at 4°C for 90 minutes to obtain the precipitate again as extracellular vesicle subgroup II.
  • the volume ratio of collagenase II to deoxyribonuclease I and the volume ratio of collagenase IV to deoxyribonuclease I are 1:1.
  • the enzymatic digestion conditions in step 1 are digestion with a constant temperature shaker at 37°C at 100-400 rpm for 20-80 minutes.
  • the concentration of collagenase II in the digestive juice is 2 mg/mL, and the concentration of deoxyribonuclease I is 0.2 mg/mL.
  • the concentration of collagenase IV in the digestive juice is 2 mg/mL, and the concentration of deoxyribonuclease I is 0.2 mg/mL.
  • the extracellular vesicle subpopulation one and the extracellular vesicle subpopulation two are two completely independent subpopulations.
  • the present invention separates and extracts multiple subpopulations of extracellular vesicles derived from gastric cancer tissue by immersing gastric cancer tissue in digestive juice for enzymatic digestion, and then performs multiple differential centrifugal extractions.
  • the cost of two enzymes used in this separation and extraction method is Low, easy to obtain reagents, simple preparation, wide range of brands to choose from, and on the basis of ensuring the quantity and quality of outer vesicles, it greatly reduces the difficulty and cost of operation.
  • the prepared digestive juice has higher extraction efficiency than other digestive juices , compared with the prior art in which a digestive enzyme formula is used for tissues from multiple sources, the present invention can more truly reflect the biological functions and mode of action of extracellular vesicles derived from gastric cancer tissues.
  • Figure 1 is a NTA particle size concentration diagram of the extracellular vesicle subpopulation EV-HD or EV-LD extracted from different digestive juices in Example 1-2.
  • A1 and A2 are collagenase II+DNase in Example 1 I digestive juice
  • B1 and B2 are the collagenase IV+DNase I digestive juice in Example 2
  • A1/B1 is the EV-HD subgroup
  • A2/B2 is the EV-LD subgroup.
  • FIG. 2 is a NTA particle size concentration diagram of the extracellular vesicle subpopulation EV-HD or EV-LD extracted from different digestive juices in Example 3-4.
  • C1 and C2 are collagenase XI+Dispase in Example 3 II digestion liquid
  • D1 and D2 are the EDTA/DTT+collagenase IV digestion liquid in Example 4
  • C1/D1 is the EV-HD subgroup
  • C2/D2 is the EV-LD subgroup.
  • Figure 3 is a structural diagram of the extracellular vesicle subpopulation EV-HD or EV-LD extracted from different digestive juices in Examples 1-4 under the visual field of 200nm and 500nm respectively.
  • A1 and A2 are the collagen in Example 1 Enzyme II+DNase I digestion solution
  • B1 and B2 are the collagenase IV+DNase I digestion solution in Example 2
  • C1 and C2 are the collagenase XI+Dispase II digestion solution in Example 3
  • D1 and D2 are the collagenase IV+DNase I digestion solution in Example 4 EDTA/DTT+collagenase IV digestion solution
  • A1/B1/C1/D1 is the EV-HD subgroup
  • A2/B2/C2/D2 is the EV-LD subgroup.
  • Figure 4 is a histogram of the protein content of the extracellular vesicle subpopulation EV-HD or EV-LD extracted from different digestive juices in Examples 1-4 and a diagram of the number of vesicles obtained per gram of tissue.
  • Scheme A is an example The collagenase II+DNase I digestion liquid in 1, the plan B is the collagenase IV+DNase I digestion liquid in embodiment 2, the plan C is the collagenase XI+Dispase II digestion liquid in embodiment 3, and plan D is the collagenase XI+Dispase II digestion liquid in embodiment 4.
  • EDTA/DTT+collagenase IV digestion solution is an example The collagenase II+DNase I digestion liquid in 1
  • the plan B is the collagenase IV+DNase I digestion liquid in embodiment 2
  • the plan C is the collagenase XI+Dispase II digestion liquid in embodiment 3
  • plan D is the collagenase XI+Dispase II digestion liquid in embodiment 4.
  • Figure 5 shows the expression of specific markers of extracellular vesicle subpopulations EV-HD or EV-LD extracted from different digestive juices in Examples 1-4.
  • A1 and A2 are collagenase II+ in Example 1 DNase I digestion liquid
  • B1 and B2 are the collagenase IV+DNase I digestion liquid in Example 2
  • C1 and C2 are the collagenase XI+Dispase II digestion liquid in Example 3
  • D1 and D2 are EDTA/DTT+ in Example 4
  • Collagenase IV digestion solution A1/B1/C1/D1 is the EV-HD subgroup
  • A2/B2/C2/D2 is the EV-LD subgroup.
  • Figure 6 is a protein expression clustering heat map of the extracellular vesicle subpopulation EV-HD or EV-LD.
  • Figure 7 is a diagram showing the biological function analysis of differentially expressed proteins in extracellular vesicle subpopulations.
  • Example 1 A method for isolating and extracting multiple subpopulations of extracellular vesicles derived from gastric cancer tissue
  • the pathological specimens used in the present invention are all fresh tumor pathological specimens from patients who underwent radical gastrectomy at the Seventh affiliated Hospital of Sun Yat-sen University (Shenzhen). The sampling is completed within 2 hours after the specimen is removed from the body. After sampling, it is briefly stored at 4°C. Packed in cold PBS buffer and shipped on ice, specimens are processed within 4 hours.
  • Separation and extraction specifically include the following steps:
  • HBSS buffer to dilute the collagenase II stock solution to 4mg/mL, use 0.15M NaCl solution to dilute the DNaseI stock solution to 400ug/mL, and finally mix the diluted collagenase II stock solution and DNase I stock solution in proportion. Mix 1:1 to obtain a digestion solution with a final concentration of collagenase II of 2 mg/mL and a final concentration of DNase I of 0.2 mg/mL.
  • step (3) Immerse the fresh gastric cancer tissue pieces minced in step (1) into 10 mL of the digestive juice prepared in step (2), then place them in a 37°C constant temperature shaker, digest at 200 rpm for 60 minutes, and filter with a 40um filter to obtain fresh Gastric cancer tissue digestion supernatant;
  • step (6) Centrifuge the filtered supernatant in step (6) in an ultracentrifuge at 4°C and 100,000g for 60 minutes to obtain the precipitated extracellular vesicle subpopulation 1 derived from gastric cancer tissue, which is named EV-HD subpopulation. and collect the supernatant;
  • step (8) Centrifuge the supernatant obtained in step (7) again in an ultracentrifuge at 4°C and 160,000g for 90 minutes to obtain the precipitated extracellular vesicle subpopulation 2 derived from gastric cancer tissue, which is named EV-LD subpopulation;
  • Example 2 A method for isolating and extracting multiple subpopulations of extracellular vesicles derived from gastric cancer tissue
  • the separation and extraction steps are the same as in Example 1.
  • the digestion liquid used is collagenase IV + deoxyribonuclease I (DNase I) digestion liquid: 100 mg of collagenase IV is dissolved in HBSS buffer and diluted to 4 mg/mL. Then use the diluted collagenase IV stock solution and the DNase I stock solution diluted to 400ug/mL in Example 1 to mix in a ratio of 1:1 to obtain a final concentration of collagenase IV of 2 mg/mL and a final concentration of DNase I of 0.2 mg/mL. mL of digestive juice.
  • DNase I deoxyribonuclease I
  • Example 3 A method for isolating and extracting multiple subpopulations of extracellular vesicles derived from gastric cancer tissue
  • the separation and extraction steps are the same as in Example 1.
  • the digestion solution used is neutral protease (Dispase II) + collagenase XI digestion solution: Collagenase XI is made into a solution with a final concentration of 1 mg/mL using HBSS buffer. , use HBSS buffer to make Dispase II into a solution with a final concentration of 1 mg/mL, and then mix Collagenase XI and Dispase II in a ratio of 1:1 to prepare a digestion solution.
  • the digestion solution used is neutral protease (Dispase II) + collagenase XI digestion solution: Collagenase XI is made into a solution with a final concentration of 1 mg/mL using HBSS buffer. , use HBSS buffer to make Dispase II into a solution with a final concentration of 1 mg/mL, and then mix Collagenase XI and Dispase II in a ratio of 1:1 to prepare a digestion solution.
  • Example 4 A method for isolating and extracting multiple subpopulations of extracellular vesicles derived from gastric cancer tissue
  • the separation and extraction steps are the same as those in Example 1.
  • the digestion liquid used is ethylenediaminetetraacetic acid (EDTA) + dithiothreitol (DTT) + collagenase IV digestion liquid.
  • EDTA ethylenediaminetetraacetic acid
  • DTT dithiothreitol
  • enzyme IV digestion solution (the preparation of collagenase IV digestion solution is the same as in Example 2), digest in a constant temperature shaker at 37°C and 200 rpm for 60 minutes, and filter with a 40um filter to obtain fresh gastric cancer tissue digestion supernatant.
  • A1, B1, C1 and D1 are EV-HD subgroups, and A2, B2, C2 and D2 are EV-LD subgroups, as shown in Figure 3, indicating that the extracellular vesicles extracted in Examples 1-4 have double-layer membranes. structure.
  • a particle size analyzer (NanoFCM, N30E) was used to measure the concentration of extracellular vesicles, and then the total volume was used to calculate the number of vesicles obtained in Examples 1-4, and then the number of vesicles obtained in Examples 1-4 per gram was calculated. The number of vesicles acquired by tumor tissue.
  • Scheme A is the extracellular vesicle subpopulation extracted from the collagenase II+DNase I digestion solution in Example 1
  • Scheme B is the extracellular vesicles extracted from the collagenase IV+DNase I digestion solution in Example 2.
  • Scheme C is the extracellular vesicle subpopulation extracted from the collagenase Bubble subgroup.
  • TBST buffer to prepare a blocking solution containing 5% skim milk powder.
  • the PVDF membrane is immersed in the blocking solution and blocked at room temperature for 1 hour. After blocking, wash it three times with TBST buffer for 10 minutes each time.
  • the first antibody is the corresponding antibody of the target protein TSG101, CD63, CD9, ⁇ -actin, APOA1, including Rabbit-TSG101 Antibody (Abcam, United States), Rabbit-CD63 Antibody (Abcam, United States), Rabbit-CD9 Antibody (Abcam, USA), Rabbit- ⁇ -actin Antibody (Proteintech, USA), Rabbit-APOA1 Antibody (Affinity, USA).
  • Primary antibody stock solution and primary antibody diluent Solarbio, China
  • a volume ratio of 1:1000 to prepare a primary antibody working solution that can specifically bind to the target protein, and then immerse the washed strip in the primary antibody working solution.
  • incubate on a shaking table at 4°C for 12-16 hours. After incubation, remove the strip and wash it three times with TBST buffer for 10 minutes each time.
  • the secondary antibodies include horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) (Beyotime Biotechnology, China), horseradish peroxidase-labeled goat anti-mouse IgG (H +L)(Biyuntian Biotechnology, China).
  • H+L horseradish peroxidase-labeled goat anti-rabbit IgG
  • H +L horseradish peroxidase-labeled goat anti-mouse IgG
  • the secondary antibody working solution Take the second antibody stock solution and the secondary antibody diluent (Solarbio, China) at a volume ratio of 1:1000 to prepare a second antibody working solution that can specifically bind to the first antibody, and then immerse the washed strip with the first antibody species. Incubate at room temperature for 1 hour in the corresponding and chromogenically labeled secondary antibody working solution, then remove the secondary antibody incubated strip and wash it 3 times with TBST buffer for 10 minutes each time.
  • A1, B1, C1 and D1 are EV-HD subpopulations
  • A2, B2, C2 and D2 are EV-LD subpopulations.
  • the two extracellular vesicle subpopulations extracted in Examples 1-4 Both EV-HD and EV-LD can see the expression of specific markers TSG101, CD63, CD9, and ⁇ -actin in extracellular vesicles, while the expression of the negative marker APOA1 is not obvious.
  • Relative quantitative proteomic analysis using the Lable-Free method includes the following steps:
  • Step 1 Extract the protein sample. Draw the standard curve and calculate the protein content of the sample. The protein content can reach 0.05ug/uL. Carry out subsequent experiments.
  • Buffer A is a 0.1% formic acid aqueous solution
  • Buffer B is a 0.1% formic acid acetonitrile aqueous solution (acetonitrile is 84%).
  • the chromatographic column is balanced with 95% A solution.
  • the sample is loaded from the automatic sampler to the loading column (Thermo ScientificAcclaim PepMap100, 100 ⁇ m ⁇ 2cm, nanoViper C18), and passes through the analytical column (Thermo scientific EASYcolumn, 10cm, ID75 ⁇ m, 3 ⁇ m, C18-A2) separation, the flow rate is 300 NL/min.
  • the mass spectrometry detection method is positive ion
  • the precursor ion scanning range is 300–1800 m/z
  • the first-level mass spectrometry resolution is 70,000at 200 m/z
  • the AGC (Automatic gain control) target is 1e6
  • Maximum IT is 50ms
  • dynamic exclusion Time is 60.0s.
  • the acquisition of mass spectrometry data uses a data-dependent top10 method to dynamically select the most abundant precursor ions from the survey scan (300-1800 m/z) for HCD fragmentation.
  • MS2 scan 20 fragment spectra (MS2 scan) are collected after each full scan (full scan)
  • MS2 Activation Type is HCD
  • Isolation window is 2 m/z
  • secondary mass spectrometry Resolution is 17,500 at 200 m/z
  • Normalized Collision Energy is 30eV
  • Underfill is 0.1%.
  • the raw data of mass spectrometry analysis are RAW files, and MaxQuant software (version number 1.5.3.17) is used for library search identification and quantitative analysis.
  • Protein expression difference analysis Normalize the mass spectrometry quantitative information of the sample protein in step (2) (normalized to the (-1,1) interval), and then analyze the protein expression amount through t test to find out whether Significantly differentially expressed proteins.
  • Figure 6 shows the identification of protein expression differences between the two subpopulations. It can be seen that the extracellular vesicle subpopulations EV-HD and EV-LD have a variety of differentially expressed proteins, and the protein expression patterns are different.
  • Figure 7 shows the biological function identification analysis of differentially expressed proteins between the two subgroups. It can be seen that the differentially expressed proteins between the two subgroups have multiple biological functions. Therefore, it can be seen from Figure 6 and Figure 7 that the extracellular vesicle subpopulations EV-HD and EV-LD are two completely independent subpopulations, with different protein expression contents and different biological functions.

Abstract

提供了一种分离提取胃癌组织来源细胞外囊泡多种亚群的方法：先将肿瘤组织浸入消化液中进行酶解消化，获得组织消化上清液，同时配制不同的消化液来优化酶解消化工艺，然后对组织消化上清液分别进行多次差速离心，从而获得组织来源细胞外囊泡及其亚群。优选用胶原酶II和DNaseI配制的消化液比用其他消化液的提取效率高。

Description

一种分离提取胃癌组织来源细胞外囊泡多种亚群的方法 技术领域
本发明涉及生物医学技术领域，特别涉及一种分离提取胃癌组织来源细胞外囊泡多种亚群的方法。
背景技术
细胞外囊泡(extracellular vesicles,EVs)的直径多为30nm-150nm，拥有双层脂质膜结构，细胞外囊泡由体内多种细胞分泌，其内包含了多种来源于母细胞的蛋白质、RNA等多种生物活性物质，能参与细胞通讯、细胞迁移、血管新生和肿瘤细胞生长等过程，广泛地存在于各种体液和组织间隙中，并稳定携带了一些重要的信号分子。细胞外囊泡进入细胞外基质后能够定向移动，并特异性地与靶细胞结合，通过与靶细胞表面受体结合而激活胞内信号级联反应，也可以通过细胞外囊泡的囊泡内化、融合作用将内部的生物活性物质直接释放入靶细胞内，导致靶细胞的生物学功能发生改变，从而达到相应的调控作用。细胞外囊泡的生物作用广泛，不同细胞来源的EVs所运载的特异性生物活性物质能够在不同的环境中起到重要的调控作用。而且细胞外囊泡由于稳定的双层脂质膜结构，能够抵御大部分的降解、破坏作用，拥有良好的运载潜力。在目前的研究中，大多数细胞外囊泡都是通过细胞系的分泌得来，而探究细胞外囊泡对机体的病理生理学功能，在肿瘤疫苗、生物标志物、化疗药物载体、靶向生物治疗等领域具有重要的指导价值。但是，人体组织中的细胞构成十分复杂，多种细胞通过一定的分布构成人体组织即可发挥正常的生理功能，因此，通过体外培养的细胞系提取的细胞外囊泡并不能真实反映细胞外囊泡在人体内的生物学功能。
近年来，由于组织来源的细胞外囊泡(tissue-derived extracellular vesicles)能直接从组织样本中分离提取得到，且通过对提取到的细胞外囊泡进行亚群分类、蛋白组学及转录组学检测，能够发现组织细胞外囊泡所包含的更多病理生理学信息，可以更加真实地反映肿瘤来源细胞外囊泡的生物学功能和作用模式，这些发现使得组织来源细胞外囊泡的概念逐渐被推广。但是作为细胞外囊泡研究的新兴领域，组织来源细胞外囊泡的提取技术还处于摸索阶段，而且多种恶性肿瘤的组织来源细胞外囊泡尚未具备准确高效的提取技术，对于组织来源外泌体的具体构成也尚未清楚。目前，组织来源细胞外囊泡的提取方法主要有酶消化法和浸出法两种，但由于使用的分子排阻色谱法(Size exclusion chromatography，SEC)、超滤管、消化酶成本过高，试剂应用复杂，操作步骤繁琐等问题，所以无法进行广泛的应用。在现有的研究中，已发现肾癌、黑色素瘤组织来源的细胞外囊泡并不是统一的整体，存在多种亚群分群的情况，而胃癌作为发病率很高的恶性肿瘤之一，还没有明确胃癌组织来源细胞外囊泡的亚群分群情况，也没有相关的提取技术标准。因此，如果能分离提取出胃癌组织来源细胞外囊泡 的多种亚群，则可以真实地反映胃癌组织来源细胞外囊泡的生物学功能和作用模式。
发明内容
本发明通过配制一种消化液优化酶解消化工艺，再结合差速离心法分离提取了胃癌组织来源的细胞外囊泡多种亚群，以解决目前胃癌组织来源细胞外囊泡亚群的分离提取方法较少以及成本过高的问题。
为了实现上述目的，本发明的技术方案如下：
一种分离提取胃癌组织来源细胞外囊泡多种亚群的方法，其特征在于，所述方法包括以下步骤：
步骤1：将胃癌组织浸入配制的消化液中进行酶解消化过滤后得到组织消化上清液，所述消化液为胶原酶II和脱氧核糖核酸酶I的混合液，或胶原酶IV和脱氧核糖核酸酶I的混合液，所述消化液也可以是乙二胺四乙酸和二硫苏糖醇的混合液与胶原酶IV的组合溶液；
步骤2：取步骤1的消化上清液以500g-1000g，4℃离心10-15分钟，再取离心后的上清液以3000g-5000g，4℃离心15-20分钟后收集上清液；
步骤3：取步骤2收集的上清液再次以15000-17000g，4℃超速离心15-30分钟，并收集上清液过滤后再以50000-120000g，4℃超速离心60-100分钟，得到沉淀即为胃癌组织来源细胞外囊泡亚群一；
步骤4：收集步骤3离心后的上清液于150000-200000g，4℃超速离心60-100分钟，再次得到沉淀为胃癌组织来源细胞外囊泡亚群二。
优选地，所述消化液优选胶原酶II和脱氧核糖核酸酶I的混合液。
优选地，所述步骤2的超速离心优选收集步骤1的上清液以500g，4℃离心10分钟，再取离心后的上清液以3000g，4℃离心20分钟后收集上清液。
优选地，所述步骤3的超速离心优选收集上清液以16500g，4℃超速离心30分钟，再收集上清液过滤后以100000g，4℃超速离心60分钟，得到沉淀为细胞外囊泡亚群一。
优选地，所述步骤4的超速离心优选收集上清液于160000g，4℃超速离心90分钟，再次得到沉淀为细胞外囊泡亚群二。
优选地，配制消化液时胶原酶II和脱氧核糖核酸酶I的体积比以及胶原酶IV和脱氧核糖核酸酶I的体积比为1：1。
优选地，所述步骤1酶解消化条件为37℃恒温摇床100-400rpm消化20-80分钟。
优选地，所述消化液中胶原酶II的浓度为2mg/mL，脱氧核糖核酸酶I的浓度为0.2mg/mL
优选地，所述消化液中胶原酶IV的浓度为2mg/mL，脱氧核糖核酸酶I的浓度为0.2mg/mL。
进一步优选地，所述细胞外囊泡亚群一和细胞外囊泡亚群二是两种完全独立的亚群。
本发明的有益效果如下：
本发明通过将胃癌组织浸入消化液中进行酶解消化，再经多次差速离心提取来分离提取胃癌组织来源的细胞外囊泡多种亚群，这种分离提取方法使用的两种酶成本低、试剂易获得、配制简单、品牌可选择范围广，而且在保证外囊泡数量和质量的基础上，极大降低了操作难度和成本，同时配制的消化液比其他消化液的提取效率高，对比现有技术中一种消化酶配方用于多种来源的组织，本发明能更加真实地反映胃癌组织来源细胞外囊泡的生物学功能和作用模式。
附图说明
为了更清楚地说明本发明的技术方案，下面将对实施方式中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是实施例1-2中不同消化液提取的细胞外囊泡亚群EV-HD或EV-LD的NTA粒径浓度图，图中：A1和A2为实施例1中胶原酶II+DNase I消化液，B1和B2为实施例2中胶原酶IV+DNase I消化液，A1/B1为EV-HD亚群，A2/B2为EV-LD亚群。
图2是实施例3-4中不同消化液提取的细胞外囊泡亚群EV-HD或EV-LD的NTA粒径浓度图，图中：C1和C2为实施例3中胶原酶XI+Dispase II消化液，D1和D2为实施例4中EDTA/DTT+胶原酶IV消化液，C1/D1为EV-HD亚群，C2/D2为EV-LD亚群。
图3是实施例1-4中不同消化液提取的细胞外囊泡亚群EV-HD或EV-LD分别在200nm和500nm视野下的结构图，图中：A1和A2为实施例1中胶原酶II+DNase I消化液，B1和B2为实施例2中胶原酶IV+DNase I消化液，C1和C2为实施例3中胶原酶XI+Dispase II消化液，D1和D2为实施例4中EDTA/DTT+胶原酶IV消化液，A1/B1/C1/D1为EV-HD亚群，A2/B2/C2/D2为EV-LD亚群。
图4是实施例1-4不同消化液提取的细胞外囊泡亚群EV-HD或EV-LD的蛋白含量柱状图和每克组织获得的囊泡数量图，图中：方案A为实施例1中胶原酶II+DNase I消化液，方案B为实施例2中胶原酶IV+DNase I消化液，方案C为实施例3中胶原酶XI+Dispase II消化液，方案D为实施例4中EDTA/DTT+胶原酶IV消化液。
图5是实施例1-4中不同消化液提取的细胞外囊泡亚群EV-HD或EV-LD的特异性标志物的表达，图中：A1和A2为实施例1中胶原酶II+DNase I消化液，B1和B2为实施例2中胶原酶IV+DNase I消化液，C1和C2为实施例3中胶原酶XI+Dispase II消化液，D1和D2为实施例4中EDTA/DTT+胶原酶IV消化液，A1/B1/C1/D1为EV-HD亚群，A2/B2/C2/D2为 EV-LD亚群。
图6是细胞外囊泡亚群EV-HD或EV-LD的蛋白表达聚类热图。
图7是细胞外囊泡亚群差异表达蛋白的生物学功能分析图。
具体实施方式
下面对本发明实施方式中的技术方案进行清楚、完整地描述。在此需要说明的是，对于这些实施方式的说明用于帮助理解本发明，但并不构成对本发明的限定。此外，下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。下述实施例中的实验方法，如无特殊说明，均为常规说法。
实施例1：一种分离提取胃癌组织来源细胞外囊泡多种亚群的方法
1、将胃癌患者术后肿瘤病理标本的新鲜肿瘤组织在术后2小时内进行酶解消化，获得组织消化上清液；
2、对消化上清液进行差速离心，获得组织来源细胞外囊泡及其亚群。
本发明使用的病理标本均来源于在中山大学附属第七医院(深圳)行胃癌根治术的患者的新鲜肿瘤病理标本，且在标本离体2小时内完成采样，采样后短暂保存于4℃预冷的PBS缓冲液中，冰盒中运输，在4小时内完成标本的处理工作。
分离提取具体包括以下步骤：
(1)取胃癌患者术后肿瘤病理标本的新鲜肿瘤组织0.5g-1g，用PBS清洗胃癌组织3遍后剔除胃癌组织上多余的脂肪组织及结缔组织，再用组织剪将胃癌组织剪碎成直径为1-2mm的小块；
(2)配制胶原酶II+脱氧核糖核酸酶I(DNase I)消化液：
①将100mg胶原酶II(上海翌圣生物)加入500uL的HBSS缓冲液配制成200mg/mL的胶原酶II储存液，在-20℃环境下长期保存；
②配置pH为6.5且包含20 mM醋酸钠(sodium acetate)、5 mM的CaCl 2、0.1 mM的苯甲基磺酰氟(PMSF)和50％甘油的混合溶液，然后取1mL混合溶液将DNase I粉末溶解制成DNase I储存液；
③用HBSS缓冲液将胶原酶II储存液稀释至4mg/mL，用0.15M的NaCl溶液将DNaseI储存液稀释至400ug/mL，最后将稀释后的胶原酶II储存液和DNase I储存液按比例1:1混合获得胶原酶II终浓度为2mg/mL，DNase I终浓度为0.2mg/mL的消化液。
(3)将步骤(1)剪碎后的新鲜胃癌组织小块浸入10mL步骤(2)配制的消化液中，然后放入37℃恒温摇床，200rpm消化60分钟，用40um滤网过滤获得新鲜胃癌组织消化上清液；
(4)将步骤(3)过滤后的新鲜胃癌组织消化上清液于冷冻离心机4℃，500g离心10分钟，收集上清液；
(5)将步骤(4)离心后的上清液于冷冻离心机4℃，3000g离心20分钟，收集上清液；
(6)将步骤(5)离心后的上清液于超速离心机4℃，16500g离心30分钟，收集上清液，然后用0.8um滤器过滤上清液，收集过滤后的上清液；
(7)将步骤(6)过滤后的上清液于超速离心机4℃，100000g离心60分钟，获得沉淀为胃癌组织来源细胞外囊泡亚群一，将其命名为EV-HD亚群，并收集上清液；
(8)将步骤(7)获得的上清液再次于超速离心机4℃，160000g离心90分钟，获得沉淀为胃癌组织来源细胞外囊泡亚群二，将其命名为EV-LD亚群；
(9)用PBS溶液将步骤(7)、(8)获得的沉淀重悬，得到胃癌组织来源细胞外囊泡的不同亚群混悬液，用于后续实验。
实施例2：一种分离提取胃癌组织来源细胞外囊泡多种亚群的方法
分离提取步骤同实施例1，不同之处在于，所用的消化液为胶原酶IV+脱氧核糖核酸酶I(DNase I)消化液：将100mg胶原酶IV用HBSS缓冲液溶解后稀释至4mg/mL，然后用稀释后的胶原酶IV储存液和实施例1中稀释至400ug/mL的DNase I储存液按比例1:1混合获得胶原酶IV终浓度为2mg/mL，DNase I终浓度为0.2mg/mL的消化液。
实施例3：一种分离提取胃癌组织来源细胞外囊泡多种亚群的方法
分离提取步骤同实施例1，不同之处在于，所用的消化液为中性蛋白酶(Dispase II)+胶原酶XI消化液：用HBSS缓冲液将胶原酶XI制成终浓度为1mg/mL的溶液，用HBSS缓冲液将Dispase II制成终浓度为1mg/mL的溶液，再将胶原酶XI和Dispase II按照比例1:1制得消化液。
实施例4：一种分离提取胃癌组织来源细胞外囊泡多种亚群的方法
分离提取步骤同实施例1，不同之处在于，所用的消化液为乙二胺四乙酸(EDTA)+二硫苏糖醇(DTT)+胶原酶IV消化液，先将剪碎后的1-2mm胃癌组织小块浸入10mL混合溶液(由0.1M的EDTA和0.1M的DTT配制而成)中，于37℃恒温摇床，200rpm消化30分钟；再次将消化后的胃癌组织小块浸入10mL胶原酶IV消化液(配制胶原酶IV消化液同实施例2)中，于37℃恒温摇床，200rpm消化60分钟，用40um滤网过滤获得新鲜胃癌组织消化上清液。
实验例1：分析不同消化液对提取细胞外囊泡亚群效率的影响
1、检测细胞外囊泡的粒径和浓度
分别取10μL实施例1-4提取的细胞外囊泡亚群EV-HD、EV-LD用PBS缓冲液稀释至 30μL，再用粒径分析仪(NanoFCM,N30E)来检测稀释后的细胞外囊泡亚群。
由图1和图2可知，实施例1-4提取到的细胞外囊泡粒径主要集中在50-200nm。
2、透射电镜检测
分别取10μL实施例1-4提取的细胞外囊泡亚群滴加于铜网上沉淀1 min，滤纸吸去浮液；再用醋酸双氧铀10μL滴加于铜网上沉淀1 min，滤纸吸去浮液，常温干燥5-10分钟后用透射电镜(Hitachi,H-7650)在100 kv电压下于200nm和500nm视野下分别进行电镜检测成像。
A1、B1、C1和D1为EV-HD亚群，A2、B2、C2和D2为EV-LD亚群，如图3所示，说明实施例1-4提取的细胞外囊泡具有双层膜结构。
3、分析细胞外囊泡亚群的蛋白含量、囊泡数以及Western Blotting验证
具体步骤包括：
(1)细胞外囊泡全蛋白的提取
按照全蛋白提取试剂盒(碧云天生物)说明书要求将1mL的冷裂解液(LysisBuffer)、10uL 100×磷酸酶抑制剂、10uL 100×蛋白酶抑制剂和5uL的PMSF混匀制备成裂解液。分别取50uL实施例1-4提取的细胞外囊泡亚群EV-HD、EV-LD，并加入10uL裂解液，于冰上裂解30分钟，且裂解期间每4分钟震荡30秒，震荡后冰上静置，然后在4℃环境下12000g离心5分钟，留取上清即为全蛋白提取液，再往蛋白提取液中加入体积为全蛋白提取液的25％的5×上样缓冲液，充分混匀后99℃金属浴5-10分钟进行蛋白变性，变性后将蛋白放置于-80℃环境保存。
(2)检测蛋白含量及囊泡数
取96孔酶标板，按照蛋白含量检测试剂盒(碧云天生物)说明书的要求加入10uL蛋白标准品试剂，并用去离子水稀释至100uL使蛋白标准品终浓度为0.5mg/mL。分别取2uL步骤(1)提取的EV-HD、EV-LD亚群的变性蛋白，用去离子水稀释至20uL；再将BCA试剂A和BCA试剂B按照50:1的比例配制BCA工作液，于96孔酶标板中每个孔加入BCA工作液200uL，充分混匀后在37℃下孵育30分钟，最后使用酶标仪(BioTek，Synergy H1M)记录吸光值并绘制标准曲线，计算细胞外囊泡亚群的蛋白含量。
另外，使用粒径分析仪(NanoFCM,N30E)来测得细胞外囊泡浓度，再通过总体积来计算出实施例1-4获得的囊泡数，进而计算得到实施例1-4从每克肿瘤组织获得的囊泡的数量。
实验结果：
由图4可知，方案A为实施例1中胶原酶II+DNase I消化液提取的细胞外囊泡亚群，方案B为实施例2中胶原酶IV+DNase I消化液提取的细胞外囊泡亚群，方案C为实施例3中胶原酶XI+Dispase II消化液提取的细胞外囊泡亚群，方案D为实施例4中EDTA/DTT+胶原 酶IV+DNase I消化液提取的细胞外囊泡亚群。
提取细胞外囊泡亚群EV-HD时，方案A的蛋白含量以及每克组织获得的囊泡数量最多，方案C最少；提取细胞外囊泡亚群EV-LD时，方案A的蛋白含量最多，方案D为最少。而方案B每克组织获得的EV-LD亚囊泡数量虽然比方案A多，但是方案A提取的亚群EV-HD和EV-LD总囊泡数要比方案B多。综上所述，实施例1用胶原酶II和DNase I配制的消化液提取细胞外囊泡亚群的效率最高，为最佳提取方案。
(3)Western Blotting蛋白免疫印迹实验
具体包括以下步骤：
①准备手动灌胶的容器及玻璃板，固定后确认无漏水，按下述成分来配制凝胶：
5％浓缩凝胶：H 2O 2.7mL、30％丙烯酰胺0.67mL、1.5M Tris-HCl(PH＝6.8)0.5mL、10％十二烷基硫酸钠(SDS)0.4mL、10％PAGE胶凝固剂(APS)0.4mL、四甲基乙二胺(TEMED)0.004mL。
8％分离凝胶：H 2O 4.6mL、30％丙烯酰胺2.7mL、1.5M Tris-HCl(PH＝8.8)2.5mL、10％十二烷基硫酸钠(SDS)0.1mL、10％PAGE胶凝固剂(APS)0.1mL、四甲基乙二胺(TEMED)0.006mL。
10％分离凝胶：H 2O 4mL、30％丙烯酰胺3.3mL、1.5M Tris-HCl(PH＝8.8)2.5mL、10％十二烷基硫酸钠(SDS)0.1mL、10％PAGE胶凝固剂(APS)0.1mL、四甲基乙二胺(TEMED)0.004mL。
12％分离凝胶：H 2O 3.3mL、30％丙烯酰胺4mL、1.5M Tris-HCl(PH＝8.8)2.5mL、10％十二烷基硫酸钠(SDS)0.1mL、10％PAGE胶凝固剂(APS)0.1mL、四甲基乙二胺(TEMED)0.006mL。
②分别将配制好的分离凝胶加入灌胶容器内至一定高度，确保凝胶上缘距离齿梳下缘约1cm，在凝胶液上层加入1mL无水乙醇，等待30分钟至分离凝胶彻底凝固后去除无水乙醇，再将5％浓缩凝胶灌注至分离凝胶上层，插入齿梳，待25分钟至浓缩凝胶彻底凝固。
③将步骤②配制好的凝胶放入电泳槽内，加入足量电泳缓冲液，在电泳缓冲液中拔出齿梳，加入免疫印迹指示剂以及每孔加入15ug步骤(1)提取的EV-HD、EV-LD亚群变性蛋白。
④设置浓缩凝胶固定电压80V，待蛋白条带跑过浓缩凝胶后切换电压。分离凝胶固定电压120V进行凝胶电泳约70分钟，至指示剂跑至目标位置后停止电泳。
⑤转膜夹两面分别放置海绵及滤纸，裁剪合适大小的0.45um PVDF膜，放入甲醇中活化1分钟，转膜液中稳定3分钟。按照电极方向放置电泳后凝胶及PVDF膜，排出气泡后组装转膜夹，并固定至转膜槽中，加入预冷的转膜缓冲液，再放置转膜槽至冰中并设置固定电流 200mA，转膜时间70分钟，取出PVDF膜，根据蛋白分子量裁剪相应位置PVDF膜，TBST缓冲液清洗3遍，每次3分钟。
⑥用TBST缓冲液配制含有5％脱脂奶粉的封闭液，PVDF膜浸没至封闭液中常温封闭1小时，封闭结束后以TBST缓冲液清洗3次，每次10分钟。
⑦配制第一抗体工作液：第一抗体为目标蛋白TSG101、CD63、CD9、β-actin、APOA1的相应抗体，包括Rabbit-TSG101 Antibody(Abcam，美国)、Rabbit-CD63 Antibody(Abcam，美国)、Rabbit-CD9 Antibody(Abcam，美国)、Rabbit-β-actin Antibody(Proteintech，美国)、Rabbit-APOA1 Antibody(Affinity，美国)。取第一抗体原液和一抗稀释液(Solarbio，中国)按1:1000的体积比配制能与目标蛋白特异结合的第一抗体工作液，再将清洗后的条带浸入第一抗体工作液中，4℃环境下摇床孵育12-16小时，孵育后取出条带，用TBST缓冲液清洗3次，每次10分钟。
⑧配制第二抗体工作液：第二抗体分别包括辣根过氧化物酶标记山羊抗兔IgG(H+L)(碧云天生物，中国)、辣根过氧化物酶标记山羊抗鼠IgG(H+L)(碧云天生物，中国)。取第二抗体原液和二抗稀释液(Solarbio，中国)按1:1000的体积比配制能与第一抗体特异结合的第二抗体工作液，再将清洗完的条带浸入与第一抗体种属相对应且带有显色标记的第二抗体工作液中，常温孵育1小时，再取出二抗孵育后条带，以TBST缓冲液清洗3遍，每次10分钟。
⑨将超敏ECL发光液A液与B液以1:1混合，配制成发光工作液，使用ChemiDoc Touch成像系统对免疫印迹条带进行显像，并进行图像处理及分析。
实验结果：
A1、B1、C1和D1为EV-HD亚群，A2、B2、C2、D2为EV-LD亚群，如图5所示，实施例1-4提取的两种的细胞外囊泡亚群EV-HD和EV-LD均可见细胞外囊泡有特异性标志物TSG101、CD63、CD9、β-actin表达，而阴性标志物APOA1表达不明显。
4、分析细胞外囊泡亚群之间蛋白表达的差异和功能
使用Lable-Free法进行相对定量蛋白质组学分析，具体包括以下步骤：
(1)蛋白质提取和肽段酶解
①将细胞外囊泡样品采用SDT【SDT裂解液由4％(w/v)SDS、100mM Tris/HCl(pH＝7.6)、0.1M DTT配制而成】裂解法提取蛋白。
②采用Bio-Rad Protein Assay Kit，并按照说明书要求在96孔酶标板上加入蛋白标准品及步骤①提取的蛋白样品，绘制标准曲线后计算样品蛋白含量，蛋白含量达到0.05ug/uL即可进行后续实验。
③EV-HD亚群或EV-LD亚群各设置四个样品，每个样品取200ug蛋白，加入30uL SDT缓冲液【由4％SDS、100 mM DTT、150 mM Tris-HCl(pH＝8.0)制得】。
④分别在步骤③制得的八个含样品蛋白的缓冲液中加入750uL的UA缓冲液【由8 M尿素(Urea)、150 mM Tris-HCl(pH＝8.0)制得】，通过滤器(Microcon units,10 kD)进行重复超滤浓缩，洗去蛋白液中的DTT和其他低分子量成分，滤器用完后以100uL的UA缓冲液洗涤滤器三遍，再以100uL的25mM NH 4HCO 3溶液洗涤滤器两遍。
⑤往步骤④超滤浓缩后的蛋白液中加入100uL被碘乙酰胺浓缩至100mM的UA缓冲液，避光孵育30分钟，以封闭残余的半胱氨酸。
⑥在40uL的25mM的NH 4HCO 3溶液中加入4ug胰蛋白酶(Promega)，再加入步骤⑤中封闭半胱氨酸之后的蛋白液，于37℃孵育过夜，最后用滤器过滤后收集的滤液为所需肽段。
⑦采用C18 Cartridges对获得的肽段进行脱盐，真空离心后获得冻干肽段。冻干肽段采用40uL的0.1％甲酸溶液复溶。
⑧根据脊椎动物蛋白质中色氨酸和酪氨酸的频率计算，以0.1％(g/L)溶液的消光系数为1.1，在280 nm处用紫外光谱密度估计肽段的含量。
(2)LC-MS/MS数据采集
①每份样品采用纳升流速的HPLC液相系统Easy nLC进行分离。
②缓冲液A液为0.1％甲酸水溶液，B液为0.1％甲酸乙腈水溶液(乙腈为84％)。
③色谱柱以95％的A液平衡，样品由自动进样器上样到上样柱(Thermo ScientificAcclaim PepMap100,100μm×2cm,nanoViper C18)，经过分析柱(Thermo scientific EASYcolumn,10cm,ID75μm,3μm,C18-A2)分离，流速为300 NL/min。
④样品经色谱分离后用Q-Exactive质谱仪进行质谱分析。
⑤质谱检测方式为正离子，母离子扫描范围为300–1800 m/z，一级质谱分辨率为70,000at 200 m/z，AGC(Automatic gain control)target为1e6，Maximum IT为50ms，动态排除时间(Dynamic exclusion)为60.0s。
⑥质谱数据的获取采用依赖于数据的top10方法，从调查扫描(300-1800 m/z)中动态选择最丰富的前驱离子用于HCD碎片。
⑦多肽和多肽碎片的质量电荷比按照下列方法采集：每次全扫描(full scan)后采集20个碎片图谱(MS2 scan)，MS2 Activation Type为HCD，Isolation window为2 m/z，二级质谱分辨率为17,500 at 200 m/z，Normalized Collision Energy为30eV，Underfill为0.1％。
(3)蛋白质鉴定、定量分析及生物信息分析
质谱分析原始数据为RAW文件，采用MaxQuant软件(版本号1.5.3.17)进行查库鉴定 及定量分析。
①蛋白表达差异分析：将步骤(2)样品蛋白的质谱定量信息进行归一化处理(归一化到(-1,1)区间)，然后通过t检验对蛋白表达量进行分析，找出存在显著性表达差异的蛋白。
②功能分析：依次通过序列比对(Blast)、GO条目提取(Mapping)、GO注释(Annotation)和InterProScan补充注释(Annotation Augmentation)来利用Blast2GO数据库对目标蛋白质集合进行GO注释。
图6为两种亚群之间蛋白质表达量差异的鉴定，可见细胞外囊泡亚群EV-HD、EV-LD具有多种差异表达蛋白，且蛋白质的表达模式不同。图7为两种亚群之间差异表达蛋白的生物学功能鉴定分析，可见两种亚群之间的差异表达蛋白具有多种生物学功能。因此由图6、图7可见细胞外囊泡亚群EV-HD、EV-LD是两种完全独立的亚群，具有不同的蛋白表达内容，存在不同的生物学功能。
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明的保护范围。

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  1. 一种分离提取胃癌组织来源细胞外囊泡多种亚群的方法，其特征在于，所述方法包括以下步骤：
    步骤1：将胃癌组织浸入配制的消化液中进行酶解消化过滤后得到组织消化上清液，所述消化液为胶原酶II和脱氧核糖核酸酶I的混合液，或胶原酶IV和脱氧核糖核酸酶I的混合液，所述消化液也可以是乙二胺四乙酸和二硫苏糖醇的混合液与胶原酶IV的组合溶液；
    步骤2：取步骤1的消化上清液以500g-1000g，4℃离心10-15分钟，再取离心后的上清液以3000g-5000g，4℃离心15-20分钟后收集上清液；
    步骤3：取步骤2收集的上清液再次以15000-17000g，4℃超速离心15-30分钟，并收集上清液过滤后再以50000-120000g，4℃超速离心60-100分钟，得到沉淀即为胃癌组织来源细胞外囊泡亚群一；
    步骤4：收集步骤3离心后的上清液于150000-200000g，4℃超速离心60-100分钟，再次得到沉淀为胃癌组织来源细胞外囊泡亚群二。
  2. 根据权利要求1所述的分离提取胃癌组织来源细胞外囊泡多种亚群的方法，其特征在于，所述消化液为胶原酶II和脱氧核糖核酸酶I的混合液。
  3. 根据权利要求1所述的分离提取胃癌组织来源细胞外囊泡多种亚群的方法，其特征在于，所述步骤2的超速离心为收集步骤1的上清液以500g，4℃离心10分钟，再取离心后的上清液以3000g，4℃离心20分钟后收集上清液。
  4. 根据权利要求1所述的分离提取胃癌组织来源细胞外囊泡多种亚群的方法，其特征在于，所述步骤3的超速离心为收集上清液以16500g，4℃超速离心30分钟，再收集上清液过滤后以100000g，4℃超速离心60分钟，得到沉淀为细胞外囊泡亚群一。
  5. 根据权利要求1所述的分离提取胃癌组织来源细胞外囊泡多种亚群的方法，其特征在于，所述步骤4的超速离心为收集上清液于160000g，4℃超速离心90分钟，再次得到沉淀为细胞外囊泡亚群二。
  6. 根据权利要求1所述的分离提取胃癌组织来源细胞外囊泡多种亚群的方法，其特征在于，配制消化液时，胶原酶II和脱氧核糖核酸酶I的体积比以及胶原酶IV和脱氧核糖核酸酶I的体积比均为1：1。
  7. 根据权利要求1所述的分离提取胃癌组织来源细胞外囊泡多种亚群的方法，其特征在于，所述步骤1酶解消化条件为37℃恒温摇床100-400rpm消化20-80分钟。
  8. 根据权利要求1-2或6所述的分离提取胃癌组织来源细胞外囊泡多种亚群的方法，其特征在于，所述消化液中胶原酶II的浓度为2mg/mL，脱氧核糖核酸酶I的浓度为0.2mg/mL。
  9. 根据权利要求1或6所述的分离提取胃癌组织来源细胞外囊泡多种亚群的方法，其特 征在于，所述消化液中胶原酶IV的浓度为2mg/mL，脱氧核糖核酸酶I的浓度为0.2mg/mL。
  10. 根据权利要求1所述的分离提取胃癌组织来源细胞外囊泡多种亚群的方法，其特征在于，所述细胞外囊泡亚群一和细胞外囊泡亚群二是两种完全独立的亚群。
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