JP7444386B2 - Method for isolating and analyzing microvesicles from human urine - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act April 30, 2019, https://www. biorxiv. org/content/10.1101/623553v1

The present invention relates to a method for efficiently separating and analyzing microvesicles present in human urine.

Exosomes (0.03 to 0.1 μm in diameter) and microvesicles (0.1 to 1 μm in diameter) are known as micromembrane fractions that separate from cells, and these are secreted by different mechanisms. It has been reported that it is secreted by many different cell types under both normal and pathological conditions (Non-Patent Document 1).

These micromembrane fractions are included in human body fluids (blood, urine, cerebrospinal fluid, etc.), and the contents contained in these micromembrane fractions are important in signal transduction functions in interactions between cells and tissues. It is believed that this role plays a role. On the other hand, it is expected that these micromembrane fractions can be used as liquid biopsy, which is a non-invasive diagnostic material, to determine the presence or absence of morbidity and disease (Patent Document 1, Patent Document 2, Non-Patent Document 2, Non-Patent Document 3).

Biomaterials used as liquid biopsies include free nucleic acids, micromembrane fractions, circulating tumor cells (CTC), and the like. The micromembrane fraction is divided into (I) exosomes: membranous vesicles with a diameter of 30 to 100 nm, and (II) ectosomes (also referred to as microvesicles and shedding microvesicles (SMVs)). (iii) Apoptotic blebs: large membranous vesicles with a diameter of 100 to 1,000 nm that are released directly from the plasma membrane; (III) apoptotic blebs: large membranous vesicles with a diameter of 50 nm that are released from dying cells ~5,000 nm vesicles.

In recent years, the clinical value of these micromembrane fractions has attracted attention. Clinical application of exosomes is progressing, and a method for diagnosing breast cancer using polynucleotides within vesicles has been developed (Patent Document 3). However, conventional diagnostic methods using exosomes have the problem that it is difficult to characterize each individual exosome and it is difficult to easily determine what tissue or cell it originates from.

Microvesicles are known to play an extremely important role in tumor invasion (Non-Patent Document 4), and microvesicles present in urine have been reported to be involved in diseases such as diabetic nephropathy. (Non-Patent Document 5), its clinical usefulness is expected. Microvesicles are found in large amounts in human body fluids compared to CTCs, play an extremely important role in tumor invasion (Non-Patent Document 4), and microvesicles present in blood have physiological effects. It has been reported that it is a major blood coagulation factor and is also involved in diseases such as sepsis (Non-Patent Document 1), so it is expected that it will be used effectively as a clinical test material, but it has not yet been realized. not present.

Although exosomes and microvesicles have different production processes and are defined as separate micromembrane fractions, conventional observation methods show that their sizes and the surface antigens present on the membranes that characterize them are very close and similar. Therefore, it was not possible to strictly distinguish and separate and observe these. For example, Patent Document 1 and Patent Document 2 describe separation methods and analysis methods that include both exosomes and microvesicles, and the distinction between the two is unclear.

In addition, the micromembrane fraction present in body fluids is generally derived from organs and tissues throughout the body, or the cells that make them up, so in order to find value in diagnostic and testing applications, it is necessary to Observation of micromembrane fractions focused on specific matched organs, tissues, and cells is required. Therefore, the clinical usefulness of micromembrane fractions would be further enhanced if an analytical method could be established to easily categorize the organ, tissue, or cell from which the observed micromembrane fractions originate.

In particular, the presence of micromembrane fractions such as exosomes has been confirmed in urine, and there is knowledge about methods for extracting and detecting them. Urine contains a large amount of Tamm-Horsfall protein (uromodulin) (hereinafter abbreviated as THP), and it is known that these proteins form high molecular weight polymers. In order to separate these high molecular weight polymers and the micromembrane fraction, a method is being considered in which the high molecular weight polymers are previously decomposed by reduction treatment and then the exosomes are extracted (Non-Patent Document 6).

As described above, in order to effectively utilize microvesicles as clinical test materials, it is necessary to separate them from exosomes and to use separation and analysis methods that can distinguish the organs, tissues, and cells from which they originate.

Patent No. 5838963 Japanese Patent Application Publication No. 2015-91251 Japanese Patent Application Publication No. 2014-117282

Andrea Piccin, William G, Owen P (2007) Circulating microparticles: pathophysiology and clinical implications. Blood Reviews 21, 157-171 Boukouris S, Mathivanan S (2015) Exosomes in bodily fluids are a highly stable resource of disease biomarkers. Proteomics Clin Appl 9(3-4): 358-367 IH Chen, L Xue, CC Hsu, (2017) Phosphoproteins in extracellular vesicles as candidate markers for breast cancer. PNAS vol. 114, no. 12, 3175-3180 James W. Clancy, Alanna Sedgwick, Carine Rosse, (2015) Regulated delivery of molecular cargo to invasive tumor-derived microvesicles. Nature Communications 6, Article number: 6919 Ai-li Sun, Jing-ti Deng, Guang-ju Guan, Shi-hong Chen, Yuantao Liu, Jing Cheng, Zhen-wei Li, Xiang-hua Zhuang, (2012) Dipeptidyl peptidase-IV is a potential molecular biomarker in diabetic kidney disease. Diabetes & Vascular Disease Research 9(4), 301-308 Patricia Ferna´ndez-Llama, Sookkasem Khositseth, Patricia A. Gonzales, Robert A. Star, Trairak Pisitkun and Mark A. Knepper (2010) Tamm-Horsfall protein and urinary exosome isolation. Kidney International 77, 736-742

As mentioned above, microvesicles are considered to be an effective clinical test material in the form of liquid biopsies, but it is extremely difficult to clearly distinguish and separate exosomes from microvesicles, and they are present in urine. Microvesicles are a mixture of microvesicles derived from various cells and tissues, and even if the entire urinary microvesicles are extracted and used as a liquid biopsy (for example, to extract nucleic acids, etc.), it will not be possible to identify disease sites or for diagnostic purposes. It is thought that it is difficult to obtain a result that matches.

Therefore, we focused on microvesicles (diameter 0.1 to 1 μm) in urine, and it is possible to easily characterize them and observe what kind of cells each microvesicle originates from. The purpose of the present invention is to construct a method for distinguishing and separating exosomes and microvesicles, which have been unclear until now, and also clarifying the cells, tissues, and organs from which the observed microvesicles originate.

For this purpose, it is necessary to efficiently concentrate microvesicles contained in urine. In addition to this, the present inventors have discovered that THP polymers present in urine are not only in close proximity to microvesicles in terms of molecular size, but also that THP polymers also interact with IgG, leading to immunochemical effects on urine-derived microvesicles. It has been found that it is necessary to remove the THP polymer as it causes problems when carrying out the characterization method. By efficiently concentrating microvesicles in urine and removing the THP polymer, we can size-fractionate the microvesicles and further characterize and analyze the proteins present on the membrane surface. It becomes possible to construct a measurement system that can estimate the cells, tissues, and organs from which vesicles originate.
In addition, in this specification, separation shall be used synonymously with fractionation, detection, etc.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have developed a process of concentrating microvesicles from urine by centrifugation, decomposing and removing THP polymers by reduction treatment, using a flow cytometer, and A step of collecting and observing a fraction of approximately 0.1 to 1 μm, a step of removing remaining THP polymer from the observed image, and a step of characterizing the microvesicles using surface antigens specific to the cells from which the microvesicles are derived. The researchers discovered that by using this process, it is possible to accurately observe which organs, tissues, and constituent cells each microvesicle was released from and present in urine. Based on this knowledge, the present invention has completed a measurement method that can individually characterize microvesicles in urine.

That is, the present invention provides:
[1] A method for separating microvesicles in a urine specimen, which includes the following steps;
(Step 1) A step of centrifuging the collected urine sample at low speed to remove cell fractions and debris,
(Step 2) high-speed centrifugation of the supernatant obtained in Step 1 to precipitate the microvesicle fraction;
(Step 3) Adding a reducing agent to the precipitated fraction obtained in Step 2,
(Step 4) high-speed centrifugation of the sample obtained in Step 3 to concentrate the microvesicle fraction and remove the reducing agent;
(Step 5) Adding (1) a staining reagent and (2) labeled IgG to the urine sample processed product obtained in Step 4 in this order, in the reverse order, or at the same time;
(Step 6) a step of subjecting the reaction product obtained in step 5 to measurement with a flow cytometer;
[2] The method of [1], which includes the following steps in flow cytometer measurement;
(Step A) A step of analyzing the acquired data and excluding (1) aggregated microvesicles and (2) particles that react with labeled IgG from the observed image in this order, in the reverse order, or at the same time. ,
(Step B) A step of sorting and characterizing the derived cells into microvesicle populations,
[3] The method of [1] or [2], wherein the reducing agent is selected from 1,4-dithiothreitol and tris(2-carboxyethyl)phosphine hydrochloride;
[4] Any method of [1] to [3], wherein the observation range in flow cytometer measurement is either 1 μm or less, or 200 nm or more and 1 μm or less,
[5] The method of any one of [1] to [4], which includes the step of setting parameters of the flow cytometer in order to converge particles of a desired diameter in the observation area of the flow cytometer;
[6] Any method of [1] to [5], which uses polystyrene beads with a particle size of 0.22 to 1.35 μm to estimate the particle size in the observation area;
[7] Any method of [1] to [6], wherein the diameter of the microvesicles to be separated is 1 μm or less,
[8] The method of any one of [1] to [7], wherein the isolated microvesicles contain the proteins listed in Table 4;
[9] A method of searching for substances specifically contained in microvesicles derived from urine using the fraction obtained by any of the methods of [1] to [8],
[10] It relates to a method of assisting in the detection of a disease, drug administration effect, or other medical condition in a subject using the fraction obtained by any of the methods of [1] to [9].

According to the present invention, microvesicles present in urine can be easily and specifically separated and observed, and furthermore, individual microvesicles can be characterized using surface antigens present on the membrane (what kind of cells and tissues are present). , whether it originates from an organ). As a result, it is expected that the value of the test will improve as a test that can be used for diagnosis and testing that focuses on specific organs, tissues, and cells that match specific diseases. Furthermore, by analyzing the fractions obtained according to the present invention, it becomes possible to estimate diseases, drug administration effects, or other medical conditions in a subject.

This is an image observed using a flow cytometer of a microvesicle fraction obtained by concentrating urine through pretreatment. This is a negative staining image of a microvesicle fraction concentrated by pretreatment using a transmission electron microscope. This is a particle size distribution histogram obtained by a nanoparticle analysis system of a microvesicle fraction concentrated by pretreatment. These are the results of protein enrichment analysis using metascape from proteins detected by shotgun proteomics analysis. These are the results of network analysis for each cluster of protein enrichment analysis results using metascape from proteins detected by shotgun proteomics analysis. This is an observation image of flow cytometer measurement data of the microvesicle fraction concentrated by pretreatment, developed in terms of pulse width and pulse area in side scattered light. This is an observation image developed by the area of APC mouse IgG and side scattered light from the gated-in population in FIG. 6. FIG. 8 is an explanatory diagram showing a process of selecting microvesicles that are positive for all of CD10, CD13, and CD26 from measurement data obtained by excluding (gating out) the APC mouse IgG positive population from the observed image shown in FIG. 7. FIG. 8 is an explanatory diagram showing a process of selecting microvesicles that are positive for MUC1 and Annexin 5 from measurement data obtained by excluding (gating out) the APC mouse IgG positive population from the observed image shown in FIG. 7. It is a diagram superimposed on a developed view of side scattered light and Annexin5 after characterization of microvesicles in the derived cell population. A diagram superimposed on the developed image of side scattered light and Annexin5 after characterization of microvesicles in the derived cell population (left side), and a flow cytometer image of Annexin5 positive population and Annexin5 negative population in each derived cell population This is an observed image (right side). It is a graph showing the results of comparing the proportions of Annexin5-positive population and Annexin5-negative population of microvesicles derived from each cell from 10 healthy individuals using a flow cytometer. Three types of fractions derived from the pellet obtained by concentrating the microvesicle fraction in Example 1 were treated with CD26 (Dipeptidyl 4 is a graph showing the results of measuring the peptide cleavage activity of peptidase 4).

Although embodiments of the present invention will be described in detail below, the usage method is not limited thereto.

The present invention is a method for separating and analyzing microvesicles in human urine. Broadly speaking, it includes the process of removing impurities and concentrating microvesicles in human urine, and the process of using a flow cytometer with observation conditions specific to urine microvesicles. In this specification, pretreatment includes the meaning of concentration and recovery, and concentration and recovery are sometimes used as almost synonymous terms.

In this specification, urine refers to collected urine, which may be used as it is, or may be dissolved or suspended in water, acidic solution, alkaline solution, buffer solution, etc., and further processed as necessary. You may. Those skilled in the art can appropriately select and use the acidic solution, alkaline solution, and buffer solution that can be used in the present invention.

An example of conditions for separating and analyzing microvesicles in urine using a flow cytometer will be described below, but the present invention is not limited thereto.

The pretreatment method for removing impurities and concentrating microvesicles that can be used in the present invention is characterized by centrifugation, and can be carried out, for example, according to the following steps.

The collected urine specimen is subjected to low-speed centrifugation to separate cell fractions, debris, etc., and the supernatant is collected (Step 1). At this time, conditions such as low-speed centrifugation can be carried out according to known methods, and a supernatant obtained by centrifugation at 2,000 x g for 20 minutes at room temperature can be used.
The supernatant obtained in step 1 may be further centrifuged at low speed to collect the supernatant. This centrifugation step is preferable because platelet-derived vesicles, apoptotic blisters, and the like can be precipitated. As in step 1, the supernatant obtained by centrifugation at 2,000 xg for 20 minutes at room temperature can be collected and used.

The supernatant obtained in step 1 is centrifuged at high speed to precipitate the microvesicle fraction (step 2). As conditions for this step, for example, a precipitate obtained by centrifugation at 20,000 x g for 30 minutes at room temperature can be used.

A reducing agent (for example, 1,4-Dithiothreitol (DTT), Tris (2-carboxyethyl) phosphine hydrochloride) is added to the precipitate fraction obtained in Step 2. : TCEP) is added and left to stand (Step 3). By carrying out this step, the disulfide bonds in the THP polymer can be reductively cleaved and the THP polymer can be decomposed.

The sample to which the reducing agent was added in step 3 is centrifuged at high speed to concentrate the microvesicle fraction and remove the reducing agent (step 4). This high-speed centrifugation allows the microvesicle fraction to be concentrated and impurities to be removed. As conditions for this step, a precipitate obtained by centrifugation at 20,000 xg for 30 minutes at room temperature can be used. This step 4 may be repeated as necessary. By performing centrifugation at high speed multiple times, impurities can be removed and the purity of the microvesicle fraction can be increased, which is preferable.

The reducing agent to be added is added for the purpose of cleaving disulfide bonds, so any reducing agent that is capable of cleaving disulfide bonds may be used. For example, protection of SH groups of proteins and disulfide bonds, such as dithiothreitol, 2-mercaptoethanol, 2-mercaptoethylamine hydrochloride, TCEP, cysteine hydrochloride, tributylphosphine (TBP), iodoacetamide, glutathione, hydrazine, etc. Those skilled in the art can appropriately select and use from among those commonly used as cutting and reducing agent reagents. Among these reducing agents, it is preferable that the reducing agent does not affect the measurement of microvesicles, and it is more preferable that the reducing agent does not have the effect of destabilizing the lipid bilayer. These reducing agents may be used alone or in combination of two or more.

In addition to carrying out each of the above steps, there are several other possible pretreatment methods that can be used as an embodiment of the present invention. For example, the desired microvesicle fraction can be extracted by combining membrane filters with various pore sizes used for filter sterilization of aqueous solutions and solutions containing proteins. In this case, a method equivalent to micro-filtration using pores with a diameter of 0.1 to 10 μm is suitable.

Other pretreatment methods include equilibrium density gradient centrifugation, in which the sample is centrifugally fractionated together with a density gradient solute, and antibodies specific to the surface antigen of the micromembrane fraction are used to transfer the micromembrane fraction to various carriers. Immunological capture method in which the membrane components of the micromembrane fraction are collected using immunological capture methods, methods in which size exclusion chromatography (gel filtration method) is used to collect fractions that elute faster than soluble proteins, and metal There are phospholipid affinity methods that use carriers that bind in the presence of ions, and polymer precipitation methods that mix high molecular weight polymers and micromembrane fractions and precipitate the desired micromembrane fractions. Those skilled in the art can select and implement these methods as appropriate; however, in these methods as well, by performing the step of adding a reducing agent to the sample, steps 1 to 5 can be performed. It can be implemented as an alternative method.

Hereinafter, the sample concentrated and impurities removed by these methods may be referred to as a processed urine sample.

Next, an example of observation conditions using a flow cytometer specialized for microvesicles will be explained, but the present invention is not limited thereto.

The flow cytometer that can be used in the present invention is not limited to a specific device, and any flow cytometer that employs any flow cell, such as a quartz cuvette type or a jet-in-air type, can be used.

The observation method using a flow cytometer that can be used in the present invention can be carried out, for example, according to the following steps.

(1) Staining of microvesicles In this step, a reducing agent is added and the concentrated processed urine specimen is mixed with a staining reagent to stain the microvesicles (Step 5). More specifically, microvesicles are stained by mixing a staining reagent, for example, a fluorescently labeled antibody specific to a surface antigen, with the processed urine specimen. As for the dyeing method, those skilled in the art can adopt a conventional method and, depending on the case, suitably examine the conditions. In order to target multiple antigens in microvesicles, detection may be performed by staining with multiple types of labeled antibodies as multicolor separation. For example, Annexin 5, which is a protein that binds to phosphatidylserine (PS), which is a membrane component of microvesicles, in the presence of metal ions, etc. can be used.

Other substances that can specifically bind to microvesicles include, for example, any substance that can bind to proteins, lipids, or sugars present on the surface of microvesicles. Substances present on the surface include, for example, proteins such as CD235a, CD59, CD44, CD33, CD45, CD144, CD66a, CD66b, CD66c, CD66e, CD41, CD61, EP-CAM, CD324, CD14, CD81, CD31, CD274. , CD63, Alix, CD105, CD133, CD279, CD15, TSG101, CD20, CD249, CD5, CD10, CD26, CD273, CD9, MUC-1, CD13, CD146, CD62E or combinations thereof can be used.

Substances that can specifically bind to microvesicles other than those listed above include, for example, substances that have binding affinity for proteins, substrates for enzymes, coenzymes, regulatory factors, substances that specifically bind to receptors, Lectins, sugars, glycoproteins, antigens, antibodies or antigen-binding fragments thereof, hormones, neurotransmitters, phospholipid-binding proteins, proteins containing pleckstrin homology (PH) domains, cholesterol-binding proteins, or these It may be a combination. Antigen-binding fragments include antigen-binding sites, and may be, for example, single-domain antibodies, Fabs, Fab's, or scFvs.

For example, a fraction that is positive for all CD10, CD13, and CD26 as microvesicles containing multiple types of membrane peptidases is combined with a fraction that is positive for Annexin5 and CD227 (MUC-1) as microvesicles derived from epithelial cells. It is preferable to use this method because it allows for more accurate separation.

(2) Addition of IgG In this step, fluorescently labeled IgG is mixed and reacted with the THP polymer in order to detect the THP polymer that could not be removed by the reduction treatment in the observed image (step 5).
Fluorescently labeled IgG is mixed with the mixture obtained by mixing the urine sample processed material and the staining reagent in the previous step, or with the urine sample processed material before being mixed with the staining reagent, and reacted. (Step 5). The origin of the IgG is not particularly limited, and for example, mouse IgG, human IgG, rat IgG, rabbit IgG, goat IgG, bovine IgG, etc. can be used. As with the microvesicle staining process, those skilled in the art can carry out the process according to standard methods. For example, it can be carried out by using allophycocyanin (APC)-labeled mouse IgG, Mix-n-Stain APC Antibody Labeling kit (Biotium), and allowing it to stand at room temperature for 30 minutes according to a regular protocol.

Proteins such as complement that supplement immunoglobulins exist in the blood, and immunocomplexes may be formed. This immunocomplex may be close in molecular size to microvesicles. Therefore, since the immunocomplex also interacts with IgG, it may pose a problem when performing an immunochemical characterization method on blood-derived microvesicles. Therefore, in order to detect the fraction that captures IgG in the observed image and remove it from the observed image, it is possible to achieve the objective by performing a step of mixing fluorescently labeled IgG and reacting with the IgG captured fraction. This method is preferable because it can remove substances other than microvesicles, and it is possible to perform more accurate measurements that do not include contaminants.
In addition, the above-mentioned step of staining microvesicles with a surface antigen-specific antibody and the step of adding IgG may be carried out first, or both steps may be carried out at the same time. It is possible to select an appropriate one according to the implementation environment.

(3) Flow cytometer settings Adjust the voltage and threshold of the photomultiplier tube for forward scattered light and side scattered light to converge particles with a specific diameter in the observation area. When setting conditions such as these parameters, it is preferable to measure unstained samples and stained samples, and determine voltage settings and sheath flow rates that satisfy the detection sensitivity for detecting each target.

Those skilled in the art can search for the optimal conditions and set the parameters of the flow cytometer as appropriate; for example, the flow rate may be 12 μL/min, the voltage of forward scattered light may be The voltage of the side scattered light can be set to 381 and 340, and the fluorescence intensity threshold can be set to 200 as the respective detection sensitivity. The wavelength and voltage of the excitation light (Ex.) and fluorescence detection filter (Em.) for each fluorescent substance can be appropriately selected and set depending on each fluorescent substance used.

Using the flow cytometer parameters set above, estimate the particle size in the observation area. Measurement can be performed according to a standard method using polystyrene beads of uniform size (eg, SPHEROTM Nano Polystyrene Size Standard Kit, Spherotech). In order to estimate the particle size in the observation area, the particle size of the polystyrene beads is preferably 0.22 μm, 0.45 μm, 0.88 μm, or 1.35 μm, for example.

The diameter set as the observation area is not particularly limited as long as it includes microvesicles, but for example, it is preferably about 1 μm or less, more preferably 100 nm to 1 μm, and even more preferably 200 nm to 1 μm. Particularly when observing these microparticles, side scattered light is preferable because it has higher resolution than forward scattered light and can be used for particle size verification.

Note that this step is not limited to the embodiment performed between the staining step and IgG addition step and the measurement/separation step described below; for example, after setting the parameters, the staining step, the IgG addition step, and the measurement/separation step are performed. Alternatively, once set, a series of steps can be performed repeatedly without setting parameters for each measurement.

(4) Measurement and separation by flow cytometry Obtain data with a flow cytometer. When multi-color measurement is performed, leakage correction is performed for the fluorescence leaking from each fluorescence to a detector other than the assigned one, and measurement results are obtained under that condition (Step 6).

(5) Exclusion of aggregated microvesicles and complexes with IgG capture fraction (Step 6-A)
Microvesicles may aggregate, but in order to exclude aggregated microvesicles from the observed image, 1) the pulse width of the side scattered light is very high and deviates from the group, 2) the forward From the plot of scattered light and side scattered light, it is possible to mark and gate out those where both are out of the group and have high values. This is particularly effective when microvesicles aggregate due to pretreatment.

Furthermore, in order to remove immune complexes with the IgG capture fraction, a positive population that reacts with fluorescently labeled IgG can be marked and gated out. This step is preferable because the positive population that reacts with fluorescently labeled IgG can be marked and gated out in order to exclude THP polymers that could not be completely removed by the reduction treatment from the observed image.

In addition, the above-mentioned step of excluding aggregated microvesicles and the step of removing immune complexes captured by IgG may be carried out first, or both may be carried out at the same time. It is possible to select an appropriate one according to the environment in which the vendor operates.

(6) Characterization of the obtained microvesicle population (Step 6-B)
By performing a gating operation on the microvesicles detected using multicolor, it is possible to separate the cells from which they originate into microvesicle populations and characterize them. Through this step, it is possible to confirm that the microvesicles are isolated in urine.

Although urinary microvesicles can be separated and characterized according to the present invention, these characterized fractions can also be further separated using a cell sorter. It is also possible to analyze the specific contents (nucleic acids, metabolites, proteins, lipids) from these separated fractions and apply them clinically. The separated fractions may be further used for frequency analysis of each microvesicle population and expression level analysis of target molecules.

In addition to the above steps, measurement techniques that can separate and observe microvesicles include, for example, the nanotracking particle size measuring device NanoSight (Malvern Panalytical), which uses NTA (Nano Tracking Analysis) technology and FFF (Field Flow Fractionation). ) techniques can also be used in combination. With NTA technology, the Brownian motion of nanoparticles in liquid can be observed in real time on a PC screen, and with FFF technology, separation is performed in a thin flow channel; Due to the special geometry, this flow forms a laminar flow with a radial cross section, and by perpendicular to this laminar flow, a separating force is generated, which separates small particles such as microvesicles by size. Therefore, more accurate separation and analysis are expected. By combining such separation/observation methods with surface antigens that characterize individual microvesicles, microvesicles can be characterized, increasing organ specificity and disease specificity, and can be used for clinical applications.

In a different embodiment of the invention, it can also be used as a method for monitoring disease progression in a subject, and for monitoring disease recurrence in an individual. In addition to the step of separating microvesicles from a urine sample, these methods include a profiling step of analyzing substances contained in the microvesicles. By analyzing the profile of a subject with a particular medical condition, it can be used, for example, to estimate the presence of a particular disease. For example, by appropriately setting the sample collection period for isolating microvesicles depending on the target disease detection, etc., it is possible to obtain a more detailed profile, observe the condition, and assist in diagnosis. It becomes possible. It can also be used as a method of monitoring disease status after drug administration.

The present invention makes it possible to effectively utilize microvesicles as clinical test materials, making it possible to easily and specifically extract and observe microvesicles present in urine, and to detect surface antigens present in membranes. It can be used to characterize individual microvesicles (what cells, tissues, and organs they originate from). As a result, it is expected that the value of the test will improve as a test that can be used for diagnosis and testing that focuses on specific organs, tissues, and cells that match specific diseases. Furthermore, by analyzing the fractions obtained according to the present invention, it becomes possible to estimate diseases, drug administration effects, or other medical conditions in a subject.

EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but these are not intended to limit the scope of the present invention.

《Example 1: Concentration of microvesicles from human urine》
~10 mL of urine was collected from a healthy individual. Urine samples were centrifuged at 2,000 x g for 10 minutes at 20°C to obtain urine, followed by centrifugation at about 2,000 x g for 10 minutes at about 20°C to remove intraurinary lipids and cellular debris. , blood-derived components, etc. were removed, and the supernatant liquid was used in the following experiment. The experiment from this urine collection was conducted under the approval of the Kyushu University Medical Department Clinical Research Ethics Review Committee (permit number 29-340).

1200 μL of the resulting urine was centrifuged at 20° C. and 18,900×g for 30 minutes. The supernatant was removed from the centrifuged product and the microvesicle fraction was concentrated in a pellet. To this pellet, 180 μL of phosphate buffered saline (PBS) containing DTT at a final concentration of 10 mg/mL was added. After stirring with a vortex, the mixture was allowed to stand at 37°C for 10 minutes. After standing, it was centrifuged at 20° C. and 18,900×g for 30 minutes. The microvesicle fraction was concentrated in a pellet from the centrifuged product.

《Example 2: Observation of urine microvesicles using a flow cytometer》
A. Immunofluorescent staining of microvesicles concentrated from urine 60 μL of PBS was added to the pellet obtained by concentrating the microvesicle fraction in Example 1, and the microvesicle fraction was dispersed in the solution by vortexing. To this solution, FITC-Annexin5 (Becton Dickinson Biosciences), APC/Cy7anti-human CD10 (Biolegend), Brilliant Violet421anti-human CD13 (Biolegend) were added. nd), PEanti-human CD26 (Biolegend), PE/Cy7anti-human CD227 MUC1 (Biolegend) ) and APC-labeled mouse IgG (purchased Normal Mouse IgG (Wako) labeled according to the standard protocol attached to the Mix-n-Stain APC Antibody Labeling kit (Biotium)) were added, and the mixture was incubated for 30 minutes at room temperature. I left it for a minute.

B. Observation of urinary microvesicles using a flow cytometer Measurements were performed using a BD FACSVerse™ (Becton Dickinson and Company) as a flow cytometer. The measurement procedure and parameter settings are as follows. The samples used were those obtained by suspending the various fluorescently stained fractions in Example 2A in 750 μL of 10 mmol/L Hepes (pH 7.4), 0.14 mol/L NaCl, and 2.5 mmol/L CaCl ₂ . The flow rate was set to 12 μL/min, the voltage of forward scattered light was set to 381, the voltage of side scattered light was set to 340, and the respective thresholds were set to 200. The wavelength and voltage of the excitation light (Ex.) and fluorescence detection filter (Em.) for each fluorescent substance are FITC:Ex. 488 nm, Em. 527/32 nm, voltage 442, PE: Ex. 488 nm, Em. 586/42 nm, voltage 411, PerCP: Ex. 488 nm, Em. 700/54 nm, voltage 556, PE/Cy7: Ex. 488 nm, Em. 783/56 nm, voltage 564.3, APC: Ex. 640nm, Em. 660/10 nm, voltage 538.2, APC/Cy7: Ex. 640nm, Em. 783/56 nm, voltage 584.8, Brilliant Violet421:Ex. 405 nm, Em. The wavelength was 448/45 nm and the voltage was 538.2.

C. Size verification measurement using polystyrene beads in the set observation area With the flow cytometer parameters set above, use polystyrene beads (SPHEROTM Nano Polystyrene Size Standard Kit, Spherotech) with a uniform size to estimate the particle size in the observation area. Measurements were taken. The polystyrene beads used had particle diameters of 0.22 μm, 0.45 μm, 0.88 μm, and 1.35 μm.

D. Interpretation of Microvesicle Measurement Results with a Flow Cytometer Figure 1 shows an observation image in which the measurement data is expanded with the horizontal axis: side scattered light (SSC) intensity and the vertical axis: FITC (Annexin 5) intensity. In this figure, the region with high fluorescence intensity was defined as the Annexin5-positive population, and the region with low fluorescence intensity was defined as the Annexin5-negative population. In addition, there is a proportional relationship between the intensity of side scattered light and the size of polystyrene beads of 0.22 to 1.35 μm in the observation region, and in particular, there is a microvesicle fraction between polystyrene beads with diameters of 0.22 μm and 0.45 μm. It was observed that the median values of

《Example 3: Electron microscopic observation of microvesicle fraction concentrated from urine》
A. Immobilization of urinary microvesicles 100 μL of PBS was added to the pellet obtained by concentrating the microvesicle fraction in Example 1, and a fixative solution (0.1 mol/L containing 4% paraformaldehyde and 4% glutaraldehyde) 100 μL of phosphate buffer (pH 7.4) was added, stirred, and left at 4° C. for 1 hour.

B. Negative Staining Method The fixed sample was adsorbed onto grid-coated formvar film and stained with 2% phosphotungstic acid (pH 7.0) for 30 seconds.

C. Observation with a transmission electron microscope The above grid was observed with a transmission electron microscope (JEM-1400; JEOL Ltd.). Acceleration voltage was set at 100 kV. A digital image (3296×2472 pixels) was acquired with a CCD camera (EM-14830RUBY2; JEOL Ltd.). The observed image is shown in Figure 2. A micromembrane fraction having the size of a microvesicle and containing a membrane structure and internal structures (organelles, etc.) inside the membrane structure was detected.

《Example 4: Particle measurement of extracted fraction using nanoparticle analysis system》
100 μL of PBS was added to the pellet obtained by concentrating the microvesicle fraction in Example 1, and the pellet was further diluted 3 times with PBS and measured using Nanosight (Malvern Panalytical). Table 1 shows the observation conditions of the device. Figure 3 shows a particle size distribution histogram obtained by the nanoparticle analysis system. In this concentration operation, the fraction with a diameter of 1 μm or more is hardly included, and the fraction with a diameter of 600 μm or less is concentrated, and the particle size mainly contains a homogeneous group with a particle size of about 200 to 300 nm. It was confirmed that the fraction contained a diameter (~100 nm).

《Example 5: Proteins detected in concentrated microvesicle fraction by shotgun proteomics analysis》
A. Extraction of protein fraction from concentrated microvesicle fraction obtained by pretreatment of urine In order to efficiently extract especially membrane proteins from the concentrated microvesicle fraction in Example 1, PTS (Phase Transfer Surfactant) was used. A protein solubilization method using a solubilizing agent) was performed. MPEX PTS Reagents for MS (GL Science Inc.) was used as a reagent kit using this principle. 250 μL of the kit reagent B was added to the pellet obtained by concentrating the microvesicle fraction in Example 1, and a cycle consisting of an operation of 1 minute 30 seconds and an interval of 30 seconds using an ultrasonic homogenizer (Bioruptor: Sonic Bio Inc.) was performed. was sonicated (power: MAX) 10 times at 10°C. After crushing, the mixture was concentrated using a centrifugal filter (Amicon ultra 3K, Merck) twice at 14,000 g for 15 min. Protein in this fraction was quantified by BCA assay (using Pierce ^™ BCA Protein Assay kit (Thermo Fisher Scientific Inc.)).

B. Enzyme Digestion of Protein Fraction The protein fraction was quantified by BCA assay and dissolved in the reagent B to a concentration of 30 μg/20 μL, which was used as a sample for membrane protein digestion. 1 μL of 100 mmol/L dithiothreitol (DTT) was added (for reductive cleavage of disulfide bonds) and incubated for 30 minutes at room temperature. To this was added 1 μL of 550 mmol/L iodoacetamide (for carbamidomethylation of Cys), and the mixture was incubated at room temperature for 30 minutes in the dark. 77 μL of reagent A of the kit was added, and 1.5 μL of trypsin (TPCK-Trypsin (Thermo Fisher Scientific Inc.: Prod #20233)) was added. Protein digestion was performed by incubating overnight at room temperature. 150 μL of Reagent C and 1.5 μL of Reagent D were added. After the addition, the mixture was vortexed for 1 minute and centrifuged at 25° C. and 15,600×g for 2 minutes to separate the two phases. Unnecessary solubilizing agent was present in the upper phase and was removed by suction with a pipette. In order to remove the remaining Reagent C, after performing centrifugal concentration, 50 μL of 5% acetonitrile, 0.1% trifluoroacetic acid (TFA) was added and vortexed.

C. Desalting and Concentration of Peptides GL-Tip SDB (GL Science Inc.) was used to desalt and concentrate the enzymatically digested peptides described above. The chip was conditioned by first adding 20 μL of 80% acetonitrile and 0.1% TFA aqueous solution and centrifuging at 3,000×g for 2 minutes at room temperature. 20 μL of 5% acetonitrile and 0.1% TFA aqueous solution was added thereto, and the column was equilibrated by centrifuging at 3,000×g at room temperature for 2 minutes. To this, the entire amount of the sample obtained in the procedure of Example 5B was added, and the peptide was adsorbed onto the column by centrifugation at 3,000×g at room temperature for 5 minutes. Furthermore, 20 μL of 5% acetonitrile and 0.1% TFA aqueous solution was added, and the column was washed by centrifugation at 3,000×g for 2 minutes at room temperature, and then 50 μL of 80% acetonitrile and 0.1% TFA aqueous solution was added. Peptides were eluted by centrifugation at 3,000 xg for 2 minutes at room temperature.

D. Mass spectrometry measurement The above sample was subjected to LC using a Fourier transform Orbitrap mass spectrometer (Q-Exactive: Thermo Fisher Scientific Inc.) connected to a nano-LC system (EASY-nLC1000: Thermo Fisher Scientific Inc.). -MS/MS An analysis was performed. 2 μL of the sample for mass spectrometry measurement prepared above was subjected to measurement. The trap column was Acclaim ^RT PepMap100 (Thermo Fisher Scientific Inc.: C18, packing diameter 3 μm, inner diameter 75 μm, column length 2 cm), and the analysis column was Acclaim ^RT PepMap RSLC (Thermo Fisher Sc). ientific Inc.: C18, filler diameter 2 μm , inner diameter 50 μm, column length 15 cm). Mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid/acetonitrile, flow rate is 200 nL/min, gradient is 0-40% mobile phase B/200 minutes, 40-100% mobile phase B /10 minutes, 100% mobile phase B/10 minutes.

The scanning conditions for MS measurement were Full MS/dd-MS2 mode, and after scanning Full MS, an MS/MS spectrum of a signal with high intensity was obtained. Table 2 shows the setting conditions.

E. Data analysis The obtained data were compiled into the human protein database Homo_sapiens_Uniprot_201210. A search was performed on ``fast'' using the SequestHT algorithm. Table 3 shows the search conditions in SequestHT.

We picked up items with a Score of 1.0 or higher from the search results. Table 4 lists the proteins detected by shotgun proteomics analysis of the extracted microvesicle fraction, and among the top-scoring proteins, the surface antigens used for characterization using a flow cytometer are shown. 5, respectively. In addition, Figure 4 shows the results of protein enrichment analysis using metascape (Tripathi et al., Cell Host & Microbe (2015), 18: 723-735) from proteins detected by shotgun proteomics analysis. The results of network analysis for each cluster are shown in FIG. 5. Many of the proteins listed in Table 4 are categorized as cell membranes or intracellular, but they also include proteins with functions such as mass transport, nutrient recycling, metabolism, and biosynthesis (Figure 4), and these detected proteins are microorganisms. It has the potential to be used as a surface marker to characterize vesicles, and its contents as a biomarker for clinical testing and diagnosis.

《Example 6: Characterization of urine microvesicles using a flow cytometer》
A. Gate out of aggregated microvesicles by pulse treatment (exclusion from observation image)
The concentrated microvesicle fraction in Example 1 was stained with each surface antigen and measured under the flow cytometer observation conditions of Example 2. At this time, an image in which multiple microvesicles appeared to aggregate together was observed (a group in which all of the various stained surface antigens were positive). In order to remove this from the observed image, we prepared an observed image (Figure 6) developed by the pulse width (vertical axis) and pulse area (horizontal axis) in side scattered light, and removed the groups that deviated greatly from the observed image. Excluded.

B. Gate-out of THP polymer (exclusion from observation image)
After the operation in Example 6A above, the APC-positive population, that is, the mouse IgG population that can be captured by the residual THP polymer, is extracted from the side scattered light and the APC mouse IgG development diagram, and the APC-positive population is extracted and excluded from the observed image. (Figure 7).

C. Characterization of microvesicles that are positive for CD10, CD13, and CD26 After the operations in Example A and Example B above, a population that is positive for both types of CD10 and CD13 is extracted from the development diagram, and the population is also positive for CD26. The group was selected. This population was defined as CD10-, CD13-, and CD26-positive microvesicles, and the proportion of this population in the entire observed population was 66%. These three types of CD markers are all peptidases present in the membrane, and it is possible that the microvesicles have the function of decomposing proteins (FIG. 8).

D. Characterization of MUC1-positive microvesicles After the operations in Example A and Example B above, a MUC1-positive population is extracted from the development diagram of CD227 (MUC1) and side scattered light, and a population that is Annexin5 positive is further selected from this population. did. This population was defined as MUC1-positive microvesicles, and the proportion of this population in the entire observed population was 2.4% (FIG. 9).

E. Two Classifications of Urinary Microvesicles In the above, an example of a diagram superimposed on a developed diagram of side scattered light and Annexin 5 after characterizing the microvesicles to the cell populations from which they are derived is shown in FIG.

F. Characterization of each cell-derived microvesicle by Annexin5 positivity and negativity After the operation in Example 6B, Annexin5 positive and negative populations were extracted from the side scattered light and the Annexin5 development diagram, and from this population, the cells shown in C and D above were extracted. Microvesicle characterization from derived cells using CD antigen was also performed (Figure 11). When the proportion of Annexin5 positive and negative microvesicles were compared for each derived vesicle, many of the microvesicles positive for CD10, CD13, and CD26 were Annexin5 negative (FIG. 12). The comparison test used the Wilcoxon signed rank test (* p < 0.05, ** p < 0.01).
Regarding conventionally used microvesicle markers (for example, Annexin 5), the above results revealed that there are many Annexin 5-negative microvesicles, and the proportion thereof varies greatly depending on the cell of origin. In other words, it has been discovered that the behavior of substances in microvesicles derived from each cell may be different from the conventional behavior. This means that the exact nature of microvesicles may be overlooked by characterization using conventionally used methods. It has been suggested that by using the present invention, it may be possible to obtain clinical test materials for identifying disease sites and obtaining results that match diagnostic purposes.

《Example 7: Measurement of peptide cleaving activity of CD26 (Dipeptidyl peptidase 4) in urine microvesicles》
The same buffer as the original sample amount was added to the concentrated microvesicle fraction in Example 1 (microvesicle fraction), and the supernatant after removing the microvesicle fraction (supernatant fraction) ), prepare 3 fractions of the original urine fraction (2000 g x 10 minutes twice to remove cell residue), and use each fraction to perform peptide cleavage of CD26 (Dipeptidyl peptidase 4). Activity was measured. The synthetic substrate H-Gly-Pro-7-amido-4-methylcoumarin hydrobromide was used for activity measurement. When H-Gly-Pro-7-amido-4-methylcoumarin hydrobromide is cleaved by peptidase activity in the solution and 7-Amino-4-methylcoumarin is produced, it develops a fluorescent color. Three fractions were measured, and it was found that when the original urine fraction was taken as 100%, the microvesicle fraction contained about 30% of the enzyme activity (FIG. 13).
From the above results, it was confirmed that enzymes such as protease contained in the microvesicle fraction separated and concentrated using the present invention maintain and reflect the properties of the original sample. Therefore, from the results of Example 7 in addition to Example 6-F, by using the present invention, it is possible to obtain clinical test materials for identifying disease sites and obtaining results that match diagnostic purposes. It was suggested that this is possible.

From the above results, we were able to establish an observation method that focuses on the microvesicle fraction by concentrating microvesicles in urine and decomposing and removing THP polymers. It was shown that the protein can be used for characterization. By using the present invention, it has become possible to identify the cells and organs from which individual microvesicles present in urine originate, and to measure their increase/decrease and content rate.

The pretreatment method for concentrating microvesicles according to the present invention can successfully remove THP polymers and efficiently extract microvesicles without involving any particularly complicated operations. By implementing this pretreatment method, it is possible to concentrate candidate substances (a list of proteins is exemplified in the example) contained in microvesicles as a biomarker, which is more sensitive than directly measuring urine. There is a possibility that measurements can be carried out.

Furthermore, according to the present invention, it is possible to easily characterize individual urinary microvesicles using surface antigens of microvesicles, and it is possible to measure/quantitate what cells and organs the microvesicles are derived from. / Can be analyzed. Micromembrane fractions such as microvesicles contain transmitters necessary for communication between cells and organs, and are expected to be used clinically as liquid biopsies. It is thought that there are clinical and physiological implications as an effect. In particular, microvesicles in urine have peptidases on their surfaces that have proteolytic properties, and their functional activity is likely to have clinical significance.

These points indicate that the observation of microvesicles according to the present invention can be applied to testing methods for determining specific diseases and patient conditions, and can be used to manage the transition of individuals from a healthy state to a pre-diseased (pre-disease) state on a daily basis. Clinical applications as preventive and predictive markers are also possible. If there is a system that can measure smaller particles together with surface antigens with performance over a wide dynamic range, the clinical application value of micromembrane fractions, including exosomes and microvesicles, can be increased.

Claims (9)

A method for separating microvesicles in a urine sample, the method comprising:
(Step 1) A step of centrifuging the collected urine sample at low speed to remove cell fractions and debris,
(Step 2) high-speed centrifugation of the supernatant obtained in Step 1 to precipitate the microvesicle fraction;
(Step 3) Adding a reducing agent to the precipitated fraction obtained in Step 2,
(Step 4) high-speed centrifugation of the sample obtained in Step 3 to concentrate the microvesicle fraction and remove the reducing agent;
(Step 5) Add (1) a labeled substance that can specifically bind to microvesicles and (2) a labeled IgG to the urine sample treated product obtained in step 4 in this order or in the reverse order. and (Step 6) subjecting the reactant obtained in Step 5 to measurement with a flow cytometer, in Step 6,
(Step A) A step of analyzing the acquired data and excluding (1) aggregated microvesicles and (2) particles that react with labeled IgG from the observed image in this order, in the reverse order, or at the same time. and (Step B) the method described above, comprising the steps of separating the derived cells into microvesicle populations and characterizing them. 2. The method of claim 1, wherein the reducing agent is selected from 1,4-dithiothreitol and tris(2-carboxyethyl)phosphine hydrochloride. The method according to claim 1 or 2, wherein the observation range in measurement by a flow cytometer is either 1 μm or less, or 200 nm or more and 1 μm or less. The method according to any one of claims 1 to 3, further comprising the step of setting parameters of the flow cytometer in order to converge particles of a desired diameter in the observation area of the flow cytometer. The method according to any one of claims 1 to 4, wherein polystyrene beads having a particle size of 0.22 to 1.35 μm are used to estimate the particle size in the observation area. The method according to any one of claims 1 to 5, wherein the diameter of the microvesicles to be separated is 1 μm or less. The method according to any one of claims 1 to 6, wherein the separated microvesicles contain any of the proteins listed in Tables 4-1 to 4-28. (Step 1) A step of centrifuging the collected urine sample at low speed to remove cell fractions and debris,
(Step 2) high-speed centrifugation of the supernatant obtained in Step 1 to precipitate the microvesicle fraction;
(Step 3) Adding a reducing agent to the precipitated fraction obtained in Step 2,
(Step 4) high-speed centrifugation of the sample obtained in Step 3 to concentrate the microvesicle fraction and remove the reducing agent;
(Step 5) Add (1) a labeled substance that can specifically bind to microvesicles and (2) a labeled IgG to the urine specimen obtained in step 4 in this order or in the reverse order. a step of adding in order or at the same time; and
(Step 6) Step of subjecting the reaction product obtained in step 5 to measurement using a flow cytometer
In the step 6,
(Step A) A step of analyzing the acquired data and excluding (1) aggregated microvesicles and (2) particles that react with labeled IgG from the observed image in this order, in the reverse order, or at the same time. ,as well as
(Step B) Step of separating and characterizing the derived cells into microvesicle populations
including, and further,
(Step 7) A step of separating the fraction characterized in step 6 using a cell sorter.
A method for searching for substances specifically contained in microvesicles derived from urine using microvesicles separated by a method for separating microvesicles in a urine sample, including : (Step 1) A step of centrifuging the collected urine sample at low speed to remove cell fractions and debris,
(Step 2) high-speed centrifugation of the supernatant obtained in Step 1 to precipitate the microvesicle fraction;
(Step 3) Adding a reducing agent to the precipitated fraction obtained in Step 2,
(Step 4) high-speed centrifugation of the sample obtained in Step 3 to concentrate the microvesicle fraction and remove the reducing agent;
(Step 5) Add (1) a labeled substance that can specifically bind to microvesicles and (2) a labeled IgG to the urine specimen obtained in step 4 in this order or in the reverse order. a step of adding in order or at the same time; and
(Step 6) Step of subjecting the reaction product obtained in step 5 to measurement with a flow cytometer
In the step 6,
(Step A) A step of analyzing the acquired data and excluding (1) aggregated microvesicles and (2) particles that react with labeled IgG from the observed image in this order, in the reverse order, or at the same time. ,as well as
(Step B) Step of separating the derived cells into microvesicle populations and characterizing them
including, and further,
(Step 7) A step of separating the fraction characterized in step 6 using a cell sorter.
A method of aiding in the detection of a disease, drug administration effect, or other medical condition in a subject using microvesicles isolated by a method of isolating microvesicles in a urine specimen, comprising :