JP6531987B2 - Exosome recovery method for renal disease diagnosis - Google Patents

Description

  The present invention relates to exosome recovery technology. More specifically, the present invention relates to a method for selectively recovering mesangial cell-derived exosomes from a biological sample, and uses thereof.

  The exosome is a 50-140 nm vesicle released by almost all cells, and contains proteins, mRNA, microRNA (miRNA) and the like possessed by the cells derived from it. Because it is resistant to RNase because it is covered by a lipid bilayer membrane, and stable and can be stored for a long time, it has attracted attention as a biomarker. Conventionally, exosomes have been separated from body fluids such as patient plasma and urine using ultracentrifugation or the like (see, for example, Non-Patent Document 1). Recently, a method of recovering using, for example, CD81, which is a general surface marker of exosomes, has also been used (for example, Patent Document 1).

  There are various cell-derived exosomes in the blood. If it is possible to recover the target (target) cell-derived exosome, there is a possibility that useful information can be obtained for diagnosis or treatment of a disease involving the cell. In practice, it is attempted to isolate and analyze exosomes specific to the cell of origin (Patent Document 2), to concentrate exosomes in urine samples using an antibody against a specific protein (Patent Document 3), etc. ing.

JP, 2013-185921, A Japanese Patent Application Publication No. 2012-50857 Japanese Patent Application Publication No. 2014-528076

Glioblastoma microvesicles transport RNA and proteins that promote tumor growth and provide diagnostic biomarkers. Nature Cell Biology 2008 Dec; 10 (12)

  It is assumed that there are 13 million patients with chronic kidney disease in Japan, and there is a need for renal replacement therapy including hemodialysis accompanied by progression of renal failure and cardiovascular diseases such as myocardial infarction as a complication From the onset etc., there is an increasing social need for early diagnosis and treatment of kidney disease. Many renal diseases are referred to a specialist in renal medicine from the indication of urinalysis abnormalities, and a definitive diagnosis is made by performing a renal biopsy. However, due to the risk of performing invasive procedures, the facilities that can perform a renal biopsy are limited, and the indication judgment of the examination is not constant.

  As mentioned above, exosomes are attracting attention as biomarkers, and their application to renal diseases is also expected. However, since all cells secrete exosomes, in the conventional recovery method, exosomes having various derived cells such as vascular endothelial cells, epithelial cells, platelets, etc. are recovered in a mixed state, and the specificity to renal disease is inferior. Therefore, an object of the present invention is to provide a method for recovering a specific exosome useful for examination of kidney disease, and a use of the recovered exosome (examination method for kidney disease etc.).

  In diabetic nephropathy, IgA nephropathy and lupus nephritis, which are frequently used as renal diseases for dialysis induction, focusing on the fact that mesangial cells are the nucleus of the lesion, aiming to selectively recover mesangial cell-derived exosomes. The If it is possible to selectively recover exosomes derived from mesangial cells, it will greatly advance toward the realization of early diagnosis such as diabetic nephropathy and early therapeutic intervention based on it.

As a result of repeated studies, it has been found that α8 integrin, which is considered to be expressed in smooth muscle cells, is a marker for separating and recovering mesangial cell-derived exosomes, and by using an antibody against α8 integrin, We succeeded in selectively recovering mesangial cell-derived exosomes from exosomes (populations of exosomes with different cell origin) prepared by the conventional method. That is, a method was developed to selectively recover mesangial cell-derived exosomes. In order to verify the effectiveness of this new recovery method, detection of markers of diabetic nephropathy was attempted using the exosome recovered by this method as a sample, and highly sensitive detection was possible. That is, while the effectiveness of the said collection | recovery method was supported, it was suggested that a diagnosis of a renal disease is possible using the collect | recovered exosome.
The following invention is mainly based on the above results [1] A method for recovering mesangial cell-derived exosomes, which comprises the following steps (1) and (2):
(1) separating exosomes from a biological sample;
(2) A step of fractionating exosomes expressing α8 integrin on the surface from the separated exosomes.
[2] The recovery method according to [1], wherein the separation of step (2) is performed by capture with an anti-α8 integrin antibody.
[3] The recovery method according to [2], wherein the anti-α8 integrin antibody is immobilized on a solid phase carrier.
[4] The recovery method according to [3], wherein the solid support is magnetic beads.
[5] The recovery method according to any one of [1] to [4], wherein the biological sample is plasma, serum or urine.
[6] The separation in step (1) is performed by one or more treatments selected from the group consisting of centrifugation, density gradient centrifugation, filtering, size exclusion chromatography and ultracentrifugation, [1] to [1] The recovery method as described in any one of 5].
[7] The recovery method according to any one of [1] to [6], wherein after step (2), exosomes are recovered while being captured by the anti-α8 integrin antibody.
[8] The recovery method according to any one of [1] to [6], wherein after step (2), exosomes are detached from the anti-α8 integrin antibody and recovered.
[9] A test method for renal disease, which comprises detecting a target molecule in or on an exosome recovered using expression of α8 integrin as an index.
[10] The test according to [9], wherein the target molecule is RNA, DNA or protein.
[11] The test according to [10], wherein the RNA is a microRNA.
[12] The test method according to any one of [9] to [11], wherein the exosome is recovered by the method according to any one of [1] to [6].
[13] The exosome is recovered by the recovery method described in [7],
The test method according to any one of [9] to [11], wherein a target molecule is detected using an exosome in a state of being captured by an anti-α8 integrin antibody as a sample.
[14] A test method characterized by detecting the amount of exosome recovered from a fixed amount of biological sample by using expression of α8 integrin as an index.

Observation of magnetic bead surface by electron microscope. Left: control, right: immunostaining with anti-α8 integrin antibody. Observation of magnetic bead surface by electron microscope. Left: magnetic beads not coated with antibody, middle: magnetic beads coated with anti-α8 integrin antibody, right: schematic drawing showing capture of exosomes by magnetic beads. Scale bar is 100 nm. Comparison of microRNA expression in diabetic nephropathy patients. Left: detection of RNA extracted from non-selectively recovered exosomes, right: detection of RNA extracted from exosomes selectively recovered by the novel recovery method.

  A first aspect of the present invention relates to a method for recovering mesangial cell-derived exosomes. "Mesangium cells" are the constituent cells of connective tissue (mesangium) that support the glomerulus of the kidney. Mesangial cells are found in the central axis of the glomerular lock. "Mesangium cell-derived" means that it is produced by mesangial cells. The present invention provides a sample useful for examination of kidney disease, determination of therapeutic effect on kidney disease, elucidation of onset or progress mechanism of kidney disease and the like by selectively recovering exosomes produced by mesangial cells.

In the recovery method of the present invention, the following steps (1) and (2) are performed.
(1) Step of separating exosomes from a biological sample (2) Step of separating exosomes expressing α8 integrin on the surface from the separated exosomes

  Step (1) is a step of preparing a sample suitable for the next step, that is, a sample in which the concentration of exosomes is increased (in other words, the amount of contaminants is reduced). Therefore, the step can be paraphrased as "a step of enriching exosomes in a sample". There is no limitation on the concentration or purity of the sample obtained in the step, that is, the separated exosome, as long as there is no problem in carrying out the step (2).

  The separation of step (1) can be carried out, for example, in a conventional manner. In the conventional method, centrifugation, density gradient centrifugation, filtration, size exclusion chromatography, ultracentrifugation, etc. are used (for example, The impact of dissection isolation methods for extracellular vesicles on downstream RNA profiling. J Extracell Vehicles. 2014 Sep 18; see 3 etc.). By combining two or more of these techniques, it is possible to prepare samples with higher concentrations of exosomes. As a specific example of the conventional method, first, a biological sample (for example, plasma) is diluted with a phosphate buffer (PBS) or the like, and then centrifuged at 300 G to 1200 G for 20 minutes to 30 minutes. Next, the supernatant is treated with a filter with a diameter of 0.1 μm to 0.22 μm and then subjected to ultracentrifugation at 100,000 G for 60 minutes to 180 minutes. Then, it is dissolved in phosphate buffer (PBS) or the like to recover sediment (pellet). Alternatively, the separation in step (1) may be performed using an apparatus or reagent for enrichment of exosomes (for example, ExoQuick provided by System Biosciences).

  Plasma, serum, urine or the like can be used as a biological sample. Above all, plasma is a particularly preferred biological sample. Plasma, unlike serum, does not contain platelets that can release exosomes. Thus, the use of plasma makes it possible to prepare mesangial cell-derived exosomes, which are the object of the present invention, in a less contaminated state.

  In step (2) following step (1), exosomes showing specific characteristics are separated from the separated exosomes. "Separating" refers to selectively recovering the target substance, ie, exosomes derived from mesangial cells. While high selectivity is desirable, it is not intended solely for recovery in the absence of contaminants. In the present invention, in order to selectively recover mesangial cell-derived exosomes, α8 integrin expressed on the surface of exosomes is used. Specifically, the expression of α8 integrin is used as an index, and exosomes in which expression of α8 integrin is observed are fractionated. As long as such separation is possible, the method, operation, etc. are not particularly limited. Preferably, immunological techniques are used. Typically, anti-α8 integrin antibodies are used to capture exosomes expressing α8 integrin. Anti-α8 integrin antibodies can be prepared by conventional methods. Anti-α8 integrin antibodies are also commercially available (eg, antibody of product number sc-365798 provided by Santa Cruz Niotechnology Inc., etc.) and can be easily obtained. The α8 integrin is known as a type 1 single-pass transmembrane protein, and has a β propeller domain structure.

  Using the anti-α8 integrin antibody immobilized on a solid phase carrier (in other words, immobilized), it is possible to efficiently fractionate the target substance (mesophagus cell-derived exosome) by simple operation It is possible. Magnetic beads, resins (polystyrene resins, polycarbonate resins, silicone resins, nylon resins, etc.), glass, etc. can be adopted as the solid phase carrier here. One preferred solid support is magnetic beads. For example, particles of a magnetic material such as iron oxide such as ferrite and magnetite, chromium oxide and cobalt can be used as magnetic beads. Although the particle size of the magnetic beads is not particularly limited, for example, magnetic beads having a particle size of 2.8 nm to 4.5 μm can be used. For binding of the anti-α8 integrin antibody to magnetic beads, for example, an antibody binding protein such as protein G or protein A (for example, Invitrogen; Exosomes Immunoprecipitatin (protein G); 10612 D) may be used. Obtaining magnetic beads on which anti-α8 integrin antibody is immobilized via antibody binding protein by contacting magnetic beads on the surface of which antibody binding protein is immobilized and anti-α8 integrin antibody under appropriate conditions Can. In addition, it is also possible to immobilize the anti-α8 integrin antibody on magnetic beads by using a binding reaction of biotin and streptavidin (see, eg, Invitrogen; Exosomes-Streptavidin for isolation / Detection; 106-08D).

  In the first aspect of the present invention, after step (2), exosomes are recovered while being captured by an anti-α8 integrin antibody. In other words, exosomes are recovered without the elimination of anti-α8 integrin antibody. In another embodiment (second embodiment), after step (2), exosomes are detached from the anti-α8 integrin antibody and recovered. In this embodiment, exosomes not bound to anti-α8 integrin antibody are obtained.

  Confirmation that exosomes can be collected can be performed by observation with an electron microscope, detection using an exosome specific marker such as CD63, or the like.

  The collected mesangial cell-derived exosomes are useful as samples used for examination for diagnosis of renal diseases, determination of therapeutic effects and the like. Therefore, in a second aspect of the present invention, there is provided a method for testing a renal disease characterized by detecting a target molecule in or on an exosome recovered using the expression of α8 integrin as an index. In the case of the exosome recovered in the first aspect described above, the target molecule can be detected using the exosome in a state of being captured by the anti-α8 integrin antibody as a sample. For example, the exosome in a state captured by an anti-α8 integrin antibody is directly subjected to analysis by FACS to detect a target molecule expressed on the surface of the exosome. Specific components (for example, RNA such as microRNA (miRNA) contained in exosome, protein, DNA) may be purified from the exosome in a state of being captured by the anti-α8 integrin antibody, and subjected to desired detection. Also in the case of the exosome recovered in the above second aspect, it is subjected to the desired operation after direct or purification operation. Purification here can be carried out, for example, by dissolving, extracting, centrifuging, filtering, chromatography and the like alone or in combination of two or more.

  Typically, a target molecule useful as an indicator of kidney disease is to be detected. The corresponding target molecules are exemplified by let-7a, let-7e, let-7g, let-7i, miR-16, miR-21, miR-29a, miR-29b, miR-29c, miR-93, miR- 126, miR-141, miR-145, miR-146a, miR-205, miR-192, miR-200a, miR-200b, miR-200c, miR-215, miR-216, miR-217, It is a protein such as IgA Fc receptor (CD89), microRNA such as miR-377, DNA mutation of TGF-β receptor and its downstream signal, and the like. The detection of the target molecule may be performed by a conventional method. For example, various methods using nucleic acid amplification reaction for detection of nucleic acid molecules (RNA and DNA), Western blot method for detection of protein, enzyme immunoassay (EIA method), fluorescence immunoassay (FIA method), radiation Immunoassay (RIA), latex agglutination, etc. can be used. Flow cytometry (FCM) is also available for membrane surface proteins. TaqMan microRNA Assay (Applied Biosystems) can be mentioned as a specific example of a detection method in the case of using microRNA as a target molecule. In addition, as an example of the nucleic acid amplification reaction, PCR (Polymerase chain reaction) method or its modification, Loop-Mediated Isothermal Amplification (LAMP) method (Tsugunori Notomi et al. Nucleic Acids Research, Vol. 28, No. 12, No. e63, 2000; Kentaro Nagamine, Keiko Watanabe et al. Clinical Chemistry, Vol. 47, No. 9, 1742-1743 (2001), ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) method (Patent No. 3433929, Patent No. 3883476), NASBA (Nucleic Acid Sequence-Based Amplification) method, LCR (Ligase Chain Reaction) method, 3SR (Self-sustained Sequence Replication) method, SDA (Standard Displacement Amplification method), TMA (Transcription Mediated Amplification) method, RCA (Rolling Circle Amplification) can be mentioned.

  The information obtained by the test method of the present invention, that is, the test results can be used for diagnosis and differentiation of kidney disease, determination of the severity of kidney disease, determination of treatment effect on kidney disease, monitoring of treatment effect on kidney disease, etc. . Examples of the renal disease here include diabetic nephropathy, IgA nephropathy, IgA angiitis, lupus nephritis, and membranoproliferative nephritis.

  By the way, it has been reported that the amount of exosome production increases in cells with high proliferative ability or proliferative ability (for example, MicroRNA signature of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer Gynecologic Oncology 110 (2008) 13-21 reference). In addition, in the exacerbation stage of kidney disease, mesangial cells increase. In consideration of these points, the amount of exosome recovered can reflect the onset or pathological condition of renal disease. Therefore, the present invention also provides a test method characterized by detecting the amount of exosome recovered from a certain amount of biological sample by using the expression of α8 integrin as an index. The above-described recovery method of the present invention can be used to recover exosomes here.

The following studies were conducted to develop a novel exosome recovery method suitable for the diagnosis of kidney disease.
1. Analysis of surface antigen of mesangial cell-derived exosome (1) Method Human cultured mesangial cells (Normal human mesangium cell) (RONZA) were cultured under conditions of 5% CO _{2 and} 37 ° C. The supernatant after culture was collected, and exosomes were concentrated by a conventional method. The obtained solution was mixed with magnetic beads Exosomes-Human CD63 Isolation / Detection (Invitrogen; 10606D) on which anti-CD63 antibody was immobilized in advance, and incubated at room temperature for 1 hour. The magnetic beads were recovered by standing on a dedicated magnet stand and subjected to immunostaining (electron microscope) using an anti-α8 integrin antibody.

(2) Results It was confirmed that α8 integrin was expressed on exosomes derived from human cultured mesangial cells as in the case of mesangial cell bodies (FIG. 1).

2. Selective recovery of mesangial cell-derived exosomes (1) Method: Magnetic beads (Exosomes Immunoprecipitation (protein G); Imvitrogen; 10612 D) on which protein G is immobilized are attached to an anti-α8 integrin antibody (Santa Cruz Niotechnology, INC) according to the manual. The magnetic beads coated with anti-α8 integrin antibody were prepared by incubation with sc-365798).

  On the other hand, 500 μl of human plasma collected by an EDTA blood collection tube was prepared, diluted with 30 ml of PBS, and centrifuged at 800 G for 20 minutes. The resulting supernatant was passed through a 0.22 μm filter (Pall). Thereafter, ultracentrifugation was performed at 4 ° C., 100,000 G for 70 minutes. The supernatant after ultracentrifugation was discarded, and the exosome pellet was recovered with 500 μl of PBS. The harvest was mixed with previously made magnetic beads and incubated for 30 minutes at ambient temperature with rotation. The treated magnetic beads were washed three times with PBS and recovered. The surface of the recovered magnetic beads was observed with an electron microscope.

(2) Results As a result of observation with an electron microscope, it was confirmed that exosomes were captured on the surface of magnetic beads (FIG. 2). That is, by using magnetic beads coated with an anti-α8 integrin antibody, mesangial cell-derived exosomes expressing α8 integrin were successfully recovered from human plasma. It was not possible to recover exosomes from endothelial cells (HUVECs) with magnetic beads coated with anti-α8 integrin antibody. That is, it was shown that there is high selectivity.

3. RNA Analysis Using Recovered Exosome (1) Method From the recovered exosome (in a state of being captured by magnetic beads), RNA was prepared by an RNA extraction method using Trizol, and subjected to various analyses.

(2) Results As a result of extracting RNA from exosomes captured and collected with magnetic beads and analyzing it with a bioanalyzer (Agilent Technologies, Inc), RNA of sufficient quality and amount could be recovered. Analysis of the obtained RNA by PCR revealed that the expression of miRNA (miR-21), which was reported as a marker for diabetic nephropathy, changed with a significant difference in plasma of patients with diabetic nephropathy with abnormalities in mesangial cells. It turned out that it is (Fig. 3 right). For comparison, the left side of FIG. 3 shows the results of the case where RNA extracted from exosomes non-selectively recovered using magnetic beads coated with anti-CD63 antibody was used as a sample.

  To date, no robust renal mesangial cell specific surface markers have been reported. According to the recovery method of the present invention, exosomes derived from renal mesangial cells can be selectively recovered. If the exosome recovered by the present invention is used as a sample, it is possible to detect kidney disease-specific biomarkers with high sensitivity and analyze with high accuracy. By using the present invention, it is possible to provide a new standard to become a Merkmar, for example, useful for diagnosis of renal diseases and determination of therapeutic effect, or to be introduced to a specialized institution.

  The present invention is not limited to the description of the embodiments and examples of the above-mentioned invention. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive without departing from the scope of the claims. The contents of articles, published patent publications, patent publications, etc. specified in the present specification are incorporated by reference in their entirety.

Claims (14)

A method of recovering exosomes from mesangial cells, comprising the following steps (1) and (2):
(1) separating exosomes from a biological sample;
(2) A step of fractionating exosomes expressing α8 integrin on the surface from the separated exosomes.   The recovery method according to claim 1, wherein the separation of step (2) is performed by capture with an anti-α8 integrin antibody.   The recovery method according to claim 2, wherein the anti-α8 integrin antibody is immobilized on a solid phase carrier.   The recovery method according to claim 3, wherein the solid support is magnetic beads.   The recovery method according to any one of claims 1 to 4, wherein the biological sample is plasma, serum or urine.   The separation of step (1) is carried out by one or more treatments selected from the group consisting of centrifugation, density gradient centrifugation, filtration, size exclusion chromatography and ultracentrifugation. The recovery method according to one item.   The recovery method according to any one of claims 1 to 6, wherein after step (2), exosomes are recovered while being captured by the anti-α8 integrin antibody.   The recovery method according to any one of claims 1 to 6, wherein after step (2), exosomes are detached from the anti-α8 integrin antibody and recovered.   A test method for renal disease, which comprises detecting a target molecule in or on an exosome recovered using the expression of α8 integrin as an index.   The test method according to claim 9, wherein the target molecule is RNA, DNA or protein.   11. The test of claim 10, wherein the RNA is a microRNA.   The test method according to any one of claims 9 to 11, wherein the exosome is recovered by the recovery method according to any one of claims 1 to 6. The exosome is recovered by the recovery method according to claim 7,
The test method according to any one of claims 9 to 11, wherein a target molecule is detected by using an exosome captured by the anti-α8 integrin antibody as a sample.   A test method characterized in that the amount of exosome recovered from a certain amount of biological sample is detected by using expression of α8 integrin as an index.

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