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    • 专利摘要:
    The invention relates to the field of biotechnology, and more particularly to a method for isolating microRNA from exosome-containing biological fluids. The method involves sequential centrifugation, ultrafiltration and ultracentrifugation of a concentrated culture medium, dissolution of the resulting precipitate in a phosphate-salt buffer, and additional centrifugation. The resulting precipitate is dissolved in water and the solution is subjected to free-flow electrophoresis using a FEE system device. Each fraction obtained then undergoes centrifugation. Each fraction is freed from the cell walls of the exosomes by filtration and centrifugation, and a reverse transcription reaction is performed using reverse transcriptase (Exiqon). The reaction is stopped by heating to 95 ° C for a period of 5 minutes.
    公开号:CH710195B1
    申请号:CH00123/16
    申请日:2014-12-12
    公开日:2017-01-31
    发明作者:Igorevich Pospelov Vadim；Andreevich Abramov Aleksandr；Aleksandrovich Murashko Dmitrij
    申请人:Marina Vasilevna Mamontova；Igorevich Pospelov Vadim；
    IPC主号:
    专利说明:

    The invention relates to the field of molecular biology, in particular the separation method of specific miRNA types of exosome-containing biological fluids, and provides to separate different miRNA types from different exosome-containing biological samples in order to analyze the individual exosomal fractions.
    Exosomes are small spherical bubbles, 20-100 nm, which are needed in the cell interaction. The exosomes contain complexes of proteins and miRNAs that are valuable as diagnostic markers and can be used to form an anti-tumor immune response in oncology. However, to use the exosomes efficiently, it is necessary to obtain a pure fraction.
    Exosomes are extremely stable in biological fluids, blood, urine, saliva, ascites fluid or cell culture supernatant in the case of cultivation. This allows a non-invasive diagnosis of the origin of a tumor by means of exosomes in plasma and urine.
    Exosomes are actively secreted by cells of the immune system-dendritic cells and B-cells, neurons, particularly secretory tissues and tumor cells (Denzer, K. et al., 2000, Huber et al., 2005, P. Wearsch, 2009). In the case of dendritic cells, the exosomes are excreted. They contain small polypeptides (8-12 amino acids) which are presented in conjunction with the antigen of the MHC-1 KIasse molecules and bind to the relevant receptors of cytotoxic CD8 + T cells, NK cells, as well as large polypeptides (12 -16 amino acids) in conjunction with the antigen of the MHC II class molecules that bind to the relevant receptors of cytotoxic CD8 + T cells. Exosomes secreted by tumor cells may interfere with the formation of the antitumor immune response of cytotoxic T cells, as they compete with DC or macrophage exosomes, considering the concentration of exosomes in the blood plasma and blood serum in the event of cancer development increases approximately 50-fold (Taylor et al 2008, lero 2008).
    Exosomes contain MHC-I class and MHC-I class histocompatibility complex molecules, Rab proteins of integrins, as well as miRNA molecules and structures defined by tissue specificity and individual characteristics of cell types. Human urine exosomes were found to contain 295 proteins (Pistikum et al, 2004). About 50 proteins are regularly detected in all exosomes (Gonzales et al., 2009). In the case of a tumor, tissue-specific protein structures and tissue-specific miRNA structures can be used for early diagnosis or to justify treatment regimens (Baj-Krzyworzeka et al, 2007)
    For the investigation of tumor-specific exosomes, the implementation of a tailor-made separation and differentiation is necessary. The concentration of specific miRNA is a criterion for the selection of exosomes of specific cells.
    [0007] According to a method used for the separation of exosomes from biological fluids (see Shtam TA et al., Tsitologyia, 2012, 54 (5), 430), culture fluid (KC) is collected and successive centrifugations at 2000 and 10 000 g performed. Thereafter, the obtained KC of 500 ml is concentrated to a final volume of 10 ml by ultrafiltration (Centrikon plus-70, 100 kDa, Millipore). Subsequently, ultracentrifugation is performed using the Avanti-301 (JA-30.50 rotor) centrifuge at 100,000 g for 2 hours to separate the exosomes from the obtained concentrated KC drugs. After centrifugation, the supernatant fluid is placed in a separate test tube and analyzed by laser correlation spectroscopy (LCS) to check for the absence of exosomally sized particles. The residue is dissolved in the maximum volume of phosphate buffered saline (PBS) and again centrifuged under the same conditions. The resulting residue is dissolved in 100 microliters of water (MiliQ) or PBS, separated into aliquots, which are frozen at -80 ° C for further proteome analysis.
    The well-known method offers no opportunity to separate exosomes into different fractions belonging to different tissues and thus to determine the miRNA content in the selected samples.
    The technical effect of the method outlined is the ability to separate individual miRNA types of exosome-containing biological fluids, in particular the possibility of separation of different types, e.g. tissue-specific and tumor-specific miRNA, in the future provide the opportunity to produce vaccines based on exosomes of tumor cells.
    The above technical effect is achieved by applying the separation method of specific miRNA types from exosome-containing biological fluids, including successive centrifugations at 2000 and 10,000 g, the ultrafiltration of the resulting supernatant liquid by means of the rotor concentrator Centrikon plus-70 , 100 kDa, Millipore to a final volume of 10 ml and ultracentrifugation using the Avanti-301 (JA-30.50 rotor) centrifuge at 100,000 g for 2 hours, dissolving the residue obtained in PBS, repeated centrifugation under the same conditions and Dissolve the residue obtained in 100 microliters of water. According to the present invention, the resulting solution is then subjected to free-flow electrophoresis in anode and cathode stabilization chambers using the FFE system for further selection of separate fractions under the following conditions: the total time of free-flow electrophoresis is 5 minutes, the applied voltage - 900 V, current - 34 mA, the sample and the marker are injected at a flow rate of 2.5 ml / h or 280 ml / h, the series is in FF-ZE cyclic interval of 85 ml / h and the fractionated sample is eluted at 320 ml / h using the following buffer for anode stabilization: 1.5 M thiourea + 6 M urea + 100 mM H2SO4 + 25 mM 2-pyridinepropanol + 300 mM 2-pyridineethanol.
    The separation medium has the following composition: 1.8 M thiourea + 6 M urea + 55 mM MOPS + 55 mM 2-pyridinepropanol; the following buffer was used for cathode stabilization: 1.8M thiourea + 76M urea + 180mM NaOH + 25mM Tris + 35mM MOPS + 280mM EPPS, then the separated fractions are again eluted with the Avanti-301 (JA-30.50 rotor ) Centrifuge at 100,000 g for 2 hours. The digestion of miRNAs from each fraction is then carried out, ie, 5 mM proteinase K is added to each fraction and incubated at 56 ° C for one hour, then applied to an Exiqon column and further centrifuged at 2000 g subjected.
    Subsequently, the column is washed three times with washing buffer. Each wash is followed by further centrifugation at 2000 g using the Eppendorf 5104 centrifuge. Thereafter, 20 microliters of nuclease-free water are applied to the column, and the column is allowed to stand for 10 minutes and then centrifuged at 3000 g. This is followed by the reverse transcription reaction using reverse transcriptase (Exiqon), adding 8 microliters of the reagent to 8 microliters of the sample and incubating at 60 ° C for one hour. Subsequently, the reaction is stopped by heating to 95 ° C for 5 minutes.
    Subsequently, the polymerase chain reaction with real-time detection using the 7500 Applied Biosystem with primers of specific types, for example, tissue-specific and tumor-specific miRNA, is used to define the specific miRNA types, which are: miR-141 , miR-200a, miR-200c, miR-203, miR-205, miR-214, miR-17-3p, miR-106a, miR-146, miR-155, miR-191, miR-192, miR-301a , miR-326, miR-331-3p, miR-432, miR-438, miR-574-3p, miR-625 *, miR-92a, miR-584, miR-517c, miR-378, miR-520f, miR-142-5p, miR-451, miR-518d, miR-29a, miR-650, miR-151, miR-19b, miR-29c.
    Then, by using the GenEx qPCR software, the number of each of the examined miRNAs separated from each fraction can be calculated. The data are then presented in the form of a table to compare the expression profiles of the miRNAs in samples of different tissues.
    Experimental examples
    600 ml of urine taken from a healthy donor is subjected to successive centrifugations at 2000 g and 10000 g, then the resulting supernatant fluid of 500 ml is concentrated by ultrafiltration (Centrikon plus-70, 100 kDa, Millipore) to a final volume of 10 ml , Subsequently, the use of ultracentrifugation using the Avanti-301 (JA-30.50 rotor) centrifuge at 100,000 g for two hours is necessary to separate the exosomes.
    After centrifugation, the supernatant fluid is placed in a separate test tube and examined by laser correlation spectroscopy (LCS) to check for the absence of particles of exosomal size. The residue is dissolved in the maximum volume of phosphate buffered saline (PBS) and again centrifuged under the same conditions. The residue obtained is dissolved in 100 microliters of water (MiliQ) or PBS. Thereafter, the analyzed sample is subjected to free-flow electrophoresis in free flow and chambers using the FFE system (FFE Service GmbH) under the following conditions: the total time of free-flow electrophoresis is 5 minutes, the applied voltage - 900 V, current - 34 mA.
    The sample and the marker are injected at a flow rate of 2.5 ml / h or 280 ml / h. The series is performed at FF-ZE cyclic interval at 85 ml / hr and the fractionated sample is eluted at 320 ml / hr. The following buffer is used for anode stabilization: 1.5 M thiourea + 6 M urea + 100 mM H 2 SO 4 + 25 mM 2-pyridinepropanol + 300 mM 2-pyridineethanol.
    The separation medium has the following composition: 1.8 M thiourea + 6 M urea + 55 mM MOPS + 55 mM 2-pyridinepropanol; the following buffer was used for cathode stabilization: 1.8 M thiourea + 76 M urea + 180 mM NaOH + 25 mM Tris + 35 mM MOPS + 280 mM EPPS, followed by collection of the separated fractions, ultracentrifugation of the samples from separate pools by means of Ulracentrifugation using the Avanti-301 (JA-30.50 rotor) centrifuge at 100,000 g for 2 hours.
    Then the digestion of the miRNAs from each fraction is carried out by the following procedure: 5 mM proteinase K is added to each fraction and incubated at 56 ° C for one hour, then it is applied to a Exiqon column (for purification of the miRNAs, the sample is applied to the column) with further centrifugation at 2000 g by means of the Eppendorf 5104 centrifuge. Thereafter, the column is washed three times with washing buffer. Each wash is followed by further centrifugation at 2000 g using the Eppendorf 5104 centrifuge. Thereafter, 20 microliters of nuclease-free water are applied to the column, and the column is allowed to stand for 10 minutes and then centrifuged at 3000 g using the Eppendorf 5104 centrifuge. This is followed by the reverse transcription reaction using reverse transcriptase (Exiqon), adding 8 microliters of the reagent to 8 microliters of the sample and incubating at 60 ° C for one hour. Subsequently, the reaction is stopped by heating to 95 ° C for 5 minutes.
    Subsequently, the polymerase chain reaction with real-time detection using the 7500 Applied Biosystem with primers of the following miRNAs is applied: miR-141, miR-200a; miR-200c, miR-203, miR-205, miR-214, miR-17-3p, miR-106a, miR-146, miR-155, miR-191, miR-192, miR-301a, miR-326, miR-331-3p, miR-432; miR-438, miR-574-3p, miR-625 *, miR-92a, miR-584, miR-517c, miR-378, miR-520f, miR-142-5p, miR-451, miR-518d, miR -29a, miR-650, miR-151, miR-19b, miR-29c. The results are analyzed using the GenEx qPCR software and presented in a table (see Figure 1) - Separation per well of miRNAs separated from 500 ml urine from a healthy donor.The concentration is logarithm based on 2 of the miRNA copies in microliters).
    600 ml of urine from a patient with breast cancer is subjected to successive centrifugations at 2000 g and 10,000 g, then the resulting supernatant fluid of 500 ml by ultrafiltration (Centrikon plus-70, 100 kDa, Millipore) to a final volume of 10 ml concentrated. Subsequently, the use of ultracentrifugation using the Avanti-301 (JA-30.50 rotor) centrifuge at 100,000 g for two hours is necessary to separate the exosomes.
    After centrifugation, the supernatant fluid is placed in a separate test tube and examined by laser correlation spectroscopy (LCS) to check for the absence of particles of exosomal size. The residue is dissolved in the maximum volume of phosphate buffered saline (PBS) and again centrifuged under the same conditions. The residue obtained is dissolved in 100 microliters of water (MiliQ) or PBS. Thereafter, the analyzed sample is subjected to free-flow electrophoresis in free flow and chambers using the FFE system (FFE Service GmbH) under the following conditions: the total time of free-flow electrophoresis is 5 minutes, the applied voltage - 900 V, current - 34 mA.
    The sample and the marker are injected at a flow rate of 2.5 ml / h or 280 ml / h. The series is performed at FF-ZE cyclic interval at 85 ml / hr and the fractionated sample is eluted at 320 ml / hr. The following buffer is used for anode stabilization: 1.5 M thiourea + 6 M urea + 100 mM H2SO4 + 25 mM 2-pyridinepropanol + 300 mM 2-pyridineethanol.
    The separation medium has the following composition: 1.8 M thiourea + 6 M urea + 55 mM MOPS + 55 mM 2-pyridinepropanol; the following buffer was used for cathode stabilization: 1.8 M thiourea + 76 M urea + 180 mM NaOH + 25 mM Tris + 35 mM MOPS + 280 mM EPPS, followed by collection of the separated fractions, ultracentrifugation of the samples from separate pools by means of Ulracentrifugation using the Avanti-301 (JA-30.50 rotor) centrifuge at 100,000 g for 2 hours.
    Thereafter, the digestion of the miRNAs from each fraction is carried out by the following procedure: 5 mM proteinase K is added to each fraction and incubated at 56 ° C for one hour, then it is applied to a Exiqon column (for purification of the miRNAs, the sample is applied to the column) with further centrifugation at 2000 g by means of the Eppendorf 5104 centrifuge. Subsequently, the column is washed three times with washing buffer. Each wash is followed by further centrifugation at 2000 g using the Eppendorf 5104 centrifuge. Thereafter, 20 microliters of nuclease-free water are applied to the column, and the column is allowed to stand for 10 minutes and then centrifuged at 3000 g using the Eppendorf 5104 centrifuge. This is followed by the reverse transcription reaction using reverse transcriptase (Exiqon), adding 8 microliters of the reagent to 8 microliters of the sample and incubating at 60 ° C for one hour. Thereafter, the reaction is stopped by heating at 95 ° C for 5 minutes.
    Subsequently, the polymerase chain reaction with real-time detection using the 7500 Applied Biosystem with primers of the following miRNAs is applied: miR-141, miR-200a, miR-200c, miR-203, miR-205, miR -214, miR-17-3p, miR-106a, miR-146, miR-155, miR-191, miR-192, miR-301a, miR-326, miR-331-3p, miR-432, miR-438 , miR-574-3p, miR-625 *, miR-92a, miR-584, miR ~ 517c, miR-378, miR-520f, miR-142-5p, miR-451, miR-518d, miR-29a, miR-650, miR-151, miR-19b, miR-29c The results are analyzed using the GenEx qPCR software and presented in the form of a table (see Figure 2). Separation per well of miRNAs separated from 500 ml urine of a patient the concentration is expressed as a logarithm on the basis of 2 miRNA copies in microliters).
    Tables 1 and 2 show that the pools of 61-66 miRNAs (miR-17-3p, miR-106a, miR-146, miR-155, miR-191, miR-192) in the patient with pulmonary adenocarcinoma and were not detected in the urine of the healthy patient. This indicates that only exosomes of tumor cells from pulmonary adenocarcinoma invade these pools. It follows that the claimed method allows separation of pure fractions of specific miRNA types, including tissue-specific and tumor-specific miRNAs of exosome-containing biological fluids.
    Brief description of the figures
    [0028]<Tb> FIG. 1 <SEP> shows in tabular form the distribution per well of microRNA separated from 500 ml urine of a healthy donor. The concentration is represented as a logarithm on the basis of 2 micro-RNA copies in microliters.<Tb> FIG. 2 <SEP> shows in tabular form the distribution per well of microRNA, separated from 500 ml urine of a patient with pulmonary adenocarcinoma. The concentration is represented as a logarithm on the basis of 2 micro-RNA copies in microliters.
    权利要求:
    Claims (2)
    [1]
    A method for isolating specific micro-RNA types of exosome-containing biological fluids, the method comprising (i) successively centrifuging the fluid at 2000 and 10,000 g, ultrafiltration of the resulting supernatant fluid to a final volume of 10 ml, (ii) ultracentrifugation at 10,000 g for 2 hours, (iii) subsequently dissolving the resulting precipitate in phosphate-saline buffer, followed by further ultracentrifugation at 100,000 g for 2 hours and dissolving the resulting precipitate in 100 microliters of water,characterized in thata) the resulting solution is then subjected to free-flow electrophoresis in individual chambers for stabilizing the anode and cathode using the free-flow electrophoresis system with subsequent collection of the fractions, whereinThe total free-flow electrophoresis time is 5 minutes,- the applied voltage is 900 V and the current is 34 mA,- the sample and the marker at a flow velocity of
    [2]
    2.5 ml / h or 280 ml / h respectively,The course was carried out in a cyclic free-flow zone electrophoresis interval of 85 ml / h and the fractionated sample was eluted at 320 ml / h,The buffer used for the anode stabilization consists of 1.5 M thiourea + 6 M urea + 100 mM H 2 SO 4 + 25 mM 2-pyridinepropanol + 300 mM 2-pyridineethanol,The medium used for the separation is composed of 1.8 M thiourea + 6 M urea + 55 mM 3- (N-morpholino) propanesulfonic acid + 55 mM 2-pyridinepropanol, andThe buffer used for cathode stabilization is 1.8 M thiourea + 76 M urea + 180 mM NaOH + 25 mM Tris + 35 mM 3- (N-morpholino) propanesulfonic acid + 280 mM 4- (2-hydroxyethyl) piperazine-1-propanesulfonic acid consists;b) then the separated fractions are again centrifuged by means of a centrifuge at 100 000 g for 2 hours, andc) then the micro-RNA is isolated from each individual fraction by5 mM proteinase K is added to each individual fraction, incubated at 56 ° C. for one hour,- then applied to a column,Followed by another centrifugation at 2000 g,- after which the column is washed three times with washing buffer and, after each washing, a centrifugation at 2000 g is carried out by means of a microcentrifuge,Finally 20 microliters of nuclease-free water are applied to the column and the column is then allowed to stand for 10 minutes at room temperature,then centrifuged at 3000 g and- Finally, a reverse transcription reaction is carried out using reverse transcriptase, 8 microliters of the reagent to 8 microliters of the sample and incubated at 60 ° C for one hour, and then the reaction is stopped by heating to 95 ° C for 5 minutes ,
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    同族专利:
    公开号 | 公开日
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2017-09-29| PCAR| Change of the address of the representative|Free format text: NEW ADDRESS: BELLERIVESTRASSE 203 POSTFACH, 8034 ZUERICH (CH) |
    2017-10-31| PUE| Assignment|Owner name: VADIM IGOREVICH POSPELOV, RU Free format text: FORMER OWNER: MARINA VASILEVNA MAMONTOVA, RU |
    2019-02-28| PUEA| Assignment of the share|Owner name: VADIM IGOREVICH POSPELOV, RU Free format text: FORMER OWNER: VADIM IGOREVICH POSPELOV, RU |
    优先权:
    申请号 | 申请日 | 专利标题
    RU2001055716|2013-12-16|
    PCT/RU2014/000941|WO2015094017A1|2013-12-16|2014-12-12|Method for isolating separate types of micro-rna from biological fluids containing exosomes|
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