WO2019022542A2 - Method for isolating extracellular vesicles using cations - Google Patents

Method for isolating extracellular vesicles using cations Download PDF

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WO2019022542A2
WO2019022542A2 PCT/KR2018/008485 KR2018008485W WO2019022542A2 WO 2019022542 A2 WO2019022542 A2 WO 2019022542A2 KR 2018008485 W KR2018008485 W KR 2018008485W WO 2019022542 A2 WO2019022542 A2 WO 2019022542A2
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endoplasmic reticulum
cation
extracellular
extracellular endoplasmic
present
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PCT/KR2018/008485
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French (fr)
Korean (ko)
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WO2019022542A3 (en
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고용송
이창진
김지현
송성현
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㈜로제타엑소좀
포항공과대학교 산학협력단
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Priority to US16/634,086 priority Critical patent/US11904259B2/en
Priority to CN201880062590.7A priority patent/CN111148828A/en
Priority to JP2020527719A priority patent/JP2020528766A/en
Priority to EP18837656.0A priority patent/EP3660142A4/en
Priority claimed from KR1020180087354A external-priority patent/KR102107844B1/en
Publication of WO2019022542A2 publication Critical patent/WO2019022542A2/en
Publication of WO2019022542A3 publication Critical patent/WO2019022542A3/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor

Definitions

  • the present invention relates to a method for separating extracellular endoplasmic reticulum from various samples using the affinity of extracellular endoplasmic reticulum for various cations.
  • Extracellular vesicles are universal cellular mechanisms, nano-sized vesicles that are naturally secreted by all living things or cells, from humans to bacteria.
  • the extracellular endoplasmic reticulum derived from eukaryotic cells is involved in the erythropoietic differentiation and regulation of the immune response. Especially, important functions of cancer progression, metastasis and angiogenesis are revealed in the cancer cell microenvironment, And has received high interest in the utilization of
  • the extracellular endoplasmic reticulum secreted from prokaryotic cells contains prokaryotic components similar to extracellular endoplasmic reticulum.
  • acute pulmonary inflammatory diseases are induced according to the pathway including systemic inflammation.
  • chronic inflammation of the local skin tissues can be induced to cause atopic dermatitis, one of the representative diseases of modern people.
  • the extracellular endoplasmic reticulum from prokaryotic cells has also attracted a great deal of attention due to the emergence of bacterial-derived extracellular endoplasmic reticulum in the human body and cancer incidence.
  • extracellular endoplasmic reticulum The function of extracellular endoplasmic reticulum is most important because they are an important component of the intercellular information exchange mechanism. Therefore, the constituents of extracellular endoplasmic reticulum have also received high interest in the basic and medical fields.
  • the extracellular endoplasmic reticulum is a bio-nanoparticle secreted from various kinds of cells in vivo or in vitro. It is present in body fluid such as blood, urine, saliva, tears, etc. and contains a cell-derived lipid bilayer. It is a vesicle of membrane structure with various sizes.
  • extracellular endoplasmic reticulum binds to other cells and tissues and acts as a transporter that transports intracellular substances such as membrane components, proteins, and RNAs, the proteins, lipids, amino acids, RNA, and so on, thus providing an important basis for understanding the physiological and pathological characteristics of the parent cells.
  • nucleic acid, growth hormone, and protein contained in the extracellular endoplasmic reticulum are protected by a phospholipid in the form of a cell membrane and can perform more stable functions than a soluble growth factor and cytokine. It is expected to be used for various purposes including diagnosis and treatment of diseases by analyzing substances contained in extracellular endoplasmic reticulum.
  • extracellular endoplasmic reticulum is small in nanometer level and numerous substances exist in body fluid and cell culture fluid in addition to extracellular endoplasmic reticulum, extracellular endoplasmic reticulum from samples such as body fluids and cell culture fluids for extracellular endoplasmic reticulum analysis It is important to separate, and it is the most important technology in all areas that utilize extracellular endoplasmic reticulum.
  • the most efficient method of material separation is to sequentially remove contaminants from the complex environment using selective binding to the target material without losing the material during the separation process.
  • the substance having such selective binding properties is limited to some antibodies or protein ligands, and separation of the extracellular endoplasmic reticulum using such an antibody or protein is not only inefficient, Ligand development is difficult, and high cost is required, which is very limited.
  • the present invention provides a method for separating extracellular endoplasmic reticulum easily and efficiently with affinity of a cation and an extracellular endoplasmic reticulum.
  • the extracellular endoplasmic reticulum and cations bind to each other in the sample to form an insoluble complex.
  • the extracellular endoplasmic reticulum-cation complex can be separated by various methods such as centrifugation, ultrafiltration, precipitation by gravity, and then the extracellular endoplasmic reticulum can be separated by desorbing the cation from the complex. This makes it possible to quickly and easily separate the extracellular endoplasmic reticulum from a variety of samples without physical and chemical modification.
  • the extracellular endoplasmic reticulum can be used for diagnosis, treatment, multi-omics studies, It is easy.
  • " extracellular < / RTI > endoplasmic reticulum " of the present invention collectively refers to a living body nanoparticle derived from cells of Archaea, Prokarya or Eukarya and includes extracellular endoplasmic reticulum (exosome), argosomes , Dexosomes, ectosomes, exovesicles, oncosomes, prominosomes, prostasomes, tolerosomes, microparticles (e. G.
  • microvesicles microparticles, microvesicles, nanovesicles, blebbing vesicles, budding vesicles, exosome-like vesicles, matrix vesicles, , Membrane vesicles, shedding vesicles, membrane particles, shedding microvesicles, membrane blebs, epididymosomes, promininosome, texosome, or archeosome, Information that is not.
  • the present invention relates to a method for preparing a biological sample, comprising the steps of (a) adding a cation to a biological sample, (b) reacting the extracellular endoplasmic reticulum contained in the biological sample with a cation to form a complex, (c) And (d) separating the cation from the complex to purify the extracellular endoplasmic reticulum.
  • FIG. 1 A method of separating the extracellular endoplasmic reticulum according to an embodiment of the present invention is schematically shown in FIG.
  • the method for separating extracellular ER of the present invention comprises the steps of adding a cation to a biological sample (step (a)) and reacting the extracellular endoplasmic reticulum contained in the biological sample with a cation to form a complex (Step (b)).
  • biological sample or " sample” of the present invention includes biological samples or cell culture fluids, tissue samples and the like, including extracellular endoplasmic reticulum, and specifically includes mammalian cell culture medium, bacterial cell culture medium, yeast culture medium, (CSF), cerebrospinal fluid (CSF), ascites, amniotic fluid, semen, milk, dust, fresh water, blood, urine, Seawater, soil, and fermented food.
  • mammalian cell culture medium bacterial cell culture medium, yeast culture medium, (CSF), cerebrospinal fluid (CSF), ascites, amniotic fluid, semen, milk, dust, fresh water, blood, urine, Seawater, soil, and fermented food.
  • CSF mammalian cell culture medium
  • CSF yeast culture medium
  • CSF cerebrospinal fluid
  • cation " of the present invention is electrically positively charged and has a specific affinity for the extracellular endoplasmic reticulum and can bind to the extracellular endoplasmic reticulum in the sample, preferably a metal cation.
  • metal cation " of the present invention may include alkali metal ions, alkaline earth metal ions, transition metal ions, and transition metal ions.
  • the cation or metal cation of the present invention may preferably be a transition metal ion or an alkaline earth metal, but is not limited thereto, so long as it is a cation having a specific affinity for an extracellular endoplasmic reticulum to be separated.
  • Alkali metal is a group of chemical elements other than hydrogen among the group 1 of the periodic table and includes lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs) Fr).
  • the alkaline earth metal is a Group 2 element of the periodic table and includes beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba) and radium (Ra).
  • the transition metal contains elements 4 to 7 and 3 to 12 of the Periodic Table of the Chemical Periods. It forms an ion-binding compound together with a nonmetal and exists in complex ion form.
  • the transition metal of the present invention is a transition metal selected from the group consisting of Sc, Y, Ti, Zr, Hf, Rf, V, Nb, Tantalum (Ta), dibonium (Db), chromium (Cr), molybdenum (Mo), tungsten (W), sb, manganese (Mn), technetium (Tc), rhenium (Re) ), Iron (Fe), ruthenium (Ru), Os, Os, cobalt, rhodium, iridium, (Pd), Pt, Ds, Cu, Ag, Au, Rg, Zn, Cd, Hg) and copper (Cn).
  • Post-transition metal means a metal element in the p-zone of the periodic table and is a metal element of aluminum (Al), gallium (Ga), indium (In), thallium (Tl), tin (Sn) Pb), bismuth (Bi), and polonium (Po).
  • the method of adding the cation of the present invention includes a method in which a solution containing a cation is added to a sample and a method in which the solution is added in a solid form to dissolve it.
  • a form capable of reacting with an extracellular endoplasmic reticulum in a sample in a cationic state It is not limited.
  • the present invention relates to a method for separating an extracellular endoplasmic reticulum associated with a cation from a sample using a property of specifically binding an extracellular endoplasmic reticulum to a cation to be separated.
  • the extracellular endoplasmic reticulum and the cation are specifically bound in the sample, and an insoluble extracellular endoplasmic reticulum-cation complex is formed.
  • insoluble complexes were formed by adding calcium ion, manganese ion, cobalt ion, copper ion or zinc ion to a culture medium sample or urine sample containing an extracellular endoplasmic reticulum. Also, it was confirmed that the insoluble complex can be easily separated because it is submerged by gravity.
  • the method for separating extracellular fibrils includes separating the extracellular endoplasmic reticulum and the cation complex from the sample (step (c)).
  • the step of separating the extracellular endoplasmic reticulum and the cation complex of the present invention is a step of separating the insoluble complex formed in the step from the sample containing various water-soluble substances, and the step of centrifuging, ultracentrifugation, filtration, ultrafiltration, gravity,
  • One or more methods may be selected from, but are not limited to, density gradient ultracentrifugation, size exclusion chromatography, ion exchange chromatography, affinity chromatography, polymer-based precipitation or organic solvent precipitation.
  • the method for separating extracellular fibrils comprises separating cations from the complex to purify the extracellular fibrils (step (d)).
  • the step of purifying the extracellular endoplasmic reticulum of the present invention can separate only the extracellular endoplasmic reticulum from the complex by eliminating the specific binding state between the extracellular endoplasmic reticulum and the cations, and it is possible to use various methods or conditions which those skilled in the art can understand Can be applied.
  • the step may comprise adding a chelating agent to the separated extracellular endoplasmic reticulum and cation complex.
  • chelate agents or " chelating ligands” of the present invention means an ion, molecule or atomic group containing two or more coordinating atoms forming a chelate complex stable to metal ions, Depending on the number of atoms, tridentate ligand, tetradentrate ligand, pentadentrate ligand, and hexadentrate ligand are also called.
  • the chelate ligand of the present invention may be selected from the group consisting of iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), tris (carboxymethyl) ethylenediamine (TED), ethylenediamine, Ethylenediamine tetraacetate (EDTA), alkylenediamine triacetic acid, diethylenetriaminepentaacetic acid (DTPA), ethylene glycol bis (beta-aminoethyl ether) -N, N, N, N ', N'-tetraacetic acid (EGTA), phosphoserine and 1,4,7-triazo (TACN, 1,4,7-triazocyclononane).
  • IDA iminodiacetic acid
  • NTA nitrilotriacetic acid
  • TED tris (carboxymethyl) ethylenediamine
  • EDTA Ethylenediamine tetraacetate
  • DTPA diethylenetriaminepentaacetic acid
  • the above step may use a method of changing the pH value of the solution containing the separated extracellular endoplasmic reticulum and the cation complex.
  • the above step further comprises the step of adding an imidazole, histidine, ethylenediamine tetraacetate (EDTA), or salt (s) to a solution containing a separated extracellular endoplasmic reticulum and a cation complex methods of purifying the extracellular endoplasmic reticulum from the complex by varying the concentration of the salts.
  • an imidazole, histidine, ethylenediamine tetraacetate (EDTA), or salt (s) to a solution containing a separated extracellular endoplasmic reticulum and a cation complex methods of purifying the extracellular endoplasmic reticulum from the complex by varying the concentration of the salts.
  • the step of purifying the extracellular endoplasmic reticulum of the present invention can be carried out by a complex selection of one or more of the above methods.
  • the purification conditions of the present invention include, but are not limited to, buffers of pH 10 or less, 0-5 M NaCl, 0-2 M imidazole, 0-2 M metal chelating agents, or combinations of the above conditions .
  • the method for separating extracellular fibrils may further comprise pretreating the sample before adding the cation to the sample.
  • the pretreatment step of the present invention is a step of partially purifying an untreated sample, which comprises centrifugation, ultracentrifugation, filtration, ultrafiltration, sonication, density gradient ultracentrifugation, size exclusion chromatography, ion exchange chromatography, affinity chromatography , Polymer-based precipitation, or organic solvent precipitation, but is not limited thereto.
  • it may further comprise post-treating the extracellular endoplasmic reticulum isolated according to the extracellular endoplasmic reticulum isolation method of the present invention.
  • the post-treatment step of the present invention is a step of purifying the separated extracellular endoplasmic reticulum by centrifugation, ultracentrifugation, filtration, ultrafiltration, sonication, density gradient ultracentrifugation, size exclusion chromatography, ion exchange chromatography, affinity
  • One or more methods may be selected from, but are not limited to, chromatography, polymer-based precipitation or organic solvent precipitation.
  • the method for separating extracellular fibrils may further include adding a polymer or salting-out ion in the step of adding a cation to the sample.
  • the rate of insoluble complex formation can be remarkably increased by adding a polymer or salting-out ion together with cations, and the efficiency and time for separation of extracellular fibrils can be remarkably improved .
  • the polymer or salting-out ion can be added to the sample simultaneously with the cation.
  • polymer or salting-out ion may be added before the cation is added to the sample.
  • polymer or salting-out ion may be added after adding the cation to the sample.
  • the polymer may be polyethylene glycol (PEG) or polyoxazoline
  • the polyoxazoline may be selected from the group consisting of polymethyloxazoline (PMOZ , poly (2-methyl-2-oxazoline), poly (2-ethyl-2-oxazoline) or poly (2-propyl-2-oxazoline)
  • PMOZ polymethyloxazoline
  • the polymer may be polyethylene glycol (PEG), poly (2-ethyl-2-oxazoline), or polyethylene oxide (PEOZ).
  • salting-out ion of the present invention refers to a kosmotropic salt for stabilizing a water structure for reducing the solubility of water in a solution to increase the strength of the hydrophobic interaction.
  • These cosmotropic salts are represented by the Hofmeister series according to their ability to affect the solubility of soluble materials in solution and the anion series are: SO 4 2- ⁇ HPO 4 2- OH - ⁇ F - ⁇ HCOO - ⁇ CH 3 COO - ⁇ Cl - ⁇ Br - ⁇ NO 3 - ⁇ I - ⁇ SCN - ⁇ ClO 4 - .
  • the cation series are: NH 4 + , Rb + , K + , Na + , Cs + , Li + , Ca 2+ , Mg 2+ , and Ba 2+ .
  • Cosmotropic salts act as salting ions for hydrophobic particles according to the Hope Meister series.
  • the salting-out ion of the present invention may be an anion stabilizing the water structure in the Hofmeister system and a cosmotropic salt of a counter cation thereof.
  • the method of separating extracellular ER according to the present invention does not require expensive equipments such as centrifugal separator and the sample is not exposed to the extreme environment during the separation process, the extracellular ER can be efficiently separated while preserving the shape or properties of extracellular ER .
  • the method of the present invention can be applied in combination with a conventional extracellular ER separation method, and can be maximized by applying the method before or after the conventional method.
  • the extracellular fibrin separation method of the present invention can separate the extracellular endoplasmic reticulum easily and effectively, it can be utilized as an important factor in the mass purification of the extracellular endoplasmic reticulum, as well as the pre- To be used for clinical diagnosis.
  • the method of separating extracellular endoplasmic reticulum of the present invention can fractionate a subset of extracellular endoplasmic reticulum with various cations using different properties of affinity for specific cations according to the type of extracellular endoplasmic reticulum.
  • the extracellular extracellular subsets can be used to diagnose a multidimensional disease.
  • FIG. 1 is a schematic diagram of a method for separating extracellular ER according to an embodiment of the present invention.
  • FIG. 2 shows the results of the separation and characterization of the extracellular endoplasmic reticulum according to an embodiment of the present invention.
  • FIG. 3 is a graph showing the results of HPLC analysis showing that extracellular endoplasmic reticulum can be isolated from cell culture medium by adding various cations (Ca 2+ , Cu 2+ , Zn 2+ ) at various concentrations according to an embodiment of the present invention.
  • FIG. 4 shows that the extracellular endoplasmic reticulum is separated from the cell culture medium by the addition of various concentrations of copper cation (copper (II) chloride) according to an embodiment of the present invention. to be.
  • FIG. 5 shows the results of the nanoparticle analysis (a) and the Western blot (b) that the extracellular endoplasmic reticulum was separated from the cell culture medium by the addition of various concentrations of copper cations (copper sulfate (II)) according to an embodiment of the present invention .
  • FIG. 6 shows the results of the nanoparticle analysis (a) and Western blot (b) showing that the extracellular endoplasmic reticulum was separated from the cell culture medium by adding cobalt cations (cobalt chloride) at various concentrations according to an embodiment of the present invention.
  • FIG. 7 is a graph showing the results of nano particle analysis (a) and Western blot (b) showing that the extracellular endoplasmic reticulum is separated from the cell culture medium by the addition of various concentrations of manganese cations (manganese chloride (II)) according to an embodiment of the present invention to be.
  • manganese cations manganese chloride (II)
  • FIG. 8 shows that the extracellular endoplasmic reticulum was separated from the cell culture medium by the addition of various concentrations of manganese cations (manganese sulfide (II)) according to an embodiment of the present invention. to be.
  • manganese cations manganese cations
  • FIG. 9 shows the results of the nanoparticle analysis (a) and Western blot (b) showing that the extracellular endoplasmic reticulum was separated from the cell culture medium by the addition of various concentrations of calcium cation (calcium chloride) according to an embodiment of the present invention.
  • FIG. 10 shows the results of the nanoparticle analysis (a) and the Western blot (b) that the extracellular endoplasmic reticulum was separated from the cell culture medium by addition of various concentrations of zinc cation (zinc chloride) according to an embodiment of the present invention.
  • Figure 11 shows the results of nano particle analysis (a) and western blot (b) showing that the extracellular endoplasmic reticulum was isolated in human urine by the addition of various concentrations of calcium cation (calcium chloride) according to one embodiment of the present invention.
  • Figure 12 shows that extracellular endoplasmic reticulum is isolated in human urine by the addition of various concentrations of manganese cations (manganese sulfate (II)) according to one embodiment of the present invention, as confirmed by nanoparticle analysis (a) and Western blot (b) to be.
  • manganese cations manganese cations
  • Figure 13 shows the results of the nanoparticle analysis (a) and western blot (b) in which the extracellular endoplasmic reticulum was isolated in human urine by the addition of various concentrations of zinc cation (zinc chloride) according to one embodiment of the present invention.
  • FIG. 14 is a graph showing the results of nanoparticle analysis in which extracellular endoplasmic reticulum is separated from a cell culture medium by adding various cations (copper (II) chloride, manganese (II) sulfate) and polymers (PEG and PEOZ) together according to an embodiment of the present invention .
  • FIG. 15 is a result of western blot analysis to confirm that extracellular endoplasmic reticulum is isolated in a cell culture solution by adding copper cation (copper sulfate (II)) and polymer (PEOZ) together according to an embodiment of the present invention.
  • FIG. 16 is a result of confirming the separation of extracellular endoplasmic reticulum by combining a conventional salting-out method (ammonium sulfide) precipitation method with a copper cation (copper sulfate (II)) method according to the present invention.
  • a conventional salting-out method ammonium sulfide
  • copper cation copper sulfate (II)
  • Figure 17 compares the separation of extracellular endoplasmic reticulum (a) with the conventional polyethylene glycol (PEG) according to one embodiment of the present invention and the result of separation (b) of extracellular endoplasmic reticulum using the method of the present invention.
  • PEG polyethylene glycol
  • Colorectal cancer cells SW480 culture was centrifuged at 500xg for 10 minutes (2 times in total) to remove remaining cells and sediments. The supernatant was centrifuged again (2 times in total) at 2,000 x g for 20 minutes to remove precipitates.
  • the sample was mixed with Optiprep (final concentration 30%) and placed in the lowest layer of the ultracentrifuge, and then 20% Optiprap, 5 The layers were stacked in order of% optimaprap.
  • OptiPrap buoyancy density gradient ultracentrifugation (30%, 20%, 5% OptiPrap triple layer) was carried out at 200,000 x g for 2 hours and the supernatant after the ultracentrifugation was subjected to an equal density of 1.08 to 1.12 g / ml ) Area were harvested.
  • FIG. 2 (a) shows the process of separating the extracellular ER from the present specimen.
  • the extracellular endoplasmic reticulum from the purified colon cancer cells was analyzed by HPLC chromatogram. As a result, molecular size-specific chromatography showed an absorption band of 280 nm at 3.6 min (FIG. 2 (b)). As a result of the nanoparticle tracking analysis (NTA) of each fraction by chromatography, it was possible to detect high nanoparticle signals in samples eluted between 3.01 and 4.5 minutes, confirming that this signal is consistent with the extinction band at 280 nm , It was found that the extracellular endoplasmic reticulum was a band detected at 3.6 minutes in the HPLC analysis (Fig. 2 (b)).
  • the extracellular endoplasmic reticulum from the colorectal cancer cell line (SW480) was confirmed by electron microscopy. As a result, as shown in Fig. 2 (d), it was confirmed that the extracellular endoplasmic reticulum derived from SW480 colon cancer cells was about 50 to 200 nm in size.
  • TSG101 and CD9 which are markers of the extracellular endoplasmic reticulum, were identified through western blotting and are shown in Fig. 2 (e).
  • the absorption band at 280 nm which was detected at 3.6 min, increased in proportion to the concentration of the cation, depending on the treatment concentration of the calcium cation, the copper cation and the zinc cation added to the culture medium for colon cancer cells, (d).
  • the extinction bands and extracellular extracellular extinction band showed the same detection time, and the yield of extracellular extracellular cells isolated from the cell culture was increased with the concentration of cation added.
  • FIG. 5 (a) shows that the signal for CD9, a typical extracellular ER marker, increases with the concentration of copper cation Is shown in Fig. 5 (b).
  • Example 5 Isolation of extracellular endoplasmic reticulum using cobalt cation (cobalt chloride)
  • Example 8 Isolation of extracellular endoplasmic reticulum using calcium cation (calcium chloride)
  • Example 9 Isolation of extracellular endoplasmic reticulum using zinc cation (zinc chloride)
  • Example 10 Isolation of extracellular endoplasmic reticulum using calcium cation (calcium chloride) in human urine
  • FIG. 11 (a) shows that the signal for CD9, a typical extracellular ER marker, increases with the calcium cation concentration Is shown in Fig. 11 (b).
  • Example 11 Isolation of extracellular endoplasmic reticulum using manganese cation (manganese sulfide (II)) in human urine
  • FIG. 12 (a) shows that the signal for CD9, a typical extracellular ER marker, increases with the concentration of calcium cation Is shown in Fig. 12 (b).
  • Example 12 Isolation of extracellular endoplasmic reticulum using zinc cation (zinc chloride) in human urine
  • Human urine was centrifuged at 2,000 x g for 15 min (total 2 replicates) to remove any remaining precipitate.
  • Various concentrations of zinc cations were added to the prepared human urine, and the mixture was centrifuged at 3,000 ⁇ g for 10 minutes.
  • the precipitates were harvested and dissolved in HEPES buffer containing 50 mM EDTA.
  • the extracellular endoplasmic reticulum isolated using the above method was confirmed by nanoparticle analysis and Western blot analysis.
  • FIG. 13 (a) shows that the signal for the normal extracellular ER marker CD9 increases with the zinc cation concentration Is shown in Fig. 13 (b).
  • Example 13 Increased efficiency of separation of extracellular endoplasmic reticulum through combination of various cations (copper (II) chloride, manganese (II) sulphide (II)) and polymer
  • extracellular endoplasmic reticulum was isolated by adding cation alone, polymer alone, or cation and polymer together.
  • polyethylene glycol (PEG) or polyethyl oxazoline (PEOZ, Poly (2-ethyl-2-oxazoline)) was used as a polymer for extracellular ER separation, Only polyethylene glycol (PEG) was added so that the concentration was 8.3% or only polyethyl oxazoline (PEOZ, Poly (2-ethyl-2-oxazoline)) was added so that the final concentration was 10%.
  • PEG polyethylene glycol
  • PEOZ Poly (2-ethyl-2-oxazoline)
  • Nanoparticle analysis was performed to compare the yield of the extracellular endoplasmic reticulum harvested under the conditions of polymer alone, cation alone, or cation-polymer blend.
  • the yield of the extracellular endoplasmic reticulum under the conditions of culturing the polymer alone for 16 hours was 10 to 20 minutes
  • the yield of extracellular ER was further increased when the reaction was performed for 10 minutes under the condition of mixed cation and polymer. From these results, it was found that the efficiency of polymer alone was very low. However, it was found that the addition of polymer in the presence of cations further increased the extracellular precipitation efficiency of cations.
  • Example 14 CD9 analysis of extracellular endoplasmic reticulum with polymer and copper cation (copper (II) sulfide) concentration
  • Example 15 Isolation of extracellular endoplasmic reticulum by combination of ammonium sulphide and copper cation (copper (II) sulfide)
  • the yield of the extracellular endoplasmic reticulum under the condition of ammonium sulphate alone for 30 minutes was extremely low, while the yield of the high extracellular endoplasmic reticulum was maintained under the conditions of only 10 mM of copper cation for 30 minutes
  • the yield of the extracellular vesicles in the sample was increased in proportion to the concentration of the copper cation in the condition of 30 minutes of reaction with the copper cation and ammonium sulphide.
  • the precipitation efficiency of the extracellular vesicle is very low in the case of ammonium sulphate alone but in the case of the copper cation alone. It was also found that when the copper cation and ammonium sulfide were added together to precipitate the extracellular endoplasmic reticulum, the extracellular ER precipitation efficiency of the copper cation was maximized.
  • Example 16 Comparison of the extracellular endoplasmic reticulum and the extracellular endoplasmic reticulum using the polymer
  • polyethylene glycol PEG, polyethylene glycol
  • HEPES buffer 20 mM HEPES, pH 7.2, 150 mM NaCl
  • copper cations were added to the same volume of cell culture medium, followed by incubation for 10 minutes.
  • the precipitates were harvested by centrifugation at 3,000 ⁇ g for 10 minutes and dissolved in HEPES buffer containing 50 mM EDTA. Samples containing extracellular endoplasmic reticulum isolated using the above method were further separated by size-exclusion chromatography and analyzed by size exclusion chromatography using an HPLC system.
  • the yield and purity of the extracellular endoplasmic reticulum were significantly improved by using the cation as compared with the conventional method of separating the extracellular endoplasmic reticulum using the polymer.

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Abstract

The present invention relates to a method for isolating extracellular vesicles using cations, and more particularly, to a method for isolating extracellular vesicles from various samples by using the affinity between the extracellular vesicles and cations. A method for isolating extracellular vesicles according to the present invention does not require expensive equipment, can be applied irrespective of sample amount, and has the advantage of being capable of efficiently isolating the extracellular vesicles while preserving the shape or characteristics thereof. Moreover, the method according to the present invention can be combined with existing isolation methods to maximize extracellular vesicle isolation efficiency, and can be applied to disease diagnosis, disease treatment, and multi-omics research using isolated extracellular vesicles, as well as to research on the properties of extracellular vesicles.

Description

양이온을 이용한 세포밖 소포체의 분리 방법Separation of extracellular endoplasmic reticulum using cation
본 발명은 양이온을 이용한 세포밖 소포체의 분리 방법에 관한 것으로서, 보다 상세하게 본 발명은 다양한 양이온에 대한 세포밖 소포체의 친화성을 이용하여 다양한 시료로부터 세포밖 소포체를 분리하는 방법에 관한 것이다.More particularly, the present invention relates to a method for separating extracellular endoplasmic reticulum from various samples using the affinity of extracellular endoplasmic reticulum for various cations.
세포밖 소포체(extracellular vesicles)는 보편적인 세포기작으로, 인간에서 박테리아에 이르기까지 모든 생명체 또는 세포에서 자연적으로 분비되는 나노 크기의 소포체이다. 진핵세포에서 유래한 세포밖 소포체의 경우, 적혈구 분화, 면역반응 조절 등에 관여하며, 특히 암세포 미세환경에서는 암의 진행, 전이, 혈관형성 등에 대한 중요한 기능들이 밝혀짐으로써 암을 포함한 다양한 질병의 진단 마커로의 활용에 있어 높은 관심을 받고 있다.Extracellular vesicles are universal cellular mechanisms, nano-sized vesicles that are naturally secreted by all living things or cells, from humans to bacteria. The extracellular endoplasmic reticulum derived from eukaryotic cells is involved in the erythropoietic differentiation and regulation of the immune response. Especially, important functions of cancer progression, metastasis and angiogenesis are revealed in the cancer cell microenvironment, And has received high interest in the utilization of
원핵세포로부터 분비되는 세포밖 소포체는 진핵세포의 세포밖 소포체와 유사하게 원핵세포의 구성물을 함유하고 있으며, 인체에서는 전신염증을 비롯, 유입 경로에 따라 급성 폐염증 질환을 유도하고, 피부의 경우, 국소 피부 조직의 염증반응을 만성적으로 유도하여 현대인의 대표 질병 중 하나인 아토피성 피부염(atopic dermatitis)의 원인이 될 수 있음이 보고되었다. 또한 인체에서 박테리아 유래 세포밖 소포체와 암 발병과의 연관성이 대두됨에 따라 원핵세포 유래 세포밖 소포체 또한 높은 관심을 받고 있다.The extracellular endoplasmic reticulum secreted from prokaryotic cells contains prokaryotic components similar to extracellular endoplasmic reticulum. In the human body, acute pulmonary inflammatory diseases are induced according to the pathway including systemic inflammation. In the case of skin, It has been reported that chronic inflammation of the local skin tissues can be induced to cause atopic dermatitis, one of the representative diseases of modern people. In addition, the extracellular endoplasmic reticulum from prokaryotic cells has also attracted a great deal of attention due to the emergence of bacterial-derived extracellular endoplasmic reticulum in the human body and cancer incidence.
세포밖 소포체의 기능으로 가장 크게는 이들이 세포간 정보 교환 메커니즘의 중요한 요소라는 점이다. 따라서 세포밖 소포체의 구성 성분 또한 기초 및 의학 분야에서 높은 관심을 받고 있다.The function of extracellular endoplasmic reticulum is most important because they are an important component of the intercellular information exchange mechanism. Therefore, the constituents of extracellular endoplasmic reticulum have also received high interest in the basic and medical fields.
세포밖 소포체는 생체 내 또는 시험관 내의 여러 종류의 세포로부터 분비되는 생체 나노입자로서, 혈액, 소변, 침, 눈물 등과 같은 체액에 존재하고 세포에서 유래한 지질이중층을 포함하며, 20 ~ 10,000 nm 범위의 다양한 크기를 갖는 막 구조의 소포체이다.The extracellular endoplasmic reticulum is a bio-nanoparticle secreted from various kinds of cells in vivo or in vitro. It is present in body fluid such as blood, urine, saliva, tears, etc. and contains a cell-derived lipid bilayer. It is a vesicle of membrane structure with various sizes.
세포밖 소포체는 다른 세포 및 조직에 결합하여 막 구성요소, 단백질, RNA 등의 세포 내 물질을 전달하는 운송체 역할을 하기 때문에 세포밖 소포체를 분비한 원래 세포(모세포)의 단백질, 지질, 아미노산, RNA 등을 그대로 포함하고 있어, 모세포의 생리적·병리적 특성을 알 수 있는 중요한 근거가 된다. 또한 세포밖 소포체에 포함되어 있는 핵산, 성장호르몬, 단백질 등은 세포막 형태의 인지질에 의해 보호되고 있어, 가용성 형태의 성장인자 및 사이토카인보다 안정적인 기능을 수행할 수 있다는 점이 알려지면서, 세포밖 소포체의 중요성이 점차 증대되고 있으며, 세포밖 소포체에 포함된 물질을 분석하여 질병의 진단, 치료를 포함한 다양한 용도로의 활용 가능성이 기대되고 있다.Since the extracellular endoplasmic reticulum (ER) binds to other cells and tissues and acts as a transporter that transports intracellular substances such as membrane components, proteins, and RNAs, the proteins, lipids, amino acids, RNA, and so on, thus providing an important basis for understanding the physiological and pathological characteristics of the parent cells. It is known that nucleic acid, growth hormone, and protein contained in the extracellular endoplasmic reticulum are protected by a phospholipid in the form of a cell membrane and can perform more stable functions than a soluble growth factor and cytokine. It is expected to be used for various purposes including diagnosis and treatment of diseases by analyzing substances contained in extracellular endoplasmic reticulum.
세포밖 소포체는 크기가 나노 미터 수준으로 작으며, 체액 내 및 세포 배양액 등에는 세포밖 소포체 이외에도 수 많은 물질이 존재하기 때문에 세포밖 소포체 분석을 위해서는 체액 내 및 세포 배양액 등의 시료로부터 세포밖 소포체를 분리하는 것이 중요하며, 세포밖 소포체를 활용하는 모든 분야에서 가장 핵심적인 기술이다.Since extracellular endoplasmic reticulum is small in nanometer level and numerous substances exist in body fluid and cell culture fluid in addition to extracellular endoplasmic reticulum, extracellular endoplasmic reticulum from samples such as body fluids and cell culture fluids for extracellular endoplasmic reticulum analysis It is important to separate, and it is the most important technology in all areas that utilize extracellular endoplasmic reticulum.
최근 비 침습적 액체 생검(liquid biopsy)을 질병 진단에 활용하는 방안이 다각도로 전개되고 있다. 이와 더불어 생체 조직 또는 체액 내 세포밖 소포체를 활용하여 새로운 질병 진단 마커를 발굴하고 이를 이용해 진단하는 노력들이 시도되고 있다. 이러한 노력의 근원적인 문제점은 생체 조직 또는 체액으로부터 세포밖 소포체를 분리하는 데에 있는데, 상대적으로 제한된 양과 높은 복잡성을 나타내는 체액에서 세포밖 소포체를 통상의 방법으로 정제하는 것이 거의 불가능하다. 따라서 통상의 세포밖 소포체 분리법과는 차별되는 효율적인 새로운 분리법이 시급히 요구되는 실정이다.Recently, there have been various ways to utilize non-invasive liquid biopsy for diagnosis of diseases. In addition, attempts have been made to diagnose new disease diagnosis markers using biopsy or extracellular endoplasmic reticulum in body fluids and to diagnose them. The fundamental problem of this effort is the separation of extracellular endoplasmic reticulum from biological tissue or body fluids, and it is nearly impossible to purify the extracellular endoplasmic reticulum in a body fluid that exhibits a relatively limited amount and high complexity. Therefore, there is a need for an efficient new separation method that is different from the conventional extracellular ER separation method.
기존의 세포밖 소포체 분리 기술로는 초원심분리(ultra-centrifugation), 크기별 제외법(size exclusion), 면역친화성 분리(immunoaffinity isolation), 미세유체칩(microfluidics chip), 또는 폴리머(Polymer)를 이용한 침전법 등이 있으며, 이중 초원심분리법이 가장 널리 사용되고 있다. 그러나 초원심분리를 이용하여 세포밖 소포체를 분리할 경우에는 단계가 복잡하여 노동력과 시간이 많이 소요되며, 고가의 장비를 필요로 할 뿐만 아니라 수율이 낮은 한계가 있어, 소량의 시료만 이용하여 신속하게 결과를 얻어야 하는 임상 진단 뿐 아니라 다량의 세포밖 소포체가 요구되는 치료제 정제 방법으로 적용하는 데는 매우 제한적이다.Existing extracellular ER separation techniques include ultra-centrifugation, size exclusion, immunoaffinity isolation, microfluidics chip, or polymer. And precipitation method. Among these, the ultracentrifugation method is most widely used. However, separation of the extracellular endoplasmic reticulum by ultracentrifugation requires complicated steps, labor and time, requires expensive equipments, and has a low yield, so that only a small amount of sample is used In addition to clinical diagnosis, which requires obtaining results, it is very limited to be applied as a therapeutic purification method requiring a large amount of extracellular endoplasmic reticulum.
물질 분리에 있어 가장 효율적인 방법은 대상 물질에 대한 선택적 결합을 이용하여 복잡성을 가지는 환경으로부터 분리 과정 중 대상 물질을 잃지 않으면서 오염체를 순차적으로 제거하는 것이다. 그러나 세포밖 소포체의 경우, 이러한 선택적 결합 성질을 가지는 물질이 일부 항체 또는 단백질 리간드(ligand)에 국한되어 있고, 이러한 항체 또는 단백질을 이용한 세포밖 소포체의 분리는 비효율적일뿐만 아니라, 고효율의 항체 및 단백질 리간드 개발이 어렵고, 고비용이 소요되어 매우 제한적이다.The most efficient method of material separation is to sequentially remove contaminants from the complex environment using selective binding to the target material without losing the material during the separation process. However, in the case of the extracellular endoplasmic reticulum, the substance having such selective binding properties is limited to some antibodies or protein ligands, and separation of the extracellular endoplasmic reticulum using such an antibody or protein is not only inefficient, Ligand development is difficult, and high cost is required, which is very limited.
따라서 세포밖 소포체에 선택적이면서 세포밖 소포체의 구조와 기능을 온전히 유지하고 효율적으로 높은 수율(yield)의 세포밖 소포체를 분리, 정제하는 기술이 시급히 요구되는 실정이다.Therefore, there is an urgent need for a technique for efficiently separating and purifying the extracellular endoplasmic reticulum with high yield while maintaining the structure and function of the extracellular endoplasmic reticulum selectively and extracellularly.
본 발명의 목적은 현재까지 보고된 바 없는 양이온과 세포밖 소포체의 친화도를 이용하여 시료 내 세포밖 소포체를 간편하면서 고효율로 분리하는 방법을 제공하고자 하는 것이다.It is an object of the present invention to provide a simple and highly efficient method of separating extracellular endoplasmic reticulum from a sample using affinity of a cation and an extracellular endoplasmic reticulum which have not been reported so far.
본 발명은 상술한 문제점을 해결하기 위한 것으로, 양이온과 세포밖 소포체의 친화도를 이용하여 시료 내 세포밖 소포체를 간편하면서 고효율로 분리하는 방법을 제공한다.Disclosure of Invention Technical Problem [8] Accordingly, the present invention provides a method for separating extracellular endoplasmic reticulum easily and efficiently with affinity of a cation and an extracellular endoplasmic reticulum.
본 발명에서는 세포밖 소포체를 포함하는 다양한 시료에 다양한 양이온을 첨가하여 반응시키면, 시료 내에서 세포밖 소포체와 양이온이 결합하여 불용성 복합체를 형성하게 된다. 상기 세포밖 소포체-양이온 복합체는 원심분리, 한외여과, 중력에 의한 침전 등 다양한 방법으로 분리할 수 있으며, 이후 상기 복합체에서 양이온을 탈착함으로써 세포밖 소포체를 분리할 수 있다. 이를 통하여 다양한 시료로부터 물리화학적 변형 없이 빠르고 쉽게 세포밖 소포체를 분리할 수 있으며, 이렇게 분리된 세포밖 소포체는 진단, 치료, 다중 오믹스(multi-omics) 연구, 세포밖 소포체의 특성 연구 등에 활용이 용이하다.In the present invention, when various cations are added to various samples including extracellular endoplasmic reticulum and reacted, the extracellular endoplasmic reticulum and cations bind to each other in the sample to form an insoluble complex. The extracellular endoplasmic reticulum-cation complex can be separated by various methods such as centrifugation, ultrafiltration, precipitation by gravity, and then the extracellular endoplasmic reticulum can be separated by desorbing the cation from the complex. This makes it possible to quickly and easily separate the extracellular endoplasmic reticulum from a variety of samples without physical and chemical modification. The extracellular endoplasmic reticulum can be used for diagnosis, treatment, multi-omics studies, It is easy.
본 발명의 용어 “세포밖 소포체”는 고세균(Archaea), 원핵생물(Prokarya) 또는 진핵생물(Eukarya)의 세포로부터 유래한 생체 나노입자를 통칭하며, 세포밖 소포체(exosome), 아그로좀(argosomes), 덱소좀(dexosomes), 엑토좀(ectosomes), 엑소베지클(exovesicle), 온코좀(oncosome), 프로미노좀(prominosome), 프로스타좀(prostasome), 톨레로좀(tolerosome), 미세입자(microparticle), 미세소포(microvesicle), 나노소포(nanovesicle), 수포성 소포(blebbing vesicle), 출아성 소포(budding vesicle), 세포밖 소포체-유사 소포(exosome-like vesicle), 매트릭스 소포(matrix vesicle), 막 소포(membrane vesicle), 탈피성 소포(shedding vesicle), 막 입자(membrane particle), 탈피성 미세소포(shedding microvesicle), 막 수포(membrane bleb), 에피디디모좀(epididimosome), 프로미니노좀(promininosome), 텍소좀(texosome) 또는 아키오좀(archeosome)을 포함할 수 있으나, 이에 한정되는 것은 아니다.The term " extracellular < / RTI > endoplasmic reticulum " of the present invention collectively refers to a living body nanoparticle derived from cells of Archaea, Prokarya or Eukarya and includes extracellular endoplasmic reticulum (exosome), argosomes , Dexosomes, ectosomes, exovesicles, oncosomes, prominosomes, prostasomes, tolerosomes, microparticles (e. G. microvesicles, microparticles, microvesicles, nanovesicles, blebbing vesicles, budding vesicles, exosome-like vesicles, matrix vesicles, , Membrane vesicles, shedding vesicles, membrane particles, shedding microvesicles, membrane blebs, epididymosomes, promininosome, texosome, or archeosome, Information that is not.
본 발명은 (a) 생물학적 시료에 양이온을 첨가하는 단계, (b) 상기 생물학적 시료에 포함된 세포밖 소포체와 양이온을 반응시켜 복합체를 형성하는 단계, (c) 상기 시료로부터 세포밖 소포체와 양이온 복합체를 분리하는 단계 및 (d) 상기 복합체로부터 양이온을 분리시켜 세포밖 소포체를 정제하는 단계를 포함하는 세포밖 소포체 분리 방법을 제공한다.The present invention relates to a method for preparing a biological sample, comprising the steps of (a) adding a cation to a biological sample, (b) reacting the extracellular endoplasmic reticulum contained in the biological sample with a cation to form a complex, (c) And (d) separating the cation from the complex to purify the extracellular endoplasmic reticulum.
본 발명의 일 구현예에 따른 상기 세포밖 소포체의 분리 방법을 도 1에 모식적으로 나타내었다.A method of separating the extracellular endoplasmic reticulum according to an embodiment of the present invention is schematically shown in FIG.
본 발명의 일 구현예에 있어서, 본 발명의 세포밖 소포체 분리 방법은 생물학적 시료에 양이온을 첨가하는 단계[(a) 단계] 및 상기 생물학적 시료에 포함된 세포밖 소포체와 양이온을 반응시켜 복합체를 형성하는 단계[(b) 단계]를 포함한다.In one embodiment of the present invention, the method for separating extracellular ER of the present invention comprises the steps of adding a cation to a biological sample (step (a)) and reacting the extracellular endoplasmic reticulum contained in the biological sample with a cation to form a complex (Step (b)).
본 발명의 용어 "생물학적 시료" 또는 "시료"는 세포밖 소포체를 포함하는 생체 시료 또는 세포 배양액, 조직 시료 등을 포함하는 것으로서, 구체적으로 포유동물 세포 배양 배지, 박테리아 세포 배양 배지, 효모 배양 배지, 조직 추출물, 암 조직, 혈청, 혈장, 침, 눈물, 땀, 소변, 대변, 뇌척수액(CSF, cerebrospinal fluid), 복수(ascite), 양수(amniotic fluid), 정액, 유(milk), 먼지, 담수, 해수, 토양 및 발효식품으로 이루어진 군에서 하나 이상이 선택될 수 있으나 이에 한정되지 않는다.The term " biological sample " or " sample " of the present invention includes biological samples or cell culture fluids, tissue samples and the like, including extracellular endoplasmic reticulum, and specifically includes mammalian cell culture medium, bacterial cell culture medium, yeast culture medium, (CSF), cerebrospinal fluid (CSF), ascites, amniotic fluid, semen, milk, dust, fresh water, blood, urine, Seawater, soil, and fermented food. However, the present invention is not limited thereto.
본 발명의 용어 "양이온"은 전기적으로 양전하를 띠는 것으로서 세포밖 소포체와 특이적인 친화력을 갖고 시료 중의 세포밖 소포체와 결합할 수 있으며, 바람직하게는 금속 양이온일 수 있다. 본 발명의 용어 "금속 양이온"은 알칼리 금속 이온, 알칼리 토금속 이온, 전이 금속 이온, 전이후 금속 이온을 포함할 수 있다. 본 발명의 양이온 또는 금속 양이온은 바람직하게 전이 금속 이온 또는 알칼리 토금속일 수 있으나, 분리하고자 하는 세포밖 소포체와 특이적인 친화성을 갖는 양이온이라면 이에 한정되지 않는다.The term " cation " of the present invention is electrically positively charged and has a specific affinity for the extracellular endoplasmic reticulum and can bind to the extracellular endoplasmic reticulum in the sample, preferably a metal cation. The term " metal cation " of the present invention may include alkali metal ions, alkaline earth metal ions, transition metal ions, and transition metal ions. The cation or metal cation of the present invention may preferably be a transition metal ion or an alkaline earth metal, but is not limited thereto, so long as it is a cation having a specific affinity for an extracellular endoplasmic reticulum to be separated.
알칼리 금속(alkali metal)은　주기율표의　1족　가운데　수소를 제외한 나머지　화학 원소를 통틀어 일컫는 표현으로　리튬(Li),　나트륨(Na),　칼륨(K),　루비듐(Rb),　세슘(Cs) 및　프랑슘(Fr)을 포함한다. 알칼리 토금속(alkaline earth metal)은　주기율표의　2족　원소로서,　베릴륨(Be),　마그네슘(Mg),　칼슘(Ca),　스트론튬(Sr),　바륨(Ba) 및　라듐(Ra)을 포함한다. 전이금속(transition metal)은 화학주기율표의 4 ~ 7주기, 3 ~ 12족 원소를 포함하는 것으로서, 비금속과 함께 이온 결합 화합물을 형성하여 착이온 형태로 존재하는 특징이 있다. 구체적으로, 본 발명의 전이금속은 스칸듐(Sc), 이트륨(Y), 티타늄(Ti), 지르코늄(Zr), 하프늄(Hf), 러더포듐(Rf), 바나듐(V), 니오븀(Nb), 탄탈룸(Ta), 더브늄(Db), 크롬(Cr), 몰리브덴(Mo), 텅스텐(W), 시보귬(Sg), 망간(Mn), 테크네튬(Tc), 레늄(Re), 보륨(Bh), 철(Fe), 루테늄(Ru), 오스뮴(Os), 하슘(Hs), 코발트(Co), 로듐(Rh), 이리듐(Ir), 마이트너륨(Mt), 니켈(Ni), 팔라듐(Pd), 백금(Pt), 다름슈타튬(Ds), 구리(Cu), 은(Ag), 금(Au), 뢴트게늄(Rg), 아연(Zn), 카드뮴(Cd), 수은(Hg) 및 코페르니슘(Cn)을 포함한다. 전이후 금속(post-transition metal)은　주기율표의　p-구역에 있는　금속　원소를 의미하며, 알루미늄(Al), 갈륨(Ga), 인듐(In), 탈륨(Tl), 주석(Sn), 납(Pb), 비스무트(Bi) 및 폴로늄(Po)을 포함한다.Alkali metal is a group of chemical elements other than hydrogen among the group 1 of the periodic table and includes lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs) Fr). The alkaline earth metal is a Group 2 element of the periodic table and includes beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba) and radium (Ra). The transition metal contains elements 4 to 7 and 3 to 12 of the Periodic Table of the Chemical Periods. It forms an ion-binding compound together with a nonmetal and exists in complex ion form. Specifically, the transition metal of the present invention is a transition metal selected from the group consisting of Sc, Y, Ti, Zr, Hf, Rf, V, Nb, Tantalum (Ta), dibonium (Db), chromium (Cr), molybdenum (Mo), tungsten (W), sb, manganese (Mn), technetium (Tc), rhenium (Re) ), Iron (Fe), ruthenium (Ru), Os, Os, cobalt, rhodium, iridium, (Pd), Pt, Ds, Cu, Ag, Au, Rg, Zn, Cd, Hg) and copper (Cn). Post-transition metal means a metal element in the p-zone of the periodic table and is a metal element of aluminum (Al), gallium (Ga), indium (In), thallium (Tl), tin (Sn) Pb), bismuth (Bi), and polonium (Po).
본 발명의 양이온을 첨가하는 방법으로는 시료에 양이온을 포함하는 용액을 첨가하는 방법과 고체 형태로 첨가하여 용해시키는 방법을 포함하나, 양이온 상태로 시료 내의 세포밖 소포체와 반응할 수 있는 형태라면 이에 한정되지 않는다.The method of adding the cation of the present invention includes a method in which a solution containing a cation is added to a sample and a method in which the solution is added in a solid form to dissolve it. However, in a form capable of reacting with an extracellular endoplasmic reticulum in a sample in a cationic state, It is not limited.
본 발명의 세포밖 소포체 분리 방법은 분리하고자 하는 세포밖 소포체가 양이온과 특이적으로 결합하는 성질을 이용하여 시료로부터 양이온과 결합한 세포밖 소포체를 분리하는 방법에 관한 것이다. 본 발명의 방법에서는 먼저 시료에 양이온을 첨가하여 반응시킴으로써 시료 중 세포밖 소포체와 양이온이 특이적으로 결합하며, 불용성의 세포밖 소포체-양이온 복합체가 형성된다. The present invention relates to a method for separating an extracellular endoplasmic reticulum associated with a cation from a sample using a property of specifically binding an extracellular endoplasmic reticulum to a cation to be separated. In the method of the present invention, by adding a cation to a sample and reacting it, the extracellular endoplasmic reticulum and the cation are specifically bound in the sample, and an insoluble extracellular endoplasmic reticulum-cation complex is formed.
본 발명의 일 실시예에서는 세포밖 소포체가 포함된 배양배지 시료 또는 소변 시료에 칼슘 이온, 망간 이온, 코발트 이온, 구리 이온 또는 아연 이온을 첨가하여 불용성 복합체가 형성됨을 확인하였다. 또한 상기 불용성 복합체는 중력에 의하여 가라앉기 때문에 손쉽게 분리가 가능함을 확인하였다.In one embodiment of the present invention, insoluble complexes were formed by adding calcium ion, manganese ion, cobalt ion, copper ion or zinc ion to a culture medium sample or urine sample containing an extracellular endoplasmic reticulum. Also, it was confirmed that the insoluble complex can be easily separated because it is submerged by gravity.
본 발명의 일 구현예에 있어서, 본 발명의 세포밖 소포체 분리 방법은 상기 시료로부터 세포밖 소포체와 양이온 복합체를 분리하는 단계[(c) 단계]를 포함한다.In one embodiment of the present invention, the method for separating extracellular fibrils includes separating the extracellular endoplasmic reticulum and the cation complex from the sample (step (c)).
본 발명의 세포밖 소포체와 양이온 복합체를 분리하는 단계는 상기 단계에서 형성된 불용성 복합체를 각종 수용성 물질이 포함된 시료로부터 분리하는 것으로서, 원심분리, 초원심분리, 여과, 한외여과, 중력, 음파처리, 밀도 구배 초원심분리, 크기 배제 크로마토그래피, 이온교환 크로마토그래피, 친화성 크로마토그래피, 폴리머 기반 침전 또는 유기 용매 침전 중에서 하나 이상의 방법이 선택될 수 있으나, 이에 한정되지 않는다.The step of separating the extracellular endoplasmic reticulum and the cation complex of the present invention is a step of separating the insoluble complex formed in the step from the sample containing various water-soluble substances, and the step of centrifuging, ultracentrifugation, filtration, ultrafiltration, gravity, One or more methods may be selected from, but are not limited to, density gradient ultracentrifugation, size exclusion chromatography, ion exchange chromatography, affinity chromatography, polymer-based precipitation or organic solvent precipitation.
본 발명의 일 구현예에 있어서, 본 발명의 세포밖 소포체 분리 방법은 상기 복합체로부터 양이온을 분리시켜 세포밖 소포체를 정제하는 단계[(d) 단계]를 포함한다.In one embodiment of the present invention, the method for separating extracellular fibrils comprises separating cations from the complex to purify the extracellular fibrils (step (d)).
본 발명의 세포밖 소포체 정제 단계는 세포밖 소포체와 양이온의 특이적 결합 상태를 제거하여 복합체로부터 세포밖 소포체만 분리할 수 있는 방법으로서 본 기술이 속한 분야의 당업자가 이해할 수 있는 다양한 방법 또는 조건을 적용할 수 있다.The step of purifying the extracellular endoplasmic reticulum of the present invention can separate only the extracellular endoplasmic reticulum from the complex by eliminating the specific binding state between the extracellular endoplasmic reticulum and the cations, and it is possible to use various methods or conditions which those skilled in the art can understand Can be applied.
본 발명의 일 실시예에 있어서, 상기 단계는 분리된 세포밖 소포체와 양이온 복합체에 킬레이트제를 첨가하는 방법을 포함할 수 있다. In one embodiment of the present invention, the step may comprise adding a chelating agent to the separated extracellular endoplasmic reticulum and cation complex.
본 발명의 용어 "킬레이트제(chelate agents)" 또는 "킬레이트 리간드(chelating ligands)"는 금속이온에 배위하여 안정된 킬레이트착물을 형성하는 2개 이상의 배위 원자를 포함한 이온, 분자 또는 원자단을 의미하며, 배위 원자 수에 따라 세자리 리간드(tridentrate ligand), 네자리 리간드(tetradentrate ligand), 다섯자리 리간드(pentadentrate ligand), 여섯자리 리간드(hexadentrate ligand) 등으로 같이 부른다. 본 발명의 킬레이트 리간드는 이미노디아세트산(IDA, iminodiacetic acid), 니트릴로트리아세트산(NTA, nitrilotriacetic acid), 트리스(카복시메틸)에틸렌디아민(TED, tris-(carboxymethyl)ethylenediamine), 에틸렌디아민(ethylenediamine), 에틸렌디아민 테트라아세테이트(EDTA, ethylendiamine tetraacetate), 알킬렌디아민 트리아세트산(alkylenediamine triacetic acid), 디에틸렌트리아민펜타아세트산(DTPA, diethylenetriaminepentaacetic acid), 에틸렌글리콜 비스(베타-아미노에틸에테르)-N,N,N',N'-테트라아세트산(EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid), 포스포세린(phosphoserine) 및 1,4,7-트리아조사이클로노난(TACN, 1,4,7-triazocyclononane)으로 이루어진 군에서 하나 이상이 선택될 수 있으나, 본 발명에서 사용되는 금속 양이온에 특이적으로 결합하여 세포밖 소포체-양이온 복합체로부터 양이온을 특이적으로 분리할 수 있는 것이라면 이에 한정되지 않는다.The term " chelate agents " or " chelating ligands " of the present invention means an ion, molecule or atomic group containing two or more coordinating atoms forming a chelate complex stable to metal ions, Depending on the number of atoms, tridentate ligand, tetradentrate ligand, pentadentrate ligand, and hexadentrate ligand are also called. The chelate ligand of the present invention may be selected from the group consisting of iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), tris (carboxymethyl) ethylenediamine (TED), ethylenediamine, Ethylenediamine tetraacetate (EDTA), alkylenediamine triacetic acid, diethylenetriaminepentaacetic acid (DTPA), ethylene glycol bis (beta-aminoethyl ether) -N, N, N, N ', N'-tetraacetic acid (EGTA), phosphoserine and 1,4,7-triazo (TACN, 1,4,7-triazocyclononane). However, it is possible to specifically bind the metal cations used in the present invention and to remove the cation from the extracellular endoplasmic reticulum-cation complex So long as it can remove the enemy is not limited to this.
본 발명의 일 실시예에 있어서, 상기 단계는 분리된 세포밖 소포체와 양이온 복합체가 포함된 용액의 pH 값을 변화시키는 방법을 사용할 수 있다.In one embodiment of the present invention, the above step may use a method of changing the pH value of the solution containing the separated extracellular endoplasmic reticulum and the cation complex.
또한 본 발명의 일 실시예에 있어서, 상기 단계는 분리된 세포밖 소포체와 양이온 복합체가 포함된 용액에서 이미다졸(imidazole), 히스티딘(histidine), 에틸렌디아민 테트라아세테이트(EDTA, ethylendiamine tetraacetate) 또는 염(salts)의 농도를 변화시킴으로써 복합체로부터 세포밖 소포체를 정제하는 방법을 사용할 수 있다. In one embodiment of the present invention, the above step further comprises the step of adding an imidazole, histidine, ethylenediamine tetraacetate (EDTA), or salt (s) to a solution containing a separated extracellular endoplasmic reticulum and a cation complex methods of purifying the extracellular endoplasmic reticulum from the complex by varying the concentration of the salts.
본 발명의 세포밖 소포체 정제 단계는 상기한 방법 중 어느 하나 또는 하나 이상의 방법을 복합적으로 선택하여 수행될 수 있다. 바람직하게 본 발명의 정제 조건은 pH 10 이하의 완충액, 0 ~ 5 M NaCl, 0 ~ 2 M 이미다졸, 0 ~ 2 M의 금속 킬레이트제 또는 상기 조건의 조합을 적용할 수 있으나, 이에 한정되지 않는다. The step of purifying the extracellular endoplasmic reticulum of the present invention can be carried out by a complex selection of one or more of the above methods. Preferably, the purification conditions of the present invention include, but are not limited to, buffers of pH 10 or less, 0-5 M NaCl, 0-2 M imidazole, 0-2 M metal chelating agents, or combinations of the above conditions .
본 발명의 일 구현예에 있어서, 본 발명의 세포밖 소포체 분리 방법은 상기 시료에 양이온을 첨가하기 전에 시료를 전처리하는 단계를 더 포함할 수 있다.In one embodiment of the present invention, the method for separating extracellular fibrils may further comprise pretreating the sample before adding the cation to the sample.
본 발명의 상기 전처리 단계는 비정제 시료의 부분 정제 단계로서 원심분리, 초원심분리, 여과, 한외여과, 음파처리, 밀도 구배 초원심분리, 크기 배제 크로마토그래피, 이온교환 크로마토그래피, 친화성 크로마토그래피, 폴리머 기반 침전 또는 유기 용매 침전 중에서 하나 이상의 방법이 선택될 수 있으나, 이에 한정되지 않는다.The pretreatment step of the present invention is a step of partially purifying an untreated sample, which comprises centrifugation, ultracentrifugation, filtration, ultrafiltration, sonication, density gradient ultracentrifugation, size exclusion chromatography, ion exchange chromatography, affinity chromatography , Polymer-based precipitation, or organic solvent precipitation, but is not limited thereto.
또한, 본 발명의 일 구현예에 있어서, 본 발명의 세포밖 소포체 분리 방법에 따라 분리된 세포밖 소포체를 후처리하는 단계를 더 포함할 수 있다.Further, in one embodiment of the present invention, it may further comprise post-treating the extracellular endoplasmic reticulum isolated according to the extracellular endoplasmic reticulum isolation method of the present invention.
본 발명의 상기 후처리 단계는 분리된 세포밖 소포체의 정제 단계로서 원심분리, 초원심분리, 여과, 한외여과, 음파처리, 밀도 구배 초원심분리, 크기 배제 크로마토그래피, 이온교환 크로마토그래피, 친화성 크로마토그래피, 폴리머 기반 침전 또는 유기 용매 침전 중에서 하나 이상의 방법이 선택될 수 있으나, 이에 한정되지 않는다.The post-treatment step of the present invention is a step of purifying the separated extracellular endoplasmic reticulum by centrifugation, ultracentrifugation, filtration, ultrafiltration, sonication, density gradient ultracentrifugation, size exclusion chromatography, ion exchange chromatography, affinity One or more methods may be selected from, but are not limited to, chromatography, polymer-based precipitation or organic solvent precipitation.
본 발명의 일 구현예에 있어서, 본 발명의 세포밖 소포체 분리 방법은 상기 시료에 양이온을 첨가하는 단계에서 고분자 또는 염석이온을 첨가하는 단계를 추가로 포함할 수 있다. 본 발명의 양이온을 이용한 세포밖 소포체 분리 방법에 있어서, 양이온과 함께 고분자 또는 염석이온을 첨가함으로써 불용성 복합체 형성 속도를 현저히 증가시킬 수 있으며, 세포밖 소포체의 분리 효율 및 분리 시간을 현저히 향상시킬 수 있다.In one embodiment of the present invention, the method for separating extracellular fibrils may further include adding a polymer or salting-out ion in the step of adding a cation to the sample. In the method for separating extracellular fibrils using the cation of the present invention, the rate of insoluble complex formation can be remarkably increased by adding a polymer or salting-out ion together with cations, and the efficiency and time for separation of extracellular fibrils can be remarkably improved .
구체적으로, 상기 고분자 또는 염석이온은 양이온과 동시에 시료에 첨가될 수 있다.Specifically, the polymer or salting-out ion can be added to the sample simultaneously with the cation.
또한, 상기 고분자 또는 염석이온은 시료에 양이온을 첨가 하기 전에 먼저 첨가될 수 있다.In addition, the polymer or salting-out ion may be added before the cation is added to the sample.
또한, 상기 고분자 또는 염석이온은 시료에 양이온을 첨가한 후에 첨가될 수 있다.Further, the polymer or salting-out ion may be added after adding the cation to the sample.
본 발명의 일 구현예에 있어서, 상기 고분자는 폴리에틸렌글리콜(PEG, poly ethylene glycol) 또는 폴리옥사졸린(polyoxazoline)일 수 있으며, 상기 폴리옥사졸린은 치환기에 따라 폴리옥사졸린은 폴리메틸옥사졸린(PMOZ, poly(2-methyl-2-oxazoline), 폴리에틸옥사졸린(PEOZ, poly(2-ethyl-2-oxazoline) 또는 폴리프로필옥사졸린(PPOZ, poly(2-propyl-2-oxazoline)일 수 있다. 바람직하게 상기 고분자는 폴리에틸렌글리콜(PEG, poly ethylene glycol) 또는 폴리에틸옥사졸린(PEOZ, poly(2-ethyl-2-oxazoline)일 수 있으나, 이에 한정되지 않는다.In one embodiment of the present invention, the polymer may be polyethylene glycol (PEG) or polyoxazoline, and the polyoxazoline may be selected from the group consisting of polymethyloxazoline (PMOZ , poly (2-methyl-2-oxazoline), poly (2-ethyl-2-oxazoline) or poly (2-propyl-2-oxazoline) Preferably, the polymer may be polyethylene glycol (PEG), poly (2-ethyl-2-oxazoline), or polyethylene oxide (PEOZ).
본 발명의 용어 "염석 이온(salting-out ion)"은 용액 중에 물의 가용성을 감소시켜 소수성 상호반응의 세기를 증가시키기 위한 것으로서, 물 구조를 안정화시키는 코스모트로픽 염(kosmotropic salt)을 지칭한다. 이러한 코스모트로픽 염은 용액에서 용해성 물질의 용해도에 영향을 미치는 정도에 대한 능력에 따라 호프마이스터 계열(Hofmeister series)로 나타내며, 음이온 계열은 다음과 같다: SO4 2- < HPO4 2- < OH- < F- < HCOO- < CH3COO- < Cl- < Br- < NO3 - < I- < SCN- < ClO4 -. 양이온 계열은 다음과 같다: NH4 +, Rb+, K+, Na+, Cs+, Li+, Ca2+, Mg2+, 및 Ba2+. 코스모트로픽 염은 호프마이스터 계열에 따라 소수성 입자에 대한 염석 이온으로 작용한다. 본 발명의 상기 염석 이온은 호프마이스터 계열에서 물 구조를 안정화시키는 음이온과 그의 카운터 양이온으로 된 코스모트로픽 염일 수 있다.The term " salting-out ion " of the present invention refers to a kosmotropic salt for stabilizing a water structure for reducing the solubility of water in a solution to increase the strength of the hydrophobic interaction. These cosmotropic salts are represented by the Hofmeister series according to their ability to affect the solubility of soluble materials in solution and the anion series are: SO 4 2- <HPO 4 2- OH - <F - <HCOO - <CH 3 COO - <Cl - <Br - <NO 3 - <I - <SCN - <ClO 4 - . The cation series are: NH 4 + , Rb + , K + , Na + , Cs + , Li + , Ca 2+ , Mg 2+ , and Ba 2+ . Cosmotropic salts act as salting ions for hydrophobic particles according to the Hope Meister series. The salting-out ion of the present invention may be an anion stabilizing the water structure in the Hofmeister system and a cosmotropic salt of a counter cation thereof.
본 발명에 따른 세포밖 소포체의 분리 방법은 원심분리기와 같은 고가 장비가 필요하지 않고, 분리 과정에서 시료가 극한의 환경에 노출되지 않기 때문에 세포밖 소포체의 형태나 성질을 보존하면서 효율적으로 분리할 수 있다는 장점이 있다. 또한 본 발명의 방법은 종래의 세포밖 소포체 분리 방법과 결합하여 적용할 수 있으며, 종래 방법의 수행 전 단계 또는 후 단계에 적용함으로써 분리 효율을 극대화할 수 있다.Since the method of separating extracellular ER according to the present invention does not require expensive equipments such as centrifugal separator and the sample is not exposed to the extreme environment during the separation process, the extracellular ER can be efficiently separated while preserving the shape or properties of extracellular ER . In addition, the method of the present invention can be applied in combination with a conventional extracellular ER separation method, and can be maximized by applying the method before or after the conventional method.
또한, 본 발명의 세포밖 소포체 분리 방법은 간단하고 효과적으로 세포밖 소포체를 분리할 수 있어, 세포밖 소포체의 대량 정제 시 중요한 요소로 활용될 수 있을 뿐만 아니라, 소량의 체액 시료의 전처리 및 후처리 단계에 적용함으로써 임상 진단에도 활용할 수 있다.In addition, since the extracellular fibrin separation method of the present invention can separate the extracellular endoplasmic reticulum easily and effectively, it can be utilized as an important factor in the mass purification of the extracellular endoplasmic reticulum, as well as the pre- To be used for clinical diagnosis.
또한, 본 발명의 세포밖 소포체 분리 방법은 세포밖 소포체의 종류에 따라 특정 양이온에 대한 친화성이 다른 성질을 이용하여, 다양한 양이온으로 세포밖 소포체의 서브셋(subset)을 분획할 수 있다. 분획된 세포밖 소포체 서브셋은 다차원적인 질병 진단에 활용할 수 있으며, 기존에 개발된 다양한 질병 진단 마커들을 본 발명에 적용함으로써 종래 진단 마커의 문제를 해결하고 다양한 활용이 가능하도록 할 수 있다. In addition, the method of separating extracellular endoplasmic reticulum of the present invention can fractionate a subset of extracellular endoplasmic reticulum with various cations using different properties of affinity for specific cations according to the type of extracellular endoplasmic reticulum. The extracellular extracellular subsets can be used to diagnose a multidimensional disease. By applying various disease diagnosis markers developed in the past to the present invention, problems of conventional diagnostic markers can be solved and various applications can be made possible.
도 1 은 본 발명의 일 실시예에 따른 세포밖 소포체 분리 방법에 대한 모식도이다.1 is a schematic diagram of a method for separating extracellular ER according to an embodiment of the present invention.
도 2 는 본 발명의 일 실시예에 따른 표본 세포밖 소포체의 분리 방법 및 특성 분석 결과이다.FIG. 2 shows the results of the separation and characterization of the extracellular endoplasmic reticulum according to an embodiment of the present invention.
도 3 은 본 발명의 일실시예에 따라 여러 농도의 다양한 양이온(Ca2+, Cu2+, Zn2+) 의 첨가로 세포 배양액에서 세포밖 소포체를 분리할 수 있음을 HPLC로 확인한 결과이다.FIG. 3 is a graph showing the results of HPLC analysis showing that extracellular endoplasmic reticulum can be isolated from cell culture medium by adding various cations (Ca 2+ , Cu 2+ , Zn 2+ ) at various concentrations according to an embodiment of the present invention.
도 4 는 본 발명의 일 실시예에 따라 여러 농도의 구리 양이온(염화구리(Ⅱ))의 첨가로 세포 배양액으로부터 세포밖 소포체가 분리됨을 나노입자 분석(a) 및 웨스턴블럿(b)으로 확인한 결과이다.FIG. 4 shows that the extracellular endoplasmic reticulum is separated from the cell culture medium by the addition of various concentrations of copper cation (copper (II) chloride) according to an embodiment of the present invention. to be.
도 5 는 본 발명의 일 실시예에 따라 다양한 농도의 구리 양이온(황산구리(Ⅱ))의 첨가로 세포 배양액으로부터 세포밖 소포체가 분리됨을 나노입자 분석(a) 및 웨스턴블럿(b)으로 확인한 결과이다.FIG. 5 shows the results of the nanoparticle analysis (a) and the Western blot (b) that the extracellular endoplasmic reticulum was separated from the cell culture medium by the addition of various concentrations of copper cations (copper sulfate (II)) according to an embodiment of the present invention .
도 6 은 본 발명의 일 실시예에 따라 다양한 농도의 코발트 양이온(염화코발트)의 첨가로 세포 배양액으로부터 세포밖 소포체가 분리됨을 나노입자 분석(a) 및 웨스턴블럿(b)으로 확인한 결과이다.FIG. 6 shows the results of the nanoparticle analysis (a) and Western blot (b) showing that the extracellular endoplasmic reticulum was separated from the cell culture medium by adding cobalt cations (cobalt chloride) at various concentrations according to an embodiment of the present invention.
도 7 은 본 발명의 일 실시예에 따라 다양한 농도의 망간 양이온(염화망간(Ⅱ))의 첨가로 세포 배양액으로부터 세포밖 소포체가 분리됨을 나노입자 분석(a) 및 웨스턴블럿(b)으로 확인한 결과이다.FIG. 7 is a graph showing the results of nano particle analysis (a) and Western blot (b) showing that the extracellular endoplasmic reticulum is separated from the cell culture medium by the addition of various concentrations of manganese cations (manganese chloride (II)) according to an embodiment of the present invention to be.
도 8 은 본 발명의 일 실시예에 따라 다양한 농도의 망간 양이온(황화망간(Ⅱ))의 첨가로 세포 배양액으로부터 세포밖 소포체가 분리됨을 나노입자 분석(a) 및 웨스턴블럿(b)으로 확인한 결과이다.FIG. 8 shows that the extracellular endoplasmic reticulum was separated from the cell culture medium by the addition of various concentrations of manganese cations (manganese sulfide (II)) according to an embodiment of the present invention. to be.
도 9 은 본 발명의 일 실시예에 따라 다양한 농도의 칼슘 양이온(염화칼슘)의 첨가로 세포 배양액으로부터 세포밖 소포체가 분리됨을 나노입자 분석(a) 및 웨스턴블럿(b)으로 확인한 결과이다.FIG. 9 shows the results of the nanoparticle analysis (a) and Western blot (b) showing that the extracellular endoplasmic reticulum was separated from the cell culture medium by the addition of various concentrations of calcium cation (calcium chloride) according to an embodiment of the present invention.
도 10 은 본 발명의 일 실시예에 따라 다양한 농도의 아연 양이온(염화아연)의 첨가로 세포 배양액으로부터 세포밖 소포체가 분리됨을 나노입자 분석(a) 및 웨스턴블럿(b)으로 확인한 결과이다.FIG. 10 shows the results of the nanoparticle analysis (a) and the Western blot (b) that the extracellular endoplasmic reticulum was separated from the cell culture medium by addition of various concentrations of zinc cation (zinc chloride) according to an embodiment of the present invention.
도 11 은 본 발명의 일 실시예에 따라 다양한 농도의 칼슘 양이온(염화칼슘)의 첨가로 사람 뇨에서 세포밖 소포체가 분리됨을 나노입자 분석(a) 및 웨스턴블럿(b)으로 확인한 결과이다.Figure 11 shows the results of nano particle analysis (a) and western blot (b) showing that the extracellular endoplasmic reticulum was isolated in human urine by the addition of various concentrations of calcium cation (calcium chloride) according to one embodiment of the present invention.
도 12 은 본 발명의 일 실시예에 따라 다양한 농도의 망간 양이온(황산망간(Ⅱ))의 첨가로 사람 뇨에서 세포밖 소포체가 분리됨을 나노입자 분석(a) 및 웨스턴블럿(b)으로 확인한 결과이다.Figure 12 shows that extracellular endoplasmic reticulum is isolated in human urine by the addition of various concentrations of manganese cations (manganese sulfate (II)) according to one embodiment of the present invention, as confirmed by nanoparticle analysis (a) and Western blot (b) to be.
도 13 은 본 발명의 일 실시예에 따라 다양한 농도의 아연 양이온(염화아연)의 첨가로 사람 뇨에서 세포밖 소포체가 분리됨을 나노입자 분석(a) 및 웨스턴블럿(b)으로 확인한 결과이다.Figure 13 shows the results of the nanoparticle analysis (a) and western blot (b) in which the extracellular endoplasmic reticulum was isolated in human urine by the addition of various concentrations of zinc cation (zinc chloride) according to one embodiment of the present invention.
도 14 은 본 발명의 일실시예에 따라 다양한 양이온(염화구리(Ⅱ), 황산망간(Ⅱ))과 폴리머(PEG, PEOZ)를 함께 첨가하여 세포 배양액에서 세포밖 소포체가 분리됨을 나노입자 분석을 통하여 확인한 결과이다.FIG. 14 is a graph showing the results of nanoparticle analysis in which extracellular endoplasmic reticulum is separated from a cell culture medium by adding various cations (copper (II) chloride, manganese (II) sulfate) and polymers (PEG and PEOZ) together according to an embodiment of the present invention .
도 15 은 본 발명의 일 실시예에 따라 구리 양이온(황산구리(Ⅱ))과 폴리머(PEOZ)를 함께 첨가하여 세포 배양액에서 세포밖 소포체가 분리됨을 웨스턴블럿 분석을 통하여 확인한 결과이다.FIG. 15 is a result of western blot analysis to confirm that extracellular endoplasmic reticulum is isolated in a cell culture solution by adding copper cation (copper sulfate (II)) and polymer (PEOZ) together according to an embodiment of the present invention.
도 16 은 본 발명의 일 실시예에 따라 종래 염석이온(황화암모늄) 침전법과 본원 발명의 구리 양이온(황산구리(II))을 이용한 방법을 조합하여 세포밖 소포체의 분리를 확인한 결과이다.FIG. 16 is a result of confirming the separation of extracellular endoplasmic reticulum by combining a conventional salting-out method (ammonium sulfide) precipitation method with a copper cation (copper sulfate (II)) method according to the present invention.
도 17 은 본 발명의 일 실시예에 따라 종래 폴리에틸렌글리콜(PEG)을 이용한 세포밖 소포체의 분리(a)와 본원 발명의 방법을 이용한 세포밖 소포체의 분리(b) 결과를 비교한 것이다.Figure 17 compares the separation of extracellular endoplasmic reticulum (a) with the conventional polyethylene glycol (PEG) according to one embodiment of the present invention and the result of separation (b) of extracellular endoplasmic reticulum using the method of the present invention.
이하, 실시예를 통하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.
실시예 1. 표본 세포밖 소포체의 정제 및 분석Example 1. Purification and analysis of extracellular endoplasmic reticulum
대장암 세포 SW480 배양액을 500 ×g에서 10 분 간 원심분리(총 2회 반복)하여 잔존하는 세포 및 침전물을 제거하였다. 상기 상층액을 다시 2,000 ×g에서 20 분 간 원심분리(총 2회 반복)하여 침전물을 제거하였다.Colorectal cancer cells SW480 culture was centrifuged at 500xg for 10 minutes (2 times in total) to remove remaining cells and sediments. The supernatant was centrifuged again (2 times in total) at 2,000 x g for 20 minutes to remove precipitates.
상기 상층액에 존재하는 세포밖 소포체를 1차 정제 및 침전하기 위하여, 세포밖 소포체 침전 유도액(8.4% Polyethylene glycol 6000, 250 mM NaCl, 20 mM HEPES, pH7.4)을 첨가하고 16시간 동안 냉장 보관한 후, 12,000 ×g에서 30 분 간 원심분리하여 침전된 세포밖 소포체를 수확하고, HEPES 완충용액(HEPES-buffered ssaline, 20 mM HEPES, 150 mM NaCl, pH7.4)에 녹여 내었다.(8.4% Polyethylene glycol 6000, 250 mM NaCl, 20 mM HEPES, pH 7.4) was added for primary purification and precipitation of the extracellular endoplasmic reticulum present in the supernatant, After storage, the extracellular endoplasmic reticulum was harvested by centrifugation at 12,000 × g for 30 minutes and dissolved in HEPES-buffered sialine (20 mM HEPES, 150 mM NaCl, pH 7.4).
밀도 및 부력을 이용하여 세포밖 소포체를 2차 정제하기 위하여 상기 시료를 옵티프랩(optiprep)과 혼합(최종 농도 30%)하여 초원심분리 용기의 가장 아래층에 위치시킨 후, 20% 옵티프랩, 5% 옵티프랩 순서로 층을 쌓았다. 200,000 ×g에서 2 시간 동안 옵티프랩 부력 밀도 구배 초원심분리(30%, 20%, 5% 옵티프랩 삼중층)를 수행하고, 초원심분리 후 세포밖 소포체와 상등 밀도(1.08 ~ 1.12 g/ml) 영역을 수확하였다.In order to secondarily purify the extracellular endoplasmic reticulum using density and buoyancy, the sample was mixed with Optiprep (final concentration 30%) and placed in the lowest layer of the ultracentrifuge, and then 20% Optiprap, 5 The layers were stacked in order of% optimaprap. OptiPrap buoyancy density gradient ultracentrifugation (30%, 20%, 5% OptiPrap triple layer) was carried out at 200,000 x g for 2 hours and the supernatant after the ultracentrifugation was subjected to an equal density of 1.08 to 1.12 g / ml ) Area were harvested.
상기 정제된 세포밖 소포체를 3차 정제하기 위하여, HPLC장비를 이용하여 세파크릴(Sephacryl) S500으로 충전된 컬럼(10 x 100 mm)에 로딩 한 후 크기 배제 크로마토그래피를 통해 최종 정제된 세포밖 소포체 분획을 수확하였다. 본 표본 세포밖 소포체 분리 과정을 도 2(a)에 나타내었다.The purified extracellular endoplasmic reticulum was loaded onto a column packed with Sephacryl S500 (10 x 100 mm) using HPLC equipment and subjected to size exclusion chromatography to obtain a final purified extracellular &lt; RTI ID = 0.0 &gt; Fractions were harvested. FIG. 2 (a) shows the process of separating the extracellular ER from the present specimen.
정제한 대장암 세포 유래 세포밖 소포체를 HPLC 크로마토그램으로 분석한 결과, 분자 크기별 크로마토그래피에서는 3.6 분에280 nm 흡광 밴드가 나타났다(도 2(b)). 크로마토그래피를 통한 각 분획의 나노입자분석(NTA, nanoparticle tracking analysis) 결과, 3.01 ~ 4.5분 사이에 용출되는 시료에서 높은 나노입자 신호를 검출할 수 있었고 이 신호는 280 nm 흡광 밴드와 일치한다는 것을 확인함으로써, 세포밖 소포체는 상기 HPLC 분석 시 3.6 분에 검출되는 밴드임을 알 수 있었다(도 2(b)).The extracellular endoplasmic reticulum from the purified colon cancer cells was analyzed by HPLC chromatogram. As a result, molecular size-specific chromatography showed an absorption band of 280 nm at 3.6 min (FIG. 2 (b)). As a result of the nanoparticle tracking analysis (NTA) of each fraction by chromatography, it was possible to detect high nanoparticle signals in samples eluted between 3.01 and 4.5 minutes, confirming that this signal is consistent with the extinction band at 280 nm , It was found that the extracellular endoplasmic reticulum was a band detected at 3.6 minutes in the HPLC analysis (Fig. 2 (b)).
상기 방법에 따라 대장암 세포주(SW480)로부터 최종 정제된 세포밖 소포체를 전자 현미경을 통하여 모양을 확인하였다. 그 결과 도 2(d)에 나타난 바와 같이 SW480 대장암 세포 유래 세포밖 소포체의 크기가 약 50 ~ 200 nm 임을 확인할 수 있었다. 또한, 웨스턴블럿을 통하여 세포밖 소포체의 마커인 TSG101 및 CD9을 확인하고 이를 도 2(e)에 나타내었다.The extracellular endoplasmic reticulum from the colorectal cancer cell line (SW480) was confirmed by electron microscopy. As a result, as shown in Fig. 2 (d), it was confirmed that the extracellular endoplasmic reticulum derived from SW480 colon cancer cells was about 50 to 200 nm in size. In addition, TSG101 and CD9, which are markers of the extracellular endoplasmic reticulum, were identified through western blotting and are shown in Fig. 2 (e).
실시예 2. 여러 농도의 다양한 양이온을 이용한 세포밖 소포체의 분리Example 2. Isolation of extracellular endoplasmic reticulum using various cations at various concentrations
여러 농도의 다양한 양이온(Ca2+, Cu2+, Zn2+)을 대장암 세포 배양액에 첨가하여 혼합한 후 3,000 ×g, 10 분 간 원심분리하여 침전물을 수확하고 50 mM EDTA가 포함된 HEPES 버퍼에 용해시켰다. 상기 분리한 세포밖 소포체를 HPLC 시스템을 이용한 크기 배제 크로마토그래피로 분석한 결과, 표본 세포밖 소포체는 3.6분에 검출되었으며 이를 도 3(a)에 나타내었다. Various concentrations of various cations (Ca 2+ , Cu 2+ , Zn 2+ ) were added to the culture medium of the colon cancer cells and mixed. The precipitates were harvested by centrifugation at 3,000 × g for 10 minutes, and HEPES Buffer. The separated extracellular endoplasmic reticulum was analyzed by size exclusion chromatography using an HPLC system. As a result, the extracellular endoplasmic reticulum was detected at 3.6 minutes, which is shown in FIG. 3 (a).
대장암 세포 배양액에 첨가한 칼슘 양이온, 구리 양이온 및 아연 양이온의 처리 농도에 따라 3.6분에 검출되는 280 nm 흡광 밴드가 양이온의 농도에 비례하여 증가함을 확인하였고 이를 도 3(b) 내지 도 3(d)에 나타내었다. 각 양이온에서 흡광 밴드들과 표본 세포밖 소포체 흡광 밴드는 동일 검출 시간을 보였으며, 양이온 첨가 농도에 따라 세포 배양액으로부터 분리되는 세포밖 소포체의 수율이 증가함을 알 수 있었다.It was confirmed that the absorption band at 280 nm, which was detected at 3.6 min, increased in proportion to the concentration of the cation, depending on the treatment concentration of the calcium cation, the copper cation and the zinc cation added to the culture medium for colon cancer cells, (d). In each cation, the extinction bands and extracellular extracellular extinction band showed the same detection time, and the yield of extracellular extracellular cells isolated from the cell culture was increased with the concentration of cation added.
실시예 3. 구리 양이온(염화구리(II))을 이용한 세포밖 소포체의 분리Example 3. Isolation of extracellular endoplasmic reticulum using copper cation (copper (II) chloride)
여러 농도의 구리 양이온을 대장암 세포 배양액에 첨가하여 혼합한 후 3,000 ×g, 10 분 간 원심분리하여 침전물을 수확하고 50 mM EDTA가 포함된 HEPES 버퍼에 용해시켰다. 상기 방법을 이용해 분리한 세포밖 소포체를 나노입자 분석 및 웨스턴블럿 분석으로 확인하였다. 나노입자 분석을 위해 Nanosight LM10 장비가 사용되었고, 카메라 레벨 10, 검출 한계 3 조건에서 60초 동안 추적 기록하였으며, 웨스턴블럿 분석을 위해 SDS 전기영동 후 통상의 세포밖 소포체 마커인 CD9에 대한 신호를 분석하였다.Various concentrations of copper cations were added to the culture medium of the colon cancer cells and mixed. The precipitates were harvested by centrifugation at 3,000 × g for 10 minutes and dissolved in HEPES buffer containing 50 mM EDTA. The extracellular endoplasmic reticulum isolated using the above method was confirmed by nanoparticle analysis and Western blot analysis. Nanosight LM10 instrument was used for the analysis of nanoparticles, followed by 60 seconds of tracking at camera level 10, detection limit 3, and for SDS electrophoresis for Western blot analysis, signals for CD9, the normal extracellular ER marker Respectively.
그 결 과, 구리 양이온의 농도가 증가함에 따라 세포밖 소포체의 수율이 증가함을 도 4(a)에 나타내었고, 이에 따라 통상의 세포밖 소포체 마커인 CD9에 대한 신호가 구리 양이온 농도에 비례하여 증가함을 도 4(b)에서 확인하였다.As a result, it was shown in FIG. 4 (a) that the yield of the extracellular endoplasmic reticulum increased as the concentration of the copper cation increased. Thus, the signal for the normal extracellular ER marker CD9 was proportional to the copper cation concentration 4 (b). &Lt; / RTI &gt;
실시예 4. 구리 양이온(황화구리(II))을 이용한 세포밖 소포체의 분리Example 4. Isolation of extracellular endoplasmic reticulum using copper cation (copper (II) sulfide)
여러 농도의 구리 양이온을 대장암 세포 배양액에 첨가하여 배양한 후 3,000 ×g, 10 분 간 원심분리하여 침전물을 수확하고 50 mM EDTA가 포함된 HEPES 버퍼에 용해시켰다. 상기 방법을 이용해 분리한 세포밖 소포체를 나노입자 분석 및 웨스턴블럿 분석으로 확인하였다.Various concentrations of copper cations were added to the culture medium of colon cancer cells, and the cells were cultured and centrifuged at 3,000 × g for 10 minutes. The precipitates were harvested and dissolved in HEPES buffer containing 50 mM EDTA. The extracellular endoplasmic reticulum isolated using the above method was confirmed by nanoparticle analysis and Western blot analysis.
그 결과, 구리 양이온의 농도가 증가함에 따라 세포밖 소포체의 농도가 증가하였음을 도 5(a)에 나타내었고, 이에 따라 통상의 세포밖 소포체 마커인 CD9에 대한 신호가 구리 양이온 농도에 따라 증가함을 도 5(b)에서 나타내었다.As a result, the concentration of the extracellular endoplasmic reticulum was increased as the copper cation concentration was increased. FIG. 5 (a) shows that the signal for CD9, a typical extracellular ER marker, increases with the concentration of copper cation Is shown in Fig. 5 (b).
실시예 5. 코발트 양이온(염화코발트)을 이용한 세포밖 소포체의 분리Example 5. Isolation of extracellular endoplasmic reticulum using cobalt cation (cobalt chloride)
여러 농도의 코발트 양이온을 대장암 세포 배양액에 첨가하여 혼합한 후 3,000 ×g, 10 분 간 원심분리하여 침전물을 수확하고 50 mM EDTA가 포함된 HEPES 버퍼에 용해시켰다. 상기 방법을 이용해 분리한 세포밖 소포체를 나노입자 분석 및 웨스턴블럿 분석으로 확인하였다. Various concentrations of cobalt cations were added to the culture medium of the colon cancer cells and mixed. The precipitates were harvested by centrifugation at 3,000 × g for 10 minutes and dissolved in HEPES buffer containing 50 mM EDTA. The extracellular endoplasmic reticulum isolated using the above method was confirmed by nanoparticle analysis and Western blot analysis.
그 결과, 코발트 양이온의 농도가 증가함에 따라 세포밖 소포체의 농도가 증가하였음을 도 6(a)에 나타내었고, 이에 따라 통상의 세포밖 소포체 마커인 CD9에 대한 신호가 코발트 양이온 농도에 따라 증가함을 도 6(b)에서 나타내었다.As a result, it was shown in FIG. 6 (a) that the concentration of the extracellular endoplasmic reticulum increased as the concentration of the cobalt cation increased. Accordingly, the signal for CD9, a typical extracellular ER marker, increased with the concentration of cobalt cation Is shown in Fig. 6 (b).
실시예 6. 망간 양이온(염화망간(II))을 이용한 세포밖 소포체의 분리Example 6. Isolation of extracellular endoplasmic reticulum using manganese cation (manganese (II) chloride)
여러 농도의 망간 양이온을 대장암 세포 배양액에 첨가하여 혼합한 후 3,000 ×g, 10 분 간 원심분리하여 침전물을 수확하고 50 mM EDTA가 포함된 HEPES 버퍼에 용해시켰다. 상기 방법을 이용해 분리한 세포밖 소포체를 나노입자 분석 및 웨스턴블럿 분석으로 확인하였다. Various concentrations of manganese cations were added to the cultures of colon cancer cells and mixed. The precipitates were harvested by centrifugation at 3,000 × g for 10 minutes and dissolved in HEPES buffer containing 50 mM EDTA. The extracellular endoplasmic reticulum isolated using the above method was confirmed by nanoparticle analysis and Western blot analysis.
그 결과, 망간 양이온의 농도가 증가함에 따라 세포밖 소포체의 농도가 증가하였음을 도 7(a)에 나타내었고, 이에 따라 통상의 세포밖 소포체 마커인 CD9에 대한 신호가 망간 양이온 농도에 따라 증가함을 도 7(b)에서 나타내었다.As a result, it was shown in FIG. 7 (a) that the concentration of extracellular ER increased with increasing concentration of manganese cation, and thus the signal for CD9, a typical extracellular ER marker, increases with the concentration of manganese cation Is shown in Fig. 7 (b).
실시예 7. 망간 양이온(황화망간(II))을 이용한 세포밖 소포체의 분리Example 7. Isolation of extracellular endoplasmic reticulum using manganese cation (manganese sulphide (II))
여러 농도의 망간 양이온을 대장암 세포 배양액에 첨가하여 혼합한 후 3,000 ×g, 10 분 간 원심분리하여 침전물을 수확하고 50 mM EDTA가 포함된 HEPES 버퍼에 용해시켰다. 상기 방법을 이용해 분리한 세포밖 소포체를 나노입자 분석 및 웨스턴블럿 분석으로 확인하였다. Various concentrations of manganese cations were added to the cultures of colon cancer cells and mixed. The precipitates were harvested by centrifugation at 3,000 × g for 10 minutes and dissolved in HEPES buffer containing 50 mM EDTA. The extracellular endoplasmic reticulum isolated using the above method was confirmed by nanoparticle analysis and Western blot analysis.
그 결과, 망간 양이온의 농도가 증가함에 따라 세포밖 소포체의 농도가 증가하였음을 도 8(a)에 나타내었고, 이에 따라 통상의 세포밖 소포체 마커인 CD9에 대한 신호가 망간 양이온 농도에 따라 증가함을 도 8(b)에서 나타내었다.As a result, it was shown in FIG. 8 (a) that the concentration of the extracellular endoplasmic reticulum increased as the concentration of manganese cation increased. Thus, the signal for CD9, a typical extracellular ER marker, increases with the concentration of manganese cation Is shown in Fig. 8 (b).
실시예 8. 칼슘 양이온(염화칼슘)을 이용한 세포밖 소포체의 분리Example 8. Isolation of extracellular endoplasmic reticulum using calcium cation (calcium chloride)
여러 농도의 칼슘 양이온을 대장암 세포 배양액에 첨가하여 혼합한 후 3,000 ×g, 10 분 간 원심분리하여 침전물을 수확하고 50 mM EDTA가 포함된 HEPES 버퍼에 용해시켰다. 상기 방법을 이용해 분리한 세포밖 소포체를 나노입자 분석 및 웨스턴블럿 분석으로 확인하였다. Various concentrations of calcium cations were added to the culture medium of colon cancer cells and mixed. The precipitates were harvested by centrifugation at 3,000 × g for 10 minutes and dissolved in HEPES buffer containing 50 mM EDTA. The extracellular endoplasmic reticulum isolated using the above method was confirmed by nanoparticle analysis and Western blot analysis.
그 결과, 칼슘 양이온의 농도가 증가함에 따라 세포밖 소포체의 농도가 증가하였음을 도 9(a)에 나타내었고, 이에 따라 통상의 세포밖 소포체 마커인 CD9에 대한 신호가 망간 양이온 농도에 따라 증가함을 도 9(b)에서 나타내었다.As a result, it was shown in FIG. 9 (a) that the concentration of the extracellular endoplasmic reticulum increased as the concentration of calcium cation increased. Thus, the signal for CD9, a typical extracellular ER marker, increased with the concentration of manganese cation Is shown in Fig. 9 (b).
실시예 9. 아연 양이온(염화아연)을 이용한 세포밖 소포체의 분리Example 9. Isolation of extracellular endoplasmic reticulum using zinc cation (zinc chloride)
여러 농도의 아연 양이온을 대장암 세포 배양액에 첨가하여 혼합한 후 3,000 ×g, 10 분 간 원심분리하여 침전물을 수확하고 50 mM EDTA가 포함된 HEPES 버퍼에 용해시켰다. 상기 방법을 이용해 분리한 세포밖 소포체를 나노입자 분석 및 웨스턴블럿 분석으로 확인하였다.Various concentrations of zinc cations were added to the culture medium of the colon cancer cells and mixed. The precipitates were harvested by centrifugation at 3,000 × g for 10 minutes and dissolved in HEPES buffer containing 50 mM EDTA. The extracellular endoplasmic reticulum isolated using the above method was confirmed by nanoparticle analysis and Western blot analysis.
그 결과, 아연 양이온의 농도가 증가함에 따라 세포밖 소포체의 농도가 증가하였음을 도 10(a)에 나타내었고, 이에 따라 통상의 세포밖 소포체 마커인 CD9에 대한 신호가 아연 양이온 농도에 따라 증가함을 도 10(b)에서 나타내었다.As a result, it was shown in FIG. 10 (a) that the concentration of the extracellular endoplasmic reticulum increased as the concentration of the zinc cation increased, and thus the signal for the normal extracellular ER marker CD9 increases with the zinc cation concentration Is shown in Fig. 10 (b).
실시예 10. 사람 뇨에서 칼슘 양이온(염화칼슘)을 이용한 세포밖 소포체의 분리Example 10. Isolation of extracellular endoplasmic reticulum using calcium cation (calcium chloride) in human urine
사람 뇨(urine)를 2,000 ×g에서 15분 간 원심분리(총 2회 반복)하여 잔존하는 침전물을 제거하였다. 상기 상층액에 여러 농도의 칼슘 양이온을 첨가하여 혼합한 후 3,000 ×g, 10 분 간 원심분리하여 침전물을 수확하고 50 mM EDTA가 포함된 HEPES 버퍼에 용해시켰다. 상기 방법을 이용해 분리한 세포밖 소포체를 나노입자 분석 및 웨스턴블럿 분석으로 확인하였다. Human urine was centrifuged at 2,000 × g for 15 min (total 2 repetitions) to remove any remaining precipitate. Various concentrations of calcium cations were added to the supernatant, and the mixture was centrifuged at 3,000 × g for 10 minutes. The precipitates were harvested and dissolved in HEPES buffer containing 50 mM EDTA. The extracellular endoplasmic reticulum isolated using the above method was confirmed by nanoparticle analysis and Western blot analysis.
그 결과, 칼슘 양이온의 농도가 증가함에 따라 세포밖 소포체의 농도가 증가하였음을 도 11(a)에 나타내었고, 이에 따라 통상의 세포밖 소포체 마커인 CD9에 대한 신호가 칼슘 양이온 농도에 따라 증가함을 도 11(b)에서 나타내었다.As a result, the concentration of the extracellular endoplasmic reticulum was increased as the concentration of calcium cation increased. FIG. 11 (a) shows that the signal for CD9, a typical extracellular ER marker, increases with the calcium cation concentration Is shown in Fig. 11 (b).
실시예 11. 사람 뇨에서 망간 양이온(황화망간(II))을 이용한 세포밖 소포체의 분리Example 11. Isolation of extracellular endoplasmic reticulum using manganese cation (manganese sulfide (II)) in human urine
사람 뇨를 2,000 ×g에서 15분 간 원심분리(총 2회 반복)하여 잔존하는 침전물을 제거하였다. 상기 준비된 사람 뇨에 여러 농도의 망간 양이온을 첨가하여 혼합한 후 3,000 ×g, 10 분 간 원심분리하여 침전물을 수확하고 50 mM EDTA가 포함된 HEPES 버퍼에 용해시켰다. 상기 방법을 이용해 분리한 세포밖 소포체를 나노입자 분석 및 웨스턴블럿 분석으로 확인하였다. Human urine was centrifuged at 2,000 x g for 15 min (total 2 replicates) to remove any remaining precipitate. Various concentrations of manganese cations were added to the prepared human urine, and the mixture was centrifuged at 3,000 × g for 10 minutes. The precipitates were harvested and dissolved in HEPES buffer containing 50 mM EDTA. The extracellular endoplasmic reticulum isolated using the above method was confirmed by nanoparticle analysis and Western blot analysis.
그 결과, 칼슘 양이온의 농도가 증가함에 따라 세포밖 소포체의 농도가 증가하였음을 도 12(a)에 나타내었고, 이에 따라 통상의 세포밖 소포체 마커인 CD9에 대한 신호가 칼슘 양이온 농도에 따라 증가함을 도 12(b)에서 나타내었다.As a result, the concentration of the extracellular endoplasmic reticulum was increased as the concentration of calcium cation increased. FIG. 12 (a) shows that the signal for CD9, a typical extracellular ER marker, increases with the concentration of calcium cation Is shown in Fig. 12 (b).
실시예 12. 사람 뇨에서 아연 양이온(염화아연)을 이용한 세포밖 소포체의 분리Example 12. Isolation of extracellular endoplasmic reticulum using zinc cation (zinc chloride) in human urine
사람 뇨를 2,000 ×g에서 15분 간 원심분리(총 2회 반복)하여 잔존하는 침전물을 제거하였다. 상기 준비된 사람 뇨에 여러 농도의 아연 양이온을 첨가하여 혼합한 후 3,000 ×g, 10 분 간 원심분리하여 침전물을 수확하고 50 mM EDTA가 포함된 HEPES 버퍼에 용해시켰다. 상기 방법을 이용해 분리한 세포밖 소포체를 나노입자 분석 및 웨스턴블럿 분석으로 확인하였다. Human urine was centrifuged at 2,000 x g for 15 min (total 2 replicates) to remove any remaining precipitate. Various concentrations of zinc cations were added to the prepared human urine, and the mixture was centrifuged at 3,000 × g for 10 minutes. The precipitates were harvested and dissolved in HEPES buffer containing 50 mM EDTA. The extracellular endoplasmic reticulum isolated using the above method was confirmed by nanoparticle analysis and Western blot analysis.
그 결과, 아연 양이온의 농도가 증가함에 따라 세포밖 소포체의 농도가 증가하였음을 도 13(a)에 나타내었고, 이에 따라 통상의 세포밖 소포체 마커인 CD9에 대한 신호가 아연 양이온 농도에 따라 증가함을 도 13(b)에서 나타내었다.As a result, the concentration of the extracellular endoplasmic reticulum was increased as the concentration of the zinc cation was increased. FIG. 13 (a) shows that the signal for the normal extracellular ER marker CD9 increases with the zinc cation concentration Is shown in Fig. 13 (b).
실시예 13. 다양한 양이온(염화구리(II), 황화망간(II))과 폴리머의 조합을 통한 세포밖 소포체의 분리 효율 증가Example 13. Increased efficiency of separation of extracellular endoplasmic reticulum through combination of various cations (copper (II) chloride, manganese (II) sulphide (II)) and polymer
다양한 양이온과 폴리머 조합에 따른 세포밖 소포체의 분리 수율을 비교하기 위해 양이온 단독, 폴리머 단독, 또는 양이온과 폴리머의 함께 첨가하여 세포밖 소포체를 분리하였다. To compare the separation yield of extracellular endoplasmic reticulum with various cation and polymer combinations, extracellular endoplasmic reticulum was isolated by adding cation alone, polymer alone, or cation and polymer together.
구체적으로, 세포밖 소포체 분리를 위한 폴리머로는 폴리에틸렌글리콜(PEG, Poly ethylene glycol) 또는 폴리에틸옥사졸린(PEOZ, Poly(2-ethyl-2-oxazoline))을 사용하였으며, 대장암 세포 배양액에 최종 농도가 8.3%가 되도록 폴리에틸렌글리콜(PEG, Poly ethylene glycol)만을 첨가하거나, 최종 농도가 10%가 되도록 폴리에틸옥사졸린(PEOZ, Poly(2-ethyl-2-oxazoline))만을 첨가하였다. 이러한 폴리머 단독 첨가군은 상온에서 10 분, 또는 4℃에서 16시간 동안 배양한 후, 원심 분리를 통하여 세포밖 소포체를 수확하였다. Specifically, polyethylene glycol (PEG) or polyethyl oxazoline (PEOZ, Poly (2-ethyl-2-oxazoline)) was used as a polymer for extracellular ER separation, Only polyethylene glycol (PEG) was added so that the concentration was 8.3% or only polyethyl oxazoline (PEOZ, Poly (2-ethyl-2-oxazoline)) was added so that the final concentration was 10%. These polymer alone groups were cultured at room temperature for 10 minutes or at 4 ° C for 16 hours, and the extracellular endoplasmic reticulum was harvested by centrifugation.
한편, 양이온 단독 첨가군은 상기 동일한 세포 배양액에 최종 농도가 20 mM가 되도록 구리 양이온(CuCl2) 또는 망간 양이온(MnSO4)만을 첨가한 후 상온에서 10 분 간 배양하고 원심분리를 통해 세포밖 소포체를 수확하였다. 또한, 상기 양이온과 폴리머를 동시에 첨가한 경우도 마찬가지로 상온에서 10 분 간 배양한 후 원심분리를 통하여 수확하였다. 이 후 침전된 세포밖 소포체를 동일 부피의 HEPES버퍼에 용해하였다.On the other hand, only the cationic addition group was added with copper cations (CuCl 2 ) or manganese cations (MnSO 4 ) only to the final concentration of 20 mM in the same cell culture medium, followed by incubation at room temperature for 10 minutes, Were harvested. When the cation and the polymer were simultaneously added, the cells were incubated at room temperature for 10 minutes and harvested by centrifugation. The resulting extracellular endoplasmic reticulum was then dissolved in the same volume of HEPES buffer.
상기 폴리머 단독, 양이온 단독, 또는 양이온-폴리머 혼합 조건에서 수확한 세포밖 소포체의 수율을 비교 분석하기 위하여 나노 입자 분석을 수행하였다. 그 결과, 도 14(a) 내지 도 14(c)에 나타낸 바와 같이, 폴리머 단독으로 16시간 동안 배양한 조건의 세포밖 소포체의 수율에 비하여 양이온 단독으로 10 분 간 반응한 조건에서 2 ~ 3배 높은 수율을 보였으며, 양이온과 폴리머를 혼합한 조건으로 10 분 간 반응한 경우 세포밖 소포체의 수율이 더욱 상승하는 것을 확인할 수 있었다. 이로부터 폴리머 단독으로는 효율이 매우 낮으나, 양이온이 존재하는 조건에서 폴리머가 첨가되면 양이온의 세포밖 소포체 침전 효율을 더욱 증가시킴을 알 수 있었다.Nanoparticle analysis was performed to compare the yield of the extracellular endoplasmic reticulum harvested under the conditions of polymer alone, cation alone, or cation-polymer blend. As a result, as shown in Fig. 14 (a) to Fig. 14 (c), the yield of the extracellular endoplasmic reticulum under the conditions of culturing the polymer alone for 16 hours was 10 to 20 minutes And the yield of extracellular ER was further increased when the reaction was performed for 10 minutes under the condition of mixed cation and polymer. From these results, it was found that the efficiency of polymer alone was very low. However, it was found that the addition of polymer in the presence of cations further increased the extracellular precipitation efficiency of cations.
실시예 14. 폴리머와 구리 양이온(황화구리(II)) 농도에 따른 세포밖 소포체의 CD9 분석Example 14. CD9 analysis of extracellular endoplasmic reticulum with polymer and copper cation (copper (II) sulfide) concentration
대장암 세포 배양액에 다양한 농도의 구리 양이온과 최종 농도 10%가 되도록 폴리에틸옥사졸린을 첨가한 후 상온에서 30 분 간 반응시킨 시료와 폴리에틸옥사졸린 단독으로 4℃ 18시간 반응시킨 시료에서 3,000 ×g, 10 분 간 원심분리하여 세포밖 소포체를 수확하였다. 각 조건에서 얻어진 세포밖 소포체의 수율을 확인하기 위하여 세포밖 소포체 마커인 CD9의 양을 웨스턴블럿을 통하여 분석하였다. In the culture of colorectal cancer cells, various concentrations of copper cations were added, and polyethyloxazoline was added to a final concentration of 10%. Then, samples were reacted at room temperature for 30 minutes and polyethyloxazoline alone at 18 ° C for 4 hours. g, for 10 minutes, and extracellular endoplasmic reticulum was harvested. The amount of CD9, an extracellular ER marker, was analyzed by western blot to confirm the yield of extracellular ER in each condition.
그 결과 도 15에 나타낸 바와 같이, 폴리에틸옥사졸린 단독으로 30 분 간 배양한 조건의 세포밖 소포체의 수율이 매우 낮은데 반해, 다양한 농도의 구리 양이온과 폴리머를 혼합한 조건으로 30 분 간 반응한 경우 구리 양이온의 농도에 비례하여 월등히 높은 세포밖 소포체가 수확됨을 알 수 있었다. 이로부터 폴리에틸옥사졸린 단독으로는 세포밖 소포체 침전 효율이 매우 낮으나, 구리 양이온이 존재하는 조건에서 폴리머가 첨가되면 세포밖 소포체의 침전 효율이 극대화 됨을 알 수 있었다.As a result, as shown in Fig. 15, the yield of the extracellular endoplasmic reticulum under the condition of culturing for 30 minutes with polyethyloxazoline alone was extremely low. However, when the reaction was carried out for 30 minutes under conditions of mixing various concentrations of copper cations and polymers Extracellular vesicles were harvested in proportion to the concentration of copper cation. From these results, it was found that the efficiency of precipitation of extracellular endoplasmic reticulum by polyethyloxazoline alone was very low, but that addition of polymer in the presence of copper cation maximizes the extracellular endocytosis efficiency.
실시예 15. 황화암모늄과 구리 양이온(황화구리(II))의 조합에 따른 세포밖 소포체의 분리Example 15 Isolation of extracellular endoplasmic reticulum by combination of ammonium sulphide and copper cation (copper (II) sulfide)
대장암 세포 배양액에 최종 농도 1.5 M이 되도록 황화암모늄을 첨가한 후 4℃에서 30 분 간 반응시킨 시료, 상기 동일한 세포 배양액에 구리 양이온(10 mM) 만을 첨가한 시료, 그리고, 상기 동일 세포 배양액에 구리 양이온과 황화암모늄을 함께 첨가한 시료로부터 3000 ×g, 10 분 간 원심분리하여 세포밖 소포체를 수확하였고, 각 조건에서 얻어진 세포밖 소포체의 수율을 확인하기 위하여 나노 입자 분석을 수행하였다. A sample obtained by adding ammonium sulphate to a final concentration of 1.5 M in a culture medium for colon cancer cells and reacting at 4 캜 for 30 minutes, a sample in which only copper cation (10 mM) was added to the same cell culture medium, The extracellular endoplasmic reticulum was harvested by centrifugation at 3000 × g for 10 min from samples containing copper cation and ammonium sulphide, and nanoparticle analysis was performed to confirm the yield of extracellular endoplasmic reticulum obtained under each condition.
그 결과 도 16에 나타낸 바와 같이, 황화암모늄 단독으로 30 분 간 배양한 조건의 세포밖 소포체의 수율은 매우 낮은데 반해, 10 mM의 구리 양이온 만을 30 분 간 반응한 조건에는 높은 세포밖 소포체의 수율을 보였으며, 구리 양이온과 황화암모늄을 혼합한 조건으로 30 분 간 반응한 조건의 경우는 구리 양이온의 농도에 비례하여 시료내 존재하는 세포밖 소포체 수율이 더욱 높아짐을 확인할 수 있었다. 이로부터 30 분의 짧은 배양을 하는 경우, 황화암모늄 단독으로는 세포밖 소포체 침전 효율이 매우 낮은 반면, 구리 양이온 단독의 경우 침전 효율이 월등하다. 또한, 구리 양이온과 황화암모늄이 함께 첨가하여 세포밖 소포체를 침전하는 경우는 구리 양이온의 세포밖 소포체 침전 효율을 극대화 시킴을 알 수 있었다.As a result, as shown in Fig. 16, the yield of the extracellular endoplasmic reticulum under the condition of ammonium sulphate alone for 30 minutes was extremely low, while the yield of the high extracellular endoplasmic reticulum was maintained under the conditions of only 10 mM of copper cation for 30 minutes The yield of the extracellular vesicles in the sample was increased in proportion to the concentration of the copper cation in the condition of 30 minutes of reaction with the copper cation and ammonium sulphide. In the case of short incubation for 30 min, the precipitation efficiency of the extracellular vesicle is very low in the case of ammonium sulphate alone but in the case of the copper cation alone. It was also found that when the copper cation and ammonium sulfide were added together to precipitate the extracellular endoplasmic reticulum, the extracellular ER precipitation efficiency of the copper cation was maximized.
실시예 16. 폴리머를 이용한 세포밖 소포체의 분리와 구리 양이온을 통한 세포밖 소포체 분리 방법의 비교Example 16: Comparison of the extracellular endoplasmic reticulum and the extracellular endoplasmic reticulum using the polymer
폴리머를 이용한 세포밖 소포체의 분리와 폴리머-양이온 조합을 통한 세포밖 소포체 분리의 수율 및 순도 차이를 분석하기 위하여, 대장암 세포 배양액 10 ml로부터 세포밖 소포체를 정제하기 위해 폴리에틸렌글리콜(PEG, Polyethylene glycol)을 최종 농도 8.3% 되게 첨가하고 4°C 18시간 동안 배양한 후 3,000 ×g, 10 분 간 원심분리 후 침전물을 HEPES 버퍼(20 mM HEPES, pH7.2, 150 mM NaCl)에 용해시켰다. 한편, 동일 부피의 세포 배양액에 구리 양이온을 첨가한 후 10 분 간 배양하고, 침전물을 3,000 ×g, 10 분 간 원심분리하여 수확하고 이를 50 mM EDTA를 포함한 HEPES 버퍼에 용해시켰다. 상기 방법을 이용해 분리한 세포밖 소포체를 포함한 시료를 크기 배제 크로마토그래피(spin-based size exclusion chromatography)로 추가 분리한 후 HPLC 시스템을 이용한 크기 배제 크로마토그래피로 분석하였다.In order to analyze the yield and purity difference of the extracellular endoplasmic reticulum and the extracellular endoplasmic reticulum from the extracellular endoplasmic reticulum using a polymer, polyethylene glycol (PEG, polyethylene glycol ) Was added to a final concentration of 8.3% and incubated at 4 ° C for 18 hours. After centrifugation at 3,000 × g for 10 minutes, the precipitate was dissolved in HEPES buffer (20 mM HEPES, pH 7.2, 150 mM NaCl). On the other hand, copper cations were added to the same volume of cell culture medium, followed by incubation for 10 minutes. The precipitates were harvested by centrifugation at 3,000 × g for 10 minutes and dissolved in HEPES buffer containing 50 mM EDTA. Samples containing extracellular endoplasmic reticulum isolated using the above method were further separated by size-exclusion chromatography and analyzed by size exclusion chromatography using an HPLC system.
그 결과 도 17에 나타낸 바와 같이, 종래의 폴리머를 이용한 세포밖 소포체의 분리 방법에 비해 양이온을 이용하면 세포밖 소포체의 수율과 순도가 크게 향상됨을 알 수 있었다.As a result, as shown in FIG. 17, the yield and purity of the extracellular endoplasmic reticulum were significantly improved by using the cation as compared with the conventional method of separating the extracellular endoplasmic reticulum using the polymer.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

  1. (a) 생물학적 시료에 양이온을 첨가하는 단계;(a) adding a cation to a biological sample;
    (b) 상기 생물학적 시료에 포함된 세포밖 소포체와 양이온을 반응시켜 복합체를 형성하는 단계;(b) reacting the extracellular endoplasmic reticulum contained in the biological sample with a cation to form a complex;
    (c) 상기 시료로부터 세포밖 소포체와 양이온 복합체를 분리하는 단계; 및(c) separating the extracellular endoplasmic reticulum and the cation complex from the sample; And
    (d) 상기 복합체로부터 양이온을 분리시켜 세포밖 소포체를 정제하는 단계(d) separating the cation from the complex to purify the extracellular endoplasmic reticulum
    를 포함하는 세포밖 소포체 분리 방법.Lt; RTI ID = 0.0 &gt; extracellular &lt; / RTI &gt;
  2. 제1항에 있어서,The method according to claim 1,
    상기 생물학적 시료는 포유동물 세포 배양 배지, 박테리아 세포 배양 배지, 효모 배양 배지, 조직 추출물, 암 조직, 혈청, 혈장, 침, 눈물, 땀, 소변, 대변, 뇌척수액(CSF, cerebrospinal fluid), 복수(ascite), 양수(amniotic fluid), 정액, 유(milk), 먼지, 담수, 해수, 토양 및 발효식품으로 이루어진 군에서 선택되는 하나 이상인 것을 특징으로 하는 세포밖 소포체 분리 방법.The biological sample may be a mammalian cell culture medium, a bacterial cell culture medium, a yeast culture medium, a tissue extract, a cancer tissue, a serum, a plasma, a needle, a tear, a sweat, a urine, a feces, a cerebrospinal fluid (CSF) Wherein the at least one extracellular matrix is at least one selected from the group consisting of amniotic fluid, semen, milk, dust, fresh water, seawater, soil and fermented food.
  3. 제1항에 있어서,The method according to claim 1,
    상기 양이온은 금속 양이온인 것을 특징으로 하는 세포밖 소포체 분리 방법.Wherein the cation is a metal cation.
  4. 제1항에 있어서,The method according to claim 1,
    상기 (c) 단계는 원심분리, 초원심분리, 여과, 한외여과, 중력, 음파처리, 밀도 구배 초원심분리, 크기 배제 크로마토그래피, 이온교환 크로마토그래피, 친화성 크로마토그래피, 폴리머 기반 침전 및 유기 용매 침전으로 이루어진 군에서 선택되는 하나 이상의 방법을 이용하는 것인 세포밖 소포체 분리 방법.The step (c) may be carried out by centrifugation, ultracentrifugation, filtration, ultrafiltration, gravity, sonication, density gradient ultracentrifugation, size exclusion chromatography, ion exchange chromatography, affinity chromatography, &Lt; RTI ID = 0.0 &gt; and / or &lt; / RTI &gt; precipitation.
  5. 제1항에 있어서,The method according to claim 1,
    상기 (d) 단계는 분리된 세포밖 소포체와 양이온 복합체에 킬레이트제를 첨가하는 방법; pH값을 변화시키는 방법; 및 이미다졸(imidazole), 히스티딘(histidine), 에틸렌디아민 테트라아세테이트(EDTA, ethylendiamine tetraacetate) 및 염(salts)으로 이루어진 군에서 선택되는 하나 이상의 농도를 변화시키는 방법으로 이루어진 군에서 선택되는 하나 이상의 방법을 포함하는 것인 세포밖 소포체 분리 방법.The step (d) comprises adding a chelating agent to the separated extracellular endoplasmic reticulum and the cation complex; a method of changing the pH value; And a method of changing at least one concentration selected from the group consisting of imidazole, histidine, ethylenediamine tetraacetate (EDTA) and salts. Lt; RTI ID = 0.0 &gt; extracellular &lt; / RTI &gt;
  6. 제5항에 있어서,6. The method of claim 5,
    상기 킬레이트제는 이미노디아세트산(IDA, iminodiacetic acid), 니트릴로트리아세트산(NTA, nitrilotriacetic acid), 트리스(카복시메틸)에틸렌디아민(TED, tris-(carboxymethyl)ethylenediamine), 에틸렌디아민(ethylenediamine), 에틸렌디아민 테트라아세테이트(EDTA, ethylendiamine tetraacetate), 알킬렌디아민 트리아세트산(alkylenediamine triacetic acid), 디에틸렌트리아민펜타아세트산(DTPA, diethylenetriaminepentaacetic acid), 에틸렌글리콜 비스(베타-아미노에틸에테르)-N,N,N',N'-테트라아세트산(EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid), 포스포세린(phosphoserine) 및 1,4,7-트리아조사이클로노난(TACN, 1,4,7-triazocyclononane)으로 이루어진 군에서 선택되는 하나 이상인 것을 특징으로 하는 세포밖 소포체 분리 방법.The chelating agent may be selected from the group consisting of iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), tris (carboxymethyl) ethylenediamine (TED), ethylenediamine, ethylenediamine Diethylenetriaminepentaacetic acid (DTPA), ethyleneglycol bis (beta-aminoethylether) -N, N, N ', N'-tetramethylethylenediamine tetraacetate (EDTA), alkylenediamine triacetic acid N, N'-tetraacetic acid (EGTA), phosphoserine and 1,4,7-triazo cyclononane (EGTA) (TACN, 1, 4,7-triazocyclononane).
  7. 제1항에 있어서,The method according to claim 1,
    상기 (a) 단계 전에 시료의 전처리 단계를 더 포함하는 것을 특징으로 하는 세포밖 소포체 분리 방법.Further comprising the step of pre-treating the sample before the step (a).
  8. 제1항에 있어서,The method according to claim 1,
    상기 (d) 단계 후에 정제된 세포밖 소포체의 후처리 단계를 더 포함하는 것을 특징으로 하는 세포밖 소포체 분리 방법.Further comprising a step of post-treatment of the extracellular endoplasmic reticulum after the step (d).
  9. 제1항에 있어서,The method according to claim 1,
    상기 (a) 단계에 고분자 또는 염석 이온을 첨가하는 단계를 더 포함하는 것을 특징으로 하는 세포밖 소포체 분리 방법.Further comprising the step of adding a polymer or a salting-out ion to the step (a).
  10. 제9항에 있어서,10. The method of claim 9,
    상기 고분자 또는 염석이온은 양이온과 동시에, 양이온 첨가 이전에, 또는 양이온 첨가 이후에 첨가되는 것을 특징으로 하는 세포밖 소포체 분리 방법.Wherein the polymer or salting-out ion is added simultaneously with the cation, before the cation addition, or after the cation addition.
  11. 제9항에 있어서,10. The method of claim 9,
    상기 고분자는 폴리에틸렌글리콜(PEG, poly ethylene glycol) 또는 폴리옥사졸린(polyoxazoline)인 것인 세포밖 소포체 분리 방법.Wherein the polymer is polyethylene glycol (PEG) or polyoxazoline.
  12. 제11항에 있어서,12. The method of claim 11,
    상기 폴리옥사졸린은 폴리메틸옥사졸린(PMOZ, poly(2-methyl-2-oxazoline), 폴리에틸옥사졸린(PEOZ, poly(2-ethyl-2-oxazoline) 또는 폴리프로필옥사졸린(PPOZ, poly(2-propyl-2-oxazoline)인 것인 세포밖 소포체 분리 방법.The polyoxazoline may be selected from the group consisting of poly (2-methyl-2-oxazoline), poly (2-ethyl-2-oxazoline) 2-propyl-2-oxazoline).
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