Luterial, and methods for isolating and culturing the same.

Abstract

This invention provides a method for enriching mitochondrial-like nano-sized particles. [Solution] A method comprising the following steps: (a) removing platelets and blood-derived substances having a size greater than or equal to platelets from blood, and passing the blood, from which platelets and blood-derived substances have been separated, through a filter having voids of 0.8 to 1.2 μm; (b) centrifuging the filtered solution to remove general microvesicles containing exosomes collected in a pellet, and providing a supernatant, thereby providing a supernatant containing mitochondrial-like nano-sized particles; (c) identifying mitochondrial-like nano-sized particles by evaluating from the liquid obtained by performing (a) to (b) whether the particles have one or more of the group (i) to (xi) having specific properties, including (i) below: (i) Shows a positive color reaction to Janus Green B, Acridine Orange, DAPI, or Rhodamine 123 in a fluorescence test.

Description

Technical Field

[0001] The present invention relates to Luterial, a mitochondria-like fine substance derived from body fluid, and the separation thereof and culture method thereof. Background Art

[0002] Fine substances such as microvesicles in blood were once recognized as substances that do not have special functions. However, various experimental data have revealed that microvesicles also have various biological activities. For example, platelet-derived microvesicles have been clarified to function to stimulate specific cells via vesicular surface proteins (CD154, RANTES and / or PF‐4; Thromb. Haemost. (1999) 82: 794; J. Biol. Chem. (1999) 2 74: 7545), it has been reported that bioactive lipids (e.g., HTET or arachidonic acid) in platelet microvesicles exhibit specific effects on specific target cells (J. Bio l. Chem. (2001) 276; 19672; Cardiovasc. Res. (2 001) 49(5): 88). As described above, characteristics of substances such as vesicles present in biological samples (e.g., size, surface antigen, origin cell determination, payload) can provide information relating to diagnosis, prognosis or therapeutic diagnosis of diseases, so there is a need for identifying biological indicators that can be used for exploring and treating diseases. Accordingly, there have also been attempts to provide RNA and other biological indicators related to vesicles and the characteristics of vesicles for diagnosis, prognosis, and therapeutic diagnosis (see WO2011 / 127219).

[0003] On the other hand, cancer is a disease in which cells proliferate uncontrollably and interfere with the function of normal cells. These include lung cancer, gastric cancer (GC), and breast cancer (GC). Ancer (BRC), colorectal cancer (CRC), etc. While representative, it can actually occur in any tissue. Early diagnosis of cancer is based on the growth of cancer cells. Although it was initially based on external changes in biological tissues, in recent years, blood, glycans (glyco ch) have been studied. (ain), diagnostics using trace amounts of biomolecules present in the tissues or cells of living organisms, such as DNA. Detection is being attempted. However, the most commonly used method for diagnosing cancer is biopsy. The diagnosis is made using tissue samples obtained through examination or by using video. Of these, living tissue The tests not only cause great distress to patients and are expensive, but they also take a long time to produce a diagnosis. It has a drawback. Also, if the patient actually has cancer, the cancer may metastasize during the biopsy process. This may induce a reaction, and in cases where a tissue sample cannot be obtained by biopsy, it may be suspected. A drawback is that it is impossible to diagnose the disease unless the tissue is surgically removed. Yes. In addition, diagnostic imaging uses X-ray images and disease target substances. Nuclear magnetic resonance (NMU) is obtained using the contrast agent applied. Cancer was diagnosed based on images (NMR, etc.). However, based on such images Diagnosis is subject to misdiagnosis depending on the skill level of the clinician or radiologist, and the precision of the imaging equipment. It has the disadvantage of being highly dependent on the degree. Furthermore, even the most accurate equipment is only capable of measuring a few meters. Tumors smaller than m are undetectable, and a drawback is that they are difficult to detect in the early stages of the disease. Furthermore, in order to obtain the image, the patient or a person who is susceptible to the disease may have a gene mutation that can be induced. Because of exposure to high-energy electromagnetic waves, there is a risk of causing other illnesses. Furthermore, it has the drawback of limiting the number of times diagnostic tests can be performed using video.

[0004] In other words, biopsies for cancer diagnosis involve a lot of time, expense, inconvenience, pain, etc. Because it is accompanied by, it is a method that can significantly reduce the number of people who are subject to unnecessary biopsies, cancer There is a need for methods to diagnose it early.

[0005] Against this backdrop, the inventors have identified minute particles present in bodily fluids already discharged from patients. We have found that by observing the qualitative characteristics, it is possible to diagnose and predict diseases, The content was filed as a patent application on July 12, 2013 (Korean Patent Application No. 10-2013-). (Patent No. 0082060). The inventors have named the above fine material "luterial )" was the name given.

[0006] However, in order to make the above-mentioned micromaterials clinically applicable, luteal can be efficiently processed. Techniques for isolation and cultivation are still unknown.

[0007] Therefore, the present inventors have investigated the presence of fine particles in bodily fluids already discharged from patients or healthy individuals. We developed a method that can effectively separate the luteal component, and by using this method, we can separate it. This invention was realized by investigating the properties of the luteal. [Overview of the project] [Problems that the invention aims to solve]

[0008] The object of the present invention is to remove luteal present in bodily fluids already discharged from patients or healthy individuals. to provide a method for separation and culture.

[0009] Another object of the present invention is to provide, between prokaryotes and Eukaryo te), have fusion characteristics at an intermediate stage, and in Immunofluorescence ) assay, Janus Green B, MitoTracker Red ( Mito‐tracker Red), Rhodamine 123 exhibit a positive immunochemical fluorescent staining reaction, have motility, and have characteristics such as ATP production to provide a luterial having the above characteristics. Means for Solving the Problem

[0010] To achieve the above object, the present invention provides a method comprising: separating platelets and blood-derived substances having a size equal to or larger than platelets from blood as a first separation step; centrifuging the blood from which said platelets and blood-derived substances having a size equal to or larger than platelets have been removed as a second separation step; separating luterial from the supernatant obtained by said centrifugation as a third separation step; and washing the separated luterial. There is provided a method for separating luterial comprising the above steps.

[0011] The present invention also provides a method for separating luterial comprising: after subjecting a body fluid to primary centrifugation, filtering the supernatant with a 2 to 5 μm filter as a step; after subjecting the filtered solution to secondary centrifugation, filtering the resulting supernatant with a 0.5 to 2 μm filter as a step. A method for separating luterial comprising the above steps is provided.

[0012] The present invention also provides a body fluid-derived luterial having one or more of the following characteristics: (a) in an immunofluorescence test, positive staining with Janus green B, acridi ​Acridine Orange and Rhodamine 123 mine123) shows a positive color reaction; (b) Under optimal conditions (pH 7.2~7.4), β-proteobacteria and γ-proteobacteria It exhibits expression characteristics of kuteria-derived genes and has a size of 30-800 nm; (c) Under acidic conditions, the genes are derived from β-proteobacteria and γ-proteobacteria. Furthermore, it produces streptophyta genes, which are eukaryotic cells. As a result, the size increases from over 400nm to over 2000nm; (d) Involved in ATP production under normal conditions; (e) It is a cell or cell analog that is entirely different from mitochondria and exosomes; (f) In the steady state, it is circular to elliptical, and in the case of patient origin, the size is different from the steady state. Mutant luteal lesions with large (longest diameter 800 nm or more) and non-uniform morphology occur; (g) It has a bilayer structure and is adhesive; (h) Can exist both inside and outside the cell; (i) Having mobility, fusion and / or fi This shows the ecological pattern of ssion; (j) Under certain conditions, the mutant luteal bursts, and bursts After Bursting, it exhibits stem cell properties; It has regulatory functions for the (k)p53 gene and telomeres.

[0013] The present invention also involves adding water to the separated luteal derived from bodily fluids and irradiating it with IR light. The present invention provides a method for culturing luteal, characterized by culturing at 18-30°C.

[0014] The present invention also provides an anticancer composition containing luterial as an active ingredient. [Brief explanation of the drawing]

[0015] [Figure 1] These are images of ruterials, which are blood-derived microparticles, taken using a confocal laser scanning microscope (Zeiss), a transmission electron microscope (Transmission Electron Microscope), a scanning electron microscope (Scanning Electron Microscope), an atomic force microscope (Atomic Force Microscope), and a confocal scanner (Leica TCS-SP8). [Figure 2]These are photographs showing the shape and morphology of ruterials at different sizes: (a): 39.6-49.0 nm, ultra-high resolution microscope (SR-GSD) image after staining with Mito-tracker Red; (b): 50.1-85.1 nm, ultra-high resolution microscope (SR-GSD) image after staining with Mito-tracker Red; (c): 76.5 nm, transmission electron microscope image; (d): 160 nm, transmission electron microscope image; (e): 170-230 nm, transmission electron microscope image, double membrane structure; (f): 234 nm, image after staining with Janus green B; (g): 250 nm, atomic force microscope image; (h): 361 nm, transmission electron microscope image Microscope image; (i): 650.1 nm, Transmission Electron Microscope image; (j): Laser Scanning Microscope image of ruterials larger than 5 μm after staining with DAPI (4',6-diamidino-2-phenylindole). [Figure 3] This is a photograph showing the presence or absence of color development after staining luteal with rhodamine 123. [Figure 4] This is a photograph showing the presence or absence of color development after staining luteal with Mito-tracker. [Figure 5] This is a photograph showing the presence or absence of color development after staining luteal with acridine orange. [Figure 6] This is a photograph showing the presence or absence of color development after staining luteal with DAPI (4',6-diamidino-2-phenylindole). [Figure 7]These are photographs showing the motility of luteal molecules measured using a nanotracker ((a) before measurement; (b) after 1 second; (c) after 3 seconds). [Figure 8] This shows the life cycle A of a normal luteal and the life cycle B of a mutated luteal. [Figure 9] This shows the life cycle and characteristics of mutant luteal. [Figure 10] The images show luteilias isolated from the bodily fluids of cancer patients. (a) shows luteilias from cancer patients with altered morphology due to the growth of long branches, and (b) shows luteilias from cancer patients stained with DAPI (4',6-diamidino-2-phenylindole), Mito-tracker, and Rhodamine 123. [Figure 11] This shows the life cycle of luteal. [Figure 12] These are photos showing the size of luteal and mutant luteal. [Figure 13] This is a photograph of a film removed by scratching using a ruterial atomic microscope probe. [Figure 14] (a) and (b) are images of mutated and fused ruterials taken with an atomic force microscope, and (c) and (d) are images of mutated ruterials after the membrane has been exfoliated with a cantilever and then taken with an atomic force microscope. [Figure 15] This image shows DNA confirmed by DAPI staining after scratching with a ruterial atomic microscope probe. [Figure 16] (a) shows the results of a bioanalyzer analysis to determine whether or not DNA is contained in the luteal, and (b) shows, using qRT-PCR, that differences in GAPDH gene expression in DNA occur depending on the size of the luteal. [Figure 17]This is the result of a bioanalyzer analysis to determine whether or not RNA is present in the luteal. [Figure 18] In culture media with varying amounts of luteal added, ATP content was measured using the luciferin-luciferase reaction and a luminometer (SSH: Sanseimaru, SSF: Fisetin, 12h: pre-activated at 37°C for 12 hours before the experiment). [Figure 19] This photograph allows us to observe the difference between luteal and exosomes. Among the 20-120 nm size, those with indistinct membrane divisions and relatively pale internal color are exosomes, while among the 50-800 nm size, those with clearly defined membrane divisions or filled interiors are luteal. [Figure 20] This photograph shows a comparison of the morphologies of luteal, exosome, and microvesicle. [Figure 21] This is an electron microscope (TEM) image of a luterial library constructed according to one embodiment of the present invention. [Figure 22] This is a confocal laser scanning microscope image showing the size change of luteal cells during culture. [Figure 23] This photograph shows the changes in morphology and size of luteal cells during cultivation. [Figure 24] This shows the homologous bacterial composition by analyzing the 16S rRNA base sequences of blood-derived luteal cells of different sizes from healthy individuals (reference: pH 7.2-7.4) ((a): ≤100 nm; (b): 100-200 nm; (c): 200-400 nm; (d): 400-800 nm). [Figure 25] This study analyzes the 16S rRNA base sequences of ruterials derived from blood and semen in fatigued and diseased states (pH 7.0 or lower), categorized by size, to show homologous similar bacterial compositions ((a): ≤100 nm; (b): 100-200 nm; (c): 200-400 nm; (d): 400-800 nm). [Figure 26](a), (b), and (c) show phylogenetic trees based on the 16S rRNA base sequences of blood-derived luteal. [Figure 27] The cell viability of ovarian cancer cell lines SKOV3 and A2780 was shown by MTT analysis after treatment with luteal particles of 100-800 nm size and the commercially available anticancer drug cisplatin at various concentrations. [Modes for carrying out the invention]

[0016] Unless otherwise defined, all technical and scientific terms used in this specification This is the same as what is commonly understood by those skilled in the art in the field to which the present invention pertains. It has one meaning. Typically, the nomenclature used in this specification and the experimental methods described below. The law is publicly known and commonly used in this technical field.

[0017] The term "luterial" used in this invention refers to a life factor present in animals. (Living organism), similar to a virus, with a range of approximately 800 nm Up to (Normal fusion step 50-800nm ​​ / Abnormal fusion step 800nm) The inventors have named fine materials having a size of (nm or larger). Luterial is, (1) A cell or cell analog that has fusion characteristics between a prokaryotic cell and a eukaryotic cell; (2) Present in bodily fluids such as blood, semen, intestinal fluid, saliva, and cell fluid; (3) In immunofluorescence tests, Janus Green B, Acridi Orange (ne Orange), and positive for Rhodamine 123 (4) Under optimal conditions (pH 7.2~7.4), β-proteobacteria It exhibits the expression characteristics of γ-proteobacteria-derived genes and has a size of 30-800 nm. (5) In acidic conditions, β-proteobacteria and γ-proteobacteria It shows the expression characteristics of not only the original gene but also the eukaryotic cell-derived gene, but mainly streptofighters (Streptophyta) gene expression, from 400nm to 2000nm and (6) The size increases; (7) It is involved in ATP production under normal conditions; and (7) Mitochondria Unlike ria, luteria are entirely different cells or cell analogs from exosomes. In animals, including humans, blood, saliva, lymphatic vessels, semen, vaginal fluid, and breast milk (especially colostrum) are used for blood analysis. It is found in umbilical cord blood, brain cells, spinal cord, and bone marrow. In addition, in animals with horns, it is also found in the horns. Luterial exists.

[0018] Normal luteal sizes are 50-800 nm, while mutant luteal fuse and mutate. They form bodies and have a size of several tens of micrometers. Luterials are mRNA and miRNs. It can refer to immature mitochondrial structures, including not only A but also DNA. Real has the characteristic of not dissolving in digestive fluids but entering the bloodstream.

[0019] Luterials are involved not only in signaling, cell differentiation, and cell death, but also in the cell cycle and fine-grained cells. It is also expected to be related to the regulation of cyst growth, but the inventors believe that luteal, in particular, is involved in the diagnosis of cancer. They found that they are closely related.

[0020] Normal luterials suppress the growth of cancer cells. It is expected to play a role in restoring a healthy immune system, and this role involves normalizing genes. This is done by RNAi (RNA interference), which has the potential to be performed. Thus, in the blood of healthy humans and animals, luteal is present in RNA. The information system deviates from its normal course and instructs the body to produce proteins that induce abnormal diseases. In some cases, artificially interfering with this can suppress the development of diseases such as cancer, and size When it matures to a wavelength of 200-500 nm or higher, it also participates in energy metabolism and is irradiated with specific wavelengths. It was then confirmed that the reaction manifests as a light energy amplification function, reacting in a manner similar to chloroplasts. Therefore, if such luteal molecules are unable to perform their normal function, homeostasis may be disrupted. It induces a critical impairment in ATP production, and in both respiration and energy metabolism. It may lead to illness.

[0021] Thus, mutant luteal cells that cannot perform their function properly are different from normal luteal cells. Al-Alfonsino differs from al-Alfonsino in ecology and characteristics, exhibiting diversity in size and morphology. Specifically, normal Ruterials do not proliferate further after forming double spores. However, mutant luteales found in the blood of cancer patients and patients with late-onset diseases are stem Because it has the characteristic of proliferating indefinitely, similar to cells, it has a size of 600-800 nm or larger. Some have a size of 200 μm (200,000 nm) or larger. Also, viruses Similarly, they invade and grow in red blood cells, white blood cells, platelets, etc., or aggregate with other luteal compounds. It exhibits characteristics such as those mentioned above.

[0022] Therefore, by observing the morphological or biochemical characteristics of luteal cells, the disease can be diagnosed. It will enable diagnosis and treatment, and its applications are expected to be limitless. However, it will not be suitable for animals, including humans. Ruterials separated from bodily fluids already discharged from the body dissolve within a short time outside the body. Because they have characteristics such as disappearing or changing form, observation itself is difficult, and in abnormal environments... If left untreated, normal luteal cells will also mutate into mutant luteal cells within 24 hours. However, there is a problem in that accurate diagnosis and treatment of diseases are difficult.

[0023] In this invention, the fine substances present in bodily fluids already discharged from a patient or a healthy person are The terrial was separated using two methods.

[0024] Therefore, in one view, the present invention relates to a method for separating luteal from bodily fluids.

[0025] The first method is a method for separating luteal from blood, wherein platelets and blood A first separation step to separate blood-derived substances having a size of platelet or larger, and the platelets The second step is to centrifuge the blood from which blood-derived substances larger than platelets have been separated. A separation step and a third separation step to separate the luteal from the supernatant obtained by the centrifugation. The procedure includes a step and a step of washing the separated ruterial.

[0026] The first separation step involves passing the blood through a filter having voids of 0.8 to 1.2 μm. The process may include a step of separating any unfiltered material. The process involves repeating centrifugal separation at 1200-5000 rpm for 5-10 minutes, which then extracts the exo This includes a step of removing general microvesicles such as exosomes to obtain the supernatant. This can be done. The third separation step involves irradiating the supernatant obtained by centrifugation with visible light. The process includes the step of separating the ruterial particles, which are mobile and aggregated, using a pipette. This can be done. The blood used in the first step above is of human origin from mammals. It is characterized by having autofluorescence and mobility, as described above. When visible light is irradiated as shown, ruterial particles can be observed in the supernatant. At that time, while observing the moving ruterial particles with a dark-field microscope or confocal microscope, the pipette... It can be separated using the following method. The ruterial separated in the third step has a diameter of 50n The material is passed through a filter with a gap of m, and only the unfiltered portion is washed with PBS. Ruterials can be obtained using this method. Ruterials have a size of 50 nm or larger in major axis. Therefore, by the above process, blood-derived microparticles other than luteal can be removed.

[0027] The second method is a method for separating luteal from bodily fluids such as blood and semen, wherein the bodily fluids The steps include: after primary centrifugation, filtering the supernatant through a 2-5 μm filter, and the filtered The solution is subjected to secondary centrifugation, and the supernatant is filtered through a 0.5-2 μm filter. Includes.

[0028] In other words, after primary centrifugation of body fluids at 2000-4000 rpm for 5-30 minutes, the supernatant is obtained. Filter the solution through a 2-5 μm filter, and then simmer the filtered solution at 3000-7000 rpm for 5-2 minutes. The process may include a step of secondary centrifugation for 0 minutes, followed by filtration through a 0.5-2 μm filter. can.

[0029] The solution obtained by filtration is irradiated with visible light, causing ruterial particles to gather and exhibit mobility. The procedure may further include the step of separating the ruterials using a pipette. Because it has light and kinetic properties, when irradiated with visible light as described above, luteinizing agent is released from the supernatant. Real particles can be observed. In this case, moving ruterial particles can be viewed with a dark-field microscope or The separated ruter can be separated using a pipette while being observed with a confocal microscope. The aluminum is passed through a filter with a 50 nm diameter void, and only the unfiltered portion is converted to P Luterials can be obtained by washing with BS. Luterials have a major diameter of 50 nm or more. Because of its size, the above process removes blood-derived fine matter other than luteal. It is possible.

[0030] The luteal separated by the two methods described above can be examined using a dark-field microscope or a confocal microscope. They can be observed using a mirror, at 200nm, 400nm, 600nm, and 800nm ​​respectively. Then, using filters of 1000 nm in sequence, depending on the size, 50-200 nm (generation period) ) / 200~400nm(mature phase) / 400~600nm(mitotic phase) / 600~800n It can be distinguished into m (hypermitotic phase).

[0031] In this invention, the properties of the separated luteal were investigated.

[0032] (1) Morphology The normal size of a ruterial is 50-800 nm (Figures 2 and 12), If John is absent, it matures up to 800 nm. In the case of patient-derived luteal, it is derived from normal This variant Ruteria is larger in size (over 800 nm in diameter) and has a non-uniform morphology compared to other species. When a rutile occurs and fusion occurs, the size of the rutile can grow to several thousand nanometers. I confirmed that it would become like that.

[0033] Furthermore, ruterials are circular to oval in shape and can be seen in SEM or TEM electron microscope images. It exhibits a bilayer structure similar to chondria, but possesses an internal cristae structure. It was not (Figure 1).

[0034] (2) Immunochemical fluorescence staining Mitochondria are detected using Janus green B and fluorescence The staining agents are Rhodamine 123 and MitoTracker. o-tracker), Acridine Orange and It is known that the color is produced by DAPI, but luteal pigments also stain the same as mitochondria. The color development by the pigment was confirmed. The luteal exhibited immunochemical fluorescence similar to that of mitochondria. In contrast to the color reaction observed by exosomes, it exhibited a reaction opposite to that of autologous cells. The characteristic of exhibiting fluorescence (autofluorescence) was confirmed from fluorescence photography (Figure 2). a), Figure 2(b), Figure 2(f), Figure 2(j), Figures 3-6).

[0035] (3) Ecological patterns Luterials are exosomes and microvesicles. Unlike le, it has adhesive and motility, and fusion or The ecological pattern of fission was shown. Under specific conditions, the ruterial variants were bar After bursting, stem cells It was confirmed that it possesses stemness and can exist either inside or outside the cell. This was confirmed (Figures 8, 9, and 11).

[0036] (4) ATP production Luciferi has shown that ATP is produced from luteal molecules with a size of 200-400 nm. The luciferin-luciferase reaction and luminometry This was verified using a luminometer. The group to which ruterial was added showed that ruterial Compared to the group without added phosphate, the ATP concentration increased. This suggests that luteal is... We can conclude that it has ATP production capacity. Addition of SSH and SSF. Regarding the difference, the SSF-added group had a higher ATP concentration than the SSH-added group. Therefore, we identified a culture medium that can efficiently increase ATP content (Figure 18).

[0037] (5) Contains nucleic acid DAPI and acridine orange (AO) staining methods identified only RNA within the luteal. It was confirmed that it also contains DNA. Specifically, RNA contains acridine oren With the di-staining agent, it appears orange at excitation levels of 460 nm and emission levels of 650 nm. The stained DNA is stained green at the excitation level of 502 nm and the emission level of 525 nm, D The presence of DNA can be confirmed by the API staining method. Using this method, we confirmed that RNA and DNA are contained in the luteal of the present invention (Figure (Figures 5 and 6). Also, luteal RNA and DNA were isolated using a kit. After purification, the bands are confirmed by agarose gel electrophoresis, and qR By confirming the degree of GAPDH expression using the T-PCR technique, the size of the luteal can be determined. We confirmed that there were differences in gene expression (Figure 2(h), Figure 16, and Figure 17).

[0038] (6)16S rRNA sequence analysis FastDNA SPIN Kit (MP Biomedicals, Cat 656 After extracting luteal gDNA using 0-200), the specific ply shown in Tables 1 and 2 The 16S rRNA gene was amplified using a primer.

[0039] Furthermore, the GeneBank database was used to analyze the 1461 amplified gene fragments mentioned above. Homology analysis using ase (NCBI database) showed that blood and semen The 16S rRNA base sequence of the ruterial is β-proteobacteria (β-Prot eobacteria), γ-Proteobacteria ), Acidobacteria, Cyanobacteria bacteria), Actinobacteria, Farm Genetics derived from Firmicutes and Eukaryotes It showed homology to its offspring and possessed fusion characteristics of an intermediate stage between prokaryotic and eukaryotic cells (Figures 24-24). twenty five).

[0040] Under optimal conditions (blood pH: 7.2-7.4), blood-derived luteal compounds are β-proteopropyl alcohol. Proteobacteria (β-Proteobacteria), γ-Proteobacteria (γ-Pro Derived from *Teobacteria* and *Bacteroidetes*. It shows homology to the gene (Figure 24) and is observed at a size of 50-800 nm.

[0041] Under steady state, semen-derived luteal compounds are found in β-proteobacteria and γ-proteobacteria. It shows homology to genes from Bacteroides and chordates.

[0042] In contrast, under acidified conditions, β-proteobacteria (β-pro teobacteria) and γ-proteobacteria (γ-Proteobacteria) In addition to genes derived from ria, bacterial genes are expressed in even greater diversity, and eukaryotic cells Eukaryote-derived genes are also expressed. Mainly streptofighters (Stre The 16S rRNA characteristics of ptophyta and planktomi were revealed. As shown in Figure 25, it grows to a size of 400-2000 nm.

[0043] [Table 1]

[0044] [Table 2]

[0045] (7) Differences from exosomes and mitochondria Table 3 summarizes the differences between exosomes, mitochondria, and luteal cells. ru.

[0046] [Table 3]

[0047] The average size of luteal is 200-800 nm, while mitochondria (400-1 It is smaller than (1,000 nm) and larger than exosomes (20-120 nm). Exosomes have indistinct membrane divisions and relatively pale internal color, whereas lutealiforms have a membrane The divisions are clear, or the interior is filled (Figure 19). Note that luteal is Their morphology is completely different from that of exosomes and microvesicles. (Figure 20).

[0048] During immunochemical fluorescence staining, luteal stains show a reaction similar to that of mitochondria, whereas e They exhibit reactions opposite to those of xosomes. Luterials exist both inside and outside the cell, and extracellularly. Exosomes are substances obtained by ingesting food, whereas mitochondria The difference is that it exists only inside cells and cannot be obtained through food intake.

[0049] Furthermore, unlike exosomes and mitochondria, luteal cells are motile. It can grow naturally, its growth can be maintained through culture, and it exhibits autofluorescence. Furthermore, luteal, exosome, and mitochondria are all forms of fusion. Although it has the formula, kiss-and-run fusion does not occur in exosomes, A TP production also does not occur. Furthermore, exosomes are located outside the cell, while mitochondria are located inside the cell. While luteal cytoplasm is present in the extracellular space, luteal cytoplasm can exist both extracellularly and intracellularly (Figure 11).

[0050] Furthermore, 16S rRNA base sequence analysis results showed that mitochondria contain α-proteobacteria. While it shows homology to A, luteal is a γ-proteobacterium (γ-Proteo bacteria), β-proteobacteria, Bacteroides, Firmicutes It shows homology to eukaryotes (tes) and other eukaryotic cells.

[0051] From another perspective, the present invention relates to a fluid-derived luteir having one or more of the following characteristics: (a) Immunofluorescence assays showed Janus green B and Acridi Acridine Orange and Rhodamine 123 mine123) shows a positive color reaction; (b) Under optimal conditions (pH 7.2~7.4), β-proteobacteria and γ-proteobacteria It exhibits expression characteristics of kuteria-derived genes and has a size of 30-800 nm; (c) Under acidic conditions, the genes are derived from β-proteobacteria and γ-proteobacteria. Furthermore, it produces streptophyta genes, which are eukaryotic cells. As a result, the size increases from over 400nm to over 2000nm; (d) Involved in ATP production under normal conditions; (e) It is a cell or cell analog that is entirely different from mitochondria and exosomes; (f) In the steady state, it is circular or elliptical, and in the case of patient origin, the size is different from the steady state. Mutant luteiliforms with large diameters (over 800 nm) and non-uniform morphology occur; (g) It has a bilayer structure and is adhesive; (h) Can exist both inside and outside the cell; (i) Having mobility, fusion and / or fi This shows the ecological pattern of ssion; (j) Under certain conditions, the mutant luteal bursts, and bursts After Bursting, it has stem cell properties; and It has regulatory functions for the (k)p53 gene and telomeres.

[0052] On the other hand, luteal lesions vary in size (diameter), area, and morphology depending on whether or not the individual has a disease. Because the nanotracking speeds differ, one or more of the above characteristics can be used to diagnose and predict the prognosis of diseases. This can be predicted. This is because luterial derived from healthy individuals without disease and human individuals with disease This can be seen from the differences in the size, morphology, and nanotracking speed of the ruterials from which they originate.

[0053] Normal luteal cells from healthy individuals simply form double spores. (fission) is all that is needed, but luteal (sudden mutation) is derived from patients with late-onset diseases or cancer patients. Heterites are formed when luteal elements fuse or coagulate with each other. They can puncture or burst, and can attach to cells such as red blood cells and cancer cells. Furthermore, its shape and size are abnormally large, differing from those of normal luteal filariae. (Figures 8-10). Mutant ruterials have high adhesion, so the cycle described above (c The fusion process is further accelerated by the ycle, and its size increases to approximately 600-8 They grow larger than 00 nm and have a size of 200 μm (200,000 nm) or larger. There are also cases where the mutated luteal form depends on the type and stage of cancer. We have confirmed that there is consistency and have filed a patent application regarding this (Korean Patent Application No. 10‐2013‐0082060).

[0054] Therefore, by observing the morphological or biochemical characteristics of luteal cells, the disease can be diagnosed. It can be used to diagnose and predict prognosis, and its applications are limitless.

[0055] The luteal morphology includes normal, flagellated, mass, rod, and The 'ta' indicates a compound form. The normal form is a different fusion or bursting form. Without undergoing deformation, it maintains a form with a ratio of major axis to minor axis of 1:1 to 3:1, and is close to a circular shape. It can be shown as a particle. When observed under a microscope, it appears as a small dot.

[0056] Flagellated form is a morphology in which the ruterial undergoes modification or fusion to acquire a flagellum on the outside. This is possible. The inventors believe that the rate at which flagellar morphology is observed increases rapidly as cancer progresses to its terminal stage. The incidence is increasing, and in stage 4 cancer, flagellated ruter is present in 99.1% of patients diagnosed with stage 4 cancer. It was confirmed that Al was observed (Korean Patent Application No. 2013-0082060). If the real morphology matches the flagellated morphology 80-100%, a tumor mammary gland is suspected to be in the terminal stage. It can be displayed using Tumor Marker. The aforementioned terminal tumor has been diagnosed. The survival period for patients is approximately 1 to 4 months, and long-term survival is impossible, especially in the case of flagellar tumors.

[0057] Mass type (M type) refers to luteal meristems that burst or fuse. It is a specimen that has been deformed from its normal size and shape, and the difference between its major and minor axes is large. It has an irregular volume shape. Preferably, the ratio of the major axis to the minor axis is 3:1 to 5:1. A variety of mass shapes can be observed.

[0058] Rod-shaped (R-shaped) ruterials are bursting, deformed, and It undergoes fusion and takes the form of a rod. The difference in length between the minor axis and the major axis is the mass It is larger than the shape. Preferably, the ratio of the major axis to the minor axis can be 5:1 to 12:1. Rod shapes include Rod 1, which consists of a single circular or elliptical chain, and Rod 1, which consists of two or more single chains. It includes a rod type 2 which is formed by combining. The rod type 1 is a single ruterial in the form of a rod. This is due to bursting and / or deformation. It can be such that the rod type 2 is formed by the joining of two or more ruterials into a rod shape. This is what resulted from bursting, deformation, and fusion. It can be due to one or more of the combinations. On the other hand, the flagellar form, due to its shape, is a large range While it can be classified as a rod-shaped organism, it differs in that its flagella are elongated. Therefore, it is possible to first determine whether it is rod-shaped and then determine whether it is flagellar-shaped. ru.

[0059] The aforementioned composite form can be a hybrid form of a rod shape and a mass shape. A form in which part of a fine substance is rod-shaped and part is mass-shaped can be called a composite form. .

[0060] The aforementioned rod shape is a rod type 1 consisting of a single chain that is circular or elliptical, and a single It can be one of the group that includes rod type 2, which is formed by the bonding of two or more single chains. It can be a hybrid form of a rod shape and a mass shape.

[0061] As described above, the morphology of luteal in the body changes as the disease develops and progresses, By observing the morphological characteristics of luteal lesions, it is possible to diagnose diseases and predict their prognosis. Furthermore, the morphological changes of the luteal are also related to the amount and sequence changes of the nucleic acids contained in the luteal. Therefore, we will analyze the nucleic acid expression pattern (16S rRNA) sequence of the luteal. This allows for the diagnosis of diseases.

[0062] For example, the 16S rRNA sequence of a normal luteal and the 16S rRNA sequence of a patient's luteal. By comparing NA sequences, diseases (especially cancer) can be diagnosed. In particular, streptococcus Streptophyta gene expression and eukaryote cells Co-expression of genes can be used as a diagnostic and predictive marker for cancer.

[0063] However, luteal, which is separated from bodily fluids already discharged from a patient or a healthy person, is ex vivo. However, because they have the characteristic of dissolving and disappearing or changing form within a short time, observation is difficult. If the body is in a difficult and abnormal state and left unattended, even normal luteal cells can suddenly appear within 24 hours. Mutations in the ruterial molecule make accurate diagnosis and treatment of the disease difficult. However, the present invention According to the culture method, luteal is cultured so that it does not exceed a specific size (500 nm). It is possible.

[0064] Therefore, from another perspective, the present invention involves adding water to luteal and irradiating it with IR light. Culture of luteal characterized by cultivation at 18-30°C (preferably 20-25°C) Regarding the method.

[0065] The water added during the culture can be saline solution or PBS solution, but It is not limited to the following. Luterials derived from bodily fluids before culture can be obtained by the separation method of the present invention. By the culture method of the present invention, a size of 50 to 200 nm can be used. The cultured blood-derived luteal can have a post-culturing size of 300-800 nm. During this process, observe under a microscope and ensure that the size of the ruterial does not exceed 500 nm. Once the culturing is complete, the cells are sorted by size, cooled to -80°C, and stored. It can be stored by filling it with nitrogen or by storing it at temperatures above 0 degrees Celsius, and preservatives can be added during storage. It is possible.

[0066] As described above, luteal cultured can be stored for a predetermined period without any change in its properties. It is capable of being effectively used in the diagnosis and prognosis prediction of diseases using luteal. Yes, it is possible. In this invention, "without changing the properties of the luteal" means the shape of the luteal. The morphology and size are maintained to be nearly identical to the state before cultivation in the culture medium. This means that. Also, the activity of ruterials such as nanotracking speed, This means maintaining values ​​similar to those before culturing.

[0067] Specifically, luteal cultured by the culture method of the present invention is used for the following purposes. Mutant luteal molecules can fuse or aggregate. Furthermore, its form and size differ from that of a normal luteal, and it has become abnormally large. (Figures 8-10) When isolated mutant luteales are cultured, luteal fusion occurs. This involves processing candidate substances or methods that can suppress aggregation, thereby preventing ruterial fusion or aggregation. By observing whether or not suppression occurs, we can identify substances that can suppress or prevent luteal mutations. It can be screened.

[0068] Furthermore, when culturing isolated luteal, conditions that promote fission are present. By processing with complementary materials or methods, the fission of mutalytic luteales can be achieved. Substances that promote this can be screened. Mutalytic luterials fuse or agglutinate. They have a collective ecological pattern (Figures 8, 9, and 11), but fission ) are transformed into a different ecological pattern, or mutated luteal are fissio n) so that it has a normal luteal size, fission (fissi By treating candidate substances that promote (on), we can suppress the mutation of luteal molecules. A substance that converts mutated luteal to have the ecological characteristics of normal luteal, In extreme cases, the goal is to screen for preventive substances against diseases that may be induced by mutated luteirials. It is possible.

[0069] The present invention will be described in more detail below with reference to examples. These examples illustrate the present invention. These embodiments are merely for the purpose of providing this information and should not be construed as limiting the scope of the present invention. This is self-evident to anyone with common knowledge in this industry.

[0070] Examples Example 1: Separation of blood-derived luteal Blood samples (50cc) were taken from a patient with terminal non-small cell lung cancer and found to contain voids with a diameter of 0.8 μm or larger. The blood was passed through a filter, and any unfiltered substances were separated. The filtered blood was then subjected to a 1200- By repeating centrifugation at 5000 rpm for 5-10 minutes, exosomes can be extracted. The supernatant was obtained by removing general microvesicles such as ome. The supernatant was irradiated with visible light. Luterial particles that were mobile and aggregated were separated using a pipette. The ruterial particles exhibited autofluorescence. Furthermore, because they possess the characteristics of mobility, when irradiated with visible light as described above, ruterial particles This can be confirmed by using a dark-field microscope or a confocal microscope to observe the moving ruterial particles. The ruterials were separated using a pipette while monitoring the process. The separated ruterials were separated into voids with a diameter of 50 nm. By passing the material through a filter containing the following properties and washing only the unfiltered portion with PBS, Ruterials were obtained. The above process yielded ruterials with a major axis of 50-800 nm, which are dark Observation and confirmation were possible using a field microscope or a confocal microscope. The obtained ruterial Depending on the size, the wavelengths are 50-200nm (developmental stage) / 200-400nm (maturation stage) / 4 The cells were divided into 00-600 nm (mitotic phase) and 600-800 nm (hypermitotic phase). Similar methods were used. This allows us to build libraries for each luterial size as shown in Figure 21, and the luterial size The morphology for each size is shown in Figure 2.

[0071] Example 2: Separation of semen-derived luteal After primary centrifugation of semen at 2000-4000 rpm for 5-30 minutes, the supernatant is collected in a 2-5 μg solution. Filter the solution through an M filter, and then perform secondary processing at 3000-7000 rpm for 5-20 minutes. After centrifugation, the sample was filtered through a 0.5–2 μm filter. The ruterial exhibited autofluorescence and luminescence. Because of their dynamic properties, ruterial particles can be observed when the filtered solution is irradiated with visible light. This can be done. In this case, the moving ruterial particles can be observed with a dark-field microscope or a confocal microscope. The separated ruterials were separated using a pipette while performing the procedure. The separated ruterials had voids with a diameter of 50 nm. By passing it through a filter and washing only the unfiltered portion with PBS, lutealization is achieved. This was obtained. This could be observed and confirmed using a dark-field microscope or a confocal microscope.

[0072] Example 3: Properties of luteal (1) Structure Ruterials obtained in Example 1 that have a size of approximately 50-400 nm This is a confocal laser scanning microscope (Confocal Laser Scanning Microscope) Icroscope (Zeiss), Transmission Electron Microscope (Transmission E (Lectron Microscope), Scanning Electron Microscope (Scanning Electron Microscope) Atomic force microscope (Atomic Force Microscope) Images were taken using a microscope and a confocal scanner (Leica TCS-SP8). As a result, luteal membranes, like mitochondria, are membrane structures with a double membrane, and the internal... It has a structure that is in an incomplete state of the cristae structure, and mitochondria It was confirmed that it could be observed in the same laser wavelength range as the rear. Furthermore, its shape was circular or We were able to observe that it was elliptical (Figure 1, Figure (e), Figure 2(h), Figure 13 and Figure 14).

[0073] (2) Dyeing characteristics Ruterials obtained in Example 1 that have a size of approximately 50-800 nm Mito-tracker, Rhodamine 123 ne123), Acridine Orange, and Yanu After staining with Janus green B, the presence or absence of color development was observed. As a result, plant-derived luteal agents, Mitotracker, Rhodamine 123, and Acridine Ole have also been found. It was confirmed that the color develops with Ang and Janus Green B (Figure 2(a), Figure 2( b), Figure 2(f), Figure 2(j), Figures 3-6).

[0074] (3) Autofluorescence Ruterials obtained in Example 1 that have a size of approximately 50-800 nm Fluorescence photography confirmed that it exhibits a photoreaction (Figure 5).

[0075] (4) Motility The motility of the ruterial obtained in Example 1 was measured using nanotracking by 3i Inc., USA. Specifically, after observing the ruterial with a bright-field microscope, a track was found in the center of the ruterial. Once you set the tracking and activate nanotracking, the real-time tracking will follow the movement of the real object. The movement trajectory of the immobile was displayed, and its speed per second was calculated (Figure 7).

[0076] As a result, the nanotracking speed of the luteal in this embodiment is approximately 13-25 μm / It was measured in seconds.

[0077] (5) Analysis of the presence or absence of RNA and DNA in luteal The 200-400 nm ruterials separated in Example 1 were imaged with an atomic microscope, and the results showed that As shown in Figures 2(h), 15, 16, and 17, RNA and DN are luteal. It can be inferred that it contains nucleic acids like A.

[0078] Total RNA and DNA were separated from the 200-400 nm luteal fragments isolated in Example 1. To do this, QIAGEN kit (RNeasy Micro Kit: Cat 7400) 4) After separation using Experion RNA (DNA) StdSens (Bi Quantitative analysis was performed using an o-Rad chip.

[0079] After recovering the luteal by centrifugation (8000g, 1 hour 30 minutes), the kit was disassembled. Buffer RLT plus (Guanidine isothiocycanate, de Add 3.5 μl of β-mercaptoethanol to 50 μl of tergents, and use a 20 gauge. The luteal was dissolved by passing it through a syringe with a needle 5 to 10 times. Sample decomposition After transferring the buffer to the AllPrep DNA spin column, centrifuge (≧8000g) (15 seconds) The buff containing the DNA packed in the column and the RNA that has passed through the column They were separated from each other.

[0080] First, add 350 μl of 70% ethanol (the same volume) to the buffer that has passed through the column. After uniform mixing, add 700 μl of the mixture to RNease MinElute spin. Transfer to a ram and centrifuge (≧8000g, 15 seconds), then remove the buffer that has passed through the column. 350 μl of RW1, 500 μl of RPE buffer and 500 μl of 80% Column washing was performed in stages using ethanol. All centrifugation used above ( The same conditions were used for the ≥8000g, 15-second procedure. 14 μl of RNea was used to obtain RNA. After placing the sy-free solution into the column, centrifuge (≧8000g, 60 seconds) is performed to extract the luteinized solution. AlRNA was isolated.

[0081] For the isolation of genomic DNA, the FastDNA SPIN Kit (MP Biome (dical) was used. After placing the separated luteal into a tube, 978 μl of phosphorus was added. Sodium phosphate buffer, 122 μl MT baff A was added. Homogenized for 40 seconds, then centrifuged (14,000g, 10 minutes) and the top After obtaining the solution, add 250 μl of PPS (Protein Precipitation Solution). Mixed for 10 minutes with lution added. After centrifugation (14000g, 5 minutes), 1 The process of transferring the supernatant to a 5ml tube is repeated twice, and DNA binding (DNA binding) is performed. g) After placing it on the rotor for 2 minutes, then place it on the silica matrix stand for 3 minutes. I left it there. I carefully collected about 600 μl of the supernatant and put it into a SPIN filter. After centrifugation (14000g, 1 minute), discard the supernatant and place the SE into a pellet. 500 μl of WS-M was added and suspended. Centrifuged for 1 minute. Next, the supernatant was discarded, and the mixture was centrifuged repeatedly to ensure no buffer remained. 50 μl Add DES (DNase / Pyrogen-Free Water) and centrifuge (14 After 000g (1 minute), genomic DNA was obtained.

[0082] Using the Experion RNA (DNA) StdSens (Bio-Rad) chip The quantitative results, as shown in Figures 16 and 17, showed that the RNA was divided into luteal groups. And we were able to confirm that it contained DNA.

[0083] (6)16S rRNA sequence analysis 16S rRNA (ribosomal ribonucleic acid) is, RNA that interacts with various proteins to constitute ribosomes. Therefore, the rate of change in the base sequence is significantly smaller than that of the base sequences of other genes in most genomes. The degree of 16S rRNA sequence similarity is recognized as reflecting the phylogenetic distance between organisms. It is.

[0084] [1] Blood-derived luteal From the blood-derived luteal obtained in Example 1, FastDNA SPIN Kit (MP After extracting gDNA using Biomedicals (Cat 6560-200), , PCR-premix (iNtRON Biotechnology, Korea) Using primers numbered 1-23, luteal 16S rRNA is extracted. It was amplified.

[0085] The amplified PCR product was processed using BigDye Terminator Cycle Seq. uencing Ready Reaction kit(applied Biosy stems, USA) and automated DNA analyzer system (PRISM 3730XL DNA analyzer, Applied Biosys The base sequence was analyzed using stems. The amplified PCR product consisted of a total of 1461 fragments. Of these, 1407 fragments are proteobacteria. ia) shows homology to genes derived from Acidobacteria, and 20 fragments are from Acidobacteria. It shows homology to genes derived from teria, and 11 fragments are from Actinobacteria (Acti It showed homology to a gene derived from nobacteria (Table 4).

[0086] The analyzed base sequence fragments were combined using SeqMan software (DNASTAR). The base sequence of 16S rRNA was obtained in combination.

[0087] Figure 24 shows the 16S r of blood-derived luteal from healthy individuals (blood pH: 7.2-7.4). This shows the composition of homologous similar bacteria by analyzing the base sequence of RNA. Analysis was performed according to the size of the luteal ((a): less than 100 nm, (b): 100-200 nm) m, (c) 200-400nm, (d) 400-800nm). There is no significant difference between sizes. Both are Proteobacteria and Firmicutes. (Firmicutes) and Bacteroidetes It showed homology with the gene.

[0088] Figure 25(c) shows luteal cells derived from blood in fatigued / illness-prone individuals (blood pH: 7.0 or lower). 16S rRNA (size: 200-400 nm) was sequenced to show homology. This shows the composition of similar bacteria. Unlike the steady state, streptofighters Further genes showing homology to (Streptophyta)-derived genes were expressed.

[0089] Figures 26(a), (b), and (c) show the 16S rRNA base pairing of blood-derived luteal. This shows a phylogenetic diagram based on columns.

[0090] [Table 4]

[0091] 16S rRNA fragments of blood-derived luteal are found in β-proteobacteria and γ-proteobacteria. Obacteria, Bacteroides, Firmicutes, and Streptophytes, etc. It was confirmed that the bacteria exhibit homology with various other bacteria.

[0092] Generally, in microbial classification systems, gDNA relatedness is less than 70%. Therefore, they are recognized as independent strains. Also, the phase of the 16S rRNA base sequence Statistically, if the same sex accounts for less than 97%, the gDNA correlation is less than 70%. This was proven by analysis. Therefore, the luteal 16S rRNA fragment and 97.0% Analysis of cells exhibiting the above homologous sequences revealed that, as shown in Tables 5 to 7, blood The derived luteal shows 100% homology with γ-proteobacteria, and Firmicute It showed 97.53% homology with *Bacteroides* and over 97% homology with *Bacteroides*.

[0093] On the other hand, luteal under abnormal acidic conditions, as shown in Table 8, is streptopha It showed over 99% homology with Ita (Streptophyta).

[0094] [Table 5] TIFF2026143646000006.tif201160TIFF2026143646000007.tif201160TIFF2026143646000008.tif180160

[0095] [Table 6]

[0096] [Table 7] TIFF2026143646000011.tif201160TIFF2026143646000012.tif201160TIFF2026143646000013.tif202160

[0097] [Table 8] TIFF2026143646000015.tif180160

[0098] [2] Semen-derived luteal gDNA extraction was performed on the semen-derived luteal obtained in Example 2 using the method described above. The samples were then subjected to PCR amplification and nucleotide sequence analysis. Figure 25 shows the fatigue / disease state (semen pH). 16S rRNA from semen-derived luteal (7.0 or less) was sequenced to determine homology. This shows the composition of similar bacteria, analyzed by luteal size. a): 100nm or less, (b): 100~200nm, (d) 400~800nm).

[0099] Steady-state semen-derived luteal, like blood-derived luteal, is similar to proteobacteria. (Proteobacteria), Firmicutes, It shows homology to genes derived from Bacteroidetes, and is unique. In particular, it showed homology to genes derived from chordates.

[0100] Furthermore, under abnormal acidic conditions, streptophyta Genes homologous to the source gene were expressed.

[0101] (7)ATP content measurement Control group, luteal, luteal (SSH 12h), and luteal (SSF 1 Place 10 mL each of four culture media composed of 2 h) into separate tubes, and add glucose (Gl Add ucose (100 mg / mL) and ADP (1 mM) substrate and bathe in a 37°C water bath. The culture was performed. After the start of the culture, 100 μl of the sample was taken at 30-minute intervals and placed in a tube, and 900 After adding μl of distilled water to dilute it 10-fold, transfer 10 μl of the sample to a new tube and then A Add 100 μl of the luciferase reagent included in the TP kit, and immediately apply luminometry. The measurement was repeated 5 times using the same method.

[0102] As a result, as shown in Figure 18, in the group to which luteal was added, The ATP concentration was increased compared to the non-added group. From these results, ruterial AT P production ability could be confirmed. By the addition of SSH and SSF Regarding the difference, the ATP concentration was higher in the SSF-added group than in the SSH-added group (Figure 18).

[0103] Example 4: Culture of ruterial (1) Among the ruterial obtained in Example 1, ruterial having a size of about 50 to 200 nm was added with PBS, irradiated with IR light, and then cultured at 18 to 30°C for about 3 hours. IR The size of ruterial was confirmed under a microscope at about 1-hour intervals immediately after irradiation with light. About 1 to After 6 hours, ruterial, which had a size of about 200 nm before culture, grew to about 500 nm This could be confirmed. Thereby, water is added to blood-derived ruterial, and IR When cultured at 18 to 30°C under light irradiation, the size can be grown to about 500 nm It was confirmed that when additionally cultured, the culture can grow up to several hundred micrometers, and in this state it was also confirmed that bursting occurs when additionally cultured (Figure 22 ).

[0104] (2) Among the ruterial obtained in Example 1, ruterial having a size of about 400 to 800 nm was added with PBS, irradiated with IR light, and then cultured at 18 to 30°C for about 3 hours. I The size and state of ruterial were confirmed under a microscope at about 1-hour intervals immediately after IR light irradiation . After about 1 to 6 hours, ruterial, which had a size of about 400 to 800 nm before culture, did not grow and it was confirmed that fission occurs.

[0105] Furthermore, when additional culture of mutant ruterial of 800 nm or larger is performed, the mutation shown in the blood of cancer patients It was observed that the morphology changed to a heteroterial form (Figure 23).

[0106] Example 5: Anticancer effect of luteal To measure the growth inhibitory effect of two ovarian cancer cell lines, SKOV3 and A2780, yellow Color Tetrazolium MTT(3-(4,5-dimethylthiazolyl-2)-2,5-dife The MTT (methyltetrazolium bromide) analysis method was performed. The MTT analysis method is used to analyze living cells. A method for measuring growth, in which dehydrogenase in the mitochondria of living cells is yellow aqueous solution This method utilizes the principle of generating purple formazan using the chemical substance MTT. The production of purple formazan is carried out by living cells that have metabolic activity. It is known to be roughly proportional to the number of cells, and is very effective in measuring cell growth and differentiation. It can be used.

[0107] Each cultured cancer cell was placed in a 96-well plate in a 5x10⁶ arrangement. 4 To make it so that it is one / ml Add 100 μl per well, and supply 5% carbon and 95% oxygen at 37°C. After culturing in a humid incubator for 24 hours, concentrate luteal particles with a size of 100-800 nm. Each well was treated at a time. After incubation for 48 hours, each well was treated with phosphate buffer solution (phos MTT (5 mg / m²) dissolved in phate-buffered saline (PBS). l) Add 15 μl of solution at a time and incubate for another 4 hours. After confirming the formation of n), completely remove the culture medium and dissolve the formazan formed at the bottom of the well. To dissolve it, 100 μl of dimethyl sulfoxide (DMSO) was added. After that, Using a cross-plate reader (GEMINI, Stratec biomedical) Absorbance was measured at 560 nm, and relative cell proliferation inhibition was observed when the control group cells were set to 100%. The rate was calculated.

[0108] As a result, the IC50 of luteal cells from SKOV3 and A2780 cell lines was 30, respectively. The IC50 values ​​are μg / ml and 60 μg / ml, and for the commercially available anticancer drug cisplatin. The concentration was 100 μM (Figure 27). Luterial was a positive control agent for two ovarian cancer cell lines. It showed stronger cytotoxicity than the control drug group, and its cytotoxicity against normal ovarian cells was similar to that of the positive control drug group. It showed its gender.

[0109] The contents of the present invention have been described in detail above, but a person with ordinary skill in the industry will find this to be... Therefore, such specific technologies are merely preferred embodiments, and this does not limit the scope of the present invention. It is clear that this is not the case. Therefore, the substantial scope of the present invention is the appendix. It can be said that it is defined by the claims and their equivalents. [Industrial applicability]

[0110] According to the present invention, luteal, which is a fine substance present in the bodily fluids of a patient or a healthy person, is effectively It can be effectively separated, and the separated luteal can be grown to a predetermined size. Because it can be cultured, it is useful in the diagnosis and treatment of diseases. In particular, luteal is useful in cancer It is useful as an anticancer agent because it exhibits a strong anticancer effect against cell lines.

Claims

1. Method for separating luteal, including the following steps: (a) A first method for separating platelets and blood-derived substances having a size greater than or equal to a platelet from blood. Separation step, (b) The blood from which the platelets and blood-derived substances having a size greater than or equal to a platelet are separated is then heated to a distance. The second separation step involves separating the heart, (c) A third separation step of separating luteal from the supernatant obtained by centrifugation, 、 (d) A step of washing the separated ruterial.

2. The separation of luteal according to claim 1, characterized in that the blood is of mammalian origin. method.

3. The method for separating luteal according to claim 2, characterized in that the blood is of human origin. 。

4. The first separation step involves passing the sample through a filter having voids of 0.8 to 1.2 μm. Claim 1 further comprises the step of separating any substances that have not been filtered. Method for separating luteal.

5. Filters for 200nm, 400nm, 600nm, 800nm, and 1000nm They are used in order, 50-200 nm, 200-400 nm, 400-600 nm, and 600 nm respectively. Further includes classifying luteiliforms into sizes of ~800 nm and 800-1000 nm. A method for separating luteal according to claim 4, characterized by the following:

6. The second separation step involves repeating centrifugation at 1200 to 5000 rpm for 5 to 10 minutes. A method for separating luteal according to claim 1, characterized by being carried out by [doing something].

7. In the second separation step, exosomes are removed. The method for separating luteal according to claim 1, characterized by its features.

8. The third separation step involves irradiating the supernatant obtained by centrifugation with visible light, and Using a pipette, ruterial particles, which have the ability to gather at the location where visible light is irradiated, are used. A method for separating luteal according to claim 1, characterized by being carried out by separation.

9. The washing step involves removing the ruterial obtained from the third separation step to a diameter of 50n Passing the material through a filter with voids of m, and washing only the unfiltered portion. The method described in claim 1, which is carried out by obtaining a luteal thereafter. Method for separating luteal.

10. Luterial derived from bodily fluids having one or more of the following characteristics: (a) Immunofluorescence assay showed Janus green B and Acridi Acridine Orange and Rhodamine 123 mine123) shows a positive color reaction; (b) Under optimal conditions (pH 7.2-7.4), β-proteobacteria and γ-proteobacteria It exhibits the expression characteristics of Obacterial-derived genes and has a size of 30–800 nm; (c) Under acidic conditions, the genes are derived from β-proteobacteria and γ-proteobacteria. Moreover, it produces the streptophyta gene, which is a eukaryotic cell. They appear, and their size increases to 400 nm to 2000 nm or more; (d) Involved in ATP production under normal conditions; (e) A cell or cell-like structure that is entirely different from mitochondria and exosomes. ru; (f) In the steady state, it is circular or elliptical, and in the case of patient origin, it is smaller than the steady state. Mutant luteal lesions with large diameters (over 800 nm) and non-uniform morphology occur; (g) It has a double-layer structure and is adhesive; (h) Can exist both inside and outside the cell; (i) Motile and capable of fusion and / or fission The modal formula is shown; (j) Under certain conditions, the mutant luteal bursts, and bursts After bursting, it possesses stem cell properties; (k) It has regulatory functions for the p53 gene and telomeres.

11. The aforementioned bodily fluid is characterized by being blood, semen, intestinal fluid, saliva, or cell fluid of mammalian origin. The luteal according to claim 10.

12. Add water to the luteal according to claim 10 and culture at 18-30°C under IR light irradiation. A method for culturing luteal, including the following.

13. Before and after the aforementioned culture, the luteal size was 50-200 nm and 300 nm, respectively. The method for culturing luteal according to claim 12, characterized in that the wavelength is ~800 nm.

14. The lute according to claim 13, characterized in that the aforementioned water is saline solution or PBS solution. Real cultivation methods.

15. Method for separating luteal, including the following steps: (a) Centrifuge the body fluid to obtain the supernatant, and filter this supernatant through a filter with a pore size of 2-5 μm. The filtering step; and, (b) The filtered solution is subjected to secondary centrifugation to obtain the supernatant, and this supernatant is divided into 0.5 to 2 μm particles. A step of filtering with a filter of a certain pore size.

16. (c) Irradiate the filtered solution in step (b) with visible light, and then see Luteral particles, which have the ability to gather at the location where light is irradiated, are separated using a pipette. The method for separating luteal according to claim 15, further comprising the step of [doing something].

17. The aforementioned bodily fluid is characterized by being blood, semen, intestinal fluid, saliva, or cell fluid of mammalian origin. The method for separating luteal according to claim 15.

18. The claim is characterized in that the primary centrifugation is performed at 2000 to 4000 rpm for 5 to 30 minutes. The method for separating luteal as described in item 15.

19. The claim is characterized in that the secondary centrifugation is performed at 3000 to 7000 rpm for 5 to 20 minutes. The method for separating luteal as described in item 15.

20. An anticancer composition containing the luterial described in claim 10 as an active ingredient.

Images