Luterial and method for isolating and culturing same

Abstract

To provide a method for concentrating mitochondria-like nano-sized particles.SOLUTION: A method includes: (a) passing blood, from which platelets and blood-derived substances having a size equal to or larger than platelets have been removed, through a filter having pores of 0.8 to 1.2 μm; (b) centrifuging the filtered solution to provide a supernatant containing mitochondria-like nano-sized particles; (c) identifying the mitochondria-like nano-sized particles from the obtained liquid, by assessing whether the particles have one or more of several properties, including: (i) exhibiting a positive color reaction with Janus Green B, Acridine Orange, DAPI or Rhodamine 123 in an immunofluorescence test; and (ii) expressing a β-proteobacteria-derived gene and a γ-proteobacteria-derived gene and having a size of less than 800 nm in a healthy state.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to mitochondrial-like microscopic substances derived from body fluids, and their isolation. and a culture method. [Background technology]

[0002] Microscopic substances such as microvesicles in the blood were once recognized as substances without any special functions. However, microvesicles also have various biological activities. This has been made clear by various experimental data. For example, It has been revealed that vesicles function to stimulate specific cells via vesicular surface proteins. (CD154, RANTES and / or PF-4; Thromb. Haemost.(1999)82:794;J.Boil.Chem.(1999)2 74:7545), bioactive lipids in platelet microvesicles (e.g., HTET or ALA It has been reported that chidonic acid has specific effects on specific target cells (J. Bio l.Chem.(2001)276;19672;Cardiovasc.Res.(2 001)49(5):88). Thus, the presence of vesicles and other materials in biological samples Characteristics (e.g., size, surface antigens, cell of origin, payload) are used to diagnose and prognose disease. or provide therapeutic diagnostic information, making it easier to detect and treat disease. There is a need to identify biological indicators that can be used. Therefore, we aim to analyze the RNA and other biological indices associated with saccular lesions and their characteristics for diagnostic, prognostic, and clinical significance. There have also been attempts to use it for therapeutic diagnosis (see WO2011 / 127219).

[0003] On the other hand, cancer is a disease in which cells grow indefinitely and interfere with the function of normal cells. and lung cancer, gastric cancer (GC), breast cancer cancer (BRC), colorectal cancer (CRC), etc. Although it is typical for esophageal cancer, it can occur in any tissue in the parenchyma. Early cancer diagnosis is based on the development of cancer cells. Recently, however, blood, glycan (glyco chain) ain), diagnostic and diagnostic methods using trace amounts of biomolecules present in biological tissues or cells, such as DNA However, the most commonly used method for diagnosing cancer is biopsy. The diagnosis is made by using tissue samples obtained by scanning or by using images. The tests are not only very painful for patients and expensive, but also take a long time to make a diagnosis. Also, if the patient does have cancer, the cancer may spread during the biopsy process. In cases where tissue samples cannot be obtained by biopsy, The drawback is that it is impossible to diagnose the disease unless the affected tissue is surgically removed. In addition, in diagnostics using images, X-ray images and disease target substances are attached. Nuclear magnetic resonance images obtained using an adsorbed contrast agent Cancer was diagnosed based on NMR (NMR) images, etc. Diagnosis may be misdiagnosed depending on the skill level of the clinician or radiologist, and the accuracy of the equipment used to obtain the images. Furthermore, even the most accurate instruments have a high degree of accuracy, with a few meters It has the disadvantage that tumors smaller than 100 mm cannot be detected and are difficult to detect in the early stages of the disease. In addition, to obtain the images, patients or potential carriers must undergo a genetic test to determine whether genetic mutations can be induced. It is possible that exposure to high-energy electromagnetic waves could lead to other illnesses. Furthermore, there is a drawback in that the number of times diagnostics can be performed using images is limited.

[0004] That is, biopsy for cancer diagnosis requires a lot of time, expense, inconvenience, and pain. This method significantly reduces the number of people who are required to undergo unnecessary biopsies. There is a need for a method that can diagnose this early.

[0005] In this context, the present inventors have investigated the microorganisms present in the body fluids already excreted from the patient. We discovered that by observing quality characteristics, we can diagnose and predict diseases. The content of this invention has been applied for as a patent on July 12, 2013 (Korean Patent Application No. 10-2013- The inventors have referred to the above fine particles as "luterials." )"

[0006] However, in order to be able to apply the above fine materials to clinical practice, The techniques for isolation and cultivation are not yet known.

[0007] Therefore, the present inventors have investigated the microorganisms present in the body fluids already excreted from patients or healthy individuals. We have developed a method that can effectively separate the lutetium, which is a substance, and separated it using this method. The present invention was made by investigating the properties of the resulting ruthenium. Summary of the Invention [Problem to be solved by the invention]

[0008] The object of the present invention is to detect the presence of lutein in body fluids already excreted from patients or healthy individuals. The present invention aims to provide a method for isolation and cultivation of the microorganism.

[0009] Another object of the present invention is to provide a method for the production of prokaryotic and eukaryotic cells. te) intermediate fusion characteristics, and immunofluorescence ) test, Janus Green B (Janus Green B), Mitotracker Red ( Mito‐tracker Red, Rhodamine 123 They showed a positive immunochemical fluorescent staining reaction to ATP, and were motile and showed characteristics such as ATP production. The object of the present invention is to provide a literal having the above-mentioned properties. [Means for solving the problem]

[0010] In order to achieve the above object, the present invention provides a method for extracting platelets and platelets or larger sizes from blood. a first separation step of separating blood-derived substances having platelets and substances more than platelets; a second separation step of centrifuging the blood from which blood-derived substances having a size have been separated; a third separation step of separating the lutetium from the supernatant obtained by centrifugation; and washing the separated lutetium.

[0011] The present invention also provides a method for filtering a supernatant after first centrifuging a body fluid through a 2 to 5 μm filter. Step 2: After the second centrifugation of the filtered solution, the supernatant is filtered through a 0.5-2 μm filter. and filtering the resulting mixture through a filter.

[0012] The present invention also provides body fluid-derived luteins having one or more of the following properties: (a) Immunofluorescence assay using Janus green B, Acrigi 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 ectoplasmic genes and has a size of 30-800 nm; (c) Under acidic conditions, genes from β-proteobacteria and γ-proteobacteria In addition, it expresses the genes of Streptophyta, a eukaryotic cell. appear and increase in size from over 400nm to over 2000nm; (d) involved in ATP generation under normal conditions; (e) are cells or cell analogues entirely distinct from mitochondria and exosomes; (f) At steady state, the shape is round or oval, and when derived from a patient, the shape is larger than that at steady state. Mutant luteal tissues with large diameters (longer than 800 nm) and non-uniform morphology occur; (g) It has a double membrane structure and is adhesive; (h) can exist both intracellularly and extracellularly; (i) Motility and fusion and / or fission ssion) ecological patterns; (j) Under certain conditions, the mutated literal will burst. After bursting, they have stemness; (k) Regulates the p53 gene and telomeres.

[0013] The present invention also provides a method for producing a cellulose-derived body fluid-derived cellulose ... The present invention provides a method for culturing Lactobacillus luteus, characterized by culturing the Lactobacillus luteus at 18 to 30°C.

[0014] The present invention also provides an anti-cancer composition containing luterial as an active ingredient. [Brief explanation of the drawings]

[0015] [Figure 1] These photographs of luteal matter, a microscopic substance derived from blood, were taken using a confocal laser scanning microscope (Zeiss), a transmission electron microscope, a scanning electron microscope, an atomic force microscope, and a confocal scanner (Leica TCS-SP8). [Figure 2]The photographs show the shape and morphology of luminal membranes by size ((a): 39.6-49.0 nm, super-resolution microscopy (SR-GSD) photograph after staining with Mitotracker Red; (b): 50.1-85.1 nm, super-resolution microscopy (SR-GSD) photograph after staining with Mitotracker Red; (c): 76.5 nm, transmission electron microscope photograph; (d): 160 nm, transmission electron microscope photograph; (e): 170-230 nm, transmission electron microscope photograph, bilayer structure; (f): 234 nm, photograph after staining with Janus green B; (g): 250 nm, atomic force microscope photograph; (h): 361 nm, transmission electron microscope photograph). (i): 650.1 nm, Transmission Electron Microscope photograph; (j): Laser Scanning Microscope photograph of lutelia of 5 μm or larger in size after staining with DAPI (4',6-diamidino-2-phenylindole). [Figure 3] These are photographs of the luteal tissue stained with Rhodamine 123 and then observed for color development. [Figure 4] These are photographs of the luminal tissue stained with Mito-tracker and then observed for color development. [Figure 5] This is a photograph of the color development observed after staining the luteal with acridine orange. [Figure 6] These photographs were taken after staining the luteal tissue with DAPI (4',6-diamidino-2-phenylindole) to observe whether or not the tissue developed color. [Figure 7]These are photographs of the motility of lutelia measured using a nanotracker ((a) before measurement; (b) after 1 second; (c) after 3 seconds). [Figure 8] The life cycle A of a normal luteal and the life cycle B of a mutated luteal are shown. [Figure 9] The life cycle and characteristics of mutant luteal are shown. [Figure 10] These images show luteal cells isolated from the body fluids of cancer patients. (a) shows luteal cells from cancer patients with long branches and altered morphology, and (b) shows luteal cells from cancer patients stained with DAPI (4',6-diamidino-2-phenylindole), Mitotracker, and Rhodamine 123. [Figure 11] This shows the life cycle of a material. [Figure 12] This is a photo confirming the size of the Luterial and mutant Luterial. [Figure 13] This is a photograph of the film being scratched and removed using a titanium atomic force microscope probe. [Figure 14] (a) and (b) are images of mutated fused luteria taken with an atomic force microscope, and (c) and (d) are images of the mutated luteria taken with an atomic force microscope after the membrane was peeled off with a cantilever. [Figure 15] This is a photograph showing DNA confirmed by DAPI staining after scratching using a literal atomic force microscope probe. [Figure 16] (a) shows the results of a bioanalyzer that analyzed whether or not lutelia contain DNA, and (b) shows the results of qRT-PCR that showed that the size of the lutelia causes differences in the GAPDH gene expression of DNA. [Figure 17]These are the results of a bioanalyzer that analyzed whether or not RNA was contained in the luteal. [Figure 18] The ATP content was measured using the luciferin-luciferase reaction and a luminometer in culture media with different amounts of fisetin added (SSH: Sanseimaru, SSF: fisetin, 12h: activated for 12 hours at 37°C before the experiment). [Figure 19] This photograph allows observation of the difference between lutells and exosomes. Exosomes are those with an unclear membrane and a relatively light internal color, within the range of 20 to 120 nm in size, while lutells are those with a clear membrane or a filled internal color, within the range of 50 to 800 nm in size. [Figure 20] Photographs showing the morphologies of luterials, exosomes, and microvesicles for comparison. [Figure 21] 1 is a transmission electron microscope (TEM) photograph of a literal library constructed in one embodiment of the present invention. [Figure 22] These are confocal laser scanning micrographs showing the change in size of luteal cells during culture. [Figure 23] Photographs showing changes in morphology and size of L. luterial during culture. [Figure 24] Analysis of the 16S rRNA base sequences of blood-derived lumenal bacteria from healthy individuals (standard: pH 7.2-7.4) by size revealed similar bacterial compositions showing homology ((a): 100 nm or less; (b): 100-200 nm; (c): 200-400 nm; (d): 400-800 nm). [Figure 25] Analysis of the 16S rRNA base sequences of blood and semen derived luteal samples from fatigued and diseased individuals (pH 7.0 or less) revealed similar bacterial compositions showing homology ((a): 100 nm or less; (b): 100-200 nm; (c): 200-400 nm; (d): 400-800 nm). [Figure 26](a), (b), and (c) show phylogenetic diagrams based on the 16S rRNA sequences of blood-borne luterials. [Figure 27] The ovarian cancer cell lines SKOV3 and A2780 were treated with various concentrations of luteal with sizes ranging from 100 to 800 nm and the commercially available anticancer drug cisplatin, and the cell viability was measured using the MTT assay. DETAILED DESCRIPTION OF THE INVENTION

[0016] Unless otherwise defined, all technical and scientific terms used in this specification is the same as commonly understood by a person skilled in the art to which the present invention pertains. Generally, the nomenclature used in this specification and the experimental methods described below The methods are those known and commonly used in the art.

[0017] The term "luterial" as used herein refers to a vital factor present in animals. (living organism) and is approximately 800 nm from the level close to the virus Up to (normal fission step 50~800nm / non-normal fusion step 800 The inventor named the microscopic material having a size of 100 nm or more. (1) A cell or cell analogue that has fusion properties intermediate between prokaryotic and eukaryotic cells; (2) It is present in body fluids such as blood, semen, intestinal fluid, saliva, and cellular fluid; (3) It is detected by immunofluorescence testing. Janus green B, Acridine orange ne Orange, and Rhodamine 123 positive (4) Under optimal conditions (pH 7.2-7.4), β-proteobacteria and γ-proteobacterial gene expression characteristics, and the size ranges from 30 to 800 nm. (5) Under acidic conditions, the β-proteobacteria and γ-proteobacteria-derived It shows the expression characteristics of not only native genes but also eukaryotic genes, but mainly streptophytes. (Streptophyta) gene and ranges from over 400nm to over 2000nm. (6) involved in ATP production under normal conditions; and (7) involved in the production of mitochondria. Unlike lysosomes, exosomes are completely different cells or cell analogues. In animals, including humans, the sources of the virus are blood, saliva, lymphatic vessels, semen, vaginal fluid, and breast milk (especially colostrum). It is found in the umbilical cord blood, brain cells, spinal cord, and bone marrow. In addition, in animals with horns, it is also found in the horns. There is a literal.

[0018] The size of normal luteal cells is 50-800 nm, and mutant luteal cells are fused and mutated. They form a membrane that is several tens of micrometers in size. It can refer to immature mitochondria that contain not only A but also DNA. Rials have the characteristic of not dissolving in digestive fluids and flowing into the blood.

[0019] The cellular fate is involved in signaling, cell differentiation, cell death, as well as the cell cycle and cellular metabolism. It is expected that lutein is also involved in the regulation of cell growth, but the present inventors have found that lutein is particularly useful for cancer diagnosis. was found to be closely related.

[0020] Normal luterial suppresses the growth of cancer cells and It is expected that this will help restore a healthy immune system, but this role is to normalize genes. RNAi (RNA interference) has the potential to Thus, in the blood of healthy people and animals, luteal is the The information system goes off track and directs the production of abnormal disease-causing proteins. In this case, artificial interference can suppress the occurrence of diseases such as cancer, and When it matures to 200-500 nm or more, it is also involved in energy metabolism and is irradiated with specific wavelengths. It was then confirmed that the reaction manifests as a light energy amplification function, and reacts like a chloroplast. Therefore, when these natriuretics are unable to perform their normal functions, homeostasis and It induces critical impairments in ATP production and both respiration and energy metabolism. There is a risk of causing illness.

[0021] Thus, mutant luterals that cannot perform their role normally are like normal luterals. The biology and characteristics of these organisms are different from those of the normal algae, and their sizes and shapes vary. After forming double spores, the luteal cells cease to grow. However, mutations found in the blood of cancer patients and those with late-onset diseases are not related to stem cells. They have the property of infinite proliferation similar to cells, and have a size of 600 to 800 nm or more. Some viruses have a size of 200 μm (200,000 nm) or more. Similarly, they invade red blood cells, white blood cells, platelets, etc., grow, and aggregate with other erythrocytes. It shows characteristics such as

[0022] Therefore, by observing the morphological or biochemical characteristics of the luteal tissue, the diagnosis of the disease can be determined. It is possible to diagnose and treat diseases, and its applications are expected to be limitless. Luteral isolated from body fluids already excreted from the body is dissolved in vitro within a short time. They tend to disappear or change form when exposed to heat, making observation difficult. If left untreated, normal luteal will be transformed into mutant luteal within 24 hours. However, there is a problem in that it is difficult to accurately diagnose and treat the disease.

[0023] In the present invention, the microscopic material present in the body fluid already excreted from a patient or a healthy person is called lubricant. Terials were separated by two methods.

[0024] Thus, the present invention in one aspect relates to a method for separating lutein from a body fluid.

[0025] The first method is a method for separating lutein from blood, which comprises extracting platelets and erythrocytes from the blood. a first separation step of separating blood-derived substances having a size equal to or larger than a platelet; The second fraction is centrifuged to separate blood from which blood-derived substances with a size larger than that of platelets are separated. and a third separation step for separating the lutein from the supernatant obtained by the centrifugation. and washing the separated lumen.

[0026] The first separation step involves passing the blood through a filter with pores of 0.8 to 1.2 μm. and separating the unfiltered material. Step 1: Repeat centrifugation at 1200-5000 rpm for 5-10 minutes to separate the exosomes. 2. A method for producing a supernatant by removing general microvesicles such as exosomes. The third separation step involves irradiating the supernatant obtained by centrifugation with visible light. and separating the motile and aggregated lunar particles using a pipette. The blood used in the first step is derived from humans among mammals. The luterial has the properties of autofluorescence and motility, and therefore, When irradiated with visible light, the lutein particles can be seen in the supernatant. While observing the moving literal particles under a dark-field or confocal microscope, The ruthenium separated in the third step can be separated using a 50n diameter. The solution was passed through a filter with pores of 100 μm, and only the unfiltered portion was washed with PBS. Luterials can be obtained by the above method. Luterials have a major axis of 50 nm or more. Therefore, by this process, microscopic substances derived from blood other than lutein can be removed.

[0027] The second method is a method for separating lutein from body fluids such as blood and semen, After the first centrifugation, a step of filtering the supernatant through a 2 to 5 μm filter; a step of subjecting the resulting solution to a second centrifugation and then filtering the supernatant through a 0.5 to 2 μm filter; Includes:

[0028] That is, the body fluid was centrifuged at 2000-4000 rpm for 5-30 minutes, and the supernatant was Filter the solution through a 2-5 μm filter and sieve the filtered solution at 3000-7000 rpm for 5-2 and filtering the mixture through a 0.5-2 μm filter after a second centrifugation for 10 minutes. can.

[0029] The solution obtained by filtration is irradiated with visible light to generate luminous particles that become mobile and aggregate. The method may further include the step of separating the fluorescent material using a pipette. Because of its photo- and motility properties, when irradiated with visible light as described above, lutein was extracted from the supernatant. The moving literal particles can be observed using a dark field microscope or The cells can be separated using a pipette while checking under a confocal microscope. The Al was passed through a filter with pores of 50 nm diameter, and only the unfiltered portion was collected as P By washing with BS, ruthenium can be obtained. Ruthenium has a long diameter of 50 nm or more. Because of this size, the above process can remove blood-derived microscopic substances other than luteal. This can be done.

[0030] The luminal regions separated by the above two methods were analyzed using dark-field or confocal microscopes. These can be observed using a mirror, and are 200nm, 400nm, 600nm, 800nm, and 1000 nm filters in sequence to separate the 50-200 nm (nascent stage) particles according to their size. ) / 200~400nm(mature phase) / 400~600nm(mitotic phase) / 600~800n m (hypermitotic phase).

[0031] In the present invention, the properties of the separated lutetium were investigated.

[0032] (1) Morphology The size of normal luminal is 50-800 nm (Fig. 2 and Fig. 12), and In the absence of IL-1, the cells mature to 800 nm. Mutant luteria are larger in size (longest diameter 800 nm or more) and have a non-uniform morphology compared to the normal luteria. When fusion occurs, the size of the luminal increases to several thousand nanometers. It was confirmed that

[0033] The crystalline is circular or elliptical, and is not visible in SEM or TEM electron microscope photographs. It has a double membrane structure similar to that of chondria, but has an internal cristae structure. (Figure 1).

[0034] (2) Immunofluorescence staining Mitochondria were stained with Janus green B and fluorescent The dyes Rhodamine 123 and Mitotracker o-tracker, Acridine Orange and It is known that the color develops with DAPI, but the luteal layer also has the same staining as mitochondria. The color development by the coloring agent was confirmed. The luteal membrane was stained with immunofluorescent staining similar to mitochondria. While the exosomes showed a color reaction, the exosomes showed the opposite reaction. The characteristics showing autofluorescence were confirmed from the fluorescence photograph (Fig. 2( a), Figure 2(b), Figure 2(f), Figure 2(j), Figures 3 to 6).

[0035] (3) Ecological pattern Luterials are exosomes and microvesicles. Unlike the le), they have adhesive and motile properties and can form fusion or The ecology of fission is shown. Under certain conditions, the mutated literal Bursting occurs, and after bursting, stem cells Confirm that the protein has stemness and can exist either intracellularly or extracellularly. We confirmed this (Figures 8, 9, and 11).

[0036] (4) ATP production Luciferases are known to produce ATP from luciferases with a size of 200-400 nm. Luciferin-luciferase reaction and luminometry The group to which luterial was added showed The ATP concentration increased compared to the group without luterial. The conclusion can be drawn that SSH and SSF have the ability to produce ATP. Regarding the difference due to the SSF addition, the ATP concentration was higher in the SSF addition group than in the SSH addition group. From these results, a culture medium capable of efficiently increasing the ATP content was identified (FIG. 18).

[0037] (5) Contains nucleic acid DAPI and acridine orange (AO) staining revealed that only RNA was present in the lumen. It was confirmed that the RNA contained not only the RNA but also DNA. The dye produces orange light at an excitation level of 460 nm and an emission level of 650 nm. The DNA is stained green at excitation 502 nm and emission 525 nm. The presence of DNA can be confirmed by the API staining method. Using this method, it was confirmed that RNA and DNA are contained in the luteal of the present invention (Figure 1). 5 and 6). In addition, RNA and DNA in the urinary tract were isolated and analyzed using a kit. After purification, the bands were confirmed by agarose gel electrophoresis. By checking the GAPDH expression level using PCR, it was possible to determine the difference in the size of the luteal membrane. We confirmed that there were differences in gene expression between the two groups (Fig. 2(h), Fig. 16 and Fig. 17).

[0038] (6)16S rRNA sequence analysis FastDNA SPIN Kit (MP Biomedicals, Cat 656 After extracting the human gDNA using a 100-mL ELISA kit (Table 1), specific primers were used. The 16S rRNA gene was amplified using a primer.

[0039] The 1,461 amplified gene fragments were analyzed using the GeneBank database. The homology analysis using ase (NCBI database) revealed that blood and semen The 16S rRNA sequences of the derived luteal species were classified as β-proteobacteria (β-Prot eobacteria, γ-Proteobacteria ), Acidobacteria, Cyanobacteria bacteria), Actinobacteria, Farmi Firmicutes and Eukaryote-derived genes It showed homology with the nucleus and possessed fusion properties intermediate between prokaryotic and eukaryotic cells (Figure 24-Figure 25). twenty five).

[0040] Under optimal conditions (blood pH: 7.2-7.4), blood-derived lutein is converted into β-proteobacteria. β-Proteobacteria, γ-Proteobacteria teobacteria, and Bacteroidetes They show homology to genes (Figure 24) and are observed to be 50 to 800 nm in size.

[0041] At steady state, seminal fluid-derived luterials were enriched for β-proteobacteria, γ-proteobacteria, and It shows homology to genes from A. cerevisiae, Bacteroidetes, and Chordata.

[0042] In contrast, under acidification conditions, the β-proteobacteria (β-pro) teobacteria and γ-Proteobacteria Not only genes derived from ria but also genes derived from bacteria are expressed in a more diverse manner, Eukaryote-derived genes are also expressed. 16S rRNA characteristics of ptophyta and planctomy The size of the crystals grows to 400 to 2000 nm (Fig. 25).

[0043] [Table 1]

[0044] [Table 2]

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

[0046] [Table 3]

[0047] The average size of the mitochondrion is 200-800 nm, and the average size of the mitochondria is 400-1 It is smaller than the globular particle (0,000 nm) but larger than the exosome (20-120 nm). Exosomes have a relatively pale internal color and no clear membrane division, whereas luteal cells have a relatively pale internal color and a clear membrane division. The division of the porcelain is clear or the interior is filled (Fig. 19). , exosomes, and microvesicles have completely different morphologies. (Figure 20).

[0048] In immunofluorescent staining, luteal cells reacted similarly to mitochondria, whereas endothelial cells reacted similarly to mitochondria. The cytoplasmic membranes are located inside and outside the cells, whereas the membranes are located outside the cells. Exosomes are also substances obtained by ingesting food, whereas mitochondria The difference is that it is found only intracellularly and cannot be obtained through food intake.

[0049] And unlike exosomes and mitochondria, luteal cells are motile. It is capable of spontaneous growth and can be maintained in culture, and exhibits autofluorescence characteristics. In addition, luteal, exosomes, and mitochondria are all living forms of fusion. Although the exosomes have the formula, kiss-and-run fusion does not occur, and A TP production also does not occur. Also, exosomes exist extracellularly, while mitochondria exist intracellularly. whereas luteal can exist both extracellularly or intracellularly (Figure 11).

[0050] In addition, the results of 16S rRNA base sequence analysis showed that mitochondria are α-proteobacteria. The γ-Proteobacteria (γ-Proteobacteria) are homologous to the γ-Proteobacteria. bacteria), β-Proteobacteria, Bacteroidetes, Firmicutes tes), and eukaryotes.

[0051] In another aspect, the present invention relates to body fluid-derived luteins having one or more of the following properties: (a) Immunofluorescence assay using Janus green B, Acrigi 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 ectoplasmic genes and has a size of 30-800 nm; (c) Under acidic conditions, genes from β-proteobacteria and γ-proteobacteria In addition, it expresses the genes of Streptophyta, a eukaryotic cell. appear and increase in size from over 400nm to over 2000nm; (d) involved in ATP generation under normal conditions; (e) are cells or cell analogues entirely distinct from mitochondria and exosomes; (f) At steady state, they are round to oval, and when derived from patients, they are larger ( Mutant luteal cells with large diameters (longest diameter 800 nm or more) and non-uniform morphology occur; (g) It has a double membrane structure and is adhesive; (h) can exist both intracellularly and extracellularly; (i) Motility and fusion and / or fission ssion) ecological patterns; (j) Under certain conditions, the mutant literal bursts and bursts. After bursting, the cells have stemness; and (k) Regulates the p53 gene and telomeres.

[0052] On the other hand, the size (diameter), area, shape, and Because of the different nanotracking speeds, one or more of these properties can be used to diagnose or prognose disease. This can be predicted by comparing the luteal fraction from healthy individuals without disease with that from individuals with disease. This can be seen from the differences in the size, shape, and nanotracking speed of the derived lutetium.

[0053] Normal luteal cells from healthy individuals only form double spores. However, the luteal (sudden mutation) derived from patients with late-onset diseases or cancer Heterogenous lutetium is a type of lutetium that is formed by fusion or coagulation. They adhere to cells such as red blood cells and cancer cells by bursting or exploding. However, its shape and size differ from normal luteal tissue and it is characterized by becoming abnormally large. (Figures 8 to 10). Because mutant luteal has high adhesiveness, the above cycle (c Fusion is further accelerated by the cycle, and the size increases to about 600-800. 00nm or larger, and have a size of 200μm (200,000nm) or larger The present inventors have investigated the mutated luteal forms depending on the type and progression of cancer. and has filed a patent application for this content (Korean Patent Application No. 10‐2013‐0082060).

[0054] Therefore, by observing the morphological or biochemical characteristics of the luteal tissue, the diagnosis of the disease can be determined. It can predict diagnosis and prognosis, and its uses are endless.

[0055] The morphology of the urtial is normal, flagellate, mass, rod, or The normal form may be a different fusion or bursting. The ratio of the major axis to the minor axis is 1:1 to 3:1 without deformation, and the shape is close to a circle. When viewed under a microscope, they appear as small dots.

[0056] Flagellate is a form in which the luteal is transformed or fused to form a flagellum on the outside. The present inventors have found that the rate at which flagellated cells are observed increases sharply as cancer progresses to the terminal stage. The percentage of patients diagnosed with stage 4 cancer was 99.1%, and flagellated lutells were found in almost all patients diagnosed with stage 4 cancer. It was confirmed that the ruthenium was observed (Korean Patent Application No. 2013-0082060). If the morphology of the cytoplasm is 80-100% identical to that of the flagellate morphology, it is suspected to be a terminal tumor. The tumor can be displayed using a tumor marker. The patient's survival period is approximately 1 to 4 months, and in the flagellate form in particular, long-term survival is impossible.

[0057] Mass type (M type) means that the literal is bursting or fusion. The size and shape are deformed from the normal shape due to the large difference between the major and minor diameters. Preferably, the ratio of the major axis to the minor axis is 3:1 to 5:1. A variety of grid shapes can be observed.

[0058] The rod type (R type) is suitable for bursting, deformation, and The difference between the length of the short diameter and the long diameter is the mass. Preferably, the ratio of the major axis to the minor axis is 5:1 to 12:1. The rod-shaped structure includes rod 1, which consists of a single circular or oval chain, and rod 2, which consists of two or more single chains. The rod type 1 includes a single literal in the form of a rod. This is due to bursting and / or deformation. The rod type 2 can be formed by combining two or more literals into a rod shape. This resulted in bursting, deformation, and melting. On the other hand, flagellates can be classified into a large range of types based on their shape. They can be contained in rod-shaped cells, but differ in that they have elongated flagella. Therefore, it is possible to determine whether it is rod-shaped first, and then whether it is flagellate-shaped. do.

[0059] The composite shape may be a combination of a rod shape and a mass shape. A form in which some of the fine material is rod-shaped and some is mass-shaped can be called a composite form. .

[0060] The rod-shaped structure includes a rod-shaped structure consisting of a single circular or elliptical chain, and a single The complex form may be one of the group including rod type 2, which is formed by linking two or more single chains. can be a hybrid of rod and mass shapes.

[0061] As mentioned above, the morphology of luteal in vivo changes with the onset and progression of the disease. Observation of the morphological characteristics of the luminal tissue can help in diagnosing and predicting the prognosis of disease. The morphological changes of the luteal also affect the amount and sequence of nucleic acids contained in the luteal. To analyze the nucleic acid expression patterns (16S rRNA) of the luteal can diagnose the disease.

[0062] For example, the 16S rRNA sequence of a normal luteal and the 16S rRNA sequence of a patient's luteal By comparing the DNA sequences, diseases (especially cancer) can be diagnosed. Streptophyta gene expression and eukaryotes Gene co-expression can be used as a diagnostic and predictive marker for cancer.

[0063] However, luteal cells isolated from body fluids already excreted from patients or healthy individuals are not suitable for in vitro In the case of quartz, it has the property of dissolving and disappearing in a short time, or changing its form, so it is difficult to observe. If the body is left in an abnormal environment, normal luteal tissue may suddenly return to normal within 24 hours. However, the present invention is not suitable for accurate diagnosis and treatment of diseases. According to the culture method, the luteal cells are cultured so that they do not grow to a specific size (500 nm). It is possible.

[0064] Therefore, in another aspect, the present invention provides a method for adding water to rutile and then irradiating the rutile with IR light. Cultivation of Lactobacillus luteum, characterized by culturing at 18 to 30°C (preferably 20 to 25°C) Regarding the method.

[0065] The water added during the culture may be a saline solution or a PBS solution. The pre-cultured body fluid-derived lutein can be obtained by the separation method of the present invention. The size of the cells can be 50 to 200 nm. The size of cultured blood-derived lutein can be 300-800 nm. At this time, observe under a microscope to ensure that the size of the ruthenium does not exceed 500 nm. After the cultivation was completed, the cells were sorted by size, cooled to -80°C, and stored. It can be stored under nitrogen or above zero temperature, and no preservatives are added during storage. It is possible.

[0066] The cultured luteal cells can be stored for a certain period without losing their properties. It is expected that this technology will be effectively utilized in the diagnosis and prognosis prediction of diseases using artificial In the present invention, "without changing the properties of the liter" means that the shape of the liter The morphology and size of the cells are maintained almost similar to the state before culturing in the medium. In addition, the activity of the luminal motility such as nanotracking speed is This means maintaining a value similar to the state before cultivation.

[0067] Specifically, the luteal cultured by the culture method of the present invention can be used for the following purposes: The mutated mutant luteal can be fused or aggregated. It is an abnormally large tumor whose shape and size differ from normal tumors. During the culture of the isolated mutant luteal cells (Figs. 8 to 10), luteal fusion or The present invention provides a method for treating luteinized fusion or aggregation by treating candidate substances or means capable of suppressing aggregation. By observing whether or not suppression occurs, we will identify substances that can suppress or prevent luteal mutations. It can be screened.

[0068] In addition, when the isolated lutelia are cultured, conditions that promote fission are By processing auxiliary materials and means, the fission of mutant literals Mutant luteal cells can be screened for substances that promote fusion or aggregation. The ecology of the fission rocks is characterized by the accumulation of crustaceans (Figs. 8, 9 and 11). ) ecology, or mutated literals are fission (fission fission (fission) to have the normal luminal size. By treating the candidate substance that promotes on, can we suppress the mutation of luteal? , a substance that converts mutated luteal to have the biology of normal luteal, Ultimately, screening for preventive agents for diseases that may be induced by mutated luteal It is possible.

[0069] The present invention will now be described in more detail with reference to the following examples. These examples are intended to illustrate the present invention. The scope of the present invention is not to be construed as being limited by these examples. This is obvious to those skilled in the art.

[0070] Example Example 1: Separation of blood-borne lutein 50cc of blood was collected from patients with terminal non-small cell lung cancer, and voids with a diameter of 0.8μm or more were identified. The filtered blood was then passed through a filter to separate unfiltered material. Repeated centrifugation at 5000 rpm for 5-10 min allowed the exosomes to be isolated. The supernatant was then irradiated with visible light to obtain the supernatant. The mobile and aggregated luminal particles were separated using a pipette. and mobility properties, the luminal particles are irradiated with visible light as described above. In this case, the moving literal particles can be observed using a dark-field microscope or a confocal microscope. The separated lutetium was separated using a pipette while checking the temperature. The unfiltered portion was washed with PBS. The above process yielded lutetium with a major axis of 50 to 800 nm, which is The obtained luster was observed and confirmed by a field microscope or a confocal microscope. Depending on the size, 50-200nm (nascent stage) / 200-400nm (mature stage) / 4 The cells were divided into 2 groups: 00-600 nm (mitotic phase) and 600-800 nm (hypermitotic phase). By doing this, we can build a library for each size of the literal as shown in Figure 21. The morphology of each size is shown in Figure 2.

[0071] Example 2: Separation of semen-derived lutein After the primary centrifugation of the semen at 2000-4000 rpm for 5-30 minutes, the supernatant was collected by 2-5 μL Filter the filtered solution through a 1000-millimeter filter and incubate it at 3000-7000 rpm for 5-20 minutes. After centrifugation, the solution was filtered through a 0.5-2 μm filter. Due to its dynamic properties, luminal particles can be identified by irradiating the filtered solution with visible light. In this case, the moving literal particles can be observed using a dark-field microscope or a confocal microscope. The separated lutetium was separated using a pipette while stirring. The unfiltered portion was washed with PBS to obtain a luteinized solution. This could be confirmed by observation using a dark-field microscope or a confocal microscope.

[0072] Example 3: Properties of Lutein (1) Structure Among the ruthenium obtained in Example 1, ruthenium having a size of about 50 to 400 nm The images were analyzed using a confocal laser scanning microscope (CLSM). icroscope (Zeiss), transmission electron microscope (Transmission E lectron microscope, scanning electron microscope electron microscope, atomic force microscope Microscope) and confocal scanner (Leica TCS‐SP8) As a result, it was found that the mitochondrion also has a double membrane structure similar to that of the mitochondria, and the inner cytoplasm The liste structure is incomplete, and the mitochondrion It was confirmed that the morphology of the nuclei was observed in the same laser wavelength range as the nuclei of the rear. It was observed that the shape was elliptical (Fig. 1, Fig. (e), Fig. 2(h), Fig. 13 and Figure 14).

[0073] (2) Dyeing properties Among the ruthenium obtained in Example 1, ruthenium having a size of about 50 to 800 nm The markers were stained with Mitotracker, Rhodamine 123, and 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 luteinizing agents such as Mitotracker, Rhodamine 123, and Acridine oleate were also found. It was confirmed that the dye was colored by Janus Green B (Fig. 2(a), Fig. 2(b)). b), Figure 2(f), Figure 2(j), Figures 3 to 6).

[0074] (3) Autofluorescence Among the ruthenium obtained in Example 1, ruthenium having a size of about 50 to 800 nm Fluorescence photographs confirmed that the cells reacted to light (Figure 5).

[0075] (4) Motility The mobility of the luteal obtained in Example 1 was measured by nanotracking from 3i Corporation in the United States. Specifically, after observing the luminal under a bright-field microscope, a track was placed at the center of the luminal. When you set the tracking and activate nanotracking, the real-time tracking will follow the movement of the object. The time movement trajectory was displayed and its speed per second was calculated (Figure 7).

[0076] As a result, the nanotracking speed of the present example is about 13 to 25 μm / s. sec was measured.

[0077] (5) Analysis of the presence or absence of RNA and DNA in luteal tissue The 200 to 400 nm ruthenium separated in Example 1 was photographed with an atomic microscope. As shown in Figure 2(h), Figure 15, Figure 16 and Figure 17, RNA and DNA It can be assumed that nucleic acid such as A is contained.

[0078] Total RNA and DNA were separated from the 200-400 nm lutein separated in Example 1. To do this, we used a QIAGEN kit (RNeasy Micro Kit: Cat 7400 4) and then analyzed by Experion RNA (DNA) StdSens (Bi Quantification was performed using a o-Rad chip.

[0079] The luteal was collected by centrifugation (8000 g, 1 hour 30 minutes), and then digested in the kit. Buffer RLT plus (Guanidine isothiocycanate, de Add 3.5 μl of β-mercaptoethanol to 50 μl of eluent and measure using a 20 gauge The lutein was dissolved by passing the sample through a syringe with a needle 5-10 times. The buffer was transferred to an AllPrep DNA spin column and then centrifuged (≥8000g 15 seconds), and then the buffer containing the DNA that has accumulated in the column and the RNA that has passed through the column is removed. They were separated from each other.

[0080] First, add 350 μl of 70% ethanol to the buffer that has passed through the column. After mixing evenly, 700 μl of the mixture was poured onto an RNease MinElute spin column. Transfer to a column and centrifuge (≥8000 g, 15 seconds). Remove the buffer that passed through the column. 350 μl of RW1, 500 μl of RPE buffer and 500 μl of 80% The column was washed stepwise with ethanol. ≥ 8000g, 15 seconds) were performed under the same conditions. After the sy-free solution was added to the column, it was centrifuged (≥8000 g, 60 seconds) to separate the lutetium. RNA was isolated.

[0081] For genomic DNA isolation, we used the FastDNA SPIN Kit (MP Biome After placing the separated luteal in the tube, 978 μl of phosphorus Sodium phosphate buffer, 122 μl of MT buffer The mixture was homogenized for 40 seconds and then centrifuged (14,000 g, 10 minutes) to separate the supernatant. After obtaining the supernatant, 250 μl of PPS (Protein Precipitation Solution) was added. After centrifugation (14000g, 5 minutes), The process of transferring the supernatant to a 5 ml tube was repeated twice to measure DNA binding. After placing it on the rotor for 2 minutes for g), it was placed on a silica matrix stand for 3 minutes. Approximately 600 μl of the supernatant was carefully collected and placed in a spin filter. After centrifugation (14000 g, 1 min), the supernatant was discarded and the pellet was treated with SE. 500 μl of WS-M was added and suspended. Centrifuged for 1 minute. After that, the supernatant was discarded and the centrifugation was repeated to ensure that no buffer remained. Add DES (DNase / Pyrogen-Free Water) and centrifuge (14 After centrifugation (1000×g, 1 min), genomic DNA was obtained.

[0082] Experion RNA (DNA) StdSens (Bio-Rad) chips were used. As shown in Figures 16 and 17, the amount of RNA in the luteal phase was 100%. It was confirmed that the DNA was contained in the sample.

[0083] (6)16S rRNA sequence analysis 16S rRNA (ribosomal ribonucleic acid) It is RNA that interacts with various proteins to form ribosomes. The rate of sequence variation is significantly smaller than that of other genes in most genomes. It is now recognized that the degree of 16S rRNA sequence similarity reflects the phylogenetic distance between organisms. are.

[0084] [1] Blood-derived luteal The blood-derived lutein obtained in Example 1 was extracted using the FastDNA SPIN Kit (MP After extracting gDNA using a ELISA kit (Biomedicals, Cat. 6560‐200) , PCR-premix (iNtRON Biotechnology, Korea) Primers numbered 1 to 23 were used to extract 16S rRNA from the lutein. Amplified.

[0085] The amplified PCR products were analyzed 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 PCR products. A total of 1,461 fragments were amplified using PCR. Of these, 1407 fragments were from Proteobacteria. ia gene, and 20 fragments were identified as Acidobacterium teria-derived genes, and 11 fragments were identified as Actinobacteria-derived genes. The gene showed homology with genes derived from B. nobacteria (Table 4).

[0086] The analyzed base sequence fragments were assembled using SeqMan software (DNASTAR). In addition, the 16S rRNA sequence was obtained.

[0087] Figure 24 shows the 16S r of blood-derived lutein from healthy individuals (blood pH: 7.2-7.4). RNA sequence analysis revealed the structure of similar bacteria showing homology. The analysis was conducted by size of the luminal ((a): 100 nm or less, (b): 100-200 nm). (c) 200-400 nm, (d) 400-800 nm). There is no significant difference between the sizes. Both belong to the Proteobacteria and Firmicutes (Firmicutes), and Bacteroidetes It showed homology with the gene.

[0088] Figure 25(c) shows blood-derived lutein in a fatigued or sick state (blood pH: 7.0 or less). (Size: 200-400nm) 16S rRNA base sequence analysis shows homology The composition of similar bacteria is shown. Unlike the steady state, streptophytes Genes showing homology to genes from (Streptophyta) were also expressed.

[0089] Figure 26(a), (b), and (c) show the 16S rRNA sequences of blood-derived luteal A column-based phylogenetic diagram is shown.

[0090] [Table 4]

[0091] The 16S rRNA fragments of blood-derived luteal bacteria were classified as β-proteobacteria, γ-proteobacteria, and Obacteria, Bacteroidetes, Firmicutes, and Streptophyta It was confirmed that it shows homology with various bacteria.

[0092] Generally, gDNA relatedness within the microbial taxonomy is less than 70% In addition, the 16S rRNA base sequences of the two strains are similar to each other, and they are recognized as independent strains. Conspecificity less than 97% is statistically indicative of gDNA relatedness less than 70%. Therefore, the 16S rRNA fragment of the lutein was 97.0%. As a result of analyzing cells having the above homologous sequences, as shown in Tables 5 to 7, The derived luteal species showed 100% homology with γ-proteobacteria and Firmicutes It showed 97.53% homology with S. spp. and over 97% homology with Bacteroides spp.

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

[0094] [Table 5] TIFF2025063161000006.tif201160TIFF2025063161000007.tif201160TIFF2025063161000008.tif180160

[0095] [Table 6]

[0096] [Table 7] TIFF2025063161000011.tif201160TIFF2025063161000012.tif201160TIFF2025063161000013.tif202160

[0097] [Table 8] TIFF2025063161000015.tif180160

[0098] [2] Semen-derived luteal The semen-derived luteal obtained in Example 2 was subjected to gDNA extraction by the method described above. The semen pH was measured using PCR amplification and sequence analysis. The 16S rRNA sequences of semen-derived luteal samples (<7.0) were analyzed to confirm the homology. The composition of similar bacteria shown is analyzed by lunar size (( a): 100nm or less, (b): 100~200nm, (d) 400~800nm).

[0099] Steady-state semen-derived luterials, like blood-derived luterials, are rich in Proteobacteria. (Proteobacteria), Firmicutes, and It shows homology with genes derived from Bacteroidetes and has a unique In particular, it showed homology with genes from chordata.

[0100] In addition, under abnormal acidic conditions, Streptophyta Genes with homology to the derived gene were expressed.

[0101] (7)ATP content measurement Control group, Luteal, Luteal (SSH 12h), and Luteal (SSF 1 10 mL of each of the four culture solutions (2 h) was placed in a tube and glucose (Gl Add 100 mg / mL of ucose (100 mg / mL) and 1 mM ADP (1 mM) substrate and place in a water bath at 37 °C. After the start of incubation, 100 μl of sample was taken at 30-minute intervals and placed in a tube. After adding μl of distilled water to dilute the sample 10 times, transfer 10 μl of the sample to a new tube and Add 100 μl of luciferase reagent included in the TP kit and immediately measure with the luminometer. The measurements were repeated five times using the same meter.

[0102] As a result, as shown in Figure 18, in the group to which ruthenium was added, The ATP concentration increased compared to the control group. It was confirmed that the soil had the ability to produce P. Regarding the difference, the ATP concentration was higher in the SSF-added group than in the SSH-added group (FIG. 18).

[0103] Example 4: Cultivation of Lactarius luteus (1) Among the ruthenium obtained in Example 1, the ruthenium having a size of about 50 to 200 nm PBS was added to the culture medium, and the medium was irradiated with IR light and then incubated at 18-30°C for approximately 3 hours. The size of the lunar particles was checked under a microscope at intervals of approximately one hour immediately after irradiation. After 6 hours, the luteal cells, which were approximately 200 nm in size before incubation, grew to approximately 500 nm. This confirmed that adding water to blood-derived lutein resulted in IR When cultivated at 18-30°C under light irradiation, the size of the cells grows to about 500 nm. Of course, it was confirmed that additional culturing could result in culturing up to several hundred μm. It was also confirmed that bursting occurred when the cells were further cultured at 1000 kJ / mL (Figure 22 ).

[0104] (2) Among the ruthenium obtained in Example 1, ruthenium having a size of about 400 to 800 nm PBS was added to the cells, and the cells were irradiated with IR light and then cultured at 18-30°C for approximately 3 hours. The size and condition of the luteal were checked under a microscope at approximately 1-hour intervals immediately after irradiation with R light. After about 1 to 6 hours, the luteal cells, which were about 400 to 800 nm in size before the culture, began to grow. It was confirmed that fission occurs without any change in the crystal structure.

[0105] In addition, when mutant luteal of 800 nm or more is cultured, the mutations shown in the blood of cancer patients It was observed that the hydroxylase was converted into a heterocyclic morphology (Figure 23).

[0106] Example 5: Anti-cancer effects of luteal To measure the growth inhibitory effect of two ovarian cancer cell lines, SKOV3 and A2780, Color tetrazolium MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl The MTT assay was performed to measure the viability of living cells. The method for measuring growth of dehydrogenase in mitochondria of living cells is shown in Fig. 1. It utilizes the principle of producing purple formazan by the reactive substance MTT. The production of purple formazan is determined by the amount of metabolically active living cells. It is known to be roughly proportional to cell number and is very effective in measuring cell growth and differentiation. It can be used.

[0107] Cultured each cancer cell in a 96-well plate at 5 x 10 4 So that it becomes 1 / ml 100 μl was added per well and incubated at 37°C with 5% carbon and 95% oxygen. After 24 hours of incubation in a humidified incubator, the luteal cells with sizes between 100 and 800 nm were concentrated. After 48 hours of incubation, each well was treated with phosphate buffer solution (phos MTT (5 mg / m ) dissolved in phosphate-buffered saline (PBS) 15 μl of formazan solution was added to each well and the mixture was further cultured for 4 hours. After confirming the formation of n), the medium was completely removed and the formazan formed at the bottom of the well was dissolved. To dissolve the mixture, 100 μl of dimethyl sulfoxide (DMSO) was added. Using a clotting plate reader (GEMINI, Stratec biomedical) Absorbance was measured at 560 nm, and the relative cell growth inhibition was calculated with control cells taken as 100%. The rate was calculated.

[0108] As a result, the IC50 of luteal in SKOV3 and A2780 cell lines was 30. The IC50 values ​​of the commercially available anticancer drug cisplatin were 60μg / ml and 60μg / ml. The activity of luteal was 100 μM (Figure 27). The cytotoxicity of the ovarian normal cells was similar to that of the positive control drug group. showed sexuality.

[0109] The present invention has been described in detail above, but it is understood by those skilled in the art that Therefore, these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is not limited to the appended claims. It is to be defined by the claims and their equivalents. [Industrial Applicability]

[0110] According to the present invention, it is possible to effectively treat luteria, which are minute substances present in the body fluids of patients or healthy individuals. The separated luminal can be effectively separated and grown to a predetermined size. Because they can be cultured, they are useful for the diagnosis and treatment of diseases. It is useful as an anticancer agent because it exhibits strong anticancer effects against cell lines.

Claims

1. The method for isolating ruthenium includes the following steps: (a) a first step for separating platelets and blood-derived substances having a size equal to or larger than platelets from blood; A separation step; (b) centrifuging the blood from which the platelets and blood-derived substances having a size equal to or larger than the platelets have been separated; a second separation step of centrifuging; (c) a third separation step of separating the luteolin from the supernatant obtained by the centrifugation; 、 (d) washing the separated ruthenium.

2. 2. The method of claim 1, wherein the blood is from a mammal. method.

3. The method for isolating lutein according to claim 2, characterized in that the blood is derived from a human. 。

4. The first separation step is to pass the mixture through a filter having a pore size of 0.8 to 1.2 μm.

2. The method of claim 1, further comprising the step of separating the unfiltered material. Method of separating luteria.

5. 200 nm, 400 nm, 600 nm, 800 nm, and 1000 nm filters These are used in order to obtain 50 to 200 nm, 200 to 400 nm, 400 to 600 nm, and 600 nm, respectively. The invention further includes categorizing the ruthenium into sizes 800 nm to 800 nm and 800 to 1000 nm. The method for separating ruthenium according to claim 4, comprising the steps of:

6. The second separation step is repeated by centrifugation at 1200-5000 rpm for 5-10 minutes. The method for separating ruthenium according to claim 1, characterized in that the method is carried out by:

7. In the second separation step, exosomes are removed. The method for separating ruthenium according to claim 1.

8. The third separation step comprises irradiating the supernatant obtained by centrifugation with visible light; Using a pipette, the luminal particles that have the ability to gather at the position where the visible light is irradiated are The method for separating ruthenium according to claim 1, characterized in that it is carried out by separating the ruthenium.

9. The washing step is performed by separating the ruthenium from the third separation step into a diameter of 50 nm.

2. Pass the mixture through a filter with pores of 1.0 m, and wash only the unfiltered portion. , whereby luterial is obtained, according to claim 1 . Methods for separating luteria.

10. Luterals derived from body fluids having one or more of the following properties: (a) Immunofluorescence assay using Janus green B, acrid Acrylidine Orange, and Rhodamine 123 mine123) shows a positive color reaction; (b) At optimal conditions (pH 7.2-7.4), β-proteobacteria and γ-proteobacteria It shows the expression characteristics of genes derived from Oobacteria and has a size of 30 to 800 nm; (c) Under acidic conditions, genes from β-proteobacteria and γ-proteobacteria In addition, it expresses the Streptophyta gene, which is a eukaryotic cell. appear and grow in size from 400 nm to 2000 nm or more; (d) responsible for ATP production under normal conditions; (e) mitochondria and exosomes are distinct cellular or cell-like structures. R; (f) At steady state, the shape is round or elliptical, and when derived from a patient, the shape is larger than that at steady state. Mutant luteal cells with large diameters (longest diameter 800 nm or more) and non-uniform morphology are generated. (g) It has a double membrane structure and is adhesive; (h) can be present both intracellularly or extracellularly; (i) It has motile properties and is capable of fusion and / or fission. The formula is shown below: (j) Under certain conditions, the mutant luteal undergoes bursting and bursty After bursting, they have stemness; (k) It has a regulatory function in the p53 gene and telomeres.

11. The body fluid is blood, semen, intestinal fluid, saliva, or cell fluid from a mammal. The ruthenium according to claim 10.

12. The ruthenium according to claim 10 is added with water and incubated at 18 to 30° C. under IR light irradiation. A method for culturing L. lutea, comprising:

13. Before and after the culture, the sizes of the luteal cells were 50-200 nm and 300 nm, respectively. The method for culturing L. lutea described in claim 12, characterized in that the nm is 800 or less.

14. The liquid according to claim 13, wherein the liquid is a saline solution or a PBS solution. Realistic cultivation methods.

15. The method for isolating ruthenium includes the following steps: (a) Centrifuge the body fluid to obtain a supernatant, which is then filtered through a filter with a pore size of 2 to 5 μm. filtering; and (b) subjecting the filtered solution to a second centrifugation to obtain a supernatant, which is then filtered through a 0.5 to 2 μm filter. Filtering through a pore size filter.

16. (c) Irradiating the filtered solution in step (b) with visible light to obtain a visible The luminal particles that have mobility and gather at the position where the light is irradiated are separated using a pipette.

16. The method for isolating ruthenium according to claim 15, further comprising the step of:

17. The body fluid is blood, semen, intestinal fluid, saliva, or cell fluid from a mammal. The method for separating ruthenium according to claim 15.

18. The primary centrifugation is performed at 2000 to 4000 rpm for 5 to 30 minutes. Item 16. The method for isolating ruthenium according to item 15.

19. The secondary centrifugation is performed at 3000 to 7000 rpm for 5 to 20 minutes. Item 16. The method for isolating ruthenium according to item 15.

20. An anti-cancer composition comprising the luteal compound according to claim 10 as an active ingredient.

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