KR102151088B1 - Composition for visualizing Corpuscle-Connected Filiform Structures and visualizing method thereof - Google Patents

Abstract

The present invention relates to a composition for visualizing a thread-shaped structure connected to a body and a method for visualizing the same. By using the visualization composition of the present invention, it is possible to visualize the thread-shaped structure (CCFS) connected to the corpuscle of a mesh structure distributed everywhere in the body of an animal, as well as the lack of reproducibility shown in the conventional method, and the visualized structure can coagulate blood. I can solve the problem of the possibility of being a by-product of the inflammatory reaction. In addition, through the visualization method of the present invention, it was confirmed that the CFFS structure is related to cell death. From this, it is expected that new biomarkers, antibody development, and research on new functions will be possible. It is expected to play an important role in developing treatment methods.

Description

BACKGROUND OF THE INVENTION Field of the Invention: Composition for visualizing Corpuscle-Connected Filiform Structures and visualizing method thereof

The present invention relates to a composition for visualizing a thread-shaped structure connected to a body and a method for visualizing the same.

Fascia (membrane) is a connective tissue composed of collagen or elastin fibers, and is a collective term for membranes that surround all internal organs of a living body, including muscles. In modern medicine, facia is made up of elastin fibers with collagen and glycosaminoglycans and has long been considered a matrix that supports internal organs. According to recent studies, there is discussion that the fascia system is related to cancer metastasis, and that it is closely related as an anatomical structure that traditional or alternative medicine affects, and the fascia system plays a new role in cancer treatment. It has been revealed that it will do.

Meanwhile, in the 1960s, Dr. Bonghan Kim announced that there is a third circulatory system different from blood circulation or lymph circulation, and this new anatomical tube was named after him as the Bonghan duct (BHD) system. However, since the method of confirming the Bonghan Gwan system was not disclosed, other researchers could not confirm the substance of the Bonghan Gwan system afterwards.

Researchers in Korea, who have begun research on this since 2002, first reported that the Bonghan-Gwan system is closely related to the Pacia system, and published the “Bonghan-Pasia model”, including some additional discoveries in the Bonghan-Gwan system. A third circulatory system called "primovascular system" was presented. However, not only is this structure difficult to identify anatomically, and the published method has also been unacceptable to orthodox biologists and doctors because of the lack of scientific access to reproducibility.

In order to visualize the Bonghan duct system anatomically by some researchers, a method of using a dye, a method of using radioactive isotopes, and a method of using electrical stimulation have been attempted. Among them, Janus Green B (JGB) dye is used to A method of visualizing Bonghan Hall was revealed. However, the conventional various visualization methods are only methods of visualizing the thread structure in the lymphatic vessel, and there has been no example of repeating and reproducibly confirming the thread-like structure existing in other organs or facia. In addition, there were suspicions that the visualized structure was a substance produced by an inflammatory reaction caused by blood coagulation or anesthetics, and a problem was raised that the reproducing experiment was not well performed. There was this.

Accordingly, the present inventors have made diligent efforts to scientifically prove the thread-shaped structure present in the internal organs of animals, and as a result, invented a highly reproducible visualization method in the absence of blood coagulation or inflammatory reactions. The present invention was completed by confirming that a new thread-shaped structure was entangled between the tent and the body wall.

Korean Patent Registration No. 10-0950246

It is an object of the present invention to provide a composition for visualizing a corpuscle-connected structure (CCFS, Corpuscle-Connected Filiform Structures) with high reproducibility and a method for visualizing a thread-shaped structure connected to a corpuscle using the same.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention provides a structure (CCFS, Corpuscle-Connected Filiform Structures) visualization composition connected to the corpuscle including Janus Green B (JGB) and heparin.

In addition, the present invention includes (a) anesthetizing animals other than humans, (b) injecting a visualization composition containing Janus Green B (JGB) and heparin, and (c) observing with a microscope It provides a method of visualizing a thread-shaped structure connected with

By using the visualization composition of the present invention, it is possible to visualize the thread-shaped structure (CCFS) connected to the corpuscle of a mesh structure distributed everywhere in the body of an animal, as well as the lack of reproducibility shown in the conventional method, and the visualized structure can coagulate blood. I can solve the problem of the possibility of being a by-product of the inflammatory reaction.

In addition, through the visualization method of the present invention, it was confirmed that the CCFS structure is related to cell death. From this, it is expected that new biomarkers, antibody development, and research on new functions will be possible. It is expected to play an important role in developing treatment methods.

1 is a corpuscular structure (CSs; blue arrow) distributed in the visceral fascia (VF) of the lung of a rat and a thread-like structure connected to the corpuscle structure (filiform structures, FSs; red arrow) ) Is the result of checking. A is the result of checking at low magnification and B is the result of checking the yellow box at high magnification. (Scale bar, A: 500 μm, B: 250 μm)
Figure 2 is a result of confirming the corpuscle structure (CSs; blue arrow) distributed in the parietal fascia (PF) of the rat lung and the thread-like structure (FSs; red arrow) connected to the corpuscle structure. A is the result of checking at low magnification and B is the result of checking the yellow box at high magnification. (Scale bar, A: 500 μm, B: 200 μm)
3 is a result of confirming the CCFS distributed in the small intestine of a mouse with a stereoscopic microscope. (Scale bar, A: 500 μm, B: 250 μm)
4 is a result of confirming the CCFS distributed in the visceral trachea of the mouse with a stereoscopic microscope. (Scale bar, A: 500 μm, B: 200 μm)
5 is a result of confirming the CCFS distributed in the adipose tissue of the rat with a stereoscopic microscope. (Scale bar, A: 500 μm, B: 200 μm)
FIG. 6 is a photograph of a CCFS structure distributed on the visceral membrane (VF) of adipose tissue around the vena cava in a deep abdominal region of a rat in an in situ, in vivo state, directly lifted using micro forceps. A: Blank arrows point to corpuscles stained with JGB, and small black arrows point to CCFS. Blood vessels distributed under the filamentous structure were not stained with JGB. B: Shows the original CCFS picture before lifting with micro forceps. (Scale bar, A: 500 μm, B: 500 μm)
Figure 7 is a result of observing the CCFS tissue visualized on the visceral membrane (VF) and diaphragm (PF) of the small intestine of a mouse with a stereoscopic microscope (SM) (a1, a2) and an optical microscope (LM) (a3, a4, a5) to be. The square area of a1 of each picture is enlarged and shown in a2, the square area of a3 is enlarged and shown in a4, and the square area of a4 is enlarged and shown in a5. Blue arrows indicate corpuscle structures (CSs), red arrows indicate filamentous structures (FSs) connecting each corpuscle, and green arrows indicate granules stained with Janus Green B in an enlarged optical microscope. (Scale bar, a1: 500 μm; a2: 150 μm; a3: 100 μm; a4: 50 μm; a5: 50 μm)
8 is a result of confirming the distribution of components of CCFS (A) and body tissue (B, control) using a fluorescence expression cell sorter (FACS). In CCFS(A), P1, P2, and P3 were 57.3%, 30% and 26%, respectively, and in the control group (B), 59.3%, 17.4% and 12% were respectively. X-axis FSC-A and Y-axis SSC-A represent the relative size and granularity of the constituents (cells, granules), respectively.
9 is a photograph (a1, a2) of the CCFS of a rat liver surface observed with an optical microscope (LM) and the results (b1 to b7) of observation with a scanning electron microscope (SEM) (scale bar, a1: 100 μm; a2: 50 μm). As a result of observation with an optical microscope, the square area of a1 is enlarged and shown in a2, the blue arrow indicates the corpuscle structure, and the red arrow indicates the thread-shaped structure connecting the corpuscle structure. The yellow square area in a2 represents the entangled CCFS over and inside the liver membrane, which shows the same pattern as the yellow square area shown in the scanning micrographs (b6 and b7). In the scanning electron micrograph, b1 is the lowest magnification image of CCFS in the liver. The blue square of b1 is enlarged to b3, and the yellow area of b1 is enlarged to b2 and b4. Here, the blue arrows in each picture indicate the same body. In b2 and b4 you can see a distinct thread-like structure (red arrow). In b3, the white and red squares were expanded to b5 and b6, respectively, and the green square of b2 was expanded to b7.
10 is a result of confirming the CCFS distributed in the outermost cornea of the small intestine continuously divided by hematotoxillin-eosin (HE) staining. The blue arrows represent the thread-shaped structures, the red arrows represent the same CCFS structures, the green arrows represent basophilic granules, and the blank arrows represent faintly visible cytoplasmic cells. (Scale bars are 50 μm (200x), 40 μm (400x), 10 μm (1,000x) in order from the left column)
11 is a result of confirming the CCFS distributed in the outermost cornea of the small intestine continuously divided by Masson Trichrome (MT) staining. The yellow square in each photo on the left is enlarged to the right. The blue arrows indicate the thread-shaped structures, and the red arrows indicate the same CCFS structure. (Scale bars are 50 μm (200x), 40 μm (400x), 10 μm (1,000x) in order from the left column) In particular, in the enlarged CCFS photo of slide number 8 (right end of number 8, 1,000x), several Darkly stained nuclei (green arrows) were observed in the outermost collagen fibers of the dog (yellow arrows) and thinly divided brown filamentous structures.
FIG. 12 is a result of immunohistochemical staining of CCFS distributed in the outermost cornea of the small intestine divided into succession with Lyve-1 (A) and CD31 (B). A: Lymphatic vessels with catheter were stained light brown (blue blank arrow) by Lyve-1, but CCFSs (red arrow) were not stained, so they appeared dark gray. B: Blood vessels (blue blank arrows) were stained light brown by CD31, but CCFSs (red arrows) were not stained, so they appeared dark gray. (Scale bar, a1: 20 μm (200x), a2: 20 μm (400x), b1: 40 μm (200x), b2: 20 μm (400x), inset: 20 μm (200x))
13 is a result of TUNEL (Terminal deoxynucleotidyl transferase (TdT) dUTP Nick End Labeling) staining of CCFSs. Cells positive for TUNEL were identified as dead cells at 1,000x magnification (red arrow). (Scale bar, 50 μm (200x), 40 μm (400x), 10 μm (1,000x))
14 is an optical fluorescence microscope (LFM) observation result of the internal film (VF) CCFS. A body (a1, blue arrow) with a filamentous structure (a3, red arrow) is clearly visible. The square of a1 is enlarged to a2 of F-actin (green). The square of a2 is enlarged to a3 showing fragmented F-actin (red arrow). A4 representing the DAPI staining result corresponds to the same part as the optical microscope (LM) of a1. The square of a4 is enlarged to a5, and a5 represents the synthesis of DAPI and bright fluorescence images. Yellow arrows in a5 indicate extracellular DNA (eDNA) on corpuscles stained with Janus Green B. The DAPI image of a6 shows a weak dotted DNA signal. (Scale bar, a1: 100 μm, a2: 50 μm, a3: 25 μm, a4: 100 μm, a5: 25 μm, a6: 25 μm)
15 is an optical fluorescence microscope (LFM) observation result of a body cavity membrane (PF) CCFS. a1 represents the composite image of the optical microscope and DAPI image for CCFS, and the four blue arrows indicate the corpuscle structure. The body is connected by a thread-shaped structure (FS). a2 and a3 represent images of DAPI-stained CCFS and F-actin in CCFS, respectively. (Scale bar, a1~a3: 40 μm)
16 is a result of observation with a confocal scanning laser microscope (CLSM) of CCFS stained with DAPI in the visceral membranes (VF, b1 to b8) and body cavity membranes (PF, b9 to b12). Optically divided into 2 μm and arranged in b1 to b4 at 12 μm intervals. Rod-shaped nuclei nuclei are arranged in corpuscles (b1, squares), and the squares of b1 are enlarged and optically incised by 1 μm to show b5 to b8 at 6 μm intervals. Yellow arrows in B5 to b8 show the granules stained with JGB, not the granules stained with DAPI. Pictures of B9 to b12 show fragmented F-actin molecules (red arrows) along with DNA molecules (eDNA, yellow arrows) stained with DAPI. These were also optically cut by 1 μm and arranged at 6 μm intervals. (Scale bar, b1~b4: 50 μm, b5~b8: 20 μm, b9~b12: 20 μm)
17 is a result of confirming the CCFS structure stained with Janus Green B solution with DAPI and then confirmed with a fluorescence microscope. Corpuscles stained with Janus Green B (red arrows) appear as black dots, while distinctly distinct from nuclei stained with DAPI (fluorescent blue). (Scale bar, 50 μm)
18 is a result of observing CCFS stained with DAPI with a fluorescence microscope, and fragmented DNA molecules (red arrows) and extracellular DNA (yellow blank arrows) could be identified. (Scale bar, 50 μm)
19 is a result of confirming the distribution of main elements in the CCFS imaged in two dimensions using a time-of-flight secondary ion mass spectrometer (TOF-SIMS). The x- and y-axes represent the 300 μm x 300 μm area of CCFS scanned using TOF-SIMS, and the colored bars next to each plot represent the relative concentrations of the elements. Several different elements have been detected in cations and anions, and the most abundant elements are Si ⁺ and O ^-, respectively. It was also confirmed that a relatively high concentration of Ca ²⁺ exists in the cations. A: 2D photo of major cations (C, Na, Ca) and overall photo of CCFS in CCFS, B: 2D photo of major anions (O, CH, NaH) and overall photo of CCFS in CCFS.
20 shows a transmission electron microscope (TEM) photograph of CCFS. a is a three-dimensional micrograph immediately after visualizing the CCFS on the abdominal wall of the mouse, and after collecting CCFS from the yellow square area, it was analyzed by TEM. b1 to b3 are the results of confirming the cross section of the thread-shaped structure (red arrow) identified in a by TEM. In b1, a circular thread-shaped structure can be seen, and the yellow squares in b1 are enlarged to b2 and b3, respectively. At b2, vacuoles (blue arrows) and adhered vacuoles are visible from the inside of the outermost membrane, and these adhered vacuoles are indicated by three dark brown arrows. In b3, clean pores surrounded by fibrous structures (dark brown arrows) and adhered vacuoles (blue arrows) are visible. c1 to c4 are the results of confirming the corpuscle structure (blue arrow) identified in a by TEM. In c1, many granules (yellow arrows) are visible from completely degraded cells (blank arrows). In c2, the disintegrated nucleus (red arrow) and the collapsed cytoplasmic membrane (blank arrow) are visible. The c3 image shows a translucent cytoplasm with pores (blank arrows). The c4 image shows the moment when the cytoplasmic membrane (red arrow) is cell degraded. In the TEM image of the corpuscle shown in c1, marrow-derived polychromatic erythroblast-like cells (red arrows) and red blood cells were found. (Scale bar, a: 500 μm, b1 to b3: 5 μm, c1 to c2: 1 μm, c3: 2 μm, c4: 1 μm)

In order to solve the problem of the conventional method for visualizing the Bonghan tube and the Bonghan corpuscle, the reproducibility of the method for visualizing the Bonghan tube and the Bonghan corpuscle is poor, there is a possibility that the visualized structure may be a by-product of blood coagulation or inflammatory reaction, and the structure was confirmed only in a specific part of the body. During the study, it was confirmed that the above problems can be solved by mixing heparin to prevent blood coagulation in the visualization composition and performing inhalation anesthesia to prevent inflammatory reactions, and the present invention was completed.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for visualizing a yarn-shaped structure (CCFS, Corpuscle-Connected Filiform Structures) connected to a corpuscle including Janus green B (JGB) and heparin.

The terms "thread-like structures connected to corpuscles", "CCFS", or "Corpuscle-Connected Filiform Structures" used in the specification of the present invention refer to the visceral system, skin, facia, brain, spinal cord, lymphatic vessels and It refers to a structure in the body that is spread in a network structure such as blood vessels, and is a structure that forms a third circulatory system that is distinguished from the vascular and lymphatic systems. CCFS is composed of small bud-shaped corpuscular structures (CSs) and filiform structures (FSs) that connect multiple corpuscles, and can exist both inside and outside organs, blood vessels, and lymphatic vessels. Because CCFS is not only very small in size, but also in a translucent color form, it is not well distinguished from the surrounding facia when observed under a microscope without special treatment. In particular, capillary lymphatic tubes distributed throughout the body are similar in shape to CCFS, making it difficult to distinguish them.

The visualization composition of the present invention comprises a mixture of Janus Green B (JGB) and heparin.

The Janus Green B may be contained in PBS (Phosphate Buffered Saline, PH 7.4) or saline at a concentration of 0.05 to 0.20%. If the amount of Janus Green B is less than 0.05%, it is difficult to observe with a microscope because the CCFS is not sufficiently stained. If it is contained in an amount exceeding 0.20%, an excessive amount of reagent may remain in the body and interfere with visualization. Preferably, the optimal concentration of Janus Green B is preferably 0.08 to 0.12%. The heparin is included for the purpose of preventing blood clotting, and is preferably included in a concentration that does not interfere with the visualization of CCFS while sufficiently exhibiting the effect of preventing blood clotting of experimental animals. Preferably, heparin may be contained in a concentration of 5 to 15 U, more preferably 8 to 12 U concentration. If the concentration of heparin is less than 5 U, blood clotting may be excessive, and if the concentration of heparin exceeds 15 U, blood clotting may not occur, leading to hemostasis and overbleeding. The heparin concentration is standardized in units of activation by biological assays, and the term "U" in the present invention is an abbreviation of "unit/ml", which means the amount of heparin contained in 1 ml of a heparin solution. .

In the visualization composition of the present invention, the Janus Green B and heparin may be mixed in a volume ratio of 5:1 to 15:1, and more preferably, the composition may be mixed in a volume ratio of 9:1 to 11:1. If the volume ratio of Janus Green B and heparin is less than 5:1 (e.g. 3:1), hemostasis may not be possible, and if the volume ratio exceeds 15:1 (e.g., 17:1), there is a problem that blood clotting is not prevented. There may be.

The visualization composition of the present invention may be for visualizing a thread-shaped structure connected to a corpuscle existing in an animal's visceral system, skin, facia, brain, spinal cord, lymphatic vessels, or blood vessels. The facia is a membrane surrounding the entire organs of animals including muscles, and is widely distributed in the body, and the CCFS structure widely present inside and outside the facia can be identified using the composition of the present invention.

The present invention also includes the steps of (a) anesthetizing animals other than humans, (b) injecting a visualization composition comprising Janus Green B (JGB) and heparin, and (c) observing under a microscope. It provides a method of visualizing a thread-shaped structure connected with

First, the visualization method of the present invention includes the step [step (a)] of anesthetizing animals other than humans.

The anesthesia step of the present invention may be using inhalation anesthesia. Inhalation anesthesia is a method of inhaling an anesthetic into the lungs through the nose or mouth to anesthetize the whole body. Compared to general anesthesia through muscle or vein, it is easier to control the absorption and discharge of an anesthetic. In addition, it can prevent inflammatory reactions that may occur during the process of injecting an anesthetic, so when checking CCFS, problems caused by artifacts caused by inflammatory reactions can be solved. Ether, halothane, nitrous oxide (N ₂ O), enproene or isoflurane may be used as an inhalation anesthetic, but is not limited thereto.

Next, the visualization method of the present invention includes the step of injecting a visualization composition comprising Janus Green B (JGB) and heparin [step (b)].

Detailed description of the visualization composition is as described above.

In one embodiment of the present invention, the visualization composition may be maintained at 35 ~ 40 ℃ before injection. This may be prepared by maintaining each solution containing Janus Green B and heparin at 35 to 40°C and then mixing, and the mixed composition may be maintained by heat treatment at 35 to 40°C. This is close to the rat's body temperature (36.5℃), so it may not cause physiological changes in the rat.

In an embodiment of the present invention, it is possible to prevent blood coagulation in advance by further comprising the step of injecting heparin before injection of the visualization composition of step (b). The description of heparin is as described above.

In one embodiment of the present invention, the visualization composition may be injected through intraperitoneal or thoracic injection. The CCFS structure to be confirmed in the present invention is confirmed to be distributed throughout the organ through Pacia.In order to achieve effective visualization of CCFS in the internal organ tissue, it is recommended to perform abdominal or thoracic injection, or concurrently with abdominal cavity and thoracic injection. desirable.

In an embodiment of the present invention, a step of waiting before observation after injection of the visualization composition may be further included. This is to ensure the time that the CCFS structure is stained with the visualization composition, and it was confirmed that waiting for 20 minutes to 70 minutes, more preferably 30 minutes to 60 minutes through repeated experiments is most effective.

Next, the visualization method of the present invention includes the step of observing with a microscope [step (c)].

The microscopic observation of the present invention is performed by a stereomicroscope, a light microscope, a confocal laser scanning microscope (CLSM), or a cryo scanning electron microscope (Cryo-SEM). It can be measured, but is not particularly limited.

In one embodiment of the present invention, the microscopic observation may be observed by connecting the observation through a stereoscopic microscope and an optical microscope in order to facilitate identification of the same CCFS structure. Specifically, the microscopic observation is performed by observing with a stereoscopic microscope in an in situ in vivo state; Separating a tissue including a thread-shaped structure connected to the corpuscular body identified by the stereoscopic microscope; And it may include the step of observing the separated tissue with an optical microscope. As described above, the structure and substance of CCFS dyed with a visualization composition including Janus Green B can be more clearly confirmed by observing the same specimen by combining a stereoscopic microscope and an optical microscope, and the observation results through a stereoscopic microscope and an optical microscope can be directly observed. It can be interpreted by connecting with

Below, The present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

<Example>

1. Experimental animals

In the present invention, rats (Wistar, 8 females, 6 males, 150-250 g) were used as animal models, and these rats were well ventilated prior to major experiments according to international guidelines of an accredited procedure. It was bred in place.

During the experiment, general anesthesia was performed with general inhalation anesthetics such as ether and halothane. The experiment was approved by KAIST's Animal Committee.

2. CCFS visualization of internal organs

As a visualization dye, Janus Green B (Sigma, JGB) was dissolved in PBS (pH 7.4) or sterilized saline to a concentration of 0.1%. Next, the dissolved Janus Green B was filtered using a 0.45 μm perforated membrane filter, and then stored in an incubator at 37 to 38°C. Next, 0.1% Janus Green B and 10 U of heparin (Sigma) solution were prepared by mixing at a volume ratio (v/v) of 10:1.

First, the experimental animal was subjected to general anesthesia by inhaling ether or halotan, and then 0.2 to 0.4 ml of heparin solution (10 U in PBS) was injected into the femoral vein of the experimental animal. Next, 0.8 to 1 ml of a mixed solution of Janus Green B and heparin was injected into the abdominal cavity of an anesthetized animal. Through repeated experiments, it was confirmed that the incubation time for the best visualization of CCFS in the internal organs of mice was between 30 minutes and 1 hour. In rare cases, about 0.2 ml of staining solution was injected into the chest cavity of the rat after the abdominal cavity experiment was completed. At the end of the experiment, the mice were immediately euthanized by ether inhalation.

3. In situ in vivo ( in situ, in vivo ) State of the internal organs CCFS under a stereoscopic microscope

After injecting a solution containing Janus Green B into the abdominal cavity and chest cavity, staining was performed while waiting for 30 to 60 minutes. Then, the abdomen was incised to prevent blood from coming out, and warm saline was gently put into the abdomen immediately after the internal organs were exposed to wash off excess Janus Green B. Next, CCFSs in the membranes of the internal organs of mice were observed with a stereoscopic microscope (STX 60X, Olympus, Japan). As a result, it was found that the CCFSs were stained well in dark blue or black unusually on Janus Green B.

Specifically, as shown in Figs. 1 and 2, a number of corpuscular structures (CSs) distributed in the visceral fascia (VF) and the parietal fascia (PF) on the outer surface of the lung tissue of the rat, and The filiform structures (FSs) connected to this corpuscle structure were identified. In addition, in addition to lung tissue, it was possible to confirm the same CCFS structure stained in dark blue or black in the small intestine, visceral trachea, and adipose tissue of mice (FIGS. 3 to 5 ).

In order to prove the relative anatomical distribution of the CCFS structure, one of the corpuscles identified in the abdominal visceral membrane (VF) of the rat was lifted using micro forceps (FIG. 6). It was confirmed that the CCFS structure was stained with Janus Green B and appeared dark blue, while the blood vessels and adipose tissues around CCFS were not stained with Janus Green B. As shown in Fig. 6B, it was found that the CCFSs were originally arranged side by side on the blood vessel before lifting with the small forceps.

In order to check whether the visualization method of the present invention is reproducible and whether CCFSs can be visualized in all organs, a total of 15 rats were tested. As a result, it was found that CCFSs were observed in 14 rats (93%), and it was found that CCFSs could be reproducibly visualized with the dyeing composition of the present invention.

Organs associated with the visualized CCFSs are summarized in Table 1 below. In order to understand the distribution of CCFSs overall, the size of the largest corpuscle and the smallest corpuscle in each tissue in which CCFSs were found were recorded and summarized, and between the two most distinct corpuscle structures when observed with a stereomicroscope. The intervals of are also summarized in Table 1 below. As can be seen in Table 1 below, the criterion for data on the number of corpuscle structures was how clearly the stereoscopic micrograph shows corpuscle structures. Therefore, there is also the possibility that there are a greater number of corpuscle structures than counting by looking directly at corpuscle structures in photos. According to Table 1, corpuscle structures were most often found in liver (138), followed by abdominal wall (114), small intestine (112), fat cells (57), mesentery (45), and large intestine (17). Dog), epididymis testis (12), lungs (12), diaphragm (10), ovary (10), body cavity wall pleura (6), and testis (5). The size of one body varied from a maximum of 1,656 µm X 186 µm to a minimum of 26 µm X 26 µm.

Location and size distribution of corpuscle structures in rat CCFS based on stereomicroscopic images No gender NF(N) BC: Lxl SC: Lxl D One M Aw(8) 821x107 107x71 357 Li(5) 252x86 60x40 320 Lv(5) 250x50 75x50 750 Ms(5) 320x180 200x60 1,180 Si(10) 800x80 133x80 133 Ts(5) 459x108 81x41 757 2 M Aw(5) 556x333 122x94 1,222 Lv(4) 442x108 38x38 480 Ms(3) 750x333 192x83 1,550 Si(4) 94x94 73x70 288 3 M Ft(2) 816x263 245x105 895 4 M Aw(14) 108x99 54x43 326 Lv(8) 207x100 67x67 293 Ft(10) 275x91 31x31 544 5 M
Lv(17) 216x182 53x26 868 Ft(22) 263x187 33x33 290 Si(10) 100x100 33x33 420 Ed(12) 1,656x186 250x125 750 6 M Aw(14) 110x51 26x26 252 Si(9) 466x308 38x35 380 7 F Aw(16) 455x455 100x91 832 Lv(5) 163x110 30x25 375 Ms(18) 150x108 113x38 300 Si(11) 167x167 105x57 145 8 F Dp(10) 267x67 50x33 467 Ft(8) 200x167 67x34 1,067 Lv(45) 667x111 56x56 1,111 Ms(14) 179x143 71x36 893 Ov(10) 233x200 33x33 333 Si(8) 471x59 30x30 647
9
F Aw(15) 500x97 156x97 306 Lv(8) 571x286 71x71 189 Ms(5) 167x125 63x63 688 Si(6) 250x229 188x63 725 10 F Si(9) 143x86 71x14 357 11 F Aw(20) 394x63 31x31 581 Lv(12) 190x133 33x33 600 Si(17) 94x56 42x28 181 12 F Aw(5) 333x146 42x21 479 Li(12) 313x227 100x67 333 Lu(12) 217x139 111x56 556 Lv(12) 133x133 48x24 333 13 F Aw(17) 125x83 42x42 542 Si(14) 45x32 32x21 223 Ft(15) 100x50 33x17 150 14 F Lv(22) 111x56 56x42 833 Si(14) 147x59 44x29 588 Pp(6) 88x59 59x29 294

※ The name of the internal organization where NF and CCFS are visible; N, the number of CCFS counted under the stereoscopic microscope; Maximum and minimum size of corpuscles in BC, SC and CCFS; L and l, the longest and shortest diameters of round and round ciliated bodies, respectively; D, the distance between the two distinct corpuscles.

※ Note: (1) All units of each number are μm. (2) The initials of each organ are tissues (membrane) that are entangled and sparsely connected and enclosed by visualized CCFS.

※ Abbreviations for internal organs: Aw (abdominal wall), Dp (diaphragm), Ed (epidial testis), Ft (adipose tissue around the tail vein), Li (large intestine), Lu (lung), Lv (liver), Ms ( Mesentery), Si (small intestine), Ts (testis), Pp (body cavity pleura), Ov (ovary).

4. Simultaneous linkage method of a stereoscopic microscope (SM) and an optical microscope (LM) for the same CCFS of visualized internal organs

In order to prove the existence of the structure of CCFSs intertwined with the inner and outer surfaces of the visceral membrane (VF) and the body wall membrane (PF), the CCFS observed in the visceral membrane (VF) and diaphragm (PF) of the small intestine was observed under a stereoscopic microscope and optical Observed under a microscope.

Specifically, after euthanizing the mice, CCFS of the intestine visceral membrane (VF) and diaphragm (PF) were observed with a stereomicroscope (STX 60X, Olympus, Japan) in situ ( in vivo ) state. Shortly thereafter, the CCFS fixed to the trachea was partially dissected and lifted using an anti-fading agent (Thermo Fisher Scientific, USA). Immediately at the site, the specimen was taken using an optical microscope (BX53, Olympus, Japan) equipped with a CCD camera (eXcope X3, DIX, Korea), and then the photographs obtained by the stereoscopic microscope and the optical microscope were compared.

As a result, as shown in FIG. 7, the structure of the CCFS distributed in the film could be more clearly confirmed by observing the CCFS structure at the same position in conjunction with the stereoscopic microscope and the optical microscope at the same time.

Specifically, it was confirmed that the black-dyed CCFSs were scattered and distributed on the surface of the reddish membrane through stereoscopic observation of the visceral membrane (VF) and body wall membrane (PF) of the small intestine at low magnification. At low magnification, the exact structure of CCFSs could hardly be identified, and the filament-like structures (FSs) connected to the corpuscle were hardly seen. However, by magnifying some areas (the square in Fig. 7, a1) at high magnification, it was possible to confirm the clear appearance of the CCFS with the thread-like structure on the VF and PF in the in situ, in vivo state. (Fig. 7, VF a2).

Immediately after observation with a stereoscopic microscope, portions of VF and PF were separated, removed, and observed with an optical microscope. Thus, the three-dimensional micrographs of the VF and PF in situ in vivo could be compared with the optical micrographs of the in vitro condition. The square dotted line (Fig. 7, a1) photograph of the stereoscopic microscope corresponds exactly to the photograph of the optical microscope (Fig. 7, a3). From the optical micrograph of CCFS, it was found that CSs and FSs have a differentiated shape of CCFS connected, and it was found that the component of CCFS consists of granules dyed with Janus Green B (Figs. 7, a4 and a5). ).

5. Fluorescent-activated cell sorting (FACS)

Pure CCFS was isolated for FACS analysis of CCFS identified in the present invention. Specifically, first, CCFS in rat liver was identified by the method described above. Immediately after the location of the CCFS was determined, only CCFS was collected using a micro forceps while observing with a stereoscopic microscope, and this was put into a 1 ml Eppendorf tube containing PBS (pH 7.4). In parallel with this procedure, we collected body tissues as a control (control variable). The volume of the collected body tissue was approximately the same as the volume of CCFS. These body tissues were placed in a 1 ml Eppendorf tube containing PBS (pH 7.4) in the same manner as CCFS. The total volume of the experimental control solution was filtered through a sieve (40-μm pore size mesh, BD Falcon), and homogenized evenly using a sterilized rubber rod. Homogenized samples were subjected to FACS (BD FACS Aris Cell Sorter) analysis. Because Janus Green B exhibits fluorescence, CCFS stained with Janus Green B does not need to be separately stained, but body tissue as a control was stained with 0.1% acridine orange before FACS analysis.

As a result, it was found that, as shown in FIG. 8, CCFS (A) is composed of components of various sizes compared to the control (body tissue) (B), and has relatively high granularity.

6. CCFS Scanning Electron Microscopy (SEM) observation

CCFS distributed in the liver of mice was stained and visualized in the same manner as described above, and immediately after observation with a stereoscopic microscope, it was fixed with 10% neutral buffered formalin (NBF). The CCFS fixed in the liver was partially excised and isolated for scanning electron microscopy. The isolated samples were stored in 10% NBF and washed with PBS (pH 7.4). Samples that were fixed and washed were gradually dehydrated using 20% to 100% ethyl alcohol in PBS. The dehydrated specimens were stored in a chamber filled with silica gel until sufficiently dehydrated, and gas was released to enable SEM inspection, and then observed using an electric field emission SEM (FE-SEM, Sirion, FEI, The Netherland).

As a result, it was possible to confirm the CCFS including the corpuscle and the thread-shaped structure connecting it with a stereoscopic microscope on the surface of the liver of the rat (Figs. 9, a1 and a2). Next, as a result of observing the same site with a scanning electron microscope, it was confirmed that CCFSs were not only entangled with the visceral membrane of the serous, but also intertwined with the loosely connected fibers of the visceral membrane. In addition, it was not observed with an optical microscope that all corpuscle structures are connected to the thread-shaped structure, but it was clearly confirmed through observation with a scanning electron microscope (FIGS. 9, b1 to b7).

7. Sample preparation for histological study of small intestine CCFS

Samples were fixed with 10% neutral buffered formalin (NBF) according to the general procedure (School of Medicine, Seoul National University). That is, the sample was washed with PBS (pH 7.4), dehydrated with multi-stage ethyl alcohol, purified with xylene, and put into paraplast (Sigma, USA) to prepare. The paraplast segment is 4 μm thick. For the distribution of CCFS related to the internal organs, we divided the specimens in succession, arranged them in order, and examined them with an optical microscope after staining. The distance between the two slides is about 32 μm, and 8 divided specimens were placed on one slide. Each slide number is indicated in the histological data of CCFS.

8. CCFS staining with hematoxylin & eosin (HE, hematoxylin & eosin) and Mattson trichrome (MT)

To confirm the histological properties of CCFSs, CCFSs were observed after staining with hematoxylin & eosin and Mattson trichrome. After paraffin removal from the fixed sample, CCFS was hydrated with PBS (pH 7.4). Hematoxylin-eosin (HE) and Masson Trichrome (MT) were applied to 4-μm CCFS slices attached to the small intestine to investigate the overall morphology and collagen fibers, respectively. These images were confirmed with an optical microscope (BX 53, Olympus, Japan) using magnifications of 200, 400, and 1000 times.

As a result of staining CCFSs on the outer surface of the small intestine with hematoxylin-eosin, it was found that CCFSs were lodged in the outermost cornea of the small intestine and the serous viceral fascia and were also distributed on the outer surface (Fig. 10). ). In addition, histologically, it was found that CCFSs had greater basophilic properties than the serous membrane. Note that when the microscope magnification was magnified to 1000 times, CCFSs were found to contain basophilic granules (green arrows in Fig. 10) in addition to cells with pale cytoplasm.

Similar to the results of the hematoxylin-eosin staining of CCFSs with Masson's Trichrome, CCFSs are distributed on the surface of the small intestine and are embedded in the small intestine membrane. In addition, through Masson's trichrome staining, it was confirmed that CCFSs were composed of different types of fibers, and the outermost fibers of them were blue-colored collagen. These collagens are typical features of the outermost cornea of the visceral membrane (Fig. 11).

9. Immunohistochemical staining

For successive sections of the sample, such as those stained with hematoxylin-eosin or Masson Trichrome, immunohistochemical staining was performed to confirm that the CCFSs identified here are new structures distinct from lymphatic vessels or blood vessels. Specifically, the paraffin of the specimen was removed and hydrated. Antigen retrieval was performed at pH 8.4, 100°C for 24 minutes. Inhibitor (inhibitor) 3% hydrogen peroxide was treated for 4 minutes at 37 ℃. As major antibodies, vascular endothelial cell marker CD31 (1:100, ab64543, Abcam) and lymphatic endothelial cell marker Lyve-1 (1:50, NB600-1008, Novus) were applied at 37° C. for 16 minutes. Linker (HQ linder) was used for 8 minutes at 37 ℃. After performing the polymer amplification at 37° C. for 8 minutes, DAB was treated at 37° C. for 8 minutes for visualization. As a final step, hematoxylin staining as a control staining was performed at 37° C. for 8 minutes (using the OPTIVIEW universal DAB kit with Ventana BenchMark XT staining system).

As a result, as shown in FIG. 12, lymphatic vessels and blood vessels exhibited positive reactions to Lyve-1 and CD31, respectively, and thus had a light brown color, but all CCFSs distributed in the small intestine membrane showed negative reactions.

10. TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) staining

In order to confirm apoptosis in CCFSs, TUNEL staining was used. Specifically, TUNEL staining was performed using the TUNEL assay kit (Chemicon, S710) with the continuously divided slices: (1) hydration after removal of paraffin, (2) protease K (20 μg/ml) Pretreatment of two specimens for 30 minutes with (3) cooling with internal peroxidase, (4) incubation for 5 minutes in dormant buffer, (5) TdT incubation at 37°C for 1 hour 30 minutes in a humidified chamber, ( 6) Stopping and washing the sample using a buffer, (7) incubation for 30 minutes and then washing after binding with anti-digoxigenin, (8) staining in a peroxidase incubator for 5 minutes Coloring and washing, (9) counter staining with Mayer's hematoxylin for 10 seconds, (10) dehydration and mounting.

As a result, as shown in Fig. 13, it was possible to clearly observe dead cells that occupy about half of the cell distribution in CCFSs, and it was confirmed that the living cells were counter-stained to Mayer's hematoxylin staining to show blue color.

11. CCFS fluorescence microscopy (FM) and confocal laser scanning microscopy (CLSM) observation

Optical fluorescence microscopy and confocal laser scanning microscopy were performed to examine the characteristics inside the CCFS, that is, extracellular DNA and fragmented F-actin. Specifically, CCFSs were isolated from the walls of the abdominal wall, which is a representative of the large intestine and body wall tissue, among the visceral tissues, and DAPI (4', 6-diamidino 2-phenyllindole) (Prolong Gold Antifade Mountant) for pure CCFS DNA molecule staining. with DAPI, Thermo Fisher Scientific) was used, and phalloidin (Alexa Fluor 488 Phalloidin, Thermo Fisher Scientific) was used for F-actin molecule staining. After staining, it was observed using a BX 53 optical microscope equipped with a fluorescent filter (Olympus, Japan), and after optical division was performed to identify the DNA signal and F-actin signal stained with DAPI and phalloidin, a confocal scanning laser It was observed using a microscope (confocal laser-scanning microscopy, CLSM; LSM 800, Carl Zeiss, Germany).

As a result, as shown in Figs. 14 to 16, it can be seen that the CCFS is composed of a corpuscle structure and connected by a thread-like structure, and these structures were visualized due to the granules dyed with Janus Green B. These granules show differences from others and can be easily observed under a bright-field optical microscope, but they were not stained with DAPI or phalloidin (FIG. 17).

Note that, as shown in a3 of FIG. 14, a fragmented signal of many F-actin molecules could be observed. A confocal scanning laser microscope was used to obtain clear pictures of fragmented F-actin molecules (Fig. 16, b9 and b10, red arrows).

Another evidence was obtained from the DAPI-stained DNA picture that dead cells were present in CCFSs. The fluorescence micrographs of Figs. 14-a5 and the confocal scanning laser micrographs of Figs. 16-b11 and b12 each show randomly distributed extracellular DNA molecules. More notable is that CCFSs contain DNA fragments stained with DAPI (Fig. 14, a5). Such fragmented DNA molecules are difficult to discriminate under a fluorescence microscope. However, through the enlargement of the digital photo, the signal of the fragmented DNA molecule could be recognized (FIG. 18).

In conclusion, extracellular DNA, fragmented F-actin molecules, and granular fragmented DNA are important information suggesting that CCFSs are composed of dead cells.

12. Time-Of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) analysis

To confirm the ionic composition in CCFSs, time-of-flight secondary ion mass spectrometry (TOF-SIMS) was performed. Specifically, the CCFS collected from the abdominal wall of the rat was fixed in 10% NBF, washed three times with PBS (pH 7.4) and washed twice with distilled water (Sigma). The washed specimen was then placed on a copper plate with carbon tape attached and dried in a chamber containing silica gel for several days before starting the TOF-SIMS test. The prepared specimen was placed in a vacuum chamber of TOF-SIMS and the CCFS surrounding the specimen and the surface of the background were observed.

Through the TOF-SIMS analysis, not only the morphological data of CCFSs but also the data on the constituents of CCFSs were confirmed. 19 shows only data on elements related to in vivo after determining overall major elements through TOF-SIMS. As a result, the cation is silicon (Si ^+), calcium (Ca ⁺⁾ in the CCFSs, negative ions of oxygen (O ^-) was found to have a major elements, carbon molecules alone are not be observed.

13. CCFS transmission electron microscopy (TEM) observation

In order to check the cross-sectional area of the CCFS collected from the abdominal wall of the rat in more detail with a transmission electron microscope (TEM), the CCFS was first fixed in situ, in vivo under a stereoscopic microscope. Next, the isolated pure CCFS was fixed in 2.5% paraformaldehyde and 2.5% glutaraldehyde in neutral 0.1 M PBS for 1 hour. Next, CCFS was dehydrated with ethanol step by step for 1 hour and then fixed in 1% (w/v) osmic acid dissolved in PBS, and inserted into Epon812 (EMS, Fort Washington, PA). The prepared sample was cut to a thickness of 1 μm and stained with 1% toluidine blue for observation. Images were obtained with an optical microscope (BX 53, Olympus) equipped with a CCD camera (eXcope X3, DIX, Korea). Next, the specimen was cut into very thin cuts, placed on a nickel grid, and double stained with uranyl acetate. This was done after staining with lead citrate. The cut surface was confirmed with a transmission electron microscope (CM20, Philips), and the results are shown in FIG. 20.

Among the transmission electron micrographs of FIG. 20, a structure that looks like a thread can be seen at b1, which is the lowest magnification, and two different parts of b1 are enlarged and shown as b2 and b3, respectively. The thread-like structure observed with a transmission electron microscope was surrounded by the outermost cornea, and many confluent vacuoles and fiber structures were observed. In the corpuscle structure, cell membranes are decomposed, phosphorus is disassembled, cytoplasm becomes transparent, and dead cells can be observed. Also, granules of various sizes could be observed around these cells. Interestingly, within the corpuscle structure, marrow-derived polychromatic erythroblast-like cells and red blood cells were also found.

Claims (15)

delete delete delete delete delete (a) anesthetizing animals other than humans;
(b) intravenously injecting 5 to 15 U (unit/ml) of heparin solution;
(c) waiting for 30 to 60 minutes after injection of the visualization composition comprising Janus Green B (JGB) and 5 to 15 U (unit/ml) of heparin; And
(d) observing the visualization structure,
The Janus Green B and heparin of the step (c) are mixed in a volume ratio of 9:1 to 11:1, wherein a thread-like structure that is present in the visceral system or facia of an animal is formed. How to visualize.
The method of claim 6,
The method of visualizing a thread-shaped structure connected to the corpuscle, characterized in that the anesthesia step of the step (a) is inhalation anesthesia.
delete The method of claim 6,
The composition of step (c) is maintained at 35 to 40° C. before injection, a method for visualizing a thread-shaped structure connected to a corpuscle.
delete The method of claim 6,
The composition of step (c) is to be injected into the abdominal cavity or thoracic cavity, the method for visualizing the thread-shaped structure connected to the corpuscle.
delete The method of claim 6,
The observation in step (d) is a stereomicroscope, a light microscope, a confocal laser scanning microscope (CLSM), or a cryo scanning electron microscope (Cryo-SEM). A method of visualizing a thread-shaped structure connected to the corpuscle.
The method of claim 6,
The observation in step (d) is
Observing with a stereoscopic microscope in an in situ, in vivo state;
Separating a tissue including a thread-shaped structure connected to the corpuscular body identified by the stereoscopic microscope; And
It comprises the step of observing the separated tissue with an optical microscope,
A method to visualize the thread-shaped structure connected to the body.
delete

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