KR100950246B1 - Visualizing agent comprising a janus green b and visualizing method by using the same - Google Patents

Abstract

The present invention relates to a visualization agent comprising Janus Green B (JGB), and a visualization method using the same. More specifically, the method relates to a method of visualizing a free intravascular sealant structure (a Bonghan duct) inside a lymphatic vessel, which comprises injecting and staining JGB into lymphoid tissue and observing under a microscope. The present invention provides a breakthrough in the field of cell anatomy and physiology. Further research into the histological characteristics and physiological function of the constructs may suggest the possibility of new concepts important to the biology and medical fields.

Description

VISUALIZING AGENT COMPRISING A JANUS GREEN B AND VISUALIZING METHOD BY USING THE SAME}

The present invention relates to a visualization agent comprising Janus Green B (JGB), and a visualization method using the same. More specifically, the method relates to a method of visualizing an intravascular seal structure liberated inside a lymphatic vessel, comprising injecting and staining JGB into lymphoid tissue and observing under a microscope.

In the early 1960s, Bonghan Kim reported the discovery of intravascular threadlike structures inside the lymphatic vessels as part of a larger network of new circulatory systems that are completely different from the vascular or nervous system. The method has not been disclosed (Ref. 1). Thus, his work has been neglected for a long time. Kim Bong-han, a Korean physiologist, reported on a physics-based needle meridian called "Bonghan duct" assuming his findings form the third circulatory system. Liquids containing specific granulocytes that regenerate cells in damaged tissues migrate through this Bonghan duct network. His research was published in the Korean Journal of Medicine (Korea) from 1962 to 1965. The ninth reference included as a reference of the present invention is an English paper summarizing some of its early work.

In contrast, Japanese anatomist Fujiwara, in part, acknowledged Kim's results (Ref. 4), but insisted on not attracting more attention. More recently, some researchers have used methods of slowly permeating blood in rats and rabbits (Ref. 6), as well as vascular structures on the visceral surfaces of rabbits and rats (Refs. 14, 15, 20). Rediscovered. Although Kim insisted on the presence of seal structures inside the lymphatic vessels, no evidence was provided for this. Also, since Kim's method was not disclosed, no one, including Fujiwara, could confirm his claim.

Early, but not completely, after the discovery of lymphatic vessels, most of the anatomical and physiological functions of the lymphatic vessels were known (Refs. 5, 16). In addition, it has become clear that there is a fluid comprising lymphocytes in the lumen of the lymphatic vessels, and no other structures in the lymphatic vessels are known.

New structures present in such lymphatic vessels are not known in the art because they are very difficult to detect under a microscope. In Western anatomy, no mention was made of vascular seal structures released inside lymphatic vessels (Refs. 5, 16). It is surprising that these structures are medium (approximately 20 μm thick) and have not been observed in many surgical procedures. Clearly, the optical transparency of such structures makes it very difficult to detect them.

1 shows a stereoscopic image of lymphatic vessels near the lower vena cava of rabbits according to Example 1;

FIG. 2 shows an optically segmented continuous image of a seal structure measured by a confocal laser scanning microscope (CLSM) of a sample stained with acridine orange according to Example 1; FIG.

FIG. 3 (A) is a photograph of the cross-section of the seal structure (arrow area) inside the lymphatic vessel stained with hematoxylin and eosin at 1,000 (x) magnification and 200 (x) magnification, and FIG. 3 (B) Is a photograph measured on the diagonal cross section of the actual structure (arrow part) according to Example 1 at 400 (x) magnification and 200 (x) magnification;

FIG. 4 (A) is a freeze-fragmented sample showing collapsed lymphatic vessels (dotted border, no microscopic view) including another substructure with characteristic outer membrane, FIG. 4 (B) An enlarged photograph of a rectangular portion of (A) according to Example 1;

FIG. 5 shows a cross-sectional view of a sample stained with hematoxylin and eosin showing lymphatic vessels with seal structures (arrow sites) measured under an optical microscope (Axiophot, Carl Zeiss, Germany) according to Example 2; And

Figure 6 shows the cross-sectional view of the new seal structure (dotted border region) inside the lymphatic vessel measured under the frozen scanning electron microscope (JSM-5410LV, JEOL, Japan) according to Example 2.

Summary of the Invention

Since the intravascular seal structures liberated inside the lymphatic vessels are difficult to identify with the naked eye, for this purpose, the present inventors, Janus Green B (JGB), a new structure that can stain a large amount of this site for direct marking. A new method was developed. Samples obtained by staining with acridine orange were confocal laser scanning microscopy (CLSM), samples obtained by staining with hematoxylin were obtained by light microscopy, and frozen samples. Were respectively measured by cryo scanning electron microscopy (Cryo-SEM). Thus, it is surprising to find the seal structures liberated inside the lymphatic vessels that do not adhere to the vessel wall. In other words, it has been incredibly inconvenient to discover a seal structure with a diameter of about 10 μm despite many surgical procedures and studies on lymphatic vessels throughout the world.

It is therefore an object of the present invention to provide a visualization agent comprising Janus Green B (JGB) which can clearly show the passage of the seal structure through the lymphatic plate inside the lymphatic vessel under stereomicroscopy. The development of a dye that specifically stains the new yarn-like tissue and its histological structure is very important for broadening the understanding of cell anatomy.

It is another object of the present invention to provide a visualization method using a visualization agent comprising JGB.

Hereinafter, the present invention will be described in more detail. BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings are constructed and appended as part of this specification in order to provide a better understanding of the present invention, and are described in detail with reference to the drawings and embodiments of the present invention in order to explain the principles of the present invention.

Visualization agent for staining of lymphatic inner Bonghan ducts containing JGB. The agent is used to visualize intravascular seal structures that are in situ or in vivo. The agent is visualized by staining a large amount of the vascular seal structure compared to other lymphoid tissues.

The visualization agent may be from 0.01 to 10 (w / v)% JGB solution, preferably from 0.1 to 5 (w / v)% JGB solution. The solvent used to prepare the JGB solution is not particularly limited, but may preferably include ethanol and saline. More preferred solvent may be ethanol.

In addition, the present invention relates to a method for visualizing an intravascular sealant structure liberated inside a lymphatic vessel using the visualization agent. More specifically, the present invention relates to a method of visualizing an intravascular seal structure liberated inside a lymphatic vessel, comprising injecting and staining JGB into lymphoid tissue, and observing under a microscope. The visualization method visualizes vascular seal structures released inside lymphatic vessels in situ or in vivo, which is a very important method for studying vascular seal structures. The JGB, a dye, can strongly stain new vascular thread-like tissues and is available for observing new thread structures in vivo and on the inside of transparent lymphatic vessels.

JGB has been known to stain mitochondria and nerves (Refs. 3, 21). In the present invention, the staining method using the JGB exposes almost transparent seal structures inside the rabbit's lymphatic vessels in phosphorus drilling and in vivo. JGB can clearly visualize the seal structure, while some other dyes did not exhibit this visualization ability.

As found in common structures, staining methods have been developed for in-situ drilling and in vivo observation of thread-like tissue liberated inside the rabbit's lymphatic vessels without attachment to the vessel wall. Such new constructs are not known in the art because they are difficult to detect when viewing the lymphatic vessels using a microscope. Accordingly, the present inventors have found a method using Janus Green B, which can dye a large amount of a new structure to enable direct display. The tissue can be measured using confocal laser scanning microscopy (CLSM), light microscopy, and cryo scanning electron microscopy (Cryo-SEM). On the CLSM obtained by staining the new tissue with acridine orange, a characteristic nucleus distribution of the tissue, ie 10-20 μm rod-shaped nuclei, arranged in the form of broken lines and stripes, was observed. In haematoxylin and eosin staining, a seal structure was observed that passes through the lymphatic valve and histologically different from the lymphatic valve. On Cryo-SEM, seal structures trapped by collapsed lymphatic vessels were observed. A spherical structure was observed inside the sinus of the rapidly frozen sample, which could be a liquid flowing through the longitudinal tube in the seal structure. This specific staining method using JGB may indicate that the silt construct inside the lymphatic vessel has a high density of mitochondria in the cells and / or the cells with neuron-like characteristics, one of which is the thread. It can provide important clues to the physiological function of the structure. JGB can visualize the seal structure more effectively than other dyes such as methylene blue, methyl green, and toluidine blue. This is because, even if the amount of the dyes is reduced or eliminated during staining, the lymphatic vessels are stained in as much amount as the seal structure.

In the following, the vascular seal structures that drained inside the lymphatic vessels were termed "Bonhan ducts", reported by Kim Bong-han as part of a larger network of new circulatory systems that are completely different from the vascular or nervous system. According to Kim Bong-han's explanation, the seal structure has a unique movement pattern that is completely different from the gastrointestinal tract, which is parasymphatomimetic (Ref. 9). This means that the Bonghan duct can be stained by JGB because it contains a large number of mitochondria to have specific peristaltic, and / or parasympathetic characteristics. Due to this feature, the Bonghan duct can be applied more and more physiologically, and further needs to be evaluated. In the present invention, the first report that there is a high density of mitochondria inside the seal structure. This may give many advantages to the visualization method using nanoparticle technology.

The stained endovascular seals leaked inside the lymphatic vessels can be observed under a microscope. The microscope is not particularly limited, and the microscope may use a microscope capable of confirming a structure stained with JGB by a conventional method. For example, the microscopic observation may be preferably performed with a stereoscopic microscope, an optical microscope, a confocal laser scanning microscope (CLSM), and a frozen scanning electron microscope (Cryo-SEM). At this time, the stereoscopic observation may be performed after injecting JGB into lymphoid tissue.

Confocal laser scanning microscopy (CLSM) observation can be performed using CLSM after injecting JGB into lymphoid tissue and staining with acridine orange. In order to prove that the JGB stained yarn construct inside the lymphatic vessel is not an optical illusion or a visible artifact that is different from the actual tissue, some of the yarn-like sample can be taken to confirm the nucleus and its distribution. After staining with 0/1% (w / v) of acridine orange, it was observed using CLSM.

Histological analysis can be observed by injecting JGB into lymphoid tissue and staining with hematoxylin and eosin and using an optical microscope. Furthermore, in order to directly demonstrate the presence of new structures inside the lymphatic vessels, the lymphatic vessels having the seal structures were examined by histological methods and by using a cryoscanning electron microscope. On the cross and oblique cross-sections of the tissues, an unusual structure was observed, and on the lyophilized scanning microscope, the liquid held by the rapid freezing of the sample was observed in detail.

The present invention has convincingly demonstrated that a new structure is present. In early studies of vascular structures, complex methods such as specific perfusion methods, fluorescence techniques using acridine orange, and staining methods with hematoxylin and eosin to demonstrate the existence and novelty of newly observed constructs Was used (Ref. 13). Therefore, developing staining techniques using JGB and methods for in situ and in vivo verification of new structures in lymphatic vessels through them are of great importance in demonstrating the existence of the novel structures.

The cross-sectional images stained with hematoxylin and eosin, and the images of freeze-fragmented samples obtained from cryoscanning electron microscopes, strongly demonstrate that certain silt structures are present inside the lymphatic vessels. The thickness of the seal construct varies depending on the subject sample, and in particular depends on the functional state of the seal construct and / or the physiological state of the subject sample animal. The diameter and bundle structure, and size and arrangement, of the nuclei of the vascular seal structures in various lymphatic vessels are similar to those found in the arteries and veins of rabbits and rats (Ref. 13). The bundle structure of the seal structure consists of tubes that are about 5 μm in size, due to CLSM and histological phase. Moreover, the specific spherical structure and separation zone observed on the frozen scanning electron microscope means that there is a viscous liquid inside the fistula of the tube. This new seal structure showed similar results in the let, and the results of this let were described in the examples of the present invention.

From the above results, Kim's theory of the physiological function of these seal structures can be considered more carefully. His theory aims to establish the physics of acupuncture meridian and acupoint. In recent years, ultrasonography of acupuncture points has revealed a structure consistent with his claim (Ref. 7). However, both his theory and his theory associated with the recently widely used neurophysiological model (Ref. 2) require further study. According to Kim, several types of liquids with granulocytes containing DNA (Ref. 10) resemble microcells and flow through ducts closely associated with cell therapy (Ref. 17). In addition, the thread-shaped tube was considered to be closely related to the developmental process, especially the lymphatic vascular seal structure (Ref. 17). Therefore, the discovery of new structures inside the lymphatic vessels may be an important cornerstone for the breakthrough of cell anatomy and cell physiology, not just curiosity. In addition, further analysis of the histological characteristics and physiological functions of the constructs may suggest the possibility of new concepts that are important in the field of biology and medicine.

Hereinafter, the present invention will be described in more detail by the following examples. At this time, a preferred embodiment of the present invention is as follows.

< Example  1> seal structure inside the rabbit's lymphatic vessel

New Zealand white rabbits (females, 12 weeks old) were purchased from Korea Central Experimental Animals Company and were acclimatized by animal protection and testing procedures in accordance with current international laws and treaties (Experimental Animal Protection and Instructions, International University Press). , 1996). Anesthesia was injected by injecting 1.5 g / kg urethane into rabbit abdominal cavity, and all surgical procedures were performed under conventional anesthesia.

The rabbit's abdomen was dissected and many lymphatic vessels near the lower vena cava exposed. In order to stain the free seal structure inside the lymphatic vessel, about 0.3 ml of JGB (100 ml of 99.9% purity ethanol and 1 g of 65% Janus Green B (Aldrich, USA) was added to 100 ml of 99.9% purity ethanol). Sample content) was loaded into a 30 1/2 dimension syringe and injected slowly into the lymph nodes or lymphatic vessels near the lower vena cava. After the needle was removed, blue JGB flowing in the lymphatic vessel was observed. Subsequently, the JGB flowing back from the sample was washed, and PBS pre-heated at 40 ° C. was injected into the lymph vessels and lymph nodes in order to maintain temperature and vitality of the sample. A stereomicroscope was used to observe the sample. The location of well stained seal structures in the lymphatic vessels varies with the lymphatic condition and degree of staining of the subject rabbit. When a well stained site was found, the connective tissue around the lymphatic vessel was carefully removed to facilitate clearer observation and easier detection of the sample targeted in the present invention.

For confocal laser scanning microscopy (CLSM) observation, the samples were taken and immediately immobilized in 10% neutral buffered formalin solution, some of which were 0/1% (w / v) of acridine orange. , Sigma-Aldrich, USA).

For histological analysis, the lymphatic vessels with seal structures were first prepared by immobilization in 10% neutral buffered formalin solution as a pretreatment procedure for embedding paraffin. The samples were cut into 5 μm thick fragments using a microtome and stained with hematoxylin and eosin. The sections were observed and printed under an optical microscope (Axiophot, Carl Zeiss, Germany).

For cryo-SEM observations, the immobilized samples were bound with rivets mounted on slotted metal stubs and then soaked in liquid nitrogen for 20 seconds for freeze-fixing. Frozen samples were placed in a freezing chamber on a freezing-delivery system (CT1500, Oxford Instruments, Oxon, UK) at -170 ° C and then transferred to the sample location on a scanning electron microscope (JSM-5410LV, JEOL, Tokyo, Japan). lost. The rivet surface in the freezing chamber was then cut with a cold knife to freeze-section to expose the internal structures of the frozen lymphatic vessels, including the yarn structures stained with JGB. The sample was heat-treated to sublimate frozen water, the sample position was kept cold at -60 ° C for 10 minutes, and the process was monitored by electron microscopy on an accelerating bolt of 5 kV. The sample was coated with gold (about 30 nm thick) in a freeze-chamber and then observed under an electron microscope on an accelerated bolt of 20 kV.

Figure 1 shows a three-dimensional image of lymphatic vessels near the lower vena cava of rabbits. At this time, (A) is a site generally observed without staining, (B, C) is a site shown by staining after injecting JGB into the lymph vessels. In (A), lymphatic plates (arrowhead sites) were observed inside the lymphatic vessels and capillaries near the lymphatic vessel wall. After injection of JGB, a distinctly stained lymph plate (arrowhead site) was observed inside the lymphatic vessel wall where the seal structure (arrow site) of (B) was weakly stained. The seal structure (arrow site) of (C) is the largest of the sites observed, and its size is about 600 μm in diameter. In the figure inserted in (C), three stained lymphatic plates (arrowhead sites) mean that the size of the lymphatic vessels and well stained new structures can be located on or through the plate. The scale bar is 1 nm.

1A shows a stereomicroscopic image of a lymphatic vessel near the lower vena cava, with a diameter of about 600 μm. Capillaries in the lymphatic wall are clearly observed and can be observed even under the lymphatic vessel wall. Also, although not clear, lymphatic plates can also be observed. On the other hand, no other structures were observed inside the lymph vessels. This means that conventional microscopic observations have difficulty detecting new tissue. However, after injection of JGB, a staining sample into the lymph vessels or lymph nodes, it was found that new structures were found inside the lymph vessels. 1B and 1C are stereograms shown after injection of JGB into lymph vessels or lymph nodes. As shown in FIG. 1B, the yarn constructs stained with JGB were clearly observed with lymphatic stained and weakly stained lymphatic wall. In addition, as shown in FIG. 1C, another very thick seal structure was observed in cylindrical form between the two lymphatic plates. As shown in FIGS. 1B and 1C, the thickness of such a seal structure varies widely depending on the target sample. The inset in FIG. 1C shows the yarn construct stained with JGB liberated through three stained lymphatic plates within the weakly stained lymphatic wall.

1B and 1C are representative views obtained through 14 experiments described in Tables 1 and 2. FIG. We investigated the lumbar nodules next to the lower vena cava and the iliac veins, and the abdominal lymphatic vessels connected to the peritoneal lymphatic vessels near the intestine. In 14 experiments, we observed the intravascular structures of the lymphatic vessels in all subject rabbits except 2 failures. Thus, it can be seen that the yarn construct stained with JGB is not an abnormal site observed in a particular subject sample. The diameter of the lymphatic vessels ranges from 130 to 2000 μm, the diameter of the filamentous structures ranges from 26 to 500 μm, on average 129 μm. As shown in Figs. 1B and 1C, the thickness of the seal structure varies depending on the target sample.

Table 1 shows the results of the 19 seal structures obtained from 14 New Zealand white rabbits. The values of the data in the following table are given as (mean ± standard deviation), and more detailed information is shown in Table 2.

Lymphatic tube location of the target sample The number of real structures Lymphatic diameter (μm) Diameter of seal structure (μm) Near the lower vena cava 15 785.6 ± 537.9 153.5 ± 142.6 Leader One 180.0 13.0 Iliac vein 3 510.0 ± 340.7 43.3 ± 7.0 all 19 719.7 ± 506.0 129.4 ± 134.6

Target Sample Injection site Lymphatic tube location of the target sample Lymphatic diameter (μm) Diameter of seal structure (μm) Length of seal structure (㎛) Date (2004) castle Weight (kg) 10.18 female 2.2 Venous end lymphatic vessels Near the end of the vena cava M: 1500 m: 600 100 4000 10.25 female 2.2 Lumbar nodules Near the end of the vena cava 2000 50 3000 11.01 female 2.2 Lumbar nodules Near the end of the vena cava 1060 80 12000 11.10 female 2.2 Lumbar nodules Near the end of the vena cava 100 16 220 880 DM: 510 Dm: 190 4000 11.11 female 2.2 Venous end lymphatic vessels Near the end of the vena cava M: 400 m: 180 30 5000 M: 880 m: 320 60 3200 11.15 female 2.2 Lumbar nodules Near the end of the vena cava 540 80 940 11.16 female 2.2 Lumbar nodules Near the end of the vena cava X X X 11.18 female 2.2 Lumbar nodules Near the end of the vena cava X X X 11.24 female 2.2 Lumbar nodules Near the end of the vena cava 180 50 1600 600 230 2700 11.25 female 2.2 Lumbar nodules Near the end of the vena cava 1500 500 5000 1300 DM: 400 Dm: 170 1700 960 170 6600 11.29 female 2.2 Colon lymphatic vessel Large intestine 360 26 1180 12.01 female 2.2 Lumbar nodules Near the end of the vena cava 130 40 2700 540 260 4540 12.06 female 2.2 Lymph node tip Iliac vein tip 270 44 2700 360 50 2180 B: 1800 12.07 female 2.2 Lymph node tip Iliac vein tip 900 36 1640

Table 2 contains more detailed information on the 14 rabbits mentioned in Table 1 above. B = branch, X = No sample collection, DM = duct maximum, Dm = duct minimum, JGB = Janus Green B.

Figure 2 shows an optically segmented continuous image of the seal structure observed with confocal laser scanning microscope (CLSM) after staining with acridine orange. The nucleus is rod-shaped, 10-20 μm in length. They are distributed in the form of broken-lines, the number of lines vary from A to D, and their depth of focus has also increased to 1 μm at the bottom of the real structure. The scale bar is 10 μm.

Nuclear distribution of the seal construct was performed by CLSM observation. One part of the seal structure was enlarged 400 times, and optical cutting was performed to a size of 1 μm. Through this fragmented phase, it was found that the seal structure had a characteristic rod-shaped shape of about 10 to 20 μm, and was distributed in a long and thin shape having a broken-line shape. The number of elongated advanced lines varied as shown in FIGS. 2A-2D, with the focal point moving by 1 μm from the bottom in turn. This feature means that the seal structure may consist of a "bundle" structure consisting of small tubes with a diameter of about 5 μm. In addition, these structures correlate with known structures within the blood vessels of rabbits and rats (Refs. 1, 13) and intestinal-surface previously known (Refs. 14, 15, 20).

Figure 3 (A) shows the cross-sectional short-circuit (arrow area) of the seal structure inside the lymphatic vessel stained with hematoxylin and eosin at 1,000 (x) magnification and 200 (x) magnification. (B) shows the diagonal cross section of a real structure measured by 400 (x) magnification and 200 (x) magnification. As shown in the figure inserted in (A), new tissue did not adhere to the lymphatic wall and lymphatic plates (arrowheads). Several large and small fistulas (starred sites), partially surrounded by nuclei (dotted arrow sites) heavily stained with hematoxylin, were observed, indicating that they continued to appear at the same site along the silt. The scale bar is 20 μm and the scale bar in the inserted figure is 50 μm.

In order to obtain information about the position of the new structure inside the lymphatic vessel, and cells and fistulas including the same, in the present invention, the cross-sectional image was analyzed after staining with hematoxylin and eosin. The cross-sectional view inserted in FIG. 3A clearly shows that there is a characteristic tissue factor that is not attached to the lymphatic vessel wall (FIG. 3A). In FIG. 3A, the new tissue has an outer membrane and shows that the vascular endothelial cells inside the lymphatic vessels contain nuclei that are specifically stained in large quantities. The extracellular matrix inside these new tissues had several large and small fistulas, which were distributed continuously at the same location on several different slides. As such, the succession of fistulas on each slide means that more effort and research is needed on the fistulas in evaluating the physiological function of new tissue. The diagonal cross-sectional view of FIG. 3B shows a characteristic view of the seal structure largely surrounded by the lymphatic plate. This figure and the inserted figure show the passage of the seal structure passing through the plate. This clearly demonstrates that the real structure actually exists.

These new tissues have many structures similar to fistulas in the extracellular matrix as well as functional features in the nucleus. In addition to morphological analytical methods such as CLSM or histological analysis by hematoxylin and eosin, cryo-SEM assays can be used to detect not only cell structures but also liquids inherent in the sample ( Reference 18). Flowable material, organelles, and macromolecules are retained in the separation zone upon freezing. In addition, this method allows more detailed observation of the shape of the seal structures inside the lymphatic vessels.

4 (A) is a diagram of a cryo-fragmented sample showing a collapsed lymphatic vessel (dotted border area, not observed under a microscope) that includes another substructure with characteristic outer membrane. (B) is an enlarged view of the rectangular part in (A). The new structure has a fistula with separation zone and spherical structure. The flowing material in the fistula enters the spherical structure (arrow area). Scale bar is 200 micrometers in FIG. (A), and 20 micrometers in FIG. (B).

In freeze-sectioned samples, the lymphatic vessels surrounded by connective tissue were found to have a wavy cortical system by sublimation after freezing, but new tissue was found to be inside the collapsed lumen of the lymphatic vessels (FIG. 4A). This new tissue has a characteristic outer membrane that does not collapse, and the size of this sample is similar to that found in the three-dimensional image of FIG. 1C. As shown in the rectangle of FIG. 4A and enlarged in FIG. 4B, it can be seen that the fistula of the new tissue has a larger separation zone than the surrounding cytoplasm (FIG. 4B). Although the fistula has a separation zone and a spherical structure, it means that the fistula is a liquid (having a liquid) containing a spherical object and other components therein.

< Example  2> Rat  Seal structure inside the lymphatic vessel

In order to analyze morphologically and histologically the seal structure inside the lymphatic vessel of the rat, it was carried out in the same manner as the experimental method using the rabbit of the first embodiment.

For this experiment, in the present Example, six Wistar rats (three males and three females) each having a weight of about 170 g per horse were purchased from the central experimental animal company. The animals were acclimated in a controlled environment (23 ° C.) with 60% relative humidity and constant temperature under a 12 hour light / dark cycle. In addition, all of these animals received feed and water without limitation. Laboratory procedures and animal protection in relation to laboratory animals have been carried out in accordance with current international laws and treaties (Experimental Animal Protection and Instructions for Use, International University Press, 1996).

The let was anesthetized by intraperitoneal injection of 1.5 g / kg urethane and all surgical procedures were performed under conventional anesthesia. The abdominal surface of the let was cut in a state of complete anesthesia, and the lumbar node was exposed by removing the peritoneum and fat near the lower vena cava.

A solution containing 1% Janus Green B (JGB) in 0.05 ml of ethanol was loaded slowly into a 30 1/2 syringe. In the rabbit experiment described in the examples of the present invention, JGB was injected into lymph nodes as well as lymph nodes, whereas in rat experiments, JGB was injected only into three types of lymph nodes, namely, lymph nodes, lymph nodes, and lumbar lymph nodes. Compared with rabbit lymphatic vessels, rat lymphatic vessels are very small and difficult to inject.

After receiving the sample, hematoxylin and eosin staining (FIG. 5) and cryoscanning electron microscope observation (FIG. 6) were performed. The results are shown in FIGS. 5, 6, and 3.

The results of rats and rabbits were very similar. The average diameter of the seal structures observed in the rats was 70 ± 57 μm smaller than that of rabbits, which were surrounded by a thin outer membrane and contained a large amount of stained nuclei and extracellular matrix (FIG. 5). Therefore, the present inventors expected that, using methods similar to those performed in rats and rabbits, the present invention could be applied to confirm the presence or absence of new seal structures inside the lymphatic vessels of other mammals. I decided it would be necessary.

FIG. 5 shows a cross-sectional view of a sample stained with hematoxylin and eosin showing lymphatic vessels with seal structures (arrow sites) measured under an optical microscope (Axiophot, Carl Zeiss, Germany). The sample was prepared via paraffin-incision to a thickness of about 5 μm. The new construct obtained therefrom has a thin outer membrane (arrow site indicated by double lines) and contains a large amount of stained nuclei (arrow site indicated by dotted lines) and an extracellular matrix. It also has several fistulas (starred areas) partially surrounded by the nucleus. The ratio of diameters between the seal structure and the lymphatic vessels varies with the sample of interest. (A) and (B) each represent a relatively thick and thin yarn structure. These are derived from the same rats but are different lymphatic vessels. The different colors in the two figures are due to the optical filter. That is, the present inventors used a blue filter (A) to obtain a clear picture of the fistula. The scale bar is 20 μm.

 Figure 6 shows the cross-sectional view of the new seal structure (dotted area) inside the lymphatic vessel under a freeze scanning electron microscope (JSM-5410LV, JEOL, Japan). In freeze-sectioned samples, the collapsed lymphatic vessels show that they contain another substructure with characteristic outer membranes. As in the case of rabbits, the lymphatic vessels collapsed but the new structures did not. The scale bar is 50 μm. FIG. 6B is an enlarged view of the rectangular portion of FIG. 6A. FIG. Through this, we could observe that the seal structure is characteristically separated from the lymphatic wall. This means that the new seal structure is freed in the lymph of lymphatic vessels. The scale bar is 10 μm. The sizes of the six seal constructs from six rats are listed in Table 3 below.

Target Sample Lymph node injection Lymph node location of target sample Lymph node diameter (μm) Diameter of seal structure (μm) Length of seal structure (㎛) Date castle Weight (g) 2004. 11.18 cock 180 Lymph node tip Near bladder 340 60 920 B: 90 B: 20 B: 680 2004. 11.18 female 160 Lymph node tip Lower lumbar nodule M: 530 m: 210 70 5100 2004. 11.20 cock 150 Intestinal lymph nodes Near the liver 550 DM: 280 3000 Dm: 80 2004. 11.22 female 180 Lymph node tip Near bladder M: 260 50 2500 m: 100 2005. 1.10 cock 170 Lumbar nodules Upper lumbar nodule M: 670 50 4000 m: 290 2005. 1.19 female 170 Lumbar nodules Lower lumbar nodule 130 10 645

The abbreviations used above are as follows: M = maximum, m = minimum, B = branch, X = No sample collection, DM = duct maximum, Dm = duct minimum, JGB = Janus Green.

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Claims (13)

Janus Green B (JGB), comprising a specific visualization of the Bonghan ducts compared to other lymphoid tissues, the visualization agent for staining the internal lymphatic Bonghan duct (Bonghan duct). The visualization agent of claim 1, wherein the agent visualizes a Bonghan duct in situ or in vivo. delete The visualization agent of claim 1, wherein the agent is a solution comprising JGB. The visualization agent of claim 4, wherein the solution contains 0.01 to 10 (w / v)% JGB in ethanol or saline. Inject lymphatic tissue with a solution containing Janus Green B (JGB) and stain, Observing with a microscope, How to visualize Bonghan ducts inside lymphatic vessels. The method of claim 6, wherein the solution contains 0.01 to 10 (w / v)% JGB. The method of claim 6, wherein the JGB stains an in situ or in vivo Bonghan duct. The method of claim 6, wherein the microscope observation is a stereomicroscope, a light microscope, a confocal laser scanning microscope (CLSM), or a cryo scanning electron microscope (Cryo-scanning microscope) Measuring by SEM). The method of claim 6, wherein the method further comprises staining the Bonghan duct with hematoxylin and eosin (H & E) after injecting JGB into the lymphoid tissue. The method of claim 10, wherein the microscopy is measured with an optical microscope. The method of claim 6, wherein the method further comprises staining the Bonghan duct with acridine orange after injecting JGB into the lymphoid tissue. The method of claim 12, wherein the microscopy is measured by confocal laser scanning microscope.

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