A visualization-based mid-focal area function evaluation system and method

Abstract

The present invention discloses a system and method for evaluating the function of the central focal region based on visualization, and relates to the technical field of diagnosis and treatment in traditional Chinese medicine. The main points of the technical solution are: 1. Visualization and functional quantification of gastric lymphatic vessels: a. Tracing of living gastric lymphatic vessels; b. Calculation of its lymphatic transport index. 2. Visualization and functional quantification of intestinal lymphatic vessels: a. Visualization of the initial capillary lymphatic vessels of the intestine using immunofluorescence staining; b. Observation of its fat transport using Oil Red O staining to quantify the function; c. Tracing of living intestinal collecting lymphatic vessels; d. Calculation of its lymphatic transport index; e. Visualization of abdominal fat to indirectly evaluate the function of intestinal collecting lymphatic vessels. The present invention realizes the visualization and quantification of the shape, quality and function of the central focal region by establishing a structural and functional detection and evaluation system for gastric and intestinal lymphatic vessels, so as to combine traditional Chinese medicine theory with modern biomedical technology, and provide new ideas and tools for the modernization and scientificization of traditional Chinese medicine.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine diagnosis and treatment, and more specifically, to a visualization-based central focal region function evaluation system and method. Background Art

[0002] The concept of "triple energizer" in TCM theory was first recorded in the TCM classic Huangdi Neijing, which said that it was one of the "six internal organs" of the five internal organs and six internal organs, and summarized its physiological functions: "Triple energizer is the official who decides the drainage, and the water channel comes out of it", clarifying that its main function is to regulate the transportation of water; then "Treatise on Warm Diseases" further developed the triple energizer theory and established the triple energizer dialectic as the outline for the diagnosis and treatment of warm diseases. In modern TCM clinical practice, the "triple energizer" theory still plays an important role in the diagnosis and treatment of various diseases, especially in the diagnosis and treatment of epidemic diseases.

[0003] The "three burners" are divided into "upper burner, middle burner, and lower burner", and the middle burner is one of the three burners. The Huangdi Neijing summarizes the physiological characteristics of the middle burner as "the middle burner is like fermentation", pointing out that the middle burner has the functions of digesting food, absorbing nutrients, absorbing water, and transporting nutrients and water throughout the body; the lymphatic vessels throughout the body are considered to be the biological morphological basis of the three burners. Combining the function and location of the middle burner, it can be judged that the stomach and intestinal lymphatic vessels are the biological morphological basis of the middle burner. The gastric lymphatic vessels have the same function as the lymphatic vessels of other organs, and their main function is to absorb the interstitial fluid that refluxes locally. In addition to the function of absorbing interstitial fluid, the intestinal lymphatic vessels have another major feature: absorbing and transporting fat from the diet. 80-90% of the body's fat formation is absorbed and transported by the intestinal lymphatic vessels; therefore, the functional characteristics of the gastrointestinal lymphatic vessels are highly consistent with the physiological characteristics of "the middle burner is like fermentation", and the stomach and intestinal lymphatic vessels are the biological morphological basis of the middle burner.

[0004] Although the triple energizer theory occupies an important position in traditional Chinese medicine, its clinical application and scientific research potential are greatly limited due to the controversy over the triple energizer's form and nature. In addition, in modern medical research, dysfunction of the middle energizer is related to a variety of diseases. Therefore, the evaluation of the middle energizer function is of great significance for the diagnosis and treatment of diseases. Therefore, the present invention aims to provide a visualization-based middle energizer regional function evaluation system and method to solve the above problems. Summary of the invention

[0005] The purpose of the present invention is to provide a visualization-based central focal region function evaluation system and method. The present invention realizes the visualization and quantification of the shape, quality and function of the central focal region by establishing a structure and function detection and evaluation system for the stomach and intestinal lymphatic vessels, thereby combining traditional Chinese medicine theory with modern biomedical technology, and providing new ideas and tools for the modernization and scientificization of Chinese medicine.

[0006] The above technical objectives of the present invention are achieved through the following technical solutions: a visualization-based mid-focal region function evaluation system, including a living gastric lymphatic tracing module, a gastric lymphatic transport function evaluation module, an intestinal initial capillary lymphatic visualization module, an intestinal initial capillary lymphatic function evaluation module, a living intestinal collecting lymphatic tracing module, an intestinal lymphatic transport function evaluation module and an abdominal fat visualization module;

[0007] The living stomach lymphatic vessel tracing module is used to inject a tracer into the living stomach and observe the lymphatic vessel structure and the tracer distribution;

[0008] The gastric lymphatic transport function evaluation module is used to evaluate the gastric lymphatic transport function;

[0009] The intestinal initial capillary lymphatic vessel visualization module is used to mark the intestinal initial capillary lymphatic vessels by immunofluorescence staining;

[0010] The intestinal initial capillary lymphatic function evaluation module is used to evaluate the function of the intestinal initial capillary lymphatic vessels;

[0011] The living intestinal collection lymphatic vessel tracing module is used to inject tracers into the living intestinal tract and observe the lymphatic vessel structure and tracer distribution;

[0012] The intestinal lymphatic vessel transport function evaluation module is used to evaluate the intestinal lymphatic vessel transport function;

[0013] The abdominal fat visualization module is used to indirectly evaluate the function of intestinal collecting lymphatic vessels by photographing abdominal fat.

[0014] The present invention also provides a method for evaluating the function of the mid-focal area based on visualization, comprising the following steps:

[0015] S1. Tracing of gastric lymphatic vessels in vivo was performed by injecting Evans blue tracer into the serosal layer of the gastric wall near the cardia at the lesser curvature of the stomach and taking time series images using a stereo microscope.

[0016] S2. Evaluate the gastric lymphatic transport function by measuring the Evans blue content and lymph node weight and calculating the lymphatic transport index;

[0017] S3. Visualize the initial intestinal lymphatic capillaries by labeling Lyve-1 with immunofluorescence staining to observe the initial intestinal lymphatic capillaries.

[0018] S4. Evaluate the initial intestinal capillary lymphatic function and observe the fat distribution using Oil Red O staining;

[0019] S5, trace the collecting lymphatic vessels in the living intestine, inject Evans blue tracer into Peyer's patches, and observe the lymphatic vessel structure and tracer distribution;

[0020] S6. Evaluate the intestinal lymphatic transport function by measuring the Evans blue content and lymph node weight and calculating the lymphatic transport index;

[0021] S7. Visualize the abdominal fat and indirectly assess the function of the intestinal collecting lymphatic vessels by exposing and photographing the abdominal fat.

[0022] The present invention is further configured as follows: in the method, by measuring the Evans blue content and the lymph node weight, the formula for calculating the lymph transport index is:

[0023] Lymphatic transport index = Evans blue content / lymph node weight;

[0024] Among them, the lymphatic transport index was negatively correlated with lymphatic transport function.

[0025] The present invention is further configured as follows: in the method, immunofluorescence staining is used to mark Lyve-1, and the process of observing the initial capillary lymphatic vessel structure of the intestine is as follows:

[0026] The samples were washed three times with TBS containing 0.1% Triton X-100 for 5 minutes each time, and then incubated with 10% serum consistent with the source of the secondary antibody at room temperature for 60 minutes. The primary antibody anti-LYVE-1 was then added to cover the specimen, incubated overnight at 4°C, and then the fluorescent secondary antibody was added to cover the specimen, incubated at room temperature for 60 minutes, and finally washed three times with PBST for 5 minutes each time, and DAPI was added to counterstain the cell nucleus, the slides were sealed with glycerol gelatin, and observed under a fluorescence microscope.

[0027] The present invention is further configured as follows: the process of observing the fat distribution by using Oil Red O staining in the method is: by comparing the fat accumulation inside and outside the initial intestinal capillary lymphatic vessels of the blank group and the model group, the fat absorption and reflux function of the intestinal capillary lymphatic vessels is evaluated.

[0028] The present invention is further configured as follows: the process of tracing the living intestinal collecting lymphatic vessels in the method is: by observing the changes in the lymphatic vessel structure and tracer distribution at different time points after the injection of Evans blue tracer, the reflux transport function of the intestinal collecting lymphatic vessels is evaluated.

[0029] The present invention is further configured as follows: the process of visualizing abdominal fat in the method is: by comparing the hyperplasia of abdominal fat in the blank group and the model group, the reflux function of the intestinal collecting lymphatic vessels is indirectly evaluated.

[0030] In summary, the present invention has the following beneficial effects:

[0031] 1. The present invention combines the concept of "triple energizer" in traditional Chinese medicine theory with modern biomedical technology, and uses visualization technology to concretize the shape, quality and function of the middle energizer area, providing new methods and tools for the modernization and scientificization of Chinese medicine theory. At the same time, by visualizing the structure and function of lymphatic vessels in the middle energizer area, the present invention can more accurately evaluate the physiological state of the middle energizer area, thereby improving the diagnosis and treatment effects of related disease models;

[0032] 2. The method for evaluating the function of the middle focal region based on visualization provided by the present invention is helpful for the research and development of traditional Chinese medicine. At the same time, the present invention also establishes an animal experimental method system involving the middle focal region, provides a standardized detection system template for traditional Chinese medicine research, and helps to improve the reliability and repeatability of experimental results;

[0033] 3. The present invention can evaluate the functional changes of the middle focal area through visualization, which is helpful for in-depth research on the disease mechanism related to the middle focal area, promote the pathological research of related diseases and the development of new therapies. At the same time, the method provided by the present invention is simple and rapid, and can evaluate the functional status of the middle focal area in a short time, thereby improving the research efficiency.

[0034] 4. The present invention enhances the repeatability of experimental results through standardized operation procedures and quantitative evaluation indicators, providing reliable data support for scientific research. Moreover, the present invention is not only applicable to the research of the middle focal area, but can also be extended to the research fields of other lymphatic systems, and has broad application prospects.

[0035] 5. The method provided by the present invention promotes cross-disciplinary cooperation between traditional Chinese medicine and biomedical engineering, imaging and other disciplines, and provides new perspectives and tools for the modernization of traditional Chinese medicine research. At the same time, the implementation of the present invention helps to enhance the international influence of traditional Chinese medicine and promote its recognition and application worldwide, which has important social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a schematic diagram of the control results of the visualization of the initial capillary lymphatic vessels in the intestine in an embodiment of the present invention;

[0037] Figure 2 is a schematic diagram of control results of the evaluation of initial intestinal capillary lymphatic function in an embodiment of the present invention;

[0038] Figure 3 is a schematic diagram of the control results of tracing the collecting lymphatic vessels of the living intestine in an embodiment of the present invention;

[0039] Figure 4 Schematic diagram of the control result of visualizing abdominal fat in an embodiment of the present invention. DETAILED DESCRIPTION

[0040] The following is combined with Figure 1-4 The present invention is described in further detail.

[0041] Embodiment 1: A visualization-based mid-focus area function evaluation system, comprising a living gastric lymphatic vessel tracing module, a gastric lymphatic vessel transport function evaluation module, an intestinal initial capillary lymphatic vessel visualization module, an intestinal initial capillary lymphatic vessel function evaluation module, a living intestinal collecting lymphatic vessel tracing module, an intestinal lymphatic vessel transport function evaluation module and an abdominal fat visualization module;

[0042] The in vivo gastric lymphatic vessel tracing module is used to inject tracers into the stomach of the living body and observe the lymphatic vessel structure and tracer distribution;

[0043] The gastric lymphatic transport function evaluation module is used to evaluate the gastric lymphatic transport function;

[0044] The intestinal initial lymphatic capillary visualization module is used to mark the intestinal initial lymphatic capillaries by immunofluorescence staining;

[0045] The intestinal initial capillary lymphatic function evaluation module is used to evaluate the function of the intestinal initial capillary lymphatic vessels;

[0046] The in vivo intestinal collection lymphatic vessel tracing module is used to inject tracers into the in vivo intestine and observe the lymphatic vessel structure and tracer distribution;

[0047] The intestinal lymphatic transport function evaluation module is used to evaluate the transport function of intestinal lymphatic vessels;

[0048] The abdominal fat visualization module is used to indirectly assess the function of the intestinal collecting lymphatic vessels by imaging the abdominal fat.

[0049] Example 2: A method for evaluating the function of the mid-focal area based on visualization

[0050] This embodiment includes visualization and quantification of gastric cardia lymphatic function detection and visualization and quantification of intestinal lymphatic function detection; wherein visualization and quantification of gastric cardia lymphatic function detection includes tracing of living gastric lymphatic vessels and evaluation of gastric lymphatic transport function, and quantification of intestinal lymphatic function detection includes visualization of intestinal initial capillary lymphatic vessels, tracing of living intestinal collecting lymphatic vessels, evaluation of intestinal lymphatic transport function and visualization of abdominal fat.

[0051] In the process of in vivo gastric lymphatic tracing, rats or mice are anesthetized, fixed in the supine position and the abdomen is opened. A 10% Evans Blue tracer (Evans Blue, solvent: PBS) is injected into the serosal layer of the gastric wall near the cardia at the lesser curvature of the stomach. The injection dose is 10 μL for rats and 5 μL for mice. After injection, the lymphatic vessels are rapidly traced, and the structure of the lymphatic vessels and the distribution of the tracer in the lymphatic vessels are observed. Time series photography is performed using a stereomicroscope.

[0052] During the evaluation of lymphatic transport function, the most obviously stained lymph nodes in the gastric and pancreatic wall were selected and collected for weighing, and the unstained lymph nodes were selected as blank controls; formamide was added to the collected lymph nodes for extraction in a water bath and centrifuged at 12000rpm for 15 minutes; the supernatant was drawn from the extraction tube of the blank control, and Evans blue was added to prepare and serially diluted standard solutions with concentrations of 16, 8, 4, 2, 1, 0.5, and 0 μg / mL; the absorbance at 562 nm was measured with an enzyme marker, and the absorbance-concentration standard curve was drawn, R 2 >0.99; calculate the Evans blue content in the stained lymph nodes, lymphatic transport index (EBTI) = Evans blue content / lymph node weight, EBTI is negatively correlated with lymphatic transport function.

[0053] In the visualization process of the initial capillary lymphatic vessels of the intestine in this embodiment, paraffin sections or frozen sections of the small intestine are used to mark the biomarker Lyve-1 of the initial capillary lymphatic vessels by immunofluorescence staining (membrane permeabilization: washing 3 times with TBS containing 0.1% Triton X-100, hereinafter referred to as TBST, 5 minutes / time; blocking: incubating with 10% serum consistent with the source of the secondary antibody at room temperature for 60 minutes; incubating with primary antibody: adding primary antibody anti-LYVE-1 to cover the specimen and incubating at 4°C overnight; incubating with secondary antibody: after taking the sections out of 4°C, washing 3 times with TBST, 5 minutes / time, adding fluorescent secondary antibody to cover the specimen, and incubating at room temperature for 60 minutes; counterstaining of cell nuclei: washing 3 times with PBST, 5 minutes / time, adding DAPI; sealing: sealing with glycerol gelatin), the structure of the initial capillary lymphatic vessels of the intestine can be directly observed under a fluorescence microscope.

[0054] like Figure 1 As shown (A. the initial lymphatic capillaries in the center of the small intestinal villi; B. the lymphatic capillaries in the mucosal layer, submucosal layer, and muscular layer of the small intestine; the white arrows point to the dilated lymphatic vessels; Muc (mucosal layer), mucosal layer; SM (submucosal layer), submucosal layer; Mus (muscularis externa.); red fluorescence: LYVE-1; blue fluorescence: DAPI), among which the Control group is a blank control, and the Model group is a hypertensive mouse model with middle-jiao dampness obstruction; when the middle-jiao area is diseased, changes in the initial lymphatic capillaries of the small intestine can be observed, which are manifested as thickening, dilation, and bifurcation. This process can clearly visualize the intestinal lymphatic capillary structure, thereby visualizing and evaluating the changes in the morphology and quality of the middle-jiao area.

[0055] In this example, in the evaluation of the initial capillary lymphatic vessels in the intestine, Oil Red O staining was used to mark the fat in the frozen sections of the small intestine to observe the distribution of fat inside and outside the lumen of the initial capillary lymphatic vessels in the small intestine.

[0056] like Figure 2 As shown in the figure (the upper row of pictures shows Oil Red O staining in the villi of the small intestine, and the lower row of pictures shows Oil Red O staining in the submucosal layer and muscular layer of the small intestine), the Control group is a blank control, and the Model group is a hypertensive mouse model with dampness obstruction in the middle focus area; when the middle focus area is diseased, it can be observed that the fat accumulates inside and outside the small intestinal lymphatic capillaries due to the initial fat absorption and reflux disorder in the small intestine. This process can clearly visualize and quantify the function of the intestinal lymphatic capillaries, thereby visualizing and evaluating the shape and function of the middle focus area.

[0057] In the process of collecting lymphatic vessels for tracing in the living intestine, rats or mice are anesthetized, fixed in a supine position, and the abdomen is opened. The Peyer's patch of the small intestine is found and a 10% Evans Blue tracer (solvent: PBS) is injected into the Peyer's patch. The injection dose is 10 μL for rats and 5 μL for mice. After injection, the lymphatic vessels are quickly traced, and the structure of the lymphatic vessels and the distribution of the tracer in the lymphatic vessels are observed. Time series photography is performed using a stereo microscope.

[0058] like Figure 3 As shown in the figure (the green arrows in the upper row of the picture indicate the lymphatic vessel structure and tracer reflux in the Control group; the red arrows in the lower row of the picture indicate the lymphatic vessel structure and tracer reflux in the Model group), where the Control group is a blank control, and the Model group is a hypertensive mouse model with dampness obstruction in the middle jiao. When the middle jiao area is lesioned, obstruction and leakage of the intestinal collecting lymphatic vessels can be observed, and the tracer reflux is delayed and leaks and spreads to the surrounding tissues. Immediately after tracer injection, intestinal collecting lymphatic vessel shadows appeared in the Control group and the Model group. The results were observed 2min, 5min, 10min, and 15min after injection, respectively. It was found that at 2min in the Control group, the intact lymphatic reticular structure could be observed in the intestinal wall indicated by the green arrow; at 5min, tracer dye began to appear in the lumen at the position indicated by the green arrow, indicating that the lymph fluid refluxed normally in the lumen; at 10min, the dye in the lumen gradually became lighter; until 15min, the tracer dye at the position indicated by the green arrow was almost completely cleared by reflux.

[0059] The results show the whole process of the normal collection of returning lymph by the intestinal collecting lymphatic vessels in the Control group. However, in the Model group, leakage and diffusion of tracer dye were immediately observed at the site indicated by the red arrow 2 minutes after injection, and a complete and clear lymphatic reticular structure could not be observed at the intestinal wall. At 5 minutes, additional leakage and diffusion of tracer dye appeared at the site indicated by the red arrow. At 10 minutes, additional leakage and diffusion of tracer dye continued to appear at the site indicated by the red arrow. At 15 minutes, the dye in the lymphatic vessel cavity was still not cleared, indicating that dye clearance was delayed and the diffused dye of the surrounding leakage still existed. This process can clearly visualize the structure and function of the intestinal lymphatic vessels, thereby visualizing the mid-focal area to evaluate its morphological and functional changes.

[0060] In the evaluation of intestinal lymphatic transport function, the most obviously stained lymph nodes on the proximal intestinal collecting lymphatic vessels were selected and collected for weighing, and then the unstained lymph nodes were selected as blank controls. Formamide was added to the collected lymph nodes for extraction in a water bath, and centrifuged at 12,000 rpm for 15 minutes. The supernatant was aspirated from the blank control extraction tube, and Evans blue was added to prepare and serially diluted standard solutions with concentrations of 16, 8, 4, 2, 1, 0.5, and 0 μg / mL. The absorbance at 562 nm was measured with an enzyme marker, and an absorbance-concentration standard curve was drawn. R 2 >0.99. The Evans blue content in the stained lymph nodes was calculated, and the lymphatic transport index (EBTI) = Evans blue content / lymph node weight. EBTI was negatively correlated with lymphatic transport function.

[0061] The comparison of EBTI in each group is shown in Table 1:

[0062] Table 1 Comparison of EBTI in each group ( n = 5, μg / g lymph node)

[0063] Group EBTI Control Group 13.64±3.62 Model Group <![CDATA[6.17±2.75 ** ]]>

[0064] The Control group was a blank control, and the Model group was a hypertensive mouse model with dampness obstruction in the middle jiao. The lymphatic transport index (EBTI) of the Model group was significantly lower than that of the Control group. This step can effectively quantify the intestinal lymphatic transport function, thereby visualizing and quantifying the middle jiao function.

[0065] During the abdominal fat visualization process, rats or mice are anesthetized, fixed in a supine position, and the abdomen is opened to fully expose the abdominal fat and take pictures. Visualizing the abdominal fat can indirectly evaluate the function of the intestinal collecting lymphatic vessels. If there is hyperplasia of abdominal fat, it can be indirectly determined that there is a functional disorder of leakage in the intestinal collecting lymphatic vessels.

[0066] like Figure 4As shown (the red dotted line in the picture is the abdominal fat tissue), the Control group is a blank control, and the Model group is a hypertensive mouse model with dampness obstruction in the middle jiao. The abdominal fat in the Model group increased significantly. This step can directly and indirectly evaluate the leakage of the intestinal collecting lymphatic vessels, thereby visually evaluating the function of the middle jiao.

[0067] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A mid-focal area function evaluation system based on visualization, characterized by: It includes a living gastric lymphatic vessel tracing module, a gastric lymphatic vessel transport function evaluation module, an intestinal initial capillary lymphatic vessel visualization module, an intestinal initial capillary lymphatic vessel function evaluation module, a living intestinal collecting lymphatic vessel tracing module, an intestinal lymphatic vessel transport function evaluation module and an abdominal fat visualization module; The living stomach lymphatic vessel tracing module is used to inject a tracer into the living stomach and observe the lymphatic vessel structure and the tracer distribution; The gastric lymphatic transport function evaluation module is used to evaluate the gastric lymphatic transport function; The intestinal initial capillary lymphatic vessel visualization module is used to mark the intestinal initial capillary lymphatic vessels by immunofluorescence staining; The intestinal initial capillary lymphatic function evaluation module is used to evaluate the function of the intestinal initial capillary lymphatic vessels; The living intestinal collection lymphatic vessel tracing module is used to inject tracers into the living intestinal tract and observe the lymphatic vessel structure and tracer distribution; The intestinal lymphatic vessel transport function evaluation module is used to evaluate the intestinal lymphatic vessel transport function; The abdominal fat visualization module is used to indirectly evaluate the function of intestinal collecting lymphatic vessels by photographing abdominal fat.

2. A method for evaluating the function of the mid-focal area based on visualization, characterized by: The following steps are involved: S1. Tracing of gastric lymphatic vessels in vivo was performed by injecting Evans blue tracer into the serosal layer of the gastric wall near the cardia at the lesser curvature of the stomach and taking time series images using a stereo microscope. S2. Evaluate the gastric lymphatic transport function by measuring the Evans blue content and lymph node weight and calculating the lymphatic transport index; S3. Visualize the initial intestinal lymphatic capillaries by labeling Lyve-1 with immunofluorescence staining to observe the initial intestinal lymphatic capillaries. S4. Evaluate the initial intestinal capillary lymphatic function and observe the fat distribution using Oil Red O staining; S5, trace the collecting lymphatic vessels in the living intestine, inject Evans blue tracer into Peyer's patches, and observe the lymphatic vessel structure and tracer distribution; S6. Evaluate the intestinal lymphatic transport function by measuring the Evans blue content and lymph node weight and calculating the lymphatic transport index; S7. Visualize the abdominal fat and indirectly assess the function of the intestinal collecting lymphatic vessels by exposing and photographing the abdominal fat.

3. The method for evaluating the function of the mid-focal area based on visualization according to claim 2, characterized in that: In the method, the formula for calculating the lymphatic transport index is as follows by measuring the Evans blue content and the lymph node weight: Lymphatic transport index = Evans blue content / lymph node weight; Among them, the lymphatic transport index was negatively correlated with lymphatic transport function.

4. The method for evaluating the function of the mid-focal area based on visualization according to claim 2, characterized in that: The method uses immunofluorescence staining to mark Lyve-1 and observes the initial capillary lymphatic structure of the intestine as follows: The samples were washed three times with TBS containing 0.1% Triton X-100 for 5 minutes each time, and then incubated with 10% serum consistent with the source of the secondary antibody at room temperature for 60 minutes. The primary antibody anti-LYVE-1 was then added to cover the specimen, incubated overnight at 4°C, and then the fluorescent secondary antibody was added to cover the specimen, incubated at room temperature for 60 minutes, and finally washed three times with PBST for 5 minutes each time, and DAPI was added to counterstain the cell nucleus, the slides were sealed with glycerol gelatin, and observed under a fluorescence microscope.

5. The method for evaluating the function of the mid-focal area based on visualization according to claim 2, characterized in that: The method uses Oil Red O staining to observe the fat distribution process as follows: by comparing the fat accumulation inside and outside the initial intestinal capillary lymphatic vessels of the blank group and the model group, the fat absorption and reflux function of the intestinal capillary lymphatic vessels is evaluated.

6. The method for evaluating the function of the mid-focal area based on visualization according to claim 2, characterized in that: The process of tracing the living intestinal collecting lymphatic vessels in the method is: by observing the changes in the lymphatic vessel structure and tracer distribution at different time points after the injection of Evans blue tracer, the reflux transport function of the intestinal collecting lymphatic vessels is evaluated.

7. The method for evaluating the function of the mid-focal area based on visualization according to claim 2, characterized in that: The process of visualizing abdominal fat in the method is: by comparing the hyperplasia of abdominal fat in the blank group and the model group, the reflux function of the intestinal collecting lymphatic vessels is indirectly evaluated.

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