Use of neutrophil extracellular traps in the preparation of a diagnostic kit for lymphangitic inflammation

Abstract

The application belongs to the technical field of biomedical diagnosis, and provides application of neutrophil extracellular traps in preparation of a lymphangitic inflammation diagnosis kit, wherein the application first discloses high expression of neutrophil extracellular traps in lymphangitic inflammation lesion tissues, and confirms that the expression level is closely related to disease occurrence and development, so that the neutrophil extracellular traps are used as an early diagnosis index to fill the technical blank of lack of early non-invasive screening means for lymphangitic inflammation in the prior art, precise diagnosis and disease monitoring of subclinical stage occult lymphangitic inflammation can be realized, misdiagnosis and missed diagnosis can be effectively avoided, reliable technical support and theoretical basis are provided for early prevention and timely intervention of serious complications such as secondary lymphedema, and thus the incidence of complications is significantly reduced and the prognosis of patients is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical diagnostic technology, and in particular relates to the application of an extracellular trap for neutrophils in the preparation of a diagnostic kit for lymphangitis. Background Technology

[0002] Lymphangitis refers to a localized or systemic inflammatory cascade pathological response triggered by pathogen invasion, tissue microenvironment damage, or immune imbalance of lymphatic endothelial cells (LECs). It is the most fundamental early pathological change in the lymphatic system and the initiating cause of secondary lymphedema and lymphatic vessel failure. Epidemiological surveys show that approximately 30% of breast cancer and gynecological malignancies will develop chronic, progressive lymphatic dysfunction due to lymphatic system damage caused by surgery or radiotherapy. However, early symptoms of lymphangitis are insidious, lack specific warning indicators and highly sensitive non-invasive diagnostic techniques, and routine imaging and blood tests are insufficient to identify subclinical endothelial activation and microinflammatory infiltration, easily leading to missed diagnoses or delayed intervention. Long-term persistent low-grade inflammation eventually progresses irreversibly to typical secondary lymphedema, accompanied by thickened and hardened skin, recurrent and refractory cellulitis, limb deformities and disabilities, and intractable chronic pain.

[0003] Extracellular neutrophil traps (NETs) are tools used by neutrophils to capture and eradicate pathogens such as bacteria, fungi, and viruses. Essentially, they are complex three-dimensional network structures released by neutrophils. Various proteins can attach to them, including histones and more than 30 primary and secondary granule components. These contain a large number of bactericidal components, such as elastase, myeloperoxidase, cathepsin G, lactoferrin, pentameric protein 3, gelatinase, protease 3, and peptidoglycan-binding proteins, as well as other antibacterial active substances capable of disrupting virulence factors.

[0004] Extraneutrophil traps exhibit a broad and complex double-edged sword effect in disease—playing a defensive role under physiological conditions, while excessive formation or clearance obstacles under pathological conditions can exacerbate tissue damage and disease progression. Therefore, in-depth exploration and precise regulation of their release mechanisms will contribute to a comprehensive understanding of their multiple roles in immune defense, inflammatory responses, autoimmune diseases, and tumorigenesis. Recent studies have shown that extraneutrophil traps can significantly enhance the proliferation and invasiveness of colorectal cancer. Furthermore, they play a decisive regulatory role in the development and progression of various tumors, including breast cancer, gastric cancer, and pancreatic cancer. Moreover, extraneutrophil traps contain citrullinated proteins and histones, making them an important source of autoantibodies such as anti-citrate protein antibodies in rheumatoid arthritis and anti-neutrophil cytoplasmic antibodies in vasculitis. In cardiovascular diseases, extraneutrophil traps have been confirmed to exist within atherosclerotic plaques, promoting plaque progression through inflammation amplification, endothelial damage, and foam cell formation. The levels of their related markers (such as myeloperoxidase-DNA complex) are closely related to plaque severity, providing important evidence for disease risk assessment and targeted intervention. Currently, research on extracellular neutrophil traps in lymphatic system diseases is still in its early stages, and there are no existing technologies reporting the application of extracellular neutrophil traps in the diagnosis of lymphangitis. Summary of the Invention

[0005] The purpose of this invention is to provide the application of extracellular neutrophil traps in the preparation of diagnostic kits for lymphangitis, thereby addressing the problems raised in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] The application of extraneutrophil traps in the preparation of diagnostic kits for lymphangitis, wherein the extraneutrophil traps serve as non-invasive screening biomarkers for diseases related to lymphangitis.

[0008] Furthermore, the diagnostic kit is used to detect the expression level of extracellular traps in neutrophils.

[0009] Furthermore, the methods for detecting the expression level of extracellular traps in neutrophils include immunofluorescence staining and real-time quantitative PCR.

[0010] Furthermore, the diagnostic kit includes reagents for detecting the expression level of extraneutrophil traps; the reagents for detecting the expression level of extraneutrophil traps include: reagents for detecting the expression level of structural component proteins of extraneutrophil traps, and / or reagents for detecting the expression level of key molecules related to the formation and function of extraneutrophil traps.

[0011] Furthermore, the extracellular trap structural component protein of neutrophils is citrullinated histone H3; the reagent for detecting the expression level of the extracellular trap structural component protein of neutrophils includes an antibody that specifically binds to citrullinated histone H3.

[0012] Furthermore, the key molecules include one or more genes selected from peptidyl arginine deiminase 3, peptidyl arginine deiminase 4, neutrophil elastase, matrix metalloproteinase 9, and myeloperoxidase.

[0013] Furthermore, the reagents for detecting the expression levels of key molecules related to the formation and function of extracellular traps in neutrophils include one or more of the following primer pairs: peptidyl arginine deiminase 3 detection primer pair, peptidyl arginine deiminase 4 detection primer pair, neutrophil elastase detection primer pair, matrix metalloproteinase 9 detection primer pair, and myeloperoxidase detection primer pair.

[0014] Furthermore, the nucleotide sequences of the peptidyl arginine deiminase 3 detection primer pair are shown in SEQ ID NO: 9-10; the nucleotide sequences of the peptidyl arginine deiminase 4 detection primer pair are shown in SEQ ID NO: 11-12; the nucleotide sequences of the neutrophil elastase detection primer pair are shown in SEQ ID NO: 13-14; the nucleotide sequences of the matrix metalloproteinase 9 detection primer pair are shown in SEQ ID NO: 15-16; and the nucleotide sequences of the myeloperoxidase detection primer pair are shown in SEQ ID NO: 17-18.

[0015] This invention provides an application of extracellular neutrophil traps in the preparation of a diagnostic kit for lymphangitis. It reveals for the first time the high expression of extracellular neutrophil traps in lymphangitis lesions and confirms that their expression level is closely related to disease development. Using them as an early diagnostic indicator fills the technological gap in the lack of non-invasive early screening methods for lymphangitis, enabling accurate diagnosis and monitoring of subclinical occult lymphangitis, effectively avoiding missed diagnoses and misdiagnoses. This provides reliable technical support and theoretical basis for the early prevention and timely intervention of serious complications such as secondary lymphedema, thereby significantly reducing the incidence of complications and improving patient prognosis. Attached Figure Description

[0016] Figure 1 The results show the successful construction of a mouse model of lymphangitis; where A represents the HE staining results and B represents the results of qRT-PCR detection of the mRNA expression levels of inflammatory factors.

[0017] Figure 2 The image shows the results of flow cytometry analysis, illustrating the expression of neutrophils in bone marrow cells of the two groups of mice.

[0018] Figure 3 The images show the results of immunofluorescence staining. By detecting the expression level of citrullinated histone H3 (CitH3), the infiltration of extraneutrophil traps can be visually displayed. In the image, A is a superimposed image of the fluorescence signals of H3Cit (green) and DAPI (blue), which can visually show the co-localization of CitH3 positive signals with cell nuclei, reflecting the distribution and cell localization of extraneutrophil traps. B is a neutrophil extraneutrophil trap marker (H3Cit) image of the control group and the model group, reflecting the core marker of neutrophil extraneutrophil trap release. Among them, the green fluorescence intensity of the model group is significantly higher than that of the control group, indicating that the infiltration of extraneutrophil traps in the tissue of the model group is significantly increased. C is a cell nuclear staining (DAPI) image of the control group and the model group, used to show the distribution of all cells in the tissue.

[0019] Figure 4 The mRNA expression levels of key molecules associated with extracellular traps in neutrophils (peptidyl arginine deiminase 3, peptidyl arginine deiminase 4, neutrophil elastase, matrix metalloproteinase 9, and myeloperoxidase) as shown by qRT-PCR results.

[0020] In the figure: ** indicates P < 0.01; *** indicates P < 0.001. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] In one embodiment of the present invention, an application of extraneutrophil traps in the preparation of a diagnostic kit for lymphangitis is provided, wherein the extraneutrophil traps serve as a non-invasive screening biomarker for lymphangitis-related diseases. The present invention aims to provide a novel application for detecting extraneutrophil traps in early screening and risk warning diagnosis of lymphangitis, overcoming the technical bottleneck of existing methods that struggle to identify subclinical, occult inflammation and easily delay intervention.

[0023] Specifically, firstly, a mouse model of lymphangitis was constructed, and its successful construction was confirmed by histological and molecular biological testing. Subsequently, bone marrow cells were isolated and extracted from the mice in the lymphangitis model group, and flow cytometry was used to analyze bone marrow cell subsets. The results showed that the proportion of neutrophils in the bone marrow of the model group (lymphangitis mouse model) was significantly higher than that of the control group (normal mice), confirming that the occurrence and development of lymphangitis in mice are closely related to the abnormal activation and infiltration of neutrophils.

[0024] Based on the above findings, immunofluorescence staining was used to detect the expression level of extraneutrophil traps in the lymphatic tissues of the two groups of mice. The results confirmed that extraneutrophil traps were highly expressed in the lesion tissues of the lymphatic inflammation model group of mice.

[0025] In addition, by detecting the expression levels of key molecules closely related to the formation and function of extraneutrophil traps (such as peptidyl arginine deiminase 3, peptidyl arginine deiminase 4, neutrophil elastase, matrix metalloproteinase 9, myeloperoxidase, etc.), we further verified and clarified the crucial regulatory role of extraneutrophil traps in the occurrence and development of lymphangitis.

[0026] Therefore, this invention uses the expression level of extracellular traps of neutrophils as an early diagnostic screening indicator for lymphangitis, providing important technical support and theoretical basis for the early and accurate diagnosis of lymphangitis, disease monitoring, and early prevention of lymphatic-related diseases.

[0027] In another embodiment of the present invention, a diagnostic kit for lymphangitis based on extraneutrophil traps is also provided. This diagnostic kit is used to detect the expression level of extraneutrophil traps. The methods for detecting the expression level of extraneutrophil traps include immunofluorescence staining and real-time quantitative PCR.

[0028] Specifically, the diagnostic kit includes reagents for detecting the expression level of extraneutrophil traps; the reagents for detecting the expression level of extraneutrophil traps include: reagents for detecting the expression level of proteins that are structural components of extraneutrophil traps, and / or reagents for detecting the expression level of key molecules related to the formation and function of extraneutrophil traps.

[0029] Among them, the extracellular trap structural component protein of neutrophils is citrullinated histone H3 (CitH3); the reagents for detecting the expression level of extracellular trap structural component protein of neutrophils include antibodies that specifically bind to citrullinated histone H3.

[0030] In addition, key molecules related to the formation of extracellular traps in neutrophils include peptidyl arginine deiminase 3 and peptidyl arginine deiminase 4; key molecules related to the function of extracellular traps (i.e., functional executive molecules) include neutrophil elastase, matrix metalloproteinase 9, and myeloperoxidase. Correspondingly, reagents for detecting the expression levels of key molecules related to the formation and function of extracellular traps in neutrophils include primer pairs for detecting peptidyl arginine deiminase 3, peptidyl arginine deiminase 4, neutrophil elastase, matrix metalloproteinase 9, and myeloperoxidase.

[0031] It should be noted that, unless otherwise specified, the experimental methods in the following embodiments are conventional methods in the art. Unless otherwise specified, all reagents and materials used are commercially available.

[0032] Example 1: This example provides a method for constructing and validating a mouse model of lymphangitis. The specific steps are as follows:

[0033] Model Construction: A mouse model of lymphangitis was successfully established by using C57BL / 6 mice and performing a tail circumcision procedure to sever the lymphatic vessels on both sides. Specifically, to establish the lymphangitis mouse model, mice were randomly divided into a control group (n=5) and a model group (n=5). Anesthesia was induced with 4% isoflurane in 100% oxygen, and then 1% isoflurane was maintained at a flow rate of 1.5 L / min throughout the procedure. All mice underwent circular incisions 15 mm and 18 mm from the base of the tail. In the model group, a 3 mm wide full-thickness skin resection was performed while preserving the integrity of the blood vessels. Subsequently, 20 μL of 0.5% patented blue dye was injected into the model group mice to identify the two lymphatic vessels accompanying the tail vein.

[0034] Histological examination: HE analysis showed that the subcutaneous tissue thickness in the tail of the model group mice was significantly increased, accompanied by significant lymphatic dilation (e.g., Figure 1 As shown in A).

[0035] Tissue samples were fixed with 4% paraformaldehyde (PFA) at 4°C, decalcified with 5% EDTA, and then embedded in paraffin to prepare 5-micron sections. For histological staining, dewaxed sections were stained with hematoxylin and eosin (H&E), dehydrated, and mounted according to standard procedures.

[0036] Molecular expression validation: The transcriptional expression levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the tail tissues of control and model mice were detected by qRT-PCR. The results showed that their mRNA expression levels were significantly upregulated in the model group (e.g., Figure 1 (As shown in B).

[0037] The primer pairs used for qRT-PCR detection of TNF-α are as follows:

[0038] TNF-α-F: 5′-AGGGTCTGGGCCATAGAACT-3′ (as shown in SEQ ID NO:1);

[0039] TNF-α-R: 5′-CAGCCTCTTCTCATTCCTGC-3′ (as shown in SEQ ID NO:2);

[0040] The primer pairs used for qRT-PCR detection of IL-1β are as follows:

[0041] IL-1β-F: 5′-GAAATGCCACCTTTTGACAGTG-3′ (as shown in SEQ ID NO:3);

[0042] IL-1β-R: 5′-TGGATGCTCTCATCAGGACAGATCGTAGC-3′ (as shown in SEQ ID NO:4);

[0043] The primer pairs used for qRT-PCR detection of IL-6 are as follows:

[0044] IL-6-F: 5′-CTGCAAGAGACTTCCATCCAG-3′ (as shown in SEQ ID NO:5);

[0045] IL-6-R: 5′-AGTGGTATAGACAGGTCTGTTGG-3′ (as shown in SEQ ID NO:6);

[0046] The primer pair for the internal reference gene β-actin used in qRT-PCR technology is as follows:

[0047] β-actin-F: 5′-GGAATCCTGTGGCATCCATGA-3′ (as shown in SEQ ID NO:7);

[0048] β-actin-R: 5′-GTCTTTACGGATGTCAACGTCACAC-3′ (as shown in SEQ ID NO:8).

[0049] Example 2: This example examines the expression level of neutrophils in the model group mice, as detailed below:

[0050] Neutrophils in the bone marrow of model mice were analyzed by flow cytometry. In short, bone marrow cells from the femur and tibia of mice were collected and incubated with neutrophil-associated antibodies FITC-CD11b and PE-Ly6G. Specifically, neutrophils were isolated from mouse bone marrow cells using a mouse neutrophil enrichment kit (Stemcell, batch number 19762) according to the manufacturer's instructions. After isolation, mouse bone marrow neutrophils were identified and analyzed by flow cytometry as follows: Bone marrow cells from the femur and tibia of model mice were collected and incubated with fluorescent antibodies FITC-CD11b (BioLegend, batch number 101205) and PE-Ly6G (BioLegend, batch number 127607); data were analyzed using a BDFACSymphony A5 flow cytometer and analyzed using FlowJo v10.8.1 software. The results are shown below. Figure 2 As shown in the figure. The results showed that neutrophils were highly expressed in the model group mice.

[0051] Example 3: This example examines the expression level of extracellular traps in neutrophils in the model group mice, as detailed below:

[0052] To confirm the release of extraneutrophil traps by highly expressed neutrophils, the expression of these traps was detected using immunofluorescence staining. Specifically, after fixation, dehydration, embedding, sectioning, and antigen retrieval (applicable to paraffin sections), tail tissue samples from model mice were incubated overnight at 4°C with anti-CitH3 (manufacturer: Abcam, batch number: ab219407) using secondary antibody. Subsequently, the samples were incubated at room temperature in the dark for immunofluorescence staining detection. The results are as follows: Figure 3 As shown in Figures A, B, and C. The results indicate that more extracellular neutrophil traps can be formed in the tail tissue of the model group mice.

[0053] Example 4: This example detects the expression levels of key molecules related to extracellular traps in neutrophils, as detailed below:

[0054] To further confirm that extraneutrophilic traps are an important pathogenic factor in lymphangitis, the expression of key molecules related to the formation of extraneutrophilic traps (peptidyl arginine deiminase 3 and peptidyl arginine deiminase 4) and key molecules related to their function (neutrophil elastase, matrix metalloproteinase 9, and myeloperoxidase) was detected by qRT-PCR. The results showed that the expression of these key molecules was significantly upregulated in the tail tissue of model mice (e.g., ...). Figure 4 (As shown).

[0055] The primer pairs used for qRT-PCR detection of peptidyl arginine deiminase 3 are as follows:

[0056] Peptidylarginine deiminase 3-F: 5′-TGTTTGAGGTCTACGGGACAC-3′ (as shown in SEQ ID NO:9);

[0057] Peptidylarginine deiminase 3-R: 5′-GGTCATTGCTAGGGGAGTTCA-3′ (as shown in SEQ ID NO: 10);

[0058] The primer pairs used for qRT-PCR detection of peptidyl arginine deiminase 4 are as follows:

[0059] Peptidylarginine deiminase 4-F: 5′-TGGTCCTCCAGTCAAGAAGAG-3′ (as shown in SEQ ID NO: 11);

[0060] Peptidylarginine deiminase 4-R: 5′-GCTTTCACCTGTAGGGTCACC-3′ (as shown in SEQ ID NO: 12);

[0061] The primer pairs used for qRT-PCR detection of neutrophil elastase are as follows:

[0062] Neutrophil elastase-F: 5′-CCTTGGCAGACTATCCAGCC-3′ (as shown in SEQ ID NO:13);

[0063] Neutrophil elastase-R: 5′-GACATGACGAAGTTCCTGGCA-3′ (as shown in SEQ ID NO:14);

[0064] The primer pairs used for qRT-PCR detection of matrix metalloproteinase 9 are as follows:

[0065] Matrix metalloproteinase 9-F: 5′-ACGGCAACGGAGAAGGCAAAC-3′ (as shown in SEQ ID NO:15);

[0066] Matrix metalloproteinase 9-R: 5′-GTCCACTCGGGTAGGGCAGAAG-3′ (as shown in SEQ ID NO:16);

[0067] The primer pairs used for qRT-PCR detection of myeloperoxidase are as follows:

[0068] Myeloperoxidase-F: 5′-AACACATACCCCCGAGACTT-3′ (as shown in SEQ ID NO:17);

[0069] Myeloperoxidase-R: 5′-GGCAGACTCCCAACCTCTA-3′ (as shown in SEQ ID NO:18).

[0070] In summary, this invention first constructs a mouse model of lymphangitis, which is confirmed to be successfully established through histological and molecular biological testing. Subsequently, flow cytometry analysis of bone marrow cell subsets in the model group mice revealed a significantly increased proportion of neutrophils, clearly indicating a close relationship between lymphangitis and neutrophils. Furthermore, immunofluorescence staining experiments confirmed high expression of extraneutrophil traps in the diseased tissues of the model group mice. By detecting the expression of key molecules related to extraneutrophil traps, the crucial regulatory role of extraneutrophil traps in the development and progression of lymphangitis was verified. Therefore, extraneutrophil traps can serve as a core potential target for the early diagnosis of lymphangitis. By detecting the expression level of extraneutrophil traps, latent inflammatory changes in the lymphatic endothelium can be detected early, clarifying the development and progression of lymphangitis, thus providing a reliable basis for the early and accurate diagnosis of lymphangitis.

[0071] This invention uses the expression level of extracellular traps of neutrophils as a screening indicator for the early diagnosis of lymphangitis. Its core advantage lies in the fact that current technologies lack early non-invasive screening methods for lymphangitis. Conventional detection methods struggle to detect subclinical, occult lymphangitis, often leading to missed diagnoses, misdiagnoses, and delayed intervention, ultimately resulting in serious complications such as secondary lymphedema. The solution provided by this invention fills this technological gap, enabling accurate early diagnosis and monitoring of lymphangitis, providing technical support and theoretical basis for the early prevention and timely intervention of lymphatic-related diseases, effectively reducing the incidence of complications and improving patient prognosis.

[0072] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. The application of extracellular neutrophil traps in the preparation of diagnostic kits for secondary lymphedema, characterized in that, The extraneutrophil trap serves as a non-invasive screening marker for secondary lymphedema; the diagnostic kit is used to detect the expression level of the extraneutrophil trap; the diagnostic kit includes reagents for detecting the expression level of the extraneutrophil trap. The reagents for detecting the expression level of extracellular traps of neutrophils include: reagents for detecting the expression level of structural component proteins of extracellular traps of neutrophils, and / or reagents for detecting the expression level of key molecules related to the formation and function of extracellular traps of neutrophils; the structural component protein of extracellular traps of neutrophils is citrullinated histone H3; the reagents for detecting the expression level of structural component proteins of extracellular traps of neutrophils include antibodies that specifically bind to citrullinated histone H3; the key molecules include one or more genes selected from peptidyl arginine deiminase 3, peptidyl arginine deiminase 4, neutrophil elastase, matrix metalloproteinase 9, and myeloperoxidase.

2. The application according to claim 1, characterized in that, The methods for detecting the expression level of extracellular traps in neutrophils include immunofluorescence staining and real-time quantitative PCR.

3. The application according to claim 1, characterized in that, The reagents used to detect the expression levels of key molecules related to the formation and function of extracellular traps in neutrophils include one or more of the following primer pairs: peptidyl arginine deiminase 3, peptidyl arginine deiminase 4, neutrophil elastase, matrix metalloproteinase 9, and myeloperoxidase.

4. The application according to claim 3, characterized in that, The nucleotide sequences of the peptidyl arginine deiminase 3 detection primer pair are shown in SEQ ID NO: 9-10; the nucleotide sequences of the peptidyl arginine deiminase 4 detection primer pair are shown in SEQ ID NO: 11-12; the nucleotide sequences of the neutrophil elastase detection primer pair are shown in SEQ ID NO: 13-14; the nucleotide sequences of the matrix metalloproteinase 9 detection primer pair are shown in SEQ ID NO: 15-16; and the nucleotide sequences of the myeloperoxidase detection primer pair are shown in SEQ ID NO: 17-18.

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