Methods for determining chemokines and mechanisms that inhibit cell migration in high-glucose microenvironment

Abstract

The present invention discloses a method for determining chemokines and mechanisms that inhibit cell migration under a high-sugar microenvironment, including: obtaining skin tissue from diabetic foot wounds or skin tissue from acute dorsal foot trauma wounds; performing single-cell transcriptome sequencing on the tissues to analyze the chemokine receptor-ligand interactions between fibroblast subpopulations and keratinocyte subpopulations; preparing fibroblast conditioned medium; treating keratinocytes with the conditioned medium to detect cell migration ability; detecting the expression level of chemokine CXCL12 in the conditioned medium; adding exogenous CXCL12 to the conditioned medium to detect the expression and migration ability of CXCR4 in keratinocytes. The results show that a high-sugar environment can inhibit cell migration by downregulating the expression of chemokines and their receptors, thereby affecting tissue repair and disease progression. This method helps to understand the regulatory mechanism of cell behavior under a high-sugar environment and provides a theoretical basis for the prevention and treatment of related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for determining chemokines and mechanisms that inhibit cell migration in a high-sugar microenvironment. Background Art

[0002] According to statistics from the International Diabetes Federation, there are about 500 million people suffering from diabetes worldwide, and it is expected that by 2045, this number will reach more than 700 million (7.8% of the world's population). Diabetic foot is one of the most serious complications of diabetes. The annual incidence of diabetic foot is 2% to 4% in high-income countries, and the lifetime prevalence of diabetic foot is 19% to 34% in low-income countries. Diabetic foot is the main cause of non-traumatic lower limb amputation. It is reported that more than 1 million people worldwide undergo amputations of varying degrees due to DFU each year.

[0003] The current conventional treatment methods for diabetic foot mainly include: 1). Control blood sugar levels by using insulin, oral hypoglycemic drugs or lifestyle adjustments to reduce the risk of diabetic foot; 2). Use antiplatelet drugs (such as aspirin), antibiotics to prevent infection, drugs to improve blood circulation (such as alprostadil), etc.; 3). Use wound dressings, debridement, negative pressure closed drainage (VAC) and other methods to promote wound healing; 4). Perform surgical treatment when necessary, including amputation, vascular reconstruction, nerve repair, etc.

[0004] After investigating relevant patent documents at home and abroad, it is found that the current treatment methods still have the following unresolved problems: 1) The efficacy of conventional Western medicine in treating diabetic foot is only 70%, and it is not effective for severe or difficult-to-heal wounds; 2) It relies on the subjective judgment of doctors or nurses and lacks objective monitoring indicators; 3) It is difficult to adapt to changing environmental conditions and individual differences, especially under extreme climatic conditions; 4) Some treatment methods (such as amputation) are costly and have a long recovery period after surgery; Summary of the invention

[0005] In view of the above defects or improvement needs of the prior art, the purpose of the present invention is to provide a method for determining the chemokines and mechanisms that inhibit cell migration under a high-sugar microenvironment. This method aims to reveal the inhibitory effect of chemokines on cell migration under a high-sugar environment and its molecular mechanism through single-cell sequencing and a variety of experimental techniques. By comparing the migration ability of cells under different conditions and the expression of related proteins, it is determined how a high-sugar environment affects cell behavior, and the role of chemokines such as CXCL12 therein. This study helps to understand the regulatory mechanism of cell behavior under a high-sugar environment and provide a theoretical basis for the prevention and treatment of related diseases.

[0006] In a first aspect, the present invention provides a method for determining chemokines and mechanisms that inhibit cell migration in a high-glucose microenvironment, comprising:

[0007] S100, obtain skin tissue from diabetic foot wounds and acute dorsum foot trauma wounds;

[0008] S200, performing single-cell transcriptome sequencing on the tissue to analyze the interaction between chemokine receptor ligands between fibroblast subpopulations and keratinocyte subpopulations;

[0009] S300, preparing fibroblast conditioned medium, wherein the fibroblasts are cultured in a high-glucose environment, and the cell supernatant cultured in a conventional culture medium serves as a control group;

[0010] S400, treating keratinocytes with the conditioned medium to detect cell migration ability;

[0011] S500, using Luminex liquid suspension chips to detect the levels of cytokines and chemokines in the conditioned medium, detecting the mRNA expression of chemokines in HFF cells by qPCR, and detecting the expression changes of CXCR4 protein by Western blotting;

[0012] S600, adding exogenous CXCL12 to the conditioned medium, and detecting the expression and migration ability of CXCR4 in keratinocytes;

[0013] S700, SPSS15.0 statistical software was used for data analysis to determine the mechanism by which high-glucose environment affects cell behavior and the role of chemokines such as CXCL12.

[0014] Furthermore, the step S200 specifically includes:

[0015] The wound edge skin of patients with type 2 diabetic foot and the wound edge skin of the dorsal part of the foot of patients with foot trauma were isolated, prepared, library constructed, and single-cell transcriptome sequencing was performed. The "cluster Profiler" package in the R package was used for GO and KEGG enrichment analysis; Cellphone DB was used to analyze cell-to-cell interactions; and the circlize v0.4.10 R software package was used to visualize the results.

[0016] Furthermore, the cell migration ability is detected by a scratch test, including:

[0017] The monolayer cells were scraped with a 200uL pipette tip and washed with PBS, 2mL of culture medium was added to each well, and the cell scratch healing was observed under a 200x optical microscope at 0, 24, 48, and 72h after scratching, and the cell migration rate was calculated at 24, 48, and 72h after scratching.

[0018] Furthermore, the scratch test in S400 needs to be repeated three times, and the results are averaged.

[0019] In a second aspect, the present invention provides an application of a CXCL12 / CXCR4 signaling pathway as a target in the preparation of a drug for treating or preventing diabetic foot.

[0020] In a third aspect, the present invention provides an application of a CXCL12 / CXCR4 signaling pathway agonist in the preparation of a medicament for treating diabetic foot, wherein the signaling pathway agonist comprises one or more of a CXCL12 agonist or a CXCR4 agonist.

[0021] Furthermore, the CXCL12 agonist includes a substance that excites CXCL12 activity or a substance that increases CXCL12 levels, and the CXCR4 agonist is selected from a substance that excites CXCR4 activity or a substance that increases CXCR4 levels.

[0022] Furthermore, the dosage form of the drug is selected from any pharmaceutically permitted dosage form;

[0023] Furthermore, the dosage form is selected from one or more of tablets, granules, capsules, pills, oral solutions, injections, and liposomes;

[0024] Furthermore, the administration method of the drug or pharmaceutical composition is selected from one or more of oral administration, topical administration, intranasal administration, systemic administration, intravenous administration, subcutaneous administration, intramuscular administration, intracerebroventricular administration, intrathecal administration or transdermal administration.

[0025] Beneficial effects of the present invention:

[0026] 1. The purpose of the present invention is to provide a method for determining chemokines and mechanisms that inhibit cell migration under a high-sugar microenvironment. This method aims to reveal the inhibitory effect of chemokines on cell migration under a high-sugar environment and its molecular mechanism through single-cell sequencing and multiple experimental techniques. By comparing the migration ability of cells under different conditions and the expression of related proteins, it is determined how a high-sugar environment affects cell behavior, and the role of chemokines such as CXCL12 in it. This study helps to understand the regulatory mechanism of cell behavior under a high-sugar environment and provide a theoretical basis for the prevention and treatment of related diseases.

[0027] 2. The present invention determined that the reduction of CXCL12 protein secreted by fibroblasts in a high-glucose environment is one of the important factors that hinder the migration of keratinocytes, providing a new target for the treatment of diabetic foot.

[0028] 3. The present invention proposes a drug that utilizes an agonist of the CXCL12 / CXCR4 signaling pathway as a drug for treating diabetic foot, providing a new treatment strategy for the treatment of diabetic foot.

[0029] Additional aspects and advantages of the present application will be partially given in the following description, which will become apparent from the following description, or will be understood through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the following description of the embodiments in conjunction with the accompanying drawings, in which:

[0031] Figure 1 is a flow chart of a method for determining chemokines and mechanisms that inhibit cell migration under a high-sugar microenvironment in an embodiment of the present invention;

[0032] Figure 2 is a schematic diagram of the interaction between chemokines and their ligands between various cell subpopulations in an embodiment of the present invention based on single-cell RNA sequencing analysis;

[0033] Figure 3 This is a schematic diagram of observation of migration of two groups of human keratinocytes (HaTaC) at different time points after scratching using an optical microscope ×200 in a scratch experiment in an embodiment of the present invention;

[0034] Figure 4 Schematic diagram of detecting the levels of cytokines and chemokines in two groups of conditioned medium using Luminex liquid suspension chip in an embodiment of the present invention;

[0035] Figure 5 Schematic diagram of qPCR detection of mRNA expression of chemokines in two groups of HFF cells in an embodiment of the present invention;

[0036] Figure 6 Schematic diagram of detecting CXCR4 protein expression in HaTaC cells of NFb-CM group and HGFb-CM group by Western blotting in an embodiment of the present invention;

[0037] Figure 7 This is a schematic diagram of observing the migration of four groups of human keratinocytes (HaTaC) at different time points after scratching using an optical microscope ×200 in an embodiment of the present invention;

[0038] Figure 8 This is a schematic diagram of using Western blotting to detect the expression of CXCR4 protein in four groups of HaTaC cells cultured for 48 hours under the HGFb-CM microenvironment using CXCL12 in an embodiment of the present invention. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention. It should also be noted that, for ease of description, only parts related to the present invention, rather than all structures, are shown in the accompanying drawings.

[0040] In the description of the present invention, unless otherwise clearly specified and limited, the terms "connected", "connected", and "fixed" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0041] Those skilled in the art will appreciate that, unless otherwise stated, the singular forms "a", "an", "said" and "the" used herein may also include plural forms. It should be further understood that the term "comprising" used in the specification of the present application refers to the presence of the features, integers, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0042] It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical terms and scientific terms) used herein have the same meaning as the general understanding of those skilled in the art to which this application belongs. It should also be understood that terms such as those defined in general dictionaries should be understood to have the same meaning as in the context of the prior art, and will not be interpreted with an idealized or overly formal meaning unless specifically defined as in the embodiments of this application.

[0043] This method aims to reveal the inhibitory effect of chemokines on cell migration under high glucose environment and its molecular mechanism through single-cell sequencing and multiple experimental techniques. By comparing the migration ability of cells and the expression of related proteins under different conditions, it can be determined how high glucose environment affects cell behavior and the role of chemokines such as CXCL12. This study helps to understand the regulatory mechanism of cell behavior under high glucose environment and provide a theoretical basis for the prevention and treatment of related diseases.

[0044] In view of the above defects or improvement needs of the prior art, the purpose of the present invention is to provide a method for determining chemokines and mechanisms that inhibit cell migration under a high-sugar microenvironment. This method uses single-cell sequencing and a variety of experimental techniques to reveal the inhibitory effect of chemokines on cell migration under a high-sugar environment and its molecular mechanism. By comparing the migration ability of cells under different conditions and the expression of related proteins, it is determined how a high-sugar environment affects cell behavior, and the role of chemokines such as CXCL12 therein. This study helps to understand the regulatory mechanism of cell behavior under a high-sugar environment and provide a theoretical basis for the prevention and treatment of related diseases.

[0045] Example 1

[0046] like Figure 1 As shown, the embodiment of the present invention provides a method for determining chemokines and mechanisms that inhibit cell migration in a high-sugar microenvironment, including:

[0047] S100, obtain skin tissue from diabetic foot wounds and acute dorsum foot trauma wounds;

[0048] S200, performing single-cell transcriptome sequencing on the tissue to analyze the interaction between chemokine receptor ligands between fibroblast subpopulations and keratinocyte subpopulations;

[0049] S300, preparing fibroblast conditioned medium, wherein the fibroblasts are cultured in a high-glucose environment, and the cell supernatant cultured in a conventional culture medium serves as a control group;

[0050] S400, treating keratinocytes with the conditioned medium to detect cell migration ability;

[0051] S500, using Luminex liquid suspension chips to detect the levels of cytokines and chemokines in the conditioned medium, detecting the mRNA expression of chemokines in HFF cells by qPCR, and detecting the expression changes of CXCR4 protein by Western blotting;

[0052] S600, adding exogenous CXCL12 to the conditioned medium, and detecting the expression and migration ability of CXCR4 in keratinocytes;

[0053] S700, SPSS15.0 statistical software was used for data analysis to determine the mechanism by which high-glucose environment affects cell behavior and the role of chemokines such as CXCL12.

[0054] The specific steps include:

[0055] 1.1 Preparation of main materials and reagents: Human keratinocyte cell line HaTaC and human fibroblast cell line HFF were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. DMEM medium, DMEM / F12 medium, and fetal bovine serum were obtained from Gibco, USA. Recombinant human SDF1 / CXCL12 protein and rabbit anti-human CXCR4 monoclonal antibody were obtained from Abcam, UK. Rabbit anti-human GAPDH monoclonal antibody, HRP-labeled goat anti-rabbit IgG antibody, primers, RT-qPCR kit, and Western blotting related reagents (kits) were obtained from Wuhan Sewell Biotechnology Co., Ltd.

[0056] 1.2 Single-cell transcriptome sequencing: The wound edge skin (CWE) of patients with type 2 diabetes and the wound edge skin (AWE) of the dorsal part of the foot of patients with foot trauma were collected and sent to Nanjing Xingeyuan Biotechnology Co., Ltd. for tissue separation, preparation, library construction, and single-cell transcriptome sequencing. The “cluster Profiler” package in the R package was used for GO and KEGG enrichment analysis; Cellphone DB was used to analyze cell-to-cell interactions. The circlize v0.4.10 R software package was used to visualize the results.

[0057] 1.3 Cell culture: HaTaC cells were placed in DMEM medium containing a mixture of 10% fetal bovine serum and 1% penicillin-streptomycin, and HFF cells were placed in DMEM / F12 medium containing a mixture of 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured in a cell constant temperature incubator at 37°C and 5% CO2.

[0058] 1.4 Preparation of conditioned media (CM): After culturing HFF with 30 mM D-glucose medium for 7 days, the cell culture supernatant (HGFb-CM) was collected and centrifuged (1000 g, 5 min), filtered with a 0.2 μm filter, and placed in a -80°C refrigerator for later use. The cell supernatant (NFb-CM) cultured with conventional culture medium was used as the control group.

[0059] 1.5 Effect of HGFb-CM on HaTaC cell migration Experimental grouping and intervention methods: HaTaC cells in the logarithmic growth phase were inoculated in 6-well plates at 1×106 / well and divided into 2 groups, namely NFb-CM group and HGFb-CM group. Among them, HaTaC cells in the NFb-CM group were inoculated in NFb-CM and cultured in a 37°C, 5% CO2 incubator; HaTaC cells in the HGFb-CM group were inoculated in HGFb-CM and cultured in a 37°C, 5% CO2 incubator.

[0060] 1.6 Scratch test: Use the tip of a 200uL pipette to scrape the monolayer cells and wash with PBS. Add 2mL of culture medium to each well and group and treat as in 1.7. Observe the healing of cell scratches under a 200x optical microscope at 0, 24, 48, and 72h after scratching and calculate the cell migration rate at 24, 48, and 72h after scratching. Cell migration rate = (scratch area at 0h after scratching - scratch area at other time points after scratching) ÷ scratch area at 0h after scratching × 100%. The experiment was repeated 3 times and the results were averaged.

[0061] 1.7 Luminex liquid suspension chip detection: The detection was performed according to the instructions of the Bio-Plex Pro Human Cytokine Screening 48-plex Panel chip. The values ​​were read using the Bio-Plex MAGPIX system (Bio-Rad). This part of the experiment was commissioned to Shanghai Wayen Biopharmaceutical Technology Co., Ltd.

[0062] 1.8 RT-qPCR: Total cell RNA was extracted according to the TRIzol instructions, and RNA was reverse transcribed into cDNA according to the instructions of the SweScript All-in-One RTSuperMix for qPCR reverse transcription kit. The primer sequences are shown in Table 1. The RT-PCR reaction system and conditions refer to the 2×Universal Blue SYBR Green qPCR Master Mix Kit, with ACTIN as the internal reference. The relative expression of mRNA was calculated using the 2-ΔCt method, and each group of experiments was repeated 3 times.

[0063] Table 1 Primer sequences and product sizes for detecting chemokine mRNA expression in human HFF fibroblasts using real-time fluorescence quantitative reverse transcription PCR

[0064]

[0065] 1.9 Western blotting to detect changes in CXCR4 protein expression: Total cell protein was extracted, and the primary antibodies added were rabbit anti-human CXCR4 monoclonal antibody and rabbit anti-human GAPDH monoclonal antibody (dilution ratio of 1:1000), and the secondary antibody was HRP-labeled goat anti-rabbit IgG antibody (dilution ratio of 1:2000). Imag J image analysis software was used to analyze the gray value of the bands. GAPDH was used as an internal reference to calculate the protein expression of CXCR4.

[0066] 1.10 Experimental grouping, intervention methods and detection indicators for the effect of CXCL12 on HaTaC cell migration: HaTaC cells in the logarithmic growth phase were seeded in 6-well plates at 1×106 cells / well and divided into 4 groups, namely NFb-CM group, NFb-CM+CXCL12 group, HGFb-CM group and HGFb-CM+CXCL12 group. Among them, HaTaC cells in the NFb-CM group were inoculated in NFb-CM and cultured in a 37°C, 5% CO2 incubator; HaTaC cells in the NFb-CM+CXCL12 group were inoculated in NFb-CM containing 100 ng / mL CXCL12 and cultured in a 37°C, 5% CO2 incubator; HaTaC cells in the HGFb-CM group were inoculated in HGFb-CM and cultured in a 37°C, 5% CO2 incubator; HaTaC cells in the HGFb-CM+CXCL12 group were inoculated in HGFb-CM containing 100 ng / mL CXCL12 and cultured in a 37°C, 5% CO2 incubator. Cell migration and CXCR4 protein expression in the four groups were detected as in 1.6 and 1.9.

[0067] 1.11 Statistical analysis

[0068] SPSS 15.0 statistical software was used for data analysis. The overall comparison among the groups was performed by one-way analysis of variance, and the multi-factor comparison among the groups was performed by LSD test. P<0.05 was considered statistically significant.

[0069] 2 Results Analysis

[0070] 2.1 Analysis of the interaction between chemokines and their ligands between cell subpopulations based on single-cell RNA sequencing

[0071] like Figure 2 As shown in the figure, compared with the skin at the edge of acute dorsal foot trauma, the interaction between chemokine ligands between fibroblast subsets and keratinocyte subsets in the skin tissue at the edge of diabetic foot wound was significantly weakened. The ligands included: CXCL1-CXCR2, CXCL2-CXCR2, CXCL3-CXCR2, CXCL8-CXCR2 and CXCL12-CXCR4.

[0072] 2.2 Effect of HGFb-CM on HaTaC migration

[0073] like Figure 3 As shown in Table 2 , 24 h and 48 h after scratching, the migration rate of HaTaC cells in the HGFb-CM group was significantly lower than that in the NFb-CM group (t values ​​were 23.5 and 15.65, respectively, P<0.01 or P<0.01).

[0074] Table 2 Comparison of migration rates of two groups of HaTaC cells at different time points after scratching (%, )

[0075]

[0076]

[0077] Note: Time factor main effect, F = 4592.04, P = 0.00; treatment factor main effect, F = 363.46, P = 0.00; interaction between the two, F = 3.36, P = 0.14; compared with the NFb-CM group, a P<0.01

[0078] 2.3 Contents of cytokines and chemokines in NFb-CM and HGFb-CM

[0079] Compared with the supernatant of HFF cells cultured in the conventional environment, there were 28 significantly differentially expressed cytokines and chemokines in the supernatant of HFF cells under the high glucose microenvironment, including chemokines CXCL1, CXCL2, CXCL8 and CXCL12 (the difference folds were 2.20, 1.42, 0.81 and 0.69, and the P values ​​were 0.0147, 0.51, 0.5969 and 0.000, respectively). Figure 3 .

[0080] 2.4 Chemokine mRNA expression in HFF cells

[0081] like Figure 5 As shown, the mRNA expressions of CXCL1, CXCL2, CXCL3 and CXCL8 in HFF cells under high glucose microenvironment were significantly higher than those in the normal control group (t values ​​were 4.246, 4.978, 4.998, 10.04, respectively, P = 0.0094 or P = 0.0132 or P = 0.0075 or P = 0.0006), and the mRNA expression of CXCL12 was significantly lower than that in the normal control group (t value was 4.099, P = 0.0149).

[0082] 2.5 Effect of HGFb-CM on CXCR4 protein expression in HaTaC

[0083] like Figure 6 The expression of CXCR4 protein in the NFb-CM group and HGFb-CM group were 0.48±0.11 and 27±0.01, respectively. Compared with the NFb-CM group, the expression of CXCR4 protein in the HGFb-CM group was significantly decreased, and the difference was statistically significant (t=5.134, P=0.007).

[0084] 2.6 Effect of CXCL12 on HaTaC cell migration in HGFb-CM microenvironment

[0085] like Figure 7 As shown in Table 3, 24h and 48h after scratching, the cell migration rate in the HGFb-CM group was significantly lower than that in the NFb-CM group and the HGFb-CM+CXCL12 group (P<0.01 or P<0.01), and the cell migration rate in the HGFb-CM+CXCL12 group was significantly higher than that in the HGFb-CM group (P<0.01).

[0086] Table 3 Comparison of migration rate of human keratinocytes in four groups at different time points after scratching (%, )

[0087]

[0088] Note: Time factor main effect, F = 1242.82, P = 0.00; treatment factor main effect, F = 646.70, P = 0.00; interaction between the two, F = 85.46, P = 0.00; compared with the NFb-CM group, a P<0.01; compared with HGFb-CM group, b P<0.01

[0089] 2.7 Effect of CXCL12 on CXCR4 protein expression in HaTaC cells under HGFb-CM microenvironment

[0090] like Figure 8 As shown in the figure, the expression of CXCR4 protein in NFb-CM group, NFb-CM+CXCL12 group, HGFb-CM group and HGFb-CM+CXCL12 group were 0.51±0.06, 0.052±0.08, 0.25±0.01 and 0.45±0.05, respectively. The overall comparison among the groups showed statistically significant differences (F=15.818, P=0.001). Compared with the NFb-CM group, the expression of CXCR4 protein in HGFb-CM group was significantly decreased (P<0.01); compared with the HGFb-CM group, the expression of CXCR4 protein in HGFb-CM+CXCL12 group was significantly increased (P=0.002).

[0091] 3. Discussion and analysis

[0092] Wound healing is a complex biological process. After skin injury, the wound healing process is initiated, and immune cells, fibroblasts, keratinocytes and other mesenchymal cells are recruited to participate in regulating the wound healing process. The wound microenvironment is composed of different types of cells. In order to play an appropriate role in the wound healing process, communication between various types of cells is necessary. Studies have shown that fibroblasts can sense and respond to tissue damage by releasing signal molecules (cytokines and growth factors, etc.) in an autocrine or paracrine manner, and form a connection network with keratinocytes, vascular endothelial cells, and macrophages in wound healing, playing an important regulatory role. Wound healing consists of several different but overlapping stages, which are generally divided into four stages: inflammatory response stage, granulation tissue formation stage, re-epithelialization stage and wound remodeling stage [7-8]. In-depth elucidation of the intercellular communication mechanism has important theoretical guiding significance for the study of wound repair strategies. Re-epithelialization is an important link in the wound healing process and is regulated by the migration and proliferation of keratinocytes. Existing reports have mostly focused on the mechanism of keratinocyte dysfunction in the high-glucose microenvironment of diabetes, but there is little exploration of the regulatory mechanism between keratinocytes and other cells.

[0093] With the continuous development of sequencing technology, single-cell transcriptome sequencing can not only identify tissue cell heterogeneity and infer cell differentiation trajectories, but also further analyze cell-to-cell interactions [9-10]. Our research group previously used this technology to analyze diabetic foot wound edge skin tissue and acute dorsal wound edge skin tissue and found that the communication between fibroblasts and keratinocytes was significantly weakened, and the cell crosstalk mechanism is still unclear. This study further analyzed the cell-to-cell ligand interaction and found that the interaction between fibroblast-derived chemokines and ligands on keratinocytes was significantly weakened. Therefore, it is speculated that changes in the expression of chemokines secreted by fibroblasts may be an important factor in the impaired migration of keratinocytes.

[0094] In this study, a high-glucose model of fibroblasts was established, and the cell culture supernatant was collected as a conditioned medium to treat keratinocytes. The scratch test confirmed that the migration rate of keratinocytes was significantly decreased. To clarify which chemokines inhibit keratinocyte migration? This research group used Luminex liquid suspension chips and qPCR technology to detect the levels of chemokine proteins and mRNA in the supernatant and cells of fibroblasts under a high-glucose microenvironment. The results showed that both CXCL12 protein secretion and mRNA expression were significantly decreased. Further experimental results showed that the expression of CXCR4 (CXCL12 receptor) protein in fibroblasts under a high-glucose microenvironment was significantly reduced, which was consistent with the change trend of CXCL12. Literature reports that CXCL12 expression is downregulated in the wound of diabetic mice

[11] , which is consistent with the experimental results of this study. In summary, this research group speculates that the reduction of CXCL12 protein secretion by fibroblasts under a high-glucose microenvironment is one of the important factors that hinder the migration of keratinocytes.

[0095] To further confirm that the CXCL12-CXCR4 signaling pathway is involved in regulating keratinocyte migration, this study used recombinant human CXCL12 protein to intervene in keratinocytes under different treatment conditions. The scratch test showed that the cell migration rate in the HGFb-CM+CXCL12 group was significantly increased compared with the HGFb-CM group, and there was no significant difference between the NFb-CM group and the NFb-CM+CXCL12 group; Western blot experiments showed that exogenous recombinant CXCL12 protein could significantly increase the expression of CXCR4 protein in keratinocytes treated with HGFb-CM. Studies have reported that the use of CXCL12 antagonists can lead to delayed aggravation of wounds in diabetic mice; overexpression of CXCL12 can shorten the wound healing time from 55 days to 23 days

[11] ; the use of CXCR4 agonist UCUF-728 can accelerate the healing of diabetic wounds and shorten the healing time of diabetic wounds by 36%

[12] . This suggests that CXCL12 protein improves the migration of HGFb-CM-treated keratinocytes by upregulating CXCR4.

[0096] In summary, this study showed that the high-glucose microenvironment inhibited the secretion of CXCL12 protein by fibroblasts, leading to downregulation of CXCR4 protein in keratinocytes and impaired migration; exogenous CXCL12 protein can promote cell migration by upregulating CXCR4 protein expression in keratinocytes. This study confirmed that the CXCL12-CXCR4 pathway is an important regulatory mechanism for crosstalk between fibroblasts and keratinocytes in diabetic wounds, providing a new target for the treatment of refractory wounds.

[0097] Example 2

[0098] An embodiment of the present invention provides an application of a CXCL12 / CXCR4 signaling pathway as a target in the preparation of a drug for treating or preventing diabetic foot.

[0099] Furthermore, an embodiment of the present invention provides an application of a CXCL12 / CXCR4 signaling pathway agonist in the preparation of a drug for treating diabetic foot, wherein the signaling pathway agonist includes one or more of a CXCL12 agonist or a CXCR4 agonist.

[0100] Furthermore, the CXCL12 agonist includes a substance that excites CXCL12 activity or a substance that increases CXCL12 levels, and the CXCR4 agonist is selected from a substance that excites CXCR4 activity or a substance that increases CXCR4 levels.

[0101] Furthermore, the dosage form of the drug is selected from any pharmaceutically permitted dosage form;

[0102] Furthermore, the dosage form is selected from one or more of tablets, granules, capsules, pills, oral solutions, injections, and liposomes;

[0103] Furthermore, the administration method of the drug or pharmaceutical composition is selected from one or more of oral administration, topical administration, intranasal administration, systemic administration, intravenous administration, subcutaneous administration, intramuscular administration, intracerebroventricular administration, intrathecal administration or transdermal administration.

[0104] It should be understood that, although the steps in the flowchart of the accompanying drawings are displayed in sequence as indicated by the arrows, these steps are not necessarily executed in sequence in the order indicated by the arrows. Unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders. Moreover, at least a part of the steps in the flowchart of the accompanying drawings may include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn or alternately with other steps or at least a part of the sub-steps or stages of other steps.

[0105] The above description is only a partial implementation method of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.

Claims

1. A method for determining chemokines and mechanisms that inhibit cell migration in a high-sugar microenvironment, characterized in that: include: S100. Obtain skin tissue from the wound edge of diabetic foot and acute dorsum of foot trauma; S200, performing single-cell transcriptome sequencing on the diabetic foot wound edge skin tissue and the acute dorsal foot trauma wound edge skin tissue to analyze the interaction between chemokine receptor ligands between fibroblast subpopulations and keratinocyte subpopulations; S300, preparing fibroblast conditioned medium, wherein the fibroblasts are cultured in a high-glucose environment, and the cell supernatant cultured in a conventional culture medium is used as a control group; S400, treating keratinocytes with the fibroblast-conditioned medium to detect the cell migration ability of the keratinocytes; S500, using Luminex liquid suspension chips to detect the levels of cytokines and chemokines in the conditioned medium, detecting the mRNA expression of chemokines in HFF cells by qPCR, and detecting the expression changes of CXCR4 protein by Western blotting; S600, adding exogenous CXCL12 to the conditioned medium, and detecting the expression and migration ability of CXCR4 in keratinocytes; S700. Analyze the data obtained from the above experiments to determine the mechanism by which high-sugar environment affects cell behavior and the role of CXCL12 chemokine therein.

2. A method for determining chemokines and mechanisms that inhibit cell migration under a high-sugar microenvironment according to claim 1, characterized in that: The step S200 specifically includes: The wound edge skin of patients with type 2 diabetic foot and the wound edge skin of the dorsal part of the foot of patients with foot trauma were isolated, prepared, library constructed, and single-cell transcriptome sequencing was performed. The cluster Profiler package in the R package was used for GO and KEGG enrichment analysis; Cellphone DB was used to analyze cell-cell interactions; and the circlize v0.4.10 R software package was used to visualize the results.

3. The method for determining chemokines and mechanisms that inhibit cell migration under a high-sugar microenvironment according to claim 1, characterized in that: The step S400 includes: The monolayer cells were scraped with a 200uL pipette tip and washed with PBS, 2mL of culture medium was added to each well, and the cell scratch healing was observed under a 200x optical microscope at 0, 24, 48, and 72h after scratching, and the cell migration rate was calculated at 24, 48, and 72h after scratching.

4. The method for determining chemokines and mechanisms that inhibit cell migration in a high-sugar microenvironment according to claim 1, characterized in that: The scratch test in step S400 needs to be repeated three times, and the results are averaged.

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