A scar diagnostic reagent and its application

Abstract

This application relates to the field of diagnostic reagents, specifically to a scar diagnostic reagent and its application. This application is the first to discover CD3+ in the peripheral blood of keloid patients. + CD8 + The study revealed a significant decrease in cytotoxic T cells, highlighting for the first time the crucial role of the immune microenvironment in the pathogenesis of keloids. Furthermore, it was the first time that sHLA-E expression was significantly different in normal individuals compared to patients with hypertrophic scars and keloids. Using sHLA-E as a diagnostic marker can differentiate between hypertrophic scars and keloids, which are traditionally considered pathological scars, and can also be used to assess the effectiveness of keloid treatment.

Description

Technical Field

[0001] This invention relates to the field of diagnostic reagents, and more specifically to a scar diagnostic reagent and its application. Background Technology

[0002] Keloids are abnormally large scar tissues that grow excessively after the healing of skin wounds or skin injuries of unknown cause. They are most common on the chest, earlobes, jaw, and back. Lesions can be single or multiple throughout the body, exhibiting non-self-limiting growth and often accompanied by itching, pain, and other discomfort. The pathological behavior of keloids involves excessive proliferation of fibroblasts and secretion of extracellular matrix, with TGF-β playing a crucial role. However, the pathogenesis of this disease is unclear. The recurrence rate of keloids after lesion excision is nearly 100%, and methods such as using TGF-β antagonists cannot inhibit their growth. Therefore, finding its pathogenesis and developing effective treatments is a clinical challenge. Currently, the classic treatment is intradermal injection of glucocorticoids. Although the recurrence rate is as high as 30-50%, lesion growth can be controlled and gradually shrunk. Glucocorticoids, as a classic immunosuppressant, regulate the proliferation and function of immune cells, such as inhibiting CD8. + T cells, enhance the function of regulatory T cells.

[0003] There are currently no reports on the pathogenesis of keloids or reliable and effective diagnostic methods. Summary of the Invention

[0004] On the one hand, this application provides CD3 + CD8 + Application of reagents for detecting cytotoxic T cells, NKG2A, KLRC1, or soluble human leukocyte antigen E in the preparation of reagents or kits for the detection and / or prognosis of keloids.

[0005] In this application, the detection of the KLRC1 gene includes the detection of intermediate products of KLRC1 gene expression; in some embodiments, the detection of NKG2A / KLRC1 is included, specifically, the detection of differences in NKG2A expression in specific cell populations using a panel composed of five proteins: CD3, CD8, T-bet, CD94, and NKG2A.

[0006] This application, using single-cell analysis, is the first to discover CD3 in the peripheral blood of keloid patients. + CD8 + Cytotoxic T cells were significantly reduced, and flow cytometry analysis showed that all cytotoxicity-related proteins in this cell population were significantly decreased. Related transcription factors, differentiation-related proteins, and proliferation proteins were all reduced, but there was no significant difference in apoptosis levels.

[0007] CD3 in single-cell analysis + CD8 + Cytotoxic T cells were isolated for further analysis. Using dotplot analysis, the top 11 differentially expressed genes between normal and patient samples were identified, revealing that the KLRC1 gene was highly expressed in patient samples. Figure 3 A).

[0008] CD3 was detected using flow cytometry. + CD8 + T-bet + Intracellular CD94 in cytotoxic T cells + NKG2A + The proportion of cells indicated that CD3+ in the peripheral blood of keloid patients was high. + CD8 + T-bet + Intracellular CD94 in cytotoxic T cells + NKG2A + The proportion of cells was significantly higher than that of normal individuals. Figure 3 B, p<0.0001), therefore CD3 + CD8 + The decrease in cytotoxic T cells may be mediated by the CD94 / NKG2A immune checkpoint.

[0009] The test results in this application indicate that the level of sHLA-E in keloid patients is significantly higher than that in normal individuals, directly proving that the high expression of sHLA-E in keloid patients interacts with CD3. + CD8 + The proportion and function of CD94 / NKG2A immune checkpoint, which is highly expressed on cytotoxic T cells, are suppressed upon binding.

[0010] On the one hand, this application provides CD3 + CD8 + Cytotoxic T cells,

[0011] NKG2A,

[0012] KLRC1, or,

[0013] Application of soluble human leukocyte antigen E (HLA) detection reagents in the preparation of diagnostic reagents or kits for the differentiation of hypertrophic scars and keloids.

[0014] On the one hand, this application provides the application of a soluble human leukocyte antigen E detection reagent in the preparation of a diagnostic reagent or kit for the differentiation of keloids and malignant fibrosarcoma protuberans.

[0015] On the one hand, this application provides CD3 + CD8 +Cytotoxic T cells,

[0016] NKG2A,

[0017] KLRC1, or,

[0018] Application of soluble human leukocyte antigen E (HLA) detection reagent in the preparation of kits for predicting the treatment efficacy of keloids.

[0019] In some embodiments, the treatment is selected from at least one of surgery, radiotherapy, laser therapy, and hormone therapy; in some embodiments, the treatment is selected from at least one of surgery, hormone therapy, a combination of surgery and radiotherapy, or a combination of hormone therapy and laser therapy; in some embodiments, the treatment is selected from hormone therapy; in some embodiments, the hormone therapy is selected from glucocorticoid + 5-fluorouracil therapy.

[0020] On the one hand, this application provides a diagnostic and / or prognostic system for keloids, comprising:

[0021] a) Detection components for the following objects:

[0022] CD3 + CD8 + Cytotoxic T cells,

[0023] NKG2A,

[0024] Soluble human leukocyte antigen E, or,

[0025] KLRC1;

[0026] b) Result judgment component; the result judgment component is used to determine the result of the test sample detected by the detection component:

[0027] CD3 + CD8 + Cytotoxic T cells,

[0028] NKG2A,

[0029] Soluble human leukocyte antigen E, or

[0030] KLRC1;

[0031] Compared with the control sample:

[0032] CD3 + CD8 + Cytotoxic T cells,

[0033] NKG2A,

[0034] Soluble human leukocyte antigen E, or

[0035] KLRC1 gene expression;

[0036] Deviations in the pathological scar can be used to assess the risk or prognosis of pathological scars.

[0037] In some implementations, when the detection component detects soluble human leukocyte antigen E, a keloid is identified when the content of soluble human leukocyte antigen E is >3 ng / ml.

[0038] In some implementations, a non-keloid is defined as a soluble human leukocyte antigen (HLA) content <3 ng / ml; in some implementations, the non-keloid is selected from hypertrophic scars or normal scars; in some implementations, a keloid is defined as non-recurrence after treatment when the soluble HLA content is <3 ng / ml.

[0039] On the one hand, this application provides the application of KLRC1 gene, NKG2A or soluble human leukocyte antigen E as therapeutic targets in the preparation of keloid treatment drugs.

[0040] On the one hand, this application provides the use of KLRC1 gene, NKG2A or soluble human leukocyte antigen E inhibitors in the preparation of drugs for treating keloids.

[0041] On one hand, this application provides a treatment system for keloids, comprising:

[0042] a) Detection systems for the following objects:

[0043] CD3 + CD8 + Cytotoxic T cells,

[0044] NKG2A,

[0045] Soluble human leukocyte antigen E, or,

[0046] KLRC1;

[0047] b) Medication system.

[0048] In some embodiments, the drug delivery system contains the KLRC1 gene, NKG2A, or a soluble human leukocyte antigen E inhibitor.

[0049] In some embodiments, the inhibitor is selected from substances that downregulate the expression of the KLRC1 gene, NKG2A, or soluble human leukocyte antigen E, degrade the KLRC1 gene, NKG2A, or soluble human leukocyte antigen E, or antagonize the function of the KLRC1 gene, NKG2A, or soluble human leukocyte antigen E.

[0050] In some implementations, the KLRC1 gene inhibitor includes a nucleic acid effector molecule.

[0051] In some implementations, the nucleic acid effector molecules include DNA, RNA, PNA, or DNA-RNA hybrids.

[0052] In some implementations, the nucleic acid effector molecule inhibits the overall or local expression of the KLRC1 gene.

[0053] In some embodiments, the nucleic acid effector molecule is selected from at least one of siRNA, dsRNA, miRNA, ribozyme, and shRNA.

[0054] In some embodiments, the NKG2A or soluble human leukocyte antigen E inhibitor is selected from at least one of antibodies and their functional fragments, or small molecule compounds.

[0055] In some embodiments, the antibody is selected from at least one of monoclonal antibodies, polyclonal antibodies, nanobodies, Fab antibodies, Fv antibodies, or single-chain antibodies.

[0056] In some implementations, the antibody is selected from monoclonal antibodies.

[0057] In some implementations, the antibody is selected from monalizumab.

[0058] In some embodiments, the test sample is selected from blood; in some embodiments, the sample is selected from peripheral blood; in some embodiments, the sample is selected from peripheral blood serum.

[0059] In this application, "detection" is synonymous with diagnosis. In addition to the early diagnosis of pathological scars (e.g., within 1 year of medical history), it also includes the diagnosis of intermediate and late-stage pathological scars, as well as the screening, risk assessment, prognosis, disease identification, diagnosis of disease stages, and selection of therapeutic targets for pathological scars.

[0060] This application found significantly reduced CD3 levels in the peripheral blood of patients with keloids. + CD8 + The presence of cytotoxic T cells suggests a systemic immune system abnormality in keloid scarring, indicating the possibility of systemic treatment approaches such as antigen stimulation, adoptive cell expansion after in vitro expansion, drug-induced cell expansion, and inhibition of CD3+. + CD8 + Cytotoxic T cells antagonize the effects of cytokines, cells, and other cytotoxic agents. Attached Figure Description

[0061] Figure 1Single-cell sequencing analysis of changes in different types of monocytes in peripheral blood. (A) Schematic diagram of single-cell sequencing. Peripheral blood was drawn from 2 normal individuals and 2 patients, and monocytes were separated for 10× single-cell sequencing and analysis. Table 1 shows the patient information used in Figure (A). (B) Single-cell sequencing analysis. t-SNE analysis showed that peripheral blood monocytes in all samples could be divided into 11 groups. (C) Trackplot analysis was used to view the expression of cell-related proteins in different cell groups. (D) t-SNE analysis was used to analyze the number of the 11 cell groups in the 4 samples. (E) Combining Figures (C) and (D), bar charts were prepared for the 4 samples to analyze the proportion of the 11 cell groups and define them. CD3 + CD8 + Cytotoxic T cells (i.e., CD8) + Cytotoxicity (T cells) was significantly reduced in patient samples. (F) CD3 + CD8 + Cytotoxic T cells (i.e., CD8) + Further analysis of the gene expression of the cytotoxicity T cell's signature proteins revealed significant expression of CD3E, CD8A, CD8B, GZMB, IFNG, and TBX21. + CD8 + Cytotoxic T cells (i.e., CD8) + Biological definition of cytotoxicity (T cell).

[0062] Figure 2 Flow cytometry validates single-cell analysis results. (A)CD3 + CD8 + There was no significant difference in T cells between normal individuals and patients. (B) CD3 + CD8 + After gating T cells, Granzyme B, Granulysin, and IFN-γ-positive cells were detected. The results showed that CD3+ cells in keloid patients... + CD8 + Reduced proteins related to the cytotoxic function of T cells. (C) CD3 + CD8 + After gating T cells, the cytotoxic transcription factor T-bet was detected, indicating a decrease in keloid samples. (D) CD3 + CD8 + After gating T cells, Granzyme B was detected. + Ki67 + (Cytotoxic cell proliferation function), Bcl2 (cytotoxic cell apoptosis) and Granzyme B+ KLRG1 + (Cytotoxic cell differentiation transcription) results in CD3 in keloid patients + CD8 + T cell cytotoxicity reduced the proliferation, differentiation, and transcriptional capacity of cells, but there was no significant difference in apoptosis. (E) CD3 + CD8 + Granzyme BT cytotoxic cell gating and subsequent detection of exhaustion-related proteins, namely PD-1 and Lag3 positive cells, showed no significant difference between normal individuals and patients. Note: HD refers to normal individuals; NTK refers to patients with keloids.

[0063] Figure 3 .10× single-cell sequencing analysis suggests that KLRC1 encoding NKG2A may be CD3. + CD8 + The reasons for the decrease in the proportion of cytotoxic T cells. (A) Single-cell sequencing will determine the CD3+ level. + CD8 + After cytotoxic T cells were selected, the top 11 differentially expressed genes were analyzed using dotplot. (B) Flow cytometry was used to verify CD3 expression. + CD8 + T-bet + Intracellular CD94 in cytotoxic T cells + NKG2A + The proportion of cell expression was significantly increased in patients with keloids. Note: HD, normal individuals; NTK, patients with keloids.

[0064] Figure 4 The CD94 / NKG2A immune checkpoint ligand HLA-E, specifically sHLA-E, is significantly increased in peripheral blood. (A) Linear fitting analysis was used to determine the standard curve of sHLA-E and to obtain machine-read OD values ​​and the conversion method for sHLA-E concentration (ng / ml). (B) t-test analysis was used to analyze the expression of sHLA-E in peripheral blood of normal and untreated keloid patients.

[0065] Figure 5 Diagnostic efficacy of sHLA-E. (A) Analysis of variance: Peripheral blood sHLA-E expression in patients with normal, hypertrophic, and untreated keloids. (B) Analysis of variance: Peripheral blood sHLA-E expression in normal individuals, untreated keloids, and patients treated with glucocorticoids. Detailed Implementation

[0066] The technical solutions of this application are further illustrated below through specific embodiments. These specific embodiments do not represent a limitation on the scope of protection of this application. Any non-essential modifications and adjustments made by others based on the concept of this application still fall within the scope of protection of this application.

[0067] General experimental procedures

[0068] Materials and methods

[0069] 1) Patient and sample information

[0070] Blood was drawn from patients using procoagulant tubes. Patient records and diagnostic information were obtained from the hospital's record system and via telephone follow-up. This study was approved by the Ethics Committee of the Ninth People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine, and patient consent was obtained prior to blood draws.

[0071] The treated patients received a combination of classic glucocorticoids and 5-FU administered via local injection into the lesions. The specific regimen was (1 ml of glucocorticoid (triamcinolone) + 0.5 ml of 5-FU + 4 ml of lidocaine), with lidocaine being a local anesthetic. Blood tests were performed on the patients 6 months after treatment.

[0072] 2) Separation of peripheral blood mononuclear cells and serum

[0073] Peripheral blood mononuclear cells were collected using anticoagulant tubes. 10 ml of Ficoll solvent was added, mixed thoroughly, and centrifuged at 800 g for 15 min to separate the cells into layers. The middle white layer was then removed. Residual red blood cells were lysed using erythrocyte lysis buffer, centrifuged again at 800 g for 15 min, the supernatant was discarded, and the cells were resuspended in 1×PBS. The cells were filtered through a 70 μm filter, centrifuged again at 800 g for 15 min, the supernatant was discarded, and the cells were resuspended in 2 ml of 1×PBS. The cells were then placed in two 2 ml EP tubes and stored at -80°C.

[0074] Peripheral blood serum was collected using a coagulation-promoting tube, with 2 ml of peripheral blood collected and allowed to stand at 4°C until complete clotting. The clotting sample was then centrifuged at 2500-3000 rpm, and the supernatant from the gel in the coagulation vessel was extracted into an EP tube and stored at -80°C for further testing.

[0075] 3) Single-cell sequencing analysis

[0076] Two patients with keloids and a history of more than 10 years, who had not received any treatment in the past 3 years, whose keloids continued to grow, had no history of colds / trauma in the past six months, no underlying diseases, no medication use, and no keloid-related complications (such as ulceration, infection, or bleeding), were selected. Two healthy individuals were also included, who had no history of colds / trauma in the past six months, no underlying diseases, and no medication use. Peripheral blood mononuclear cells underwent 10× single-cell sequencing and analysis (this part was performed by Beijing Novogene Technology Co., Ltd.).

[0077] 4) Flow cytometry analysis

[0078] Prepare flow cytometry antibodies for CD3, CD8, Granzyme B, IFN-γ, Granulysin, T-bet, Ki67, Bcl2, KLRG1, PD-1, and Lag3. Perform membrane protein analysis on peripheral blood mononuclear cells. + Nuclear staining was performed, followed by flow cytometry analysis (this part was performed by Shanghai Unimicron Biotechnology Co., Ltd.).

[0079] 5) ELISA testing

[0080] a) Before starting the ELISA test, remove all reagents from the ELISA kit and bring them to room temperature; remove the patient's serum stored at -80℃ and bring it to room temperature.

[0081] b) Preparation of working solution for standard: Centrifuge the standard at 10000×g for one minute. Add 1.0 ml of standard and sample diluent to the lyophilized standard, tighten the cap, and let stand for 10 minutes. Invert the tube several times to ensure thorough mixing and dissolution, preparing a 20 ng / ml working solution for standard. During preparation, mix thoroughly and avoid foaming as much as possible. Then, perform serial dilutions as needed. The concentrations prepared are as follows: 20, 10, 5, 2.5, 1.25, 0.63, 0.31, 0 ng / ml. The serial dilution method is as follows: Take 7 EP tubes, add 500 μl of standard and sample diluent to each tube, and then pipette 500 μl of the 20 ng / ml working solution into one of the EP tubes and mix well to prepare a 10 ng / ml working solution. Repeat this process for the remaining tubes.

[0082] c) Sample addition: Set up blank wells, standard wells, and sample wells. Add 100 μl of sample diluent to the blank wells, and add 100 μl of standard or sample to the remaining wells. Cover the ELISA plate with a membrane and incubate at 37°C for 90 minutes.

[0083] d) Preparation of biotinylated antibody working solution: Prepare the biotinylated antibody diluent 15 minutes before the start of the next experiment. Calculate the required volume for each test level (based on 100 μl / well). Prepare an additional 100-200 μl during actual preparation. 15 minutes before use, centrifuge the concentrated biotinylated antibody at 800×g for 1 minute, and dilute the 100× concentrated biotinylated antibody to a 1× working concentration using the biotinylated antibody diluent.

[0084] e) Discard the liquid, shake dry, do not wash, add 100 μl of biotinylated antibody working solution (prepared within 15 minutes before use) to each well, cover the plate with a membrane, and incubate at 37°C for 1 hour.

[0085] f) Preparation of washing solution: Dilute the concentrated washing solution with double-distilled water (1:24).

[0086] g) Discard the liquid in the wells, spin dry, wash the plate 3 times with washing solution, soaking for 1-2 minutes each time, about 350μl / well, spin dry and gently pat dry the liquid in the wells on absorbent paper.

[0087] h) Preparation of enzyme conjugate working solution: Begin preparation 15 minutes before use. Calculate the required volume for each test level before the experiment (based on 100 μl / well). Prepare an additional 100-200 μl during actual preparation. 15 minutes before use, centrifuge the concentrated HRP enzyme conjugate at 800×g for 1 minute, and dilute the 100× concentrated HRP enzyme conjugate to 1× working concentration with enzyme conjugate dilution buffer.

[0088] i) Add 100 μl of HRP enzyme conjugate working solution (prepared within 15 minutes before use) to each well, cover with a membrane, and incubate at 37°C for 30 minutes.

[0089] j) Discard the liquid in the well, spin dry, and wash the plate 5 times, following the same procedure as step g).

[0090] k) Add 90 μl of substrate solution to each well, cover the plate with a membrane, and incubate at 37°C in the dark for 15 minutes.

[0091] l) Add 50 μl of stop solution to each well to stop the reaction. At this time, the blue color will immediately turn yellow.

[0092] m) Measure the optical density (OD value) of each well immediately or within half an hour using an ELISA reader at a wavelength of 450 nm. The ELISA reader should be turned on in advance to preheat the instrument and the detection program should be set.

[0093] 6) Calculation of sHLA-E concentration

[0094] Linear fitting analysis was performed using SPSS software (version 16.0, SPSS Inc, Chicago, IL) based on standard concentrations and corresponding OD values ​​to obtain linear curves and methods for converting concentrations to OD values. Then, the sHLA-E concentrations were calculated in normal individuals, patients with hypertrophic scars, and patients with keloids.

[0095] 7) Data Analysis

[0096] Statistical analysis was performed using SPSS software (version 16.0, SPSS Inc., Chicago, IL). Differences between two variables were analyzed using a two-tailed t-test. Comparisons of three or more samples were performed using one-way ANOVA (using the F-distribution). When the data did not follow a Gaussian distribution, the Wilcoxon paired-signed mean-rank test and the Mann-Whitney test were used as two nonparametric tests. All measurement data are expressed as mean ± SD. P < 0.05 was considered statistically significant.

[0097] Example 1: 10× single-cell sequencing analysis of changes in different types of mononuclear cells in peripheral blood

[0098] Patient and sample information:

[0099] Peripheral blood was drawn from two healthy individuals and two patients with keloids. Mononuclear cells were isolated and sequenced at 10× for analysis. All four individuals had no other underlying diseases, no history of long-term medication use, and no history of colds or trauma within the past year. The two patients had medical histories of 14 and 10 years, respectively, and had not received any treatment in the past 3 years. They also had no local infections, ulcers, or other complications. Figure 1 A and Table 1).

[0100] Table 1. Single-cell sequencing sample information

[0101]

[0102] t-SNE analysis showed that peripheral blood mononuclear cells in all samples could be divided into 11 cell subsets, ranging from 0 to 10. Figure 1 B). After using the trackplot analysis method to view the expression of cell-related proteins within different cell populations, cells were defined ( Figure 1 C). Simultaneously, t-sne analysis was used to analyze the number of 11 cell populations in the four samples. Figure 1 D). Following this, bar charts of four samples were prepared using Figures (C) and (D) to analyze and define the proportions of 11 cell populations, revealing that CD3... + CD8 + Cytotoxic T cells (i.e., CD8) + Cytotoxicity T cells (CTLs) were significantly reduced in patient samples. Figure 1 E). To further confirm that the reduced cell population in the patient samples was CD3... + CD8 + Cytotoxic T cells (i.e., CD8) + The expression of the characteristic proteins of cytotoxicity T cells (CD3E, CD8A, CD8B, GZMB, IFNG, and TBX21) was analyzed. Significant expression was found, consistent with CD3E expression. + CD8 + Cytotoxic T cells (i.e., CD8) + Biological definition of cytotoxicity T cell (Cytotoxicity T cell) Figure 1 F).

[0103] Example 2: Flow cytometry validation of single-cell analysis results

[0104] Patient and sample information:

[0105] The study included 34 patients with keloids and 36 normal patients who received treatment at the Ninth People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine.

[0106] CD3 + CD8 + There was no significant difference in T cell counts between healthy individuals and patients. Figure 2 A). CD3 + CD8 + After gating T cells, the presence of Granzyme B, Granulysin, and IFN-γ-positive cells (i.e., cytotoxic cytokines) was detected. The results showed that CD3+ cells in keloid patients... + CD8 + Decreased T cell cytotoxicity-related proteins Figure 2 B). CD3 + CD8 + After gating T cells, the cytotoxic transcription factor T-bet was detected, indicating a decrease in T-bet in keloid samples. Figure 2 C). Further analysis of the general function of the cell population, including CD3... + CD8 + After gating T cells, Granzyme B was detected. + Ki67 + (Cytotoxic cell proliferation function), Granzyme B + Bcl2 (apoptosis in cytotoxic cells) and Granzyme B + KLRG1 + (Cytotoxic cell differentiation transcription) results in CD3 in keloid patients + CD8 + T cell cytotoxicity was reduced, and the proliferation, differentiation, and transcriptional abilities of T cells were decreased, but there was no significant difference in apoptosis. Figure 2 D). Guess CD3 + CD8 + Does the reduction of cytotoxic T cells relate to cellular exhaustion? Therefore, CD3... + CD8 + After gating and selecting Granzyme BT cytotoxic cells, exhaustion-related proteins, namely PD-1 and Lag3 positive cells, were detected, and no significant differences were found between normal individuals and patients. Figure 2 E).

[0107] Example 3: Single-cell sequencing analysis to mine CD3 + CD8 + Possible reasons for the decrease in cytotoxic T cells

[0108] Patient and sample information: Same as in Example 1.

[0109] CD3 in single-cell analysis + CD8 + Cytotoxic T cells were isolated for further analysis. Using dotplot analysis, the top 11 differentially expressed genes between normal and patient samples were identified. It was found that the KLRC1 gene was highly expressed in patient samples. Figure 3 The KLRC1 circle corresponding to the keloid in Figure A is larger (the size of the circle indicates the proportion of expression, and the color indicates the intensity of expression).

[0110] KLRC1 encodes the protein NKG2A, which typically works in combination with CD94 as an immune checkpoint. When it functions, CD3... + CD8 + Cytotoxic T cells are suppressed. This mechanism may explain CD3. + CD8 + The decrease in cytotoxic T cells in keloid patients. Therefore, flow cytometry was used to detect CD3. + CD8 + T-bet + Intracellular CD94 in cytotoxic T cells + NKG2A + The proportion of cells indicated that CD3+ in the peripheral blood of keloid patients was high. + CD8 + T-bet + Intracellular CD94 in cytotoxic T cells + NKG2A + The proportion of cells was significantly higher than that of normal individuals. Figure 3 B, p<0.0001), therefore CD3 + CD8 + The decrease in cytotoxic T cells may be mediated by the CD94 / NKG2A immune checkpoint.

[0111] Example 4: Detection of CD94 / NKG2A Immune Checkpoint Ligands

[0112] Patient and sample information: Same as in Example 2.

[0113] The specific ligand for the CD94 / NKG2A immune checkpoint is human lymphocyte antigen E (HLA-E), which exists in peripheral blood in a free form (soluble HLA-E; sHLA-E). It is assumed that CD3... + CD8 + The decrease in cytotoxic T cells is regulated by the CD94 / NKG2A immune checkpoint, therefore sHLA-E will change accordingly, that is, it will be significantly elevated in patients.

[0114] First, a linear fitting analysis was performed based on the standard concentration samples and their corresponding OD values ​​to obtain the standard curve and the conversion method between concentration and OD value. Figure 4 A) The conversion formula is Y = 0.1916 * X + 0.08524 (Y is the OD value, X is the concentration (ng / ml)), R2 = 0.9948, indicating a strong linear fit. Next, this conversion method will be used to calculate the concentration values ​​of all samples.

[0115] According to this conversion method, a comparison between normal individuals and patients with untreated keloids revealed that the expression level in patients with untreated keloids was 5.61±1.54 ng / ml, which was significantly higher than that in normal individuals (0.50±0.46 ng / ml) (p<0.0001). Figure 4 B).

[0116] Since sHLA-E is a specific ligand for CD94 / NKG2A, their binding can significantly inhibit CD3 expression of CD94 / NKG2A. + CD8 + Cytotoxic T cells. The results of this embodiment suggest that sHLA-E levels in keloid patients are significantly higher than in normal individuals, directly demonstrating that the high expression of sHLA-E in keloid patients interacts with CD3+. + CD8 + The proportion and function of CD94 / NKG2A immune checkpoint, which is highly expressed on cytotoxic T cells, are suppressed upon binding.

[0117] Example 5: sHLA-E values ​​can be used to differentiate between normal individuals, patients with hypertrophic scars, and patients with keloids.

[0118] Patient and sample information:

[0119] The study included 34 patients with untreated keloids and 36 patients with normal skin, as well as 27 patients with hypertrophic scars, all admitted to the Ninth People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine.

[0120] When comparing normal individuals, patients with hypertrophic scars, and patients with untreated keloids, the level of sHLA-E in the peripheral blood of patients with hypertrophic scars was significantly higher than that of normal individuals (1.49±0.65 vs. 0.50±0.46 ng / ml; p<0.0001); the expression level in patients with untreated keloids was 5.61±1.54 ng / ml, which was significantly different from that in both normal individuals and patients with hypertrophic scars (p<0.0001). Figure 5 A).

[0121] This example demonstrates that hypertrophic scars, which belong to the same category of pathological scars in the traditional classification, have significantly lower sHLA-E expression than keloid patients, but are still higher than normal individuals.

[0122] Example 6: sHLA-E values ​​can be used to assess the condition of keloid patients after receiving glucocorticoid treatment.

[0123] Patient and sample information: Same as in Example 2.

[0124] When comparing normal individuals, untreated keloid patients, and patients treated with glucocorticoids, the expression level in untreated keloid patients was 5.61±1.54 ng / ml, which was significantly higher than that in normal individuals (0.50±0.46 ng / ml) (p<0.0001); the sHLA-E expression in keloid patients treated with classic glucocorticoids was 2.15±1.73 ng / ml, which was significantly lower than that in untreated patients (p<0.0001). Figure 5 B), but still higher than normal.

[0125] The results of Examples 5 and 6 demonstrate a significant correlation between sHLA-E and the occurrence of pathological scars, with different expression levels observed between different types of pathological scars: hypertrophic scars and keloids. This suggests that the two types of scars share similarities in their pathogenesis, but the intensity of the causative factors differs significantly. This may explain the different clinical manifestations: hypertrophic scars are self-limiting, not exceeding the original wound boundary; while keloids are not self-limiting, progressing beyond the original wound boundary into surrounding normal tissue. The significant decrease in sHLA-E after keloid treatment indicates that the effectiveness of glucocorticoid + 5-FU treatment is reflected in peripheral blood serum.

[0126] Example 7: Validating the discriminative effect of sHLA-E in a population sample.

[0127] Patient and sample information: 512 healthy donors, 100 interfering cases (patients diagnosed with other diseases (i.e., non-keloids)) and 104 keloid patients (untreated).

[0128] The serum sHLA-E level in healthy donors was 1.26±1.00 ng / ml, and in patients with interference cases it was 2.05±2.41 ng / ml, both of which were significantly lower than those in patients with keloids (6.34±2.58; P<0.0001; Table 2). We set the cutoff value for sHLA-E at 3 ng / ml to assess the sensitivity (82.69%) and specificity (non-keloid (healthy donor + interfering cases) vs. keloid = 92.16%; healthy donor (non-keloid) vs. keloid = 94.73% (specificity); interfering cases (non-keloid) vs. keloid cases = 79.00% (specificity) for sHLA-E diagnosis of keloids (Table 2). We followed 61 patients with keloids who received >3 intralesional treatments (triamcinolone + 5-FU). After treatment interruption, 13 patients (21.31%) experienced keloid recurrence within 6 months. The serum sHLA-E level in these 13 patients was 4.06 ± 1.70 ng / ml after the last treatment, significantly higher than in other patients (1.73 ± 1.07; P < 0.0002; Table 2).

[0129] Table 2. Soluble human leukocyte antigen E (sHLA-E) levels in healthy donors, patients with interference, and keloids.

[0130]

[0131] Example 8: Detection effect of malignant dermatofibrosarcoma protuberans

[0132] Malignant dermatofibrosarcoma protuberans is a common type of soft tissue sarcoma of the skin. Treatment primarily involves surgery, but due to the large area of ​​surgical resection and high recurrence rate, it often results in significant damage to tissue structure and morphology. In particular, malignant dermatofibrosarcoma protuberans on the face and neck often causes deformities or even loss of facial organs and structures, significantly impacting the patient's psychological well-being and frequently hindering their reintegration into society. The pathogenesis, development, and appearance of malignant dermatofibrosarcoma protuberans are highly similar to keloids, frequently leading to misdiagnosis and delayed treatment. This results in excessive growth and increased extent of the sarcoma, requiring further surgical resection and causing greater soft tissue destruction. Given the current lack of simple and feasible differential diagnostic methods besides local biopsy or excision for pathological examination, this embodiment proposes diagnostic markers to aid diagnosis, which has significant clinical application value in the above-mentioned clinical scenarios.

[0133] Patient and sample information:

[0134] 512 healthy donors, 14 patients with malignant dermatofibrosarcoma protuberans and 104 patients with keloids (untreated).

[0135] Tests revealed that the serum sHLA-E level in healthy donors was 1.26 ± 1.00 ng / ml, while that in keloid patients was 6.34 ± 2.58 (Table 3). The results were consistent with those in Example 5. Further comparison showed that the sHLA-E level in malignant dermatofibrosarcoma protuberans was 1.24 ± 0.59 ng / ml, significantly lower than that in keloid patients (P < 0.0001, Table 3). The significant difference in sHLA-E between malignant dermatofibrosarcoma protuberans and keloid patients provides a new approach for the diagnosis of malignant dermatofibrosarcoma protuberans.

[0136] Table 3

[0137]

[0138] Given the many possible embodiments to which the principles of the disclosed invention can be applied, it should be understood that the illustrated embodiments are merely preferred examples of this application and should not be considered as limiting the scope of this application. Rather, the scope of this application is defined by the appended claims. Therefore, we claim protection for all inventions falling within the scope and spirit of these claims.

Claims

1. Use of a detection agent of soluble human leukocyte antigen E in the preparation of a detection agent or a kit for detecting keloids.

2. Use according to claim 1, wherein The detection sample for the detection is selected from peripheral blood or serum of peripheral blood.

3. A diagnostic system for keloids, characterized by, comprising: a) a detection means for soluble human leukocyte antigen E; b) a result judging means for judging the risk of pathological scarring according to the deviation of the content of soluble human leukocyte antigen E in the sample to be detected by the detection means from the content of soluble human leukocyte antigen E in the control sample.

4. The system of claim 3, wherein, When the detection means detects soluble human leukocyte antigen E, when the content of soluble human leukocyte antigen E is > 3 ng / ml, it is judged as high risk of keloids, and when the content of soluble human leukocyte antigen E is < 3 ng / ml, it is judged as low risk of keloids.

5. The system of claim 4, wherein, The low risk of keloids is selected from proliferative scars or normal.

6. The system of claim 4 or 5, wherein, The detection sample for the detection is selected from peripheral blood or serum of peripheral blood.

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