Application of CDCP1 protein in preparation of product for evaluating severity of body alopecia

Abstract

The invention provides application of CDCP1 protein in preparation of a product for evaluating the severity of body alopecia, and belongs to the technical field of alopecia prevention and treatment. The invention firstly proposes that the CDCP1 protein can be used as a marker for evaluating the severity of the body alopecia, and firstly proposes that the CDCP1 level and the severity of the alopecia of a male AGA patient are in significant positive correlation, and the method for evaluating the severity of the body alopecia according to the concentration of the CDCP1 protein has high specificity, sensitivity and accuracy.

Description

Technical Field

[0001] The present invention belongs to the technical field of hair loss prevention and treatment, and in particular relates to the application of CDCP1 protein in the preparation of a product for assessing the severity of hair loss in an organism. Background Art

[0002] Androgenetic Alopecia (AGA) is a progressive hair follicle miniaturization disease. It is a common and frequently occurring disease in dermatology and one of the intractable diseases. With the improvement of people's living standards, hair loss and hair loss anxiety have received increasing attention. Genetic factors and androgens are key factors in the pathogenesis of AGA. In AGA patients, hair growth is affected by genetic, hormonal, inflammatory, metabolic and other factors, which leads to the dysregulation of multiple signal transduction pathways in cells and the body's inappropriate immune and inflammatory responses, which in turn shortens the hair growth period and eventually leads to the clinical characteristics of hair follicle miniaturization, that is, the terminal hair gradually becomes thinner and softer, and is eventually replaced by short vellus hair. AGA is a progressive hair loss disease with a trend of younger people. Early identification and treatment can help prevent the further development of AGA, thereby retaining as much hair as possible, and almost all patients require lifelong treatment to achieve sustained improvement. Therefore, exploring biomarkers that predict the severity of AGA can help to detect risk individuals early and start preventive intervention or treatment for the development of AGA.

[0003] Cub domain-containing protein 1 (CDCP1) is a transmembrane glycoprotein with three CUB domains in its extracellular region. Recent studies have found that CDCP1 expression is significantly increased in a variety of solid tumors and hematological tumors such as prostate cancer, lung cancer, bladder cancer, colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, and acute myeloid leukemia, and is associated with tumor progression, drug resistance, and poor survival prognosis. At present, there are no reports in this field on the relationship between CDCP1 and the prediction of the severity of hair loss. Summary of the invention

[0004] In view of this, the purpose of the present invention is to propose for the first time that CDCP1 protein can be used as a marker for assessing the severity of hair loss in an organism.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides application of CDCP1 protein in preparing a product for assessing the severity of hair loss in a body.

[0007] Preferably, the organism includes a male patient with androgenic alopecia.

[0008] Preferably, the product comprises a kit.

[0009] Preferably, the severity of hair loss in an organism is determined based on the concentration of CDCP1 protein in the biological sample.

[0010] Preferably, the biological sample comprises blood.

[0011] Preferably, when the CDCP1 concentration in the blood is less than 2.52 ng / ml, it indicates mild hair loss; when the CDCP1 concentration in the blood is 2.52-9.78 ng / ml, it indicates moderate hair loss; when the CDCP1 concentration in the blood is greater than 9.78 ng / ml, it indicates severe hair loss.

[0012] The present invention also provides a kit for evaluating the severity of hair loss in a body, wherein the kit comprises a reagent for detecting the concentration of CDCP1 protein in a sample.

[0013] Preferably, the sample comprises blood.

[0014] Preferably, when the CDCP1 concentration in the blood is less than 2.52 ng / ml, it indicates mild hair loss; when the CDCP1 concentration in the blood is 2.52-9.78 ng / ml, it indicates moderate hair loss; when the CDCP1 concentration in the blood is greater than 9.78 ng / ml, it indicates severe hair loss.

[0015] Beneficial effects of the present invention:

[0016] The present invention proposes for the first time that CDCP1 protein can be used as a marker for assessing the severity of hair loss in an organism, and proposes for the first time that the CDCP1 level is significantly positively correlated with the severity of hair loss in male AGA patients. Using the concentration of CDCP1 protein to assess the severity of hair loss in an organism has high specificity, sensitivity, and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the molecular structure of CDCP1 protein;

[0018] Figure 2 The principle of PEA technology, where A is the immune antibody pair, labeled with DNA oligonucleotides, binding to the target antigen in the solution, B is the adjacent DNA oligonucleotide chains hybridized and extended by DNA polymerase, C is the newly generated DNA barcode block for PCR amplification, and D is the quantitative detection of each DNA barcode by microfluidic qPCR;

[0019] Figure 3 Loading position for incubation plate;

[0020] Figure 4 Analyze the experimental data process;

[0021] Figure 5 This is the Spearman correlation analysis diagram of the severity of AGA and CDCP1 level in males;

[0022] Figure 6 ROC curves for AGA severity and CDCP1 levels in males;

[0023] Figure 7 Preparation process of diluted standards for ELISA test;

[0024] Figure 8 It is the standard curve of ELISA test;

[0025] Fig. 9 This is the Spearman correlation analysis diagram between the severity of AGA in males and the CDCP1 concentration measured by ELISA;

[0026] Fig.10 ROC curve of the severity of AGA in males and CDCP1 concentration measured by ELISA. DETAILED DESCRIPTION

[0027] The present invention provides application of CDCP1 protein in preparing a product for assessing the severity of hair loss in a body.

[0028] In the present invention, CDCP1 is a transmembrane glycoprotein, the gene is located on chromosome 3p21-p23, the molecular weight is about 135kDa, and it is composed of 836 amino acids, including a signal peptide, an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain contains three CUB domains and multiple glycosylation sites. The specific molecular structure is shown in Figure 1 , the mRNA sequence of CDCP1 can be found at the website: https: / / www.ncbi.nlm.nih.gov / nuccore / XM_011534024.2. In the present invention, the organism preferably includes male androgenic alopecia patients, and the product preferably includes a kit. In the present invention, the severity of hair loss in the organism is preferably judged based on the concentration of CDCP1 protein in the biological sample. When the CDCP1 concentration in the blood is <2.52ng / ml, it indicates mild hair loss; when the CDCP1 concentration in the blood is 2.52-9.78ng / ml, it indicates moderate hair loss; when the CDCP1 concentration in the blood is >9.78ng / ml, it indicates severe hair loss. In the present invention, the ELISA method is preferably used to detect the protein concentration of CDCP1 in the blood.

[0029] The present invention also provides a kit for assessing the severity of hair loss in an organism, the kit comprising a reagent for detecting the concentration of CDCP1 protein in a sample. In the present invention, the reagent for detecting the concentration of CDCP1 protein in a sample preferably comprises an ELISA detection reagent.

[0030] In the present invention, the sample preferably includes blood. Preferably, when the CDCP1 concentration in the blood is less than 2.52 ng / ml, it indicates mild hair loss; when the CDCP1 concentration in the blood is 2.52-9.78 ng / ml, it indicates moderate hair loss; when the CDCP1 concentration in the blood is greater than 9.78 ng / ml, it indicates severe hair loss.

[0031] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0032] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0033] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0034] Example 1

[0035] In accordance with the principle of informed consent, a total of 88 male AGA patients aged 18-55 years who visited the hair disease clinic of Huashan Hospital Affiliated to Fudan University from June to November 2022 were collected. The age, course of disease, family history and other medical history data of each patient were recorded, and at least 3 dermatologists evaluated the severity of hair loss according to the Hamilton-Norwood grading method based on the patient's clinical photos.

[0036] Venous blood samples were collected from patients for Olink proteomics research. The blood samples were centrifuged, incubated, extended, and tested using Fluidigm Biomark TM The instrument will carry the designed oligonucleotide sequence antibody to immunolink with the target protein (oligonucleotide sequence antibody is the labeled antibody used in Olink proteomics research. The principle of Olink technology is proximity extension assay (PEA), which is a dual recognition immunoassay, that is, two matching antibodies bind to a target at the same time and are labeled with a unique DNA oligonucleotide ( Figure 2 A in the figure). This brings the two antibodies into close proximity, allowing their DNA oligonucleotides to hybridize and serve as templates for the DNA polymerase-dependent extension step ( Figure 2 B in Figure 1). This creates a double-stranded DNA "barcode" that is unique to the specific antigen and is present in quantity proportional to the initial concentration of the target protein. Hybridization and extension are followed by PCR amplification ( Figure 2 C), and finally the amplicon was quantified by microfluidic qPCR ( Figure 2 The specific operations of D) are as follows:

[0037] (1) Collect patient plasma samples: draw 5 ml of venous blood from each patient into an anticoagulant-treated blood collection tube; centrifuge at 1000-2000 × g for 10 min in a refrigerated centrifuge (2-8°C) to remove cells in the plasma; immediately transfer the plasma to a clean tube and divide it into EP tubes; store at -80°C for later use.

[0038] (2) Incubation: Prepare the incubation mixture according to the method in Table 1; all reagents in Table 1 were purchased from Shanghai Jingzhou Gene Technology Co., Ltd.

[0039] Table 1 Preparation method of incubation mixture

[0040]

[0041] Vortex and centrifuge the prepared incubation mixture. Use the PCR eight-tube strip and add 47μL of the incubation mixture to each test tube. Use the reverse pipetting method to transfer 3μL of the incubation mixture from the eight-tube strip to each well of a new 96-well plate and mark this plate as the incubation plate. Use a multichannel pipette to transfer 1μL of the sample and add it to the bottom of each well of the incubation plate. According to the sample distribution diagram ( Figure 3 ), transfer 1 μL of the control group to the three wells marked with light green in the 12th vertical row of the incubation plate, and transfer 1 μL of the negative control group to the three wells marked with red in the 12th vertical row. Use the mixed plasma sample as the sample control group and add it to the two wells marked with orange in the 12th vertical row of the incubation plate. Seal the incubation plate with a heat seal and centrifuge at room temperature for 1 minute at a speed of 400×g. Then incubate the incubation plate at 4°C overnight.

[0042] (3) Extension: Prepare the extension mixture according to the method in Table 2.

[0043] Table 2 Preparation method of extension mixture

[0044]

[0045] Remove the incubation plate and centrifuge for 1 minute after the temperature returns to room temperature. Preheat the PCR instrument. Vortex the extension mixture and pour it into the reagent tank. Set the timer for 5 minutes and use the reverse pipetting method to add 96μL of the extension mixture to each well of the incubation plate. After sealing the incubation plate with a heat plastic seal, vortex thoroughly to ensure that the sample is evenly mixed. Then centrifuge at room temperature for 1 minute. Place the incubation plate in the PCR instrument and start the proximity extension assay (PEA) program according to the instrument instructions: 50℃20min, 95℃5min, (95℃30s, 54℃1min, 60℃1min)×17, store at 10℃.

[0046] (4) Detection: Prepare a 96.96 Dynamic Array chip according to the chip instructions. TM Integrated Fluidic Circuit (IFC) chip (purchased from Shanghai Jingzhou Gene Technology Co., Ltd.). Add control line fluid to the two value areas of the chip, then load it into the IFC controller and pre-treat for 20 minutes. Thaw the primer plate, vortex and centrifuge. Prepare the detection mixture according to the method in Table 3.

[0047] Table 3 Preparation method of detection mixture

[0048]

[0049] Vortex and centrifuge the prepared detection mixture. Take a new PCR eight-tube strip and add 95μL of the detection mixture to each test tube. Take a new 96-well plate and add 7.2μL of the detection mixture to each well and mark it as the sample plate. Remove the incubation plate from the PCR instrument, vortex and centrifuge. Use forward pipetting to transfer 2.8μL of the extended sample from the upper surface of the liquid to the sample plate. Seal the sample plate with heat plastic and centrifuge it at room temperature for 1 minute at 400×g. From each well of the sample plate, use reverse pipetting to transfer 5μL of the amplified sample to the reagent compartment on the right side of the IFC chip. From each well of the primer plate, use reverse pipetting to transfer 5μL of primer to the reagent compartment on the left side of the IFC chip. Load the chip into the Fluidigm Integrated Fluidic Pathway Controller HX and perform the following steps according to the Fluidigm Biomark TM According to the instrument instructions, place the loaded IFC chip into the instrument, and then start the Olink inflammation and metabolism-related protein expression detection program (50℃120s, 70℃1800s, 25℃600s, 95℃300s, (95℃15s, 60℃60s)×40).

[0050] (5) Data processing: Fluidigm Biomark TM The instrument carries out immunological connection with the target protein by carrying the designed oligonucleotide sequence antibody. After the two oligonucleotide sequences connected to the protein are complementary to each other in base pairing, they are extended under the action of DNA polymerase. The amplified PCR sequence is tested by qPCR. The data generated by qPCR test is converted into counts file and output Olink normalized protein expression (NPX) data. NPX Manager is used to preprocess the data and obtain the input file required for the bioinformatics analysis process after quality control: NPX Data ( Figure 4 ).

[0051] Statistical analysis: Based on protein NPX Data, bioinformatics data analysis and data plotting were performed using R language (R-4.1.3) software and GraphPad Prism9.0.2 software. The test level α was set at 0.05, and P < 0.05 was considered statistically significant. Spearman correlation analysis was performed on the hair loss grade of male AGA patients and the expression levels of 92 inflammation-related proteins. The results showed that a total of 5 inflammation-related proteins were significantly correlated with the hair loss grade of male AGA patients. Among them, as the severity of hair loss in patients increased, the level of CDCP1 also increased (correlation coefficient r = 0.352, P < 0.01). Figure 5 As shown in Figure 2, the ROC curve shows that CDCP1 has a high accuracy in predicting the degree of male AGA hair loss (AUC = 0.685, P < 0.01). Figure 6 , with sensitivity = 58% and specificity = 74%, which suggests that CDCP1 can be used as a biomarker to predict the severity of hair loss in male AGA patients.

[0052] Example 2

[0053] According to the principle of informed consent, a total of 80 male AGA patients aged 18-60 years who visited the hair disease clinic of Huashan Hospital Affiliated to Fudan University from January to December 2023 were collected. The age, course of disease, family history and other medical history data of each patient were recorded, and at least 3 dermatologists evaluated the severity of hair loss based on the clinical photos of the patients.

[0054] Venous blood samples were collected from patients to determine CDCP1 concentration. The method for determining CDCP1 concentration in the blood was as follows: using a human CDCP1 ELISA kit (purchased from RayBiotech) and performing the test according to the instructions. The reagents required for the ELISA test are shown in Table 4:

[0055] Table 4 ELISA reagents required

[0056]

[0057] ELISA test steps:

[0058] (1) Before use, keep all reagents and samples at room temperature (18-25°C).

[0059] (2) Remove the plate and arrange the order of samples and standards.

[0060] (3) Add 100 μL of sample diluent to the sample well. Cover the well and incubate at room temperature for 2.5 h. The diluted standard should be prepared in advance. The preparation steps are as follows: Figure 7 : Mix a vial of standard protein by oscillation, add 400μL 1× reagent diluent B (dilute 5 times with deionized or distilled water before use) to the standard protein bottle to prepare a 50ng / mL standard solution. Mix gently to fully dissolve the powder. Use a pipette to add 270μL 1× reagent diluent B to each of the remaining tubes, use the 50ng / mL standard solution to make a diluted standard, mix well before the next pipetting. 1× reagent diluent B is used as the zero standard (0ng / mL).

[0061] (4) Discard the liquid in the plate and wash 4 times with washing buffer. Use a multichannel pipette to add washing buffer (300 μL) to each well for washing. After the last wash, remove all remaining washing buffer. Invert the plate and blot dry with a clean paper towel.

[0062] (5) Add 100 μL of 1× prepared biotinylated antibody to each well and incubate at room temperature for 1 h.

[0063] (6) Discard the liquid and repeat the washing steps in step (4).

[0064] (7) Add 100 μL of the prepared HRP-streptavidin solution to each well and incubate at room temperature for 45 min.

[0065] (8) Discard the liquid and repeat the washing steps in step (4).

[0066] (9) Add 100 μL of TMB one-step substrate reagent to each well and incubate in the dark at room temperature for 30 min.

[0067] (10) Add 50 μL of stop solution to each well and immediately read the plate at 450 nm.

[0068] (11) Use regression analysis to fit the standard curve (see Figure 8). The concentration of CDCP1 in the sample was calculated using the formula of the standard curve fitting, and then multiplied by the dilution factor to obtain the actual concentration of CDCP1.

[0069] Test results:

[0070] Through ELISA test and Spearman correlation analysis, it was found that as the severity of hair loss increased, the concentration of CDCP1 also increased (correlation coefficient r = 0.421, P < 0.001). Fig. 9 ( Fig. 9 The hair loss level in the test is divided according to the Hamilton-Norwood or Sinclair grading method). Hamilton-Norwood or Sinclair grade ≥ 4 is defined as severe hair loss, 3 ≤ Hamilton-Norwood or Sinclair grade < 4 is defined as moderate hair loss, and Hamilton-Norwood or Sinclair grade < 3 is defined as mild hair loss. The detection accuracy of the present invention for 80 samples was calculated based on the correlation between the CDCP1 concentration in the blood and the severity of AGA (as shown in Table 5). The results are shown in the ROC curve (see Fig.10 ), indicating that CDCP1 has a high accuracy in predicting severe hair loss in male AGA patients (AUC = 0.704, P < 0.01), with sensitivity = 57% and specificity = 81%, which also verifies that CDCP1 can be used as a biomarker to predict the severity of hair loss in male AGA patients.

[0071] Table 5 Correlation between CDCP1 concentration in blood and severity of AGA in men

[0072]

[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of CDCP1 protein in the preparation of products for assessing the severity of hair loss in the body.

2. The use according to claim 1, It is characterized in that The organism includes male androgenic alopecia patients.

3. The use according to claim 1, It is characterized in that The products include kits.

4. The use according to claim 1, It is characterized in that The severity of hair loss in the body is determined based on the concentration of CDCP1 protein in the biological sample.

5. The use according to claim 4, It is characterized in that The biological sample includes blood.

6. The use according to claim 5, It is characterized in that When the CDCP1 concentration in the blood is less than 2.52ng / ml, it indicates mild hair loss; when the CDCP1 concentration in the blood is 2.52-9.78ng / ml, it indicates moderate hair loss; when the CDCP1 concentration in the blood is greater than 9.78ng / ml, it indicates severe hair loss.

7. A kit for assessing the severity of hair loss in a body, It is characterized in that The kit includes reagents for detecting the concentration of CDCP1 protein in a sample.

8. The kit according to claim 7, It is characterized in that The sample includes blood.

9. The kit according to claim 8, It is characterized in that When the CDCP1 concentration in the blood is less than 2.52ng / ml, it indicates mild hair loss; when the CDCP1 concentration in the blood is 2.52-9.78ng / ml, it indicates moderate hair loss; when the CDCP1 concentration in the blood is greater than 9.78ng / ml, it indicates severe hair loss.

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