Application of ANGPTL1 in predicting severity of male alopecia

Abstract

The invention provides application of ANGPTL1 in predicting severity of male alopecia, and belongs to the technical field of alopecia prevention and treatment. The invention firstly proposes that ANGPTL1 can be used as a marker for evaluating the severity of body alopecia, and firstly proposes that the ANGPTL1 level is remarkably positively correlated with the severity of alopecia of a male MPHL patient, and the method for evaluating the severity of body alopecia according to the concentration of ANGPTL1 in blood 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 use of ANGPTL1 in predicting the severity of male pattern hair loss. Background Art

[0002] Androgenetic Alopecia (AGA) is a polygenic, hereditary, non-scarring hair loss disease, which is divided into male pattern hair loss (MPHL) and female pattern hair loss (FPHL) according to the hair loss pattern. Currently, oral finasteride, topical minoxidil and low-energy laser therapy are FDA-approved treatments for male AGA, but clinical findings show that the effects of traditional therapies on different patients are inconsistent, and the exact pathogenesis of the disease itself has not been fully elucidated, so early intervention and treatment of AGA remain challenging.

[0003] Angiopoietin-related protein (ANGPTL) is a family of proteins that are structurally similar to angiopoietin. Eight types of ANGPTL have been discovered, namely ANGPTL1-8, which play an important role in regulating various physiological and pathological processes such as angiogenesis, inflammation, lipid metabolism, glucose metabolism, anti-aging, tumorigenesis and metastasis. Angiopoietin-related protein 1 (ANGPTL1), also known as angiostatin, is the first member of the ANGPTL family to be discovered. It can inhibit the proliferation, migration and adhesion of vascular endothelial cells. In addition, ANGPTL1, as a tumor suppressor gene, can inhibit the generation, invasion and metastasis of tumor cells, thereby improving the prognosis of malignant tumors.

[0004] MPHL is the most common hair loss disease in clinic and is becoming younger. The mechanisms of action of existing treatments include vasodilation, inhibition of dihydrotestosterone production, and induction of hair growth. Exploring biomarkers to predict the severity of MPHL and taking early intervention or preventive measures will help prevent the miniaturization of hair follicles and the irreversible loss of hair follicle function, increase hair density, and select the best treatment plan based on the patient's degree of hair loss. Summary of the invention

[0005] In view of this, an object of the present invention is to provide a use of ANGPTL1 in the preparation of a product for assessing the severity of hair loss in a body.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides the use of ANGPTL1 in preparing a product for predicting the severity of hair loss in an organism.

[0008] Preferably, the organism comprises a patient suffering from male pattern baldness.

[0009] Preferably, the product comprises a kit.

[0010] Preferably, the severity of hair loss in an organism is determined based on the concentration of ANGPTL1 in the biological sample.

[0011] Preferably, the biological sample comprises blood.

[0012] Preferably, when the ANGPTL1 concentration in the blood is less than 525.31 ng / mL, it indicates mild hair loss; when the ANGPTL1 concentration in the blood is 525.31-579.36 ng / mL, it indicates moderate hair loss; when the ANGPTL1 concentration in the blood is greater than 579.36 ng / mL, it indicates severe hair loss.

[0013] The present invention also provides a kit for evaluating the severity of hair loss in an organism, wherein the kit comprises a reagent for detecting the concentration of ANGPTL1 in a sample.

[0014] Preferably, the sample comprises blood.

[0015] Preferably, when the ANGPTL1 concentration in the blood is less than 525.31 ng / mL, it indicates mild hair loss; when the ANGPTL1 concentration in the blood is 525.31-579.36 ng / mL, it indicates moderate hair loss; when the ANGPTL1 concentration in the blood is greater than 579.36 ng / mL, it indicates severe hair loss.

[0016] Beneficial effects of the present invention:

[0017] The present invention proposes for the first time that ANGPTL1 can be used as a marker for assessing the severity of hair loss in an organism, and proposes for the first time that the level of ANGPTL1 is significantly positively correlated with the severity of hair loss in male MPHL patients. Assessing the severity of hair loss in an organism by the concentration of ANGPTL1 in the blood has high specificity, sensitivity, and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the molecular structure of ANGPTL1;

[0019] Figure 2The 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;

[0020] Figure 3 Loading position for incubation plate;

[0021] Figure 4 Analyze the experimental data process;

[0022] Figure 5 This is the Spearman correlation analysis diagram of male MPHL severity and ANGPTL1 level;

[0023] Figure 6 ROC curves for MPHL severity and ANGPTL1 levels in males;

[0024] Figure 7 It is the ELISA test procedure;

[0025] Figure 8 Preparation process of diluting standards for ELISA tests;

[0026] Fig. 9 It is the standard curve of ELISA test;

[0027] Fig.10 This is the Spearman correlation analysis diagram between the severity of male MPHL and the ANGPTL1 concentration measured by ELISA;

[0028] Fig.11 ROC curve of MPHL severity and ANGPTL1 concentration measured by ELISA in males. DETAILED DESCRIPTION

[0029] The present invention provides the use of ANGPTL1 in preparing a product for predicting the severity of hair loss in an organism.

[0030] In the present invention, ANGPTL1 is a secretory glycoprotein widely expressed in adult tissues. The gene is located on chromosome 1q24.2, consists of 491 amino acid residues, and has three conserved domains: an N-terminal signal peptide, an N-terminal coiled-coil domain, and a C-terminal fibrinogen-like domain. The specific molecular structure is as follows Figure 1 shown.

[0031] In the present invention, the organism preferably includes a patient with male pattern baldness, and the product preferably includes a kit. In the present invention, the severity of hair loss in the organism is preferably determined based on the concentration of ANGPTL1 in a biological sample, and the biological sample preferably includes blood. In the present invention, when the concentration of ANGPTL1 in the blood is <525.31 ng / mL, it indicates mild hair loss; when the concentration of ANGPTL1 in the blood is 525.31-579.36 ng / mL, it indicates moderate hair loss; when the concentration of ANGPTL1 in the blood is >579.36 ng / mL, it indicates severe hair loss. In the present invention, the concentration of blood ANGPTL1 is preferably detected by ELISA method.

[0032] 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 ANGPTL1 in a sample. In the present invention, the sample preferably comprises blood. In the present invention, the reagent for detecting the concentration of ANGPTL1 in a sample preferably comprises an ELISA detection reagent. When the kit of the present invention is used, when the concentration of ANGPTL1 in the blood is <525.31 ng / mL, it indicates mild hair loss; when the concentration of ANGPTL1 in the blood is 525.31-579.36 ng / mL, it indicates moderate hair loss; when the concentration of ANGPTL1 in the blood is >579.36 ng / mL, it indicates severe hair loss.

[0033] 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.

[0034] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0035] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0036] Example 1

[0037] In accordance with the principle of informed consent, a total of 46 male MPHL 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.

[0038] Venous blood samples were collected from patients for Olink proteomics research. The blood samples were centrifuged, incubated, extended, and tested using Fluidigm Biomark TMThe 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:

[0039] (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 dispense it into EP tubes; store at -80°C until needed.

[0040] (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.

[0041] Table 1 Preparation method of incubation mixture

[0042] Incubation mixture Volume required per 96-well plate (μL) Incubation solution 280.0 Incubation Stabilizer 40.0 A-probes 40.0 B-probes 40.0 Total volume 400.0

[0043] 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.

[0044] (3) Extension: Prepare the extension mixture according to the method in Table 2.

[0045] Table 2 Preparation method of extension mixture

[0046] Extension Mix Volume required per 96-well plate (μL) high purity water 9385 PEA solution 1100 PEA enzyme 55 PCR polymerase 22 Total volume 10562

[0047] Take out 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 it thoroughly to ensure that the sample is evenly mixed. Then centrifuge it at room temperature for 1 minute. Place the incubation plate in the PCR instrument and start the proximity extension analysis (PEA) program according to the instrument instructions: 50℃20min, 95℃5min, (95℃30s, 54℃1min, 60℃1min)×17, store at 10℃.

[0048] (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.

[0049] Table 3 Preparation method of detection mixture

[0050] Detection mixture Volume required per 96-well plate (μL) Detection solution 550.0 high purity water 230.0 Detection enzyme 7.8 PCR polymerase 3.1 Total volume 790.9

[0051] 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 TMAccording 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).

[0052] (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 base-paired, they are extended under the action of DNA polymerase. The amplified PCR sequence is tested by qPCR. The data generated by qPCR detection is converted into counts files and output Olink normalized protein expression (NPX) data. NPX Manager is used to pre-process the data and obtain the input files required for the bioinformatics analysis process after quality control: NPX Data ( Figure 4 ).

[0053] (6) Statistical analysis: Based on the protein NPX data, bioinformatics data analysis and data plotting were performed using R language (R-4.1.3) and GraphPad Prism 9.0.2. The test level was 0.05, and P < 0.05 was considered statistically significant.

[0054] Spearman correlation analysis was performed on the severity of hair loss in male MPHL patients and the expression levels of 92 metabolic-related proteins. The results showed that ANGPTL1 was significantly correlated with the hair loss grade in male MPHL patients. As the severity of hair loss increased, the level of ANGPTL1 also increased (correlation coefficient r=0.476, P<0.001). Figure 5 Hamilton-Norwood grade ≥ 4 was defined as severe hair loss, 3 ≤ Hamilton-Norwood grade < 4 was defined as moderate hair loss, and Hamilton-Norwood grade < 3 was defined as mild hair loss. According to the ROC curve, ANGPTL1 has a high accuracy in predicting severe hair loss in male AGA (AUC = 0.712, P < 0.05). Figure 6 , with sensitivity = 91% and specificity = 50%, which suggests that ANGPTL1 can be used as a biomarker to predict the severity of hair loss in male MPHL patients.

[0055] Example 2

[0056] According to the principle of informed consent, a total of 42 male MPHL patients aged 18-60 years who visited the hair disease clinic of Huashan Hospital Affiliated to Fudan University from January 2023 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 according to the Hamilton-Norwood grading method based on the patient's clinical photos. Hamilton-Norwood grade ≥ 4 was defined as severe hair loss, 3 ≤ Hamilton-Norwood grade < 4 was defined as moderate hair loss, and Hamilton-Norwood grade < 3 was defined as mild hair loss.

[0057] Venous blood samples were collected from patients to measure the ANGPTL1 concentration. The method for measuring the ANGPTL1 concentration in the blood was as follows: using a human ANGPTL1 ELISA kit (purchased from MyBioSource) and performing the test according to the instructions. The reagents required for the ELISA test are shown in Table 4:

[0058] Table 4 Reagents for human ANGPTL1 ELISA kit (MyBioSource)

[0059] Reagents quantity Detection plate (96 wells) 1 Standard (lyophilized) 2 Biotin conjugate (concentrate 100x) 1x120μL Streptavidin-Horseradish Peroxidase (Concentrate 100x) 1x120μL Washing buffer (concentrate 25x) 1x20mL Stop Solution 1x10mL Sample diluent 1x20mL Biotin-conjugate diluent 1x20mL Streptavidin-horseradish peroxidase dilution 1x20mL Substrate solution 1x10mL Adhesive film 4

[0060] ELISA test steps are as follows Figure 7 As shown:

[0061] (1) Before use, keep all reagents and samples at room temperature (37°C).

[0062] (2) Remove the plate and arrange the order of samples and standards.

[0063] (3) Add 100 μL of standard and sample to each well. Cover with adhesive film. Incubate at 37°C for 2 h. The diluted standard should be prepared in advance. The preparation steps are as follows: Figure 8 : Use a pipette to transfer 250 μL of the sample diluent into each test tube. Prepare the dilution series using the stock solution. Mix each test tube thoroughly before the next pipetting. The undiluted standard solution is used as the high standard solution (200 pg / mL). The sample diluent is used as the zero standard solution (0 pg / mL).

[0064] (4) Pipette each well and wash, repeating the washing process three times. Wash by adding wash buffer (250 μL) to each well using a squirt bottle, multichannel pipette, manifold dispenser, or automated washer. Complete removal of liquid at each step is critical to the assay. After the final wash, remove any remaining wash buffer by pipetting or pouring. Invert the plate and blot dry with a clean paper towel.

[0065] (5) Add 100 μL of biotin conjugate (1x) to each well. Cover with adhesive film. Incubate at 37°C for 1 hour. (Biotin conjugate (1x) may look turbid. Warm to room temperature and stir gently until the solution looks homogeneous.)

[0066] (6) Aspirate each well and wash, repeating the washing process three times (the specific washing method is the same as step 4).

[0067] (7) Add 100 μL of streptavidin-horseradish peroxidase (1x) to each well. Cover the microtiter plate with adhesive film. Incubate at 37°C for 1 h.

[0068] (8) Aspirate each well and wash, repeating the washing process five times (the specific washing method is the same as step 4).

[0069] (9) Add 100 μL of substrate solution to each well. Incubate at 37°C for 15 min. Place the plate in a dark place away from drafts and temperature fluctuations, and avoid placing the plate in direct light.

[0070] (10) When the first four wells containing the highest concentration of standard are visibly blue, add 50 μL of stop solution to each well. If the color change is uneven, gently tap the plate to ensure adequate mixing.

[0071] (11) Using a microplate reader set at 450 nm, measure the OD (optical density) value of each well within 5 min.

[0072] (12) The standard curve was prepared using the “Curve Exert 1.4” software (see Fig. 9 ). The concentration of ANGPTL1 in the sample was calculated using the formula of the standard curve fitting, and the actual concentration of ANGPTL1 was obtained by multiplying it by the dilution factor.

[0073] Test results:

[0074] Through ELISA test and Spearman correlation analysis, it was found that as the severity of hair loss in MPHL patients increased, the blood concentration of ANGPTL1 also increased (correlation coefficient r = 0.529, P < 0.001). Fig.10 ( Fig.10 The hair loss level is classified according to the Hamilton-Norwood scale.)

[0075] Hamilton-Norwood grade ≥ 4 is defined as severe hair loss, 3 ≤ Hamilton-Norwood grade < 4 is defined as moderate hair loss, and Hamilton-Norwood grade < 3 is defined as mild hair loss. The detection accuracy of the present invention for 42 MPHL patient samples was calculated based on the correlation between the concentration of ANGPTL1 in the blood and the severity of MPHL (as shown in Table 5). The results are shown in the ROC curve (see Fig.11 ), according to the ROC curve, ANGPTL1 has a high accuracy in predicting severe hair loss in male MPHL patients (AUC=0.781, P<0.01), with sensitivity=88% and specificity=61% (maximum Youden index is 0.49), which also verifies that ANGPTL1 can be used as a biomarker to predict the severity of hair loss in male AGA patients.

[0076] Table 5 Correlation between ANGPTL1 concentration in blood and severity of MPHL

[0077] Severity of MPHL in men ANGPTL1 serum concentration (pg / mL) Mild hair loss ＜525.31 Moderate hair loss 525.31-579.36 Severe hair loss ＞579.36

[0078] 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 ANGPTL1 in the preparation of products for predicting the severity of hair loss in the body.

2. The use according to claim 1, characterized in that: The organism includes a patient with male pattern baldness.

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

4. The use according to claim 1, characterized in that: The severity of hair loss in the body is determined based on the concentration of ANGPTL1 in the biological sample.

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

6. The use according to claim 5, characterized in that: When the ANGPTL1 concentration in the blood is less than 525.31 ng / mL, it indicates mild hair loss; when the ANGPTL1 concentration in the blood is 525.31-579.36 ng / mL, it indicates moderate hair loss; when the ANGPTL1 concentration in the blood is greater than 579.36 ng / mL, it indicates severe hair loss.

7. A kit for assessing the severity of hair loss in an organism, characterized in that: The kit includes reagents for detecting the concentration of ANGPTL1 in a sample.

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

9. The kit according to claim 8, characterized in that When the ANGPTL1 concentration in the blood is less than 525.31 ng / mL, it indicates mild hair loss; when the ANGPTL1 concentration in the blood is 525.31-579.36 ng / mL, it indicates moderate hair loss; when the ANGPTL1 concentration in the blood is greater than 579.36 ng / mL, it indicates severe hair loss.

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