Biomarker for early warning or diagnosing early Parkinson's disease and application thereof

Abstract

The invention discloses a biomarker for early warning or diagnosis of early-stage PD (Parkinson's Disease) and application thereof, belongs to the technical field of biological medicines, and proposes that the level of peripheral blood P-LCK cells in a blood sample is taken as the biomarker for the first time, and the biomarker has extremely high accuracy, specificity and sensitivity for early warning or diagnosis of early-stage PD.

Description

Technical Field

[0001] The present invention relates to a biomarker for early warning or diagnosing Parkinson's disease and its application, specifically to using peripheral blood P-LCK cells (phospho-LCK, that is, phosphorylated LCK or P-LCK) as a biomarker for early warning or diagnosing early Parkinson's disease (PD), and related applications of this biomarker in Parkinson's disease, belonging to the field of biomedical technology. Background Art

[0002] Immune activation and neuroinflammatory responses play important roles in the pathological process of PD and have received increasing attention, especially in the application of developing disease biomarkers and new therapies. Epidemiological studies have shown that patients with autoimmune diseases have a higher risk of developing PD, while patients receiving immunosuppressive therapy have a lower risk of developing PD. Research has shown that the neuroinflammatory process of PD involves the dual participation of the innate and adaptive immune systems. Peripheral immunity has also been confirmed to be involved in the pathogenesis of PD, including innate immunity, adaptive immunity, gut immunity, etc. Peripheral immune activation in PD patients is related to disease severity, disease course, cognition, etc. Peripheral chronic inflammation can further form a vicious cycle of the "peripheral-central immune axis" and promote the pathological progression of PD. Among them, the adaptive immune response mediated by T cells may be the key to causing the pathology of PD. The aggregation of CD8+ T cells in the substantia nigra was found in the autopsy of preclinical PD patients, suggesting that CD8+ T cells are the key to participating in the pathology of PD, especially playing an important role in the early stage of PD. The role of CD8+ T cells in the pathogenesis of PD suggests that CD8+ T cells may be potential targets for PD treatment. However, whether CD8+ T cells formed by the peripheral immune response have an impact on the pathology of PD needs further study.

[0003] LCK is one of the eight members of the tyrosine kinase Src family, and its expression is limited to immune cells derived from lymphocytes. It plays a crucial role in the maturation of thymic lymphocytes and the activation and proliferation of mature T cells. LCK is involved in several key processes related to T cell development and proliferation. LCK controls several downstream molecular targets in T cells after antigen stimulation, including other targets such as the transcription factors GATA3, STAT3, NFATc1, and NF-κB. The development of thymocytes to the double-negative stage requires the participation of LCK. Studies have shown that there are very few circulating mature T cells in LCK gene knockout mice. The LCK kinase is related to various carcinogenic processes, especially in colorectal cancer, chronic lymphocytic leukemia, and thymoma. CD8 + Abnormal differentiation and regulation of CD8+ T cells may be related to the pathological mechanism of PD, and LCK can regulate T cell activation and differentiation. The LCK protein kinase is activated through the Y-394 site to function as phosphorylated P-LCK.

[0004] Currently, the research on Parkinson's disease mainly focuses on CD8 + T cells. For example, the peripheral blood CD8 + T cells reported in Chinese Patent CN116953247A can be used as biomarkers for the early diagnosis of neurodegenerative diseases such as Alzheimer's disease. However, regarding the CD8+T cell subtypes, especially the relationship between the proportion of peripheral blood P-LCK cells and Parkinson's disease, and whether the proportion of peripheral blood P-LCK cells can be used to diagnose early Parkinson's disease, there has been no in-depth exploration in previous studies.

[0005] It can be seen that there is currently no study investigating the relationship between the proportion of peripheral blood P-LCK cells and PD patients, and whether this biomarker can be used to diagnose early Parkinson's disease remains unknown. Summary of the Invention

[0006] The present invention aims to solve the existing blank for biomarkers for the diagnosis of early PD, and provides a biomarker for warning or diagnosing early Parkinson's disease and its application. It is proposed for the first time to use the level of peripheral blood P-LCK cells in a blood sample as a biomarker, which has extremely high accuracy, specificity, and sensitivity for warning or diagnosing early PD.

[0007] The present invention is achieved through the following technical solutions: A biomarker for warning or diagnosing early Parkinson's disease, by detecting the level of P-LCK cells in a blood sample of a subject, the proportion of P-LCK / CD8+T cells in the peripheral blood mononuclear cells of the subject is obtained.

[0008] The proportion of P-LCK / CD8+T cells in the peripheral blood mononuclear cells of the subject is greater than 17.70%.

[0009] Meanwhile, the present invention also provides the application of a substance for detecting P-LCK cells in the preparation of a warning or diagnostic product for early Parkinson's disease. By detecting the level of P-LCK cells in a blood sample of a subject, the proportion of P-LCK / CD8+T cells in the peripheral blood mononuclear cells of the subject is obtained.

[0010] The detection method includes flow cytometry.

[0011] The substance for detecting P-LCK cells includes detection reagents, flow cytometers, or other substances that can be achieved in medicine.

[0012] The blood sample includes whole blood.

[0013] The proportion of P-LCK / CD8+T cells in the peripheral blood mononuclear cells of the subject is greater than 17.70%.

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) By researching and exploring relevant biomarkers for diagnosing early Parkinson's disease patients, the present invention first discovers that the level of P-LCK cells in peripheral blood is related to the diagnosis of early Parkinson's disease patients. Therefore, it can be used as a biomarker for diagnosing early Parkinson's disease, and through verification with a large number of clinical samples, this diagnostic biomarker has extremely high accuracy, specificity, and sensitivity.

[0015] (2) The present invention can use flow cytometry detection technology to detect the level of P-LCK cells in the peripheral blood of a subject, thereby obtaining the cell ratio of P-LCK / CD8+T in peripheral blood, which can be quickly and efficiently applied to clinical diagnosis to improve the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the ratio of P-LCK / CD8+T cells in peripheral blood mononuclear cells of early PD patients and healthy controls.

[0017] Figure 2 It is the receiver operating characteristic curve of diagnosing early PD patients by the proportion of P-LCK in peripheral blood. DETAILED DESCRIPTION OF THE INVENTION

[0018] Hereinafter, the object of the invention, technical solutions, and beneficial effects of the present invention will be further described in detail.

[0019] It should be noted that the following detailed description is exemplary and is intended to provide further description of the claimed invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0020] Parkinson's disease (PD) is a neurodegenerative disease of the nervous system. The main cause of PD is the degeneration and death of dopaminergic neurons in the substantia nigra, which may be related to multiple factors such as genetics, environmental factors, and aging of the nervous system. The symptoms of PD vary, mainly manifested as two categories: motor and non-motor symptoms. Motor symptoms include resting tremor, muscle rigidity, bradykinesia, and postural balance disorder; non-motor symptoms mainly include constipation, olfactory disorder, sleep disorder, autonomic dysfunction, and mental and cognitive disorders, etc. Early diagnosis of Parkinson's disease is currently a difficult point in clinical work, but currently, there is no report of using P-LCK cells in peripheral blood as a biomarker for diagnosing early PD.

[0021] The present invention for the first time uses peripheral blood P-LCK cells as biomarkers for the diagnosis of early Parkinson's disease, with the level content of peripheral blood P-LCK / CD8+ T cells as the detection index. Specifically, by detecting the proportion content (17.70%) of P-LCK / CD8+ T cells in peripheral blood mononuclear cells in the blood sample of a subject, it is indicated that the subject is a patient with early Parkinson's disease, with extremely high accuracy, specificity and sensitivity.

[0022] In some alternative embodiment cases, flow cytometry can be selected as the detection method. Known detection reagents, flow cytometers or other medically achievable substances can be used to detect peripheral blood P-LCK cells in the blood sample of a subject. The blood sample of the subject can be whole blood.

[0023] The present invention will be further described in detail below in conjunction with embodiments, but the implementation manners of the present invention are not limited thereto.

[0024] Example 1: In this example, flow cytometry is used to detect the level content of P-LCK cells in the blood sample of a subject, so as to determine the proportion of P-LCK / CD8+ T cells in the peripheral blood mononuclear cells of the subject.

[0025] Source of the blood sample of the subject: whole blood.

[0026] Carry out the detection work on the flow cytometer according to the instructions.

[0027] (1) Preparation of peripheral blood PBMC.

[0028] (2) Take the PBMC resuspension, and the number of cells in each tube is about 1×10 6 cells.

[0029] (3) Use Fc blocking, add 5 uL to each tube, and incubate in the refrigerator for 20 min.

[0030] (4) Cell surface staining. Add 1 uL of CD3, CD4, CD8, CD45RA, and CCR7 to each tube respectively, vortex and mix well. Set single stains and blank controls for CD3, CD4, CD8, CD45RA, and CCR7 respectively, and incubate in the dark for 30 minutes.

[0031] (5) Add 1 mL of wash working solution for washing, centrifuge at 400 rpm for 5 min, aspirate and discard the supernatant, add 200 uL of fixation / permeabilization working solution (Cat: 554714, BD Pharmingen), vortex and mix again, and incubate at 4°C in the dark environment for 20 min.

[0032] (6) Resuspend the cells in 100 μL of PBS buffer, add 5 μL of P-LCK flow antibody to each tube for intracellular cell staining, vortex the cells to mix well, and incubate the samples in the dark at 4 °C for 40 min.

[0033] (7) Subsequently, add 2 mL of wash working solution to wash the cells, discard the supernatant, and repeat the washing step.

[0034] (8) Add 300 μL of PBS to resuspend the cells, and detect CD8+ Tem and P-LCK cells by flow cytometry. The detection results can show the proportions of CD8+ Tem and P-LCK cells in peripheral blood, and thus the proportion of LCK cells in peripheral blood mononuclear cells can be obtained.

[0035] Example 2: This example involves using the level of P-LCK cells in peripheral blood as a detection marker, detecting the blood samples of PD patients and healthy controls respectively, and using the proportion of P-LCK / CD8+ T cells in peripheral blood mononuclear cells as a judgment criterion to verify the diagnosis of early PD patients.

[0036] A total of 60 early PD patients and 30 healthy controls were enrolled in this study. The proportions of P-LCK / CD8+ T cells in peripheral blood mononuclear cells in the blood samples of 60 early PD patients and 30 healthy controls were detected by flow cytometry respectively (the detection method refers to Example 1).

[0037] The test results are shown in Table 1 below, Figure 1 and Figure 2 as shown.

[0038] Table 1 Demographic data, clinical characteristics and hematological markers of the testers The above Table 1 shows the clinical data of healthy controls (HC) and early PD patients (PD). It can be seen from Table 1 that the age of PD patients is 58.82 (11.29), and the age of healthy controls is 57.63 (9.88), and there is no statistical difference in age.

[0039] Figure 1 are the proportions of P-LCK cells in peripheral blood mononuclear cells of early PD patients and healthy controls. Figure 1 In (A) is the flow cytometry diagram, and (B) is the scatter plot of early PD patients and healthy controls. It can be seen from Figure 1 that the proportion of P-LCK / CD8+ T cells in peripheral blood mononuclear cells of early PD patients is higher than that of healthy controls (20.45 ± 5.83 vs 14.09 ± 3.86, P <0.001).

[0040] Figure 2 Survival curves for early PD patients and healthy controls. By Figure 2 It can be seen that when the proportion of P-LCK / CD8+ T cells in peripheral blood mononuclear cells is greater than 17.70%, the probability of diagnosing early PD patients is 0.819, the sensitivity is 63.33%, and the specificity is 83.33%.

[0041] As described above, it is only a preferred embodiment of the present invention and does not impose any formal limitations on the present invention. Any simple modifications and equivalent changes made to the above embodiments based on the technical essence of the present invention all fall within the protection scope of the present invention.

Claims

1. A biomarker for early warning or diagnosis of Parkinson's disease, characterized in that: By detecting the level of P-LCK kinase in the blood sample of a subject, the ratio of P-LCK / CD8+ T cells in the peripheral blood of the subject is obtained.

2. The biomarker according to claim 1, wherein: The ratio of P-LCK / CD8+ T cells in the peripheral blood mononuclear cells of the subject is greater than 17.70%.

3. Use of a substance for detecting P-LCK cells in the preparation of a product for early warning or diagnosis of Parkinson's disease, characterized in that: By detecting the level of P-LCK cells in the blood sample of a subject, the ratio of P-LCK / CD8+ T cells in the peripheral blood mononuclear cells of the subject is obtained.

4. The application according to claim 3, characterized in that: The detection methods include flow cytometry.

5. The application according to claim 3, characterized in that: The substances for detecting P-LCK cells include detection reagents, flow cytometers or other substances that can be achieved medically.

6. The application according to claim 3, wherein: The blood sample includes whole blood.

7. The application according to claim 3, wherein: The ratio of P-LCK / CD8+ T cells in the peripheral blood mononuclear cells of the subject is greater than 17.70%.

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