Application of migration capability of chemokine CXCL12 to chemotaxis of peripheral blood immune cells of subject to diagnosis of Alzheimer's disease

Abstract

The invention provides application of a method for detecting the migration ability of peripheral blood immune cells of a chemotactic factor CXCL12 chemotactic subject in preparation of a product for diagnosing Alzheimer's disease. A migration chemotactic experiment proves that under the chemotactic action of CXCL12, peripheral blood immune cells of an AD patient are more easily chemotactic by CXCL12, and the number of cells on the lower surface of an upper ventricular membrane is obviously increased compared with that of cells on a healthy control. Therefore, auxiliary diagnosis of the Alzheimer's disease can be achieved through the migration ability of chemotactic immune cells of the chemotactic factor CXCL12, and a new thought is provided for diagnosis of the Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of Alzheimer's disease, and in particular relates to the use of the migration function of peripheral blood immune cells under the action of chemokine CXCL12 for diagnosing Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease with progressive memory loss and cognitive dysfunction as the main clinical features. At present, the diagnosis of AD mainly adopts the ATN biomarker classification system, which includes three core indicators: β-amyloid protein (A), pathological Tau protein (including total Tau and phosphorylated Tau) (T), and neurodegeneration (N). By evaluating the abnormalities of these three biomarkers, AD patients can be diagnosed and the pathological changes at different stages of the disease process can be described. However, even with the combination of advanced diagnostic technologies such as pathology and imaging, about 20-25% of clinical AD cases cannot be clearly diagnosed. In addition, because AD is characterized by complex disease and long course, and patients show significant heterogeneity at different stages of the disease, it is urgent to discover more imaging and fluid biomarkers to further improve the diagnosis and treatment system of AD.

[0003] At present, there is no research exploring the application value of cell migration experimental technology in AD diagnosis, nor any related research reports targeting the CXCL12 / CXCR4 axis. This provides an important research direction for the development of new AD diagnostic strategies. The content of this invention is supported by the Shanghai Ministry of Science and Technology Project (2023YFC3603200). Summary of the invention

[0004] The purpose of the present invention is to overcome at least one disadvantage of the above-mentioned prior art and to provide a peripheral blood immune cell migration function under the action of chemokine CXCL12 for diagnosing Alzheimer's disease.

[0005] In order to solve the above technical problems, the present invention provides a reagent for detecting the migration ability of chemokine CXCL12 in chemotactic peripheral blood immune cells of a subject for use in preparing a product for diagnosing Alzheimer's disease.

[0006] Preferably, the migration function of the subject's peripheral blood immune cells under the action of chemokine CXCL12 is detected by counting the number of cells on the lower surface of the upper chamber membrane.

[0007] Preferably, the product is a kit.

[0008] The present invention confirms through migration chemotaxis experiments that under the chemotactic effect of CXCL12, the peripheral immune cells of AD patients are more susceptible to chemotaxis of CXCL12, which is manifested by a significant increase in the number of cells on the lower surface of the upper chamber membrane compared with the healthy control. Therefore, the present invention provides that the migration ability of chemotactic immune cells by chemokine CXCL12 can assist in the diagnosis of Alzheimer's disease, providing a new idea for the diagnosis of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Schematic diagram of the immune cell migration chemotaxis experiment in this example.

[0010] Figure 2 The migration of peripheral blood immune cells from different subjects differs in response to the chemokine CXCL12. DETAILED DESCRIPTION

[0011] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments are conventionally commercially available. Unless otherwise specified, the relevant operations in the embodiments and test examples are conventional technical means in the field.

[0012] Preliminary studies by the team of the present invention have confirmed that the expression of CXCR4 receptor protein in peripheral blood cells of AD patients is significantly increased compared with the healthy control group. CXCR4, as a chemokine receptor, is mainly expressed on the surface of various immune cells and plays an important role in immune regulation. In order to study the role of the CXCL12 / CXCR4 signaling axis in AD pathology, the present invention uses the Transwell cell migration experiment to evaluate the function of immune cells. The experiment divides the culture system into two independent spaces, upper and lower, through a special porous membrane. The ability of cells to pass through the porous membrane from the upper chamber to the lower chamber is observed and quantitatively analyzed, thereby evaluating the migration ability of cells. The Transwell experiment has been widely used in many research fields such as tumor metastasis, immune cell function, wound healing and reproductive medicine due to its advantages such as standardized operation, reliable results and good repeatability.

[0013] Experimental methods

[0014] like Figure 1 As shown in FIG. 1 , a schematic diagram of the process of the migration chemotaxis experiment of this embodiment is shown. Fresh peripheral blood of healthy subjects and Alzheimer's disease patients is collected in anticoagulation collection tubes. Direct Human PBMC Isolation Kit (kit) is used to extract peripheral blood mononuclear cells (PBMC). For detailed operation steps, please refer to the kit instructions.

[0015] The specific operation is as follows: take 1ml of human blood sample into a 5ml flow tube, add 6nM EDTA and mix well, add 50ul Isolation Cocktail into the flow tube, and leave it at room temperature for 5 minutes. Buffer, gently mix 2 to 3 times. After 30 seconds, add 50ul of After mixing, place the flow tube in the magnetic stand for 5 minutes. Use a pipette to transfer the clear liquid (non-magnetic bead adsorption part) in the flow tube to a new 5ml flow tube and add 50ul Mix well and immediately place the flow tube in a magnetic stand for 5 minutes. Use a pipette to transfer the clear liquid (non-magnetic bead adsorption part) in the flow tube to a new 15ml test tube, add 10ml PBS and mix well, centrifuge at 350g and 4 degrees for 5 minutes. Discard the supernatant, add 10ml PBS and mix well, centrifuge at 350g and 4 degrees for 5 minutes. PBMCs are obtained.

[0016] After cell counting, 200,000 PBMCs were placed in the upper chamber of the cell migration chamber, and PBS / chemokine CXCL12 was added to the lower chamber of different cell chambers. Cultured in a cell culture incubator (37 degrees, 5% CO2) for 24 hours. The chamber was fixed in 4% paraformaldehyde fixative for 15 minutes, washed thoroughly with PBS, stained with crystal violet staining solution for 10 minutes, and counted under a microscope after being washed thoroughly with PBS. The number of cells on the lower surface of the upper chamber membrane was counted, and the difference in migration function of PBMCs between healthy people and patients with Alzheimer's disease under the action of chemokine CXCL12 was compared. The data of each group are expressed as mean ± standard error (SEM). The t test was used to compare the two groups of data. The result was considered to be significantly different if the P value was less than 0.05.

[0017] Experimental Results

[0018] like Figure 2 As shown, the number of cells on the lower surface of the upper chamber membrane of different subjects under the action of chemokine CXCL12. Among them, NC is a healthy control, and AD is an Alzheimer's disease patient. The results show that under the chemotactic effect of CXCL12, the peripheral immune cells of AD patients are more susceptible to the chemotactic effect of CXCL12, which is manifested by a significant increase in the number of cells on the lower surface of the upper chamber membrane compared with the healthy control.

[0019] In this specification, the present invention has been described with reference to specific embodiments thereof. However, it is apparent that various modifications and variations may be made without departing from the spirit and scope of the present invention. Therefore, the description should be regarded as illustrative rather than restrictive.

Claims

1. Use of a reagent for detecting the migration ability of chemokine CXCL12 in chemotactic peripheral blood immune cells of a subject in the preparation of a product for diagnosing Alzheimer's disease.

2. The use according to claim 1, characterized in that The migration ability of peripheral blood immune cells of subjects induced by chemokine CXCL12 was detected by counting the number of cells on the lower surface of the upper chamber membrane.

3. The use according to claim 1, characterized in that The product described is a test kit.

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