Application of SLAMF7 expressed CD4+T cell in preparation of tuberculosis diagnosis or treatment reagent

A technology for tuberculosis and reagents, which is applied in the field of preparation of tuberculosis diagnosis or treatment reagents, can solve problems such as drug resistance of Mycobacterium tuberculosis, and achieve the effects of reducing inflammation, easy review, and pain relief

Inactive Publication Date: 2021-05-28
THE FIFTH AFFILIATED HOSPITAL SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, long-term chemotherapy can easily lead to drug resistance of Mycobacterium tuberculosis
With the high rate of drug resistance and the spread of drug-resistant bacteria, the treatment of tuberculosis is becoming more and more severe. At present, there is no specific drug for the treatment of drug-resistant tuberculosis.

Method used

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  • Application of SLAMF7 expressed CD4+T cell in preparation of tuberculosis diagnosis or treatment reagent
  • Application of SLAMF7 expressed CD4+T cell in preparation of tuberculosis diagnosis or treatment reagent
  • Application of SLAMF7 expressed CD4+T cell in preparation of tuberculosis diagnosis or treatment reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 The expression level of SLAMF7 on the surface of CD4+ T cells and the diagnosis of tuberculosis

[0034] Peripheral blood was collected from subjects, including 58 healthy subjects and 57 tuberculosis patients. The tuberculosis patients included 22 cases of active tuberculosis (Active TB), 16 cases of latent infection of tuberculosis (Latent TB), and 19 cases of cured tuberculosis (Cured TB).

[0035] Collect 5 mL of peripheral blood from healthy people and tuberculosis patients, lyse red blood cells to prepare single cell suspension, and adjust the total amount of cells to 5×10 6 First, add SLAMF7 antibody and CD4 antibody to 100 μL of the system, mix well and protect from light for 30 minutes, flow cytometry detection, FCS / SSC set a gate to analyze the proportion of SLAMF7 positive cells to CD4 positive cells.

[0036] The result is as figure 1 as shown, figure 1 A It can be seen that the proportion of SLMF7-positive cells in tuberculosis patients is much...

Embodiment 2

[0037]Example 2 The effect of recombinant protein activation of SLAMF7 on the secretion of IFN-γ and TNF-a by CD4+ T cells

[0038] Collect 10ml of peripheral blood from 10 active tuberculosis patients, separate PBMCs with lymphocyte separation medium; treat PBMCs with SLAMF7 recombinant protein and isotype control IgG, transfer the cells to a flow tube after 24 hours, add 1ml of reaction buffer, 1500 rpm Centrifuge for 5 min; discard supernatant.

[0039] (1) Surface staining reaction: 100 μl of reaction buffer, 2 μl of SLAMF7 antibody or isotype control antibody, and 1 μl of CD4 antibody. Incubate the above reaction tube on ice (0-4° C.) in the dark for 30 minutes. Add 1ml of reaction buffer, centrifuge at 1500 rpm for 5 minutes, repeat 3 times;

[0040] (2) Fix with 4% paraformaldehyde at room temperature for 15 minutes, and incubate for 30 minutes on ice (0-4° C.) in the dark with 1X membrane breaking agent (Permeabilization, manufacturer ebioscience).

[0041] (3) Intra...

Embodiment 3

[0044] Example 3 Effect of SLAMF7 Gene Deficiency on Cytokines Secreted by CD4+ T Cells

[0045] Splenic CD4+ T cells were sorted by magnetic beads using 4-6 week old SPF grade female C57BL / 6 wild-type (WT) mice and SLAMF7 gene deficient (SLAMF7 KO) mice, cultured in 96-well plates, and added with anti-CD3 , anti-CD28, IL-2 stimulation, cultured for three days. The expression levels of IFN-γ and TNF-a in CD4+ were detected and analyzed by flow cytometry (the method is the same as in Example 2). The concentration of anti-CD3 and CD28 is 1 ug / ml; the concentration of IL-2 is 0.25 mg / ml.

[0046] The result is as image 3 As shown in A and 3B, SLAMF7 gene deficiency significantly inhibited the expression levels of inflammatory factors IFN-γ and TNF-a produced by CD4+ T cells, indicating that the loss of SLAMF7 would inhibit the production of inflammatory factors.

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Abstract

The invention belongs to the technical field of biology. The research finds that the expression of SLAMF7 in peripheral blood CD4+T cells of tuberculosis patients is obviously increased and is closely related to the disease progress. SLAMF7 can be used as a tuberculosis diagnosis and prognosis marker, the marker can be from peripheral blood, and compared with complex procedures such as biochemical, blood coagulation and blood sample saturation degree multi-index detection, the SLAMF7 has the advantages that the pain of a patient is relieved, the wound is small, and the sensitivity is high. In addition, an SLAMF7+CD4+T cell is applied to the treatment of tuberculosis for the first time. Immunotherapy of tuberculosis can be realized by utilizing the T cell subgroup and the method, and the method has the advantages of reducing pulmonary mycobacterium tuberculosis, relieving inflammation and the like, and is suitable for comprehensive treatment of tuberculosis and clinical popularization and application.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, the application of CD4+T cells expressing SLAMF7 in the preparation of tuberculosis diagnosis or treatment reagents. Background technique [0002] Pulmonary tuberculosis is one of the more common clinical diseases, with a high incidence rate, and it is on the rise. According to the survey, the incidence rate of tuberculosis in my country ranks second in the world. The disease is mainly caused by Mycobacterium tuberculosis infection. Chronic diseases are highly contagious, and the main vector of infection is droplets. If effective treatment is not carried out in time, it will seriously affect the patient's health, and the key to treatment is early detection and prevention, which is also one of the important links to improve the prognosis. The gold standard for TB diagnosis is bacteriological examination, including acid-fast staining of sputum smear and isolation and culture of Mycob...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577A61K39/00A61P31/06
CPCG01N33/6893G01N33/6854G01N33/577A61K39/001102A61P31/06G01N2333/70503G01N2800/26G01N2800/52A61K2039/5158
Inventor 黄曦劳娟凤吴永坚唐惠姝明思奇
Owner THE FIFTH AFFILIATED HOSPITAL SUN YAT SEN UNIV
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