A marker for active pulmonary tuberculosis and its use

Abstract

The application relates to an active pulmonary tuberculosis marker and application thereof, the active pulmonary tuberculosis marker is PCBP1, the expression of the PCBP1 in peripheral blood mononuclear cells of an active pulmonary tuberculosis patient is obviously down-regulated, the down-regulated PCBP1 has certain significance for diagnosis of the active pulmonary tuberculosis patient. Moreover, the expression amount of the PCBP1 in the APTB patient is detected by using an RT-qPCR and western blot method, the disease can be diagnosed, and then the active pulmonary tuberculosis diagnosis reagent can be prepared. The application discloses that the PCBP1 can be used as the active pulmonary tuberculosis marker for the first time, and provides a new direction for clinical diagnosis of the active pulmonary tuberculosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to PCBP1 as a marker for active tuberculosis and its application. BACKGROUND

[0002] Tuberculosis (TB) is a chronic infectious disease that seriously affects human life and health, caused by Mtb infection, known as the "world's most deadly infectious disease killer", and is the thirteenth leading cause of death globally. Tuberculosis is highly contagious, and when patients expel bacteria into the air, tuberculosis spreads, and patients with pulmonary tuberculosis may infect others in the environment through coughing and sneezing. Therefore, the prevention and control of tuberculosis is very severe. Although effective drug therapy is widely used at present, with the emergence of drug-resistant (especially multidrug-resistant) strains of tuberculosis, the number of patients with poor or no response to treatment is also increasing. At present, the main basis for laboratory diagnosis of TB is sputum smear microscopy and sputum culture, but this diagnostic technique has obvious defects, such as too low sensitivity of sputum smear microscopy, easy to miss detection; sputum culture takes too long, and it is difficult to use for TB screening. As can be seen from the above, it is urgent to further study the pathogenesis of tuberculosis and seek more rapid and effective detection methods.

[0003] The test method relying on biomarkers as a means of diagnosis of tuberculosis is widely recognized and strongly supported by the World Health Organization, and is currently one of the research focuses of tuberculosis diagnosis technology. Monocytes / macrophages, as innate immune cells, can non-specifically phagocytose and kill a variety of pathogenic microorganisms, and play an important role in the body's resistance to Mtb infection. Understanding the interaction between Mtb and macrophages is of great significance for effective control of Mtb infection. At present, in the research of diagnosis of active tuberculosis, miRNA has been found to be a good biomarker. However, there has been no report on PCBP1 as a marker for active tuberculosis. SUMMARY

[0004] One of the purposes of the present application is to provide PCBP1 as a marker for active tuberculosis, which can be used for disease diagnosis in patients with active tuberculosis, in view of the deficiencies of the prior art.

[0005] The second purpose of the present application is to provide the use of PCBP1 in the preparation of a diagnostic reagent for active tuberculosis, in view of the deficiencies of the prior art.

[0006] The purposes of the present application are achieved by the following technical solutions:

[0007] A marker for active tuberculosis, the marker being PCBP1.

[0008] Further, the PCBP1 is obviously down-regulated in peripheral blood mononuclear cells of active pulmonary tuberculosis patients.

[0009] Further, the PCBP1 indicates early active pulmonary tuberculosis.

[0010] The application of the active pulmonary tuberculosis marker, the application of the PCBP1 in preparation of an active pulmonary tuberculosis diagnosis reagent.

[0011] Further, the active pulmonary tuberculosis diagnosis reagent contains a reagent capable of quantifying the expression amount of PCBP1.

[0012] Compared with the prior art, the application has the beneficial effects that:

[0013] (1) The application discloses an active pulmonary tuberculosis marker, the marker is PCBP1, the PCBP1 is obviously down-regulated in peripheral blood mononuclear cells (PBMCs) of active pulmonary tuberculosis (APTB) patients, and the obviously down-regulated PCBP1 can be used as a good biomarker for diagnosing APTB, and has important significance for early diagnosis of APTB patients.

[0014] (2) The expression amount of PCBP1 in APTB patients is detected by using RT-qPCR and western blot methods, the disease can be diagnosed, and the active pulmonary tuberculosis diagnosis reagent can be prepared, and new directions and theoretical guidance are provided for technical research on clinical diagnosis of active pulmonary tuberculosis. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is the expression difference of PCBP1 in clinical specimens PBMCs;

[0016] A: RT-qPCR is used to detect the relative expression of PCBP1 mRNA in peripheral blood PBMCs of 45 APTB patients and 40 healthy persons;

[0017] B: PCBP1 is used for diagnosing active pulmonary tuberculosis;

[0018] C: The relative expression level of PCBP1 mRNA in peripheral blood PBMCs of pulmonary tuberculosis patients before and after treatment;

[0019] D: Western blot is used to detect the protein expression of PCBP1 in peripheral blood PBMCs of 4 APTB patients and 4 healthy persons;

[0020] E: FCM is used to analyze the protein expression of peripheral blood PBMCs of 16 APTB patients and 14 healthy persons.

[0021] Figure 2PCBP1 expression in BCG infected macrophages;

[0022] A-B: PCBP1 mRNA (A) and protein (B) expression levels in THP-1 macrophages infected with different multiplicity of infection (MOI) of BCG for 24 h or uninfected;

[0023] C-E: PCBP1 mRNA (C) and protein (D-E) expression levels in THP-1 macrophages infected with BCG (MOI = 10) for different time points or uninfected. DETAILED DESCRIPTION

[0024] For the convenience of those skilled in the art, the present application will be further described below in conjunction with examples, and the content mentioned in the embodiments is not a limitation of the present application.

[0025] The embodiment of the present application provides an active tuberculosis marker, and the marker is PCBP1.

[0026] Further, the expression of PCBP1 in peripheral blood mononuclear cells of the active tuberculosis patient is obviously down-regulated.

[0027] Further, the PCBP1 indicates early active tuberculosis.

[0028] The embodiment of the present application also provides an application of an active tuberculosis marker, and the application of PCBP1 in preparation of an active tuberculosis diagnostic reagent.

[0029] Further, the active tuberculosis diagnostic reagent contains a reagent capable of quantifying the expression amount of PCBP1.

[0030] Embodiment one, collection of clinical samples, extraction of PBMCs and sorting of mononuclear cells

[0031] 1. Collection of clinical samples

[0032] Peripheral blood samples of 45 APTB patients and 40 healthy persons for physical examination were collected respectively as an experimental group and a control group, and the demographic characteristics and clinical baseline characteristics are shown in Table 1.

[0033] Table 1 Demographic characteristics and clinical baseline characteristics

[0034] Groups APTB (n=45) HC (n=40) Age (years) (12～91) (22～54) Age (Mean ± SEM) 53.58±18.63 36.33±9.33 Gender (Male / Female) 35 / 10 20 / 20 Newly / Relapsed 21 / 14 - / - Sputum smear (+ / -) 13 / 32 - / -

[0035] 2. Extraction of peripheral blood mononuclear cells (PBMCs)

[0036] The density gradient centrifugation method is used to extract human peripheral blood mononuclear cells (PBMCs), and the extraction steps of PBMCs are as follows: ① 5 mL of EDTA anticoagulant venous blood is collected, and PBMCs are separated within 2 h; ② 5 mL of EDTA anticoagulant blood is placed in a centrifuge, and after centrifugation at 20℃, 250g for 10 min, the plasma is removed; ③ 2-3 times the volume of blood cells of 1×PBS is added to dilute the blood cells, and mix well. Use a disposable pipette to slowly drop the diluted blood cells along the wall of the tube into the centrifuge tube containing the lymphocyte separation medium, keep the interface between the lymphocyte separation medium and the blood cells clear, centrifuge at 20℃, 450g for 25 min; ④ after centrifugation, the blood in the centrifuge tube is divided into four layers. The second layer is a narrow band of cloud layer mainly composed of mononuclear cells, including mononuclear cells and lymphocytes. Discard the supernatant above the mononuclear cell layer 0.5 cm, and suck the mononuclear cell layer cells into a new centrifuge tube, add 10 mL of PBS, and centrifuge at 250g for 10 min; ⑤ discard the supernatant, resuspend the cells with 5 mL of 1×PBS, and centrifuge at 20℃, 250g for 10 min. Repeat the above steps. After resuspending the cells with 1×PBS, directly detect or resuspend 1 mL of cryopreservation solution and store at -80℃ for long-term preservation.

[0037] About 5 ml of peripheral blood of each subject was collected in an EDTA anticoagulant tube, and PBMCs in fresh blood were separated by standard Ficoll (GE Healthcare, Little Chalfont, UK) density gradient centrifugation. Cell viability was determined by trypan blue staining experiment (all experiments >95%), and then PBMCs were immediately detected or stored at -80℃ for the following experiments.

[0038] Example 2, Construction of a Tuberculosis-Infected Human Monocyte Cell Line THP-1 Model

[0039] 1. Induction of human THP-1-derived macrophages

[0040] Prepare THP-1 cells, add PMA (Union Biotechnology) to make the final concentration 50 ng / mL, and induce THP-1-derived macrophages after 24 h of treatment. Wash three times with 37℃ pre-warmed PBS for subsequent experiments.

[0041] 2. Construction of a Tuberculosis-Infected Human Monocyte Cell Line THP-1 Model

[0042] BCG is used to infect THP-1-derived macrophages, and after 6 h of infection, 1×PBS is used to wash away extracellular unphagocytosed Mycobacterium tuberculosis, fresh culture medium (containing 10% fetal bovine serum 1640 culture medium) is added, and the cells are cultured for a certain period of time according to the experimental requirements.

[0043] Example 3, RT-qPCR, western blot and FCM verification of PCBP1

[0044] As shown in the following Figures 1-2 , the expression of PCBP1 mRNA in PBMCs of 45 active TB patients and 40 healthy individuals was detected by RT-qPCR method. The results showed that the expression of PCBP1 mRNA in PBMCs of TB patients was significantly lower than that of the normal control group (such as Figure 1 A). Down-regulated PCBP1 can be used as a biomarker for early diagnosis of APTB (such as Figure 1 B). Among them, 15 TB patients were treated by standardization, and the PCBP1 mRNA level in PBMCs was significantly up-regulated (such as Figure 1 C). In addition, the expression of PCBP1 protein in PBMCs of 4 active TB patients and 4 healthy individuals was detected by Western blot, and the results showed that the expression of PCBP1 protein in TB patients was decreased (such as Figure 1 D).

[0045] Further, the protein expression in PBMCs of 16 active TB patients and 14 healthy individuals was analyzed by flow cytometry, and the results showed that PCBP1 was mainly down-regulated in B cells and monocytes (such as Figure 1 E).

[0046] Subsequently, the THP-1 macrophage model infected with BCG was further used to verify that PCBP1 was down-regulated in BCG-infected macrophages (such as Figure 2 A-E).

[0047] The results of the above Example 3 show that down-regulated PCBP1 can be used as a biomarker for early diagnosis of APTB. At the same time, the THP1-derived macrophage experiment further confirms that TB infection can down-regulate PCBP1, and PCBP1 may be closely related to the occurrence and development of TB.

[0048] The above specific embodiments are further illustrations of the technical solutions and beneficial effects of the present application, and are not limitations of the embodiments. Any obvious replacement without departing from the concept of the present application is within the protection scope of the present application.

Claims

1. Use of a reagent for detecting PCBP1 gene expression in the preparation of a diagnostic reagent for active tuberculosis.

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