WO2018196011A1 - Use of mfsd2a in preparing product for diagnosing purulent meningitis - Google Patents

Abstract

A use of Mfsd2a in preparing a product for diagnosing purulent meningitis. A biological product enables detection of a change of the Mfsd2a concentration. A method employing a fluorescent marker uses Mfsd2a as a detection marker, and employs brain microvascular endothelial cells in circulation to perform an experiment in vitro and diagnose purulent meningitis, the brain microvascular endothelial cells being isolated out of human peripheral blood by means of UEA I magnetic beads. The present invention provides a novel molecule, Mfsd2a, for clinically diagnosing purulent meningitis, and employs Mfsd2a as a marker to develop a method of diagnosing purulent meningitis in vitro on the basis of a peripheral blood sample.

Description

Application of Mfsd2a in the preparation of products for diagnosis of purulent meningitis Technical field

The invention belongs to the technical field of molecular diagnosis, and in particular relates to the application of Mfsd2a in preparing a product for diagnosing purulent meningitis.

Background technique

Suppurative meningitis is a central nervous system infectious disease caused by purulent bacteria, which is mainly caused by meningeal inflammation. It is common in infants and young children. Common pathogens are Escherichia coli, Streptococcus pneumoniae, Haemophilus influenzae and meningitis. Diplococcus and the like. Purulent meningitis causes pathological changes such as blood-brain barrier function destruction and neutrophil infiltration. Neonatal encephalitis is one of the three most common diseases in newborns, causing approximately 393,000 neonatal deaths worldwide each year.

The diagnosis of purulent meningitis mainly depends on the examination of cell counts in cerebrospinal fluid, but it is sometimes difficult to diagnose purulent meningitis in clinical work because early clinical symptoms are sometimes mild (especially for infants who cannot self-report symptoms) Cerebrospinal fluid obtained by invasive examinations such as lumbar puncture is clinically limited, especially for preterm birth, fat or low birth weight. Therefore, it is urgent to explore and develop a non-invasive diagnostic method for separating blood-brain barrier cells from peripheral blood as a new biomarker, thereby effectively diagnosing central nervous system infection and reducing mortality and disability.

Summary of the invention

The present invention provides the use of Mfsd2a in the preparation of a diagnostic purulent meningitis article to compensate for the deficiencies of the prior art.

One aspect of the present invention provides a novel use of Mfsd2a, that is, in the preparation of a diagnostic septic meningitis article;

Another aspect of the present invention provides a biological product capable of detecting a change in concentration of Mfsd2a;

Another aspect of the present invention provides a method of using fluorescent labeling, using Mfsd2a as a detection target The cerebral vascular endothelial cells isolated from human peripheral blood by the UEA I magnetic bead method were used for experimental diagnosis of purulent meningitis in vitro.

The invention provides a novel molecule Mfsd2a for clinical diagnosis of purulent meningitis, and develops a method for in vitro diagnosis of purulent meningitis based on peripheral blood specimens using Mfsd2a as a marker.

DRAWINGS

Figure 1. Changes in the expression of Mfsd2a after E. coli stimulation of HBMECs (A-B).

Figure 2 Western blot analysis of changes in Mfsd2a (A-C) in blood and cerebrospinal fluid of C57BL/6J mice infected with Escherichia coli.

Figure 3. Changes in cBMECs (A-B) isolated from mouse peripheral blood using UEA magnetic beads after stimulation with Escherichia coli.

Figure 4 shows the change of Mfsd2a in cerebrospinal fluid (A-B) in children with neonatal encephalitis infected with Escherichia coli by Western blot.

Figure 5 is a technical roadmap for the separation of cBMECs from peripheral blood.

detailed description

Mfsd2a used in the present invention is a major facilitator superfamily domain-containing protein 2a (Mfsd2a).

Example 1:

In vitro, Escherichia coli, which is the main pathogen causing bacterial meningitis in children, stimulates human brain microvascular endothelial cells (HBMECs) in vitro, confirming that Escherichia coli stimulates HBMECs in vitro (0.5916± The expression of Mfsd2a was significantly increased in 0.041 compared with the control group (0.278±0.043) (P=0.0060) (Fig. 1).

In vivo, Escherichia coli was injected into the neonatal C57BL/6J mouse meningitis animal model to detect changes in Mfsd2a levels in blood and cerebrospinal fluid of mice, confirming Escherichia coli-stimulated newborn C57BL/6J mice ( 1.824±0.323 Int/mm ² ) The amount of Mfsd2a in peripheral blood was significantly higher than that in the control group (0.314±0.141 Int/mm ² ) (P=0.0055). At the same time, the Mfsd2a content in the cerebrospinal fluid of Escherichia coli-stimulated newborn C57BL/6J mice (2567.0±434.8 Int/mm ² ) was significantly higher than that of the control group (702.2±297.1 Int/mm ² ). (P = 0.0122) (Figure 2).

Escherichia coli was injected into the neonatal C57BL/6J mouse meningitis animal model, and the blood Mfsd2a content in the mice was significantly increased.

Using fluorescent labeling method, Mfsd2a was used as a molecular marker for detection. After stimulation by Escherichia coli, the circulating cerebral vascular endothelial cells (HUV) were isolated from mouse peripheral blood by UEA I magnetic bead method. Cells, cBMECs) (AB), confirmed the content of cBMECs labeled with Mfsd2a in peripheral blood of Escherichia coli-stimulated newborn C57BL/6J mice (2567.0±434.8 Int/mm ² ) (106.3±6.3/ml The number of cells of cBMECs marked with Mfsd2a was significantly higher than that of the control group (10.0 ± 1.29/ml) (P < 0.001) (Fig. 3).

Example 2:

Further preferably, in the human cerebrospinal fluid of Escherichia coli infected with Escherichia coli, the change of Mfsd2a content in human cerebrospinal fluid is detected, and the content of Mfsd2a in human cerebrospinal fluid infected with Escherichia coli (12650±2734 Int/mm ² ) and the control are confirmed. The group (3953 ± 424 Int / mm ² ) showed a significant increase in the expression of Mfsd2a (P = 0.0348)) (Fig. 4).

Western blot was used to detect the expression levels of Mfsd2a protein in HBMECs, mouse brain and CSF, and clinical cerebrospinal fluid samples. Brain tissue was suspended in a buffer containing 1% protease inhibitor (Sigma-Aldrich) lysate (Cell Signaling Technology), and then centrifuged at 12000 g for 15 minutes at 4 °C. All protein products were stored at -80 °C. 30 μL of murine CSF was diluted with 400 μL of PBS for protein electrophoresis. Clinical cerebrospinal fluid samples also need to be in a 4 ° C environment Next, centrifuge at 12000g for 15 minutes and store at -80 °C for later use. The cerebrospinal fluid protein was separated by SDS-PAGE on a 10% Tris-glycine gel. After the sample ran out of the sample well, the voltage was adjusted to 180 V, and the gel was collected after about 2 hours of running. The blade cuts the rubber block near the target strip and then transfers it to the semi-dry film transfer machine. The rubber block is placed on the transfer film (PVDF film); the two are placed in the middle of the 6-layer filter paper. The filter paper is first soaked in methanol and then bubbled into the Transfer buffer. After the transfer is completed, the film transfer device is energized, and the current is about 8 mA and transferred to the nitrocellulose membrane. At the end of the transfer, the PVDF membrane was removed and immersed in 5% skim milk powder (prepared with PBS pH 7.2) for 90 min at 4 °C. The transfer film was taken out and placed in rabbit anti-Mfsd2a (1:500) and rabbit anti-β-actin (1:10000) primary antibody, and incubated on a shaker at 4 ° C overnight. After aspirating the primary antibody, 5 times the volume of PBST was added to wash away the non-specific binding, and the shaker speed was doubled; once every 15 minutes, a total of 4 times. Wash the membrane with the horseradish peroxidase-labeled secondary antibody (rabbit anti-mouse IgG) for 1 h, shake gently for 45 min on a 4 °C shaker; aspirate the secondary antibody and add 5 volumes of PBST to wash away non-specific binding. The speed of the shaker is doubled; once every 15 minutes, a total of 4 times. The combined transfer film was taken out and placed in PBS, and then developed according to Roche's Lumi-light Western Blotting Substrate product description; the exposure time in the dark room was adjusted. Relative mass analysis of Mfsd2a was performed using a Quantity One instrument (Version 4.6.2, Bio-Rad Laboratories, Hercules, USA).

Another aspect of the present invention provides a method for fluorescent labeling, using Mfsd2a as a detection marker, and circulating brain vascular endothelial cells (cBMECs) isolated from human peripheral blood by UEA I magnetic bead method. ), used in vitro for the diagnosis of purulent meningitis (Figure 5).

Separating and counting cBMECs

The peripheral blood cBMECs were bound to Ulex europaeus I (UEA I) lectin (L-1060) coated Dynabeads magnetic beads, which were prepared and resuspended in Hanks Balanced Salt Solution (HBSS) according to the manufacturer's instructions (Invitrogen). Invitrogen Corp, Carlsbad, CA, USA ) plus 5% fetal calf serum (HBSS + 5% FCS), the final concentration was adjusted to 4xl0 ⁸ beads / ml. The UEA I-coated Dynabeads were used to affinity capture cBMECs from whole blood at 4 °C. The non-specific cells were washed to bind to the cells on the beads, and then the cells were resuspended in PBS by using a magnetic powder concentrator. The cells were released from the magnetic beads by incubation with 0.5 M fucose for 10 minutes at room temperature. The magnetic beads were washed 5 times with HBSS + 5% FBS cleaning solution. The suspension was collected, the supernatant was discarded after centrifugation, and the underlying cells were retained. Mfsd2a pre-coated Dynabeads magnetic beads were affinity captured at 4 °C. After blocking and washing, the cells were fixed with 2% paraformaldehyde and 0.25% glutaraldehyde for 30 minutes. FITC-conjugated CD146 antibodies and CD44 antibodies (BMECs-specific markers for cBMECs recognition) were then incubated for 1 hour (dark room), DAPI stains were added and cells were incubated for 30 minutes. Finally, the cells (10 ul) were transferred to a cell counter and counted under a fluorescence microscope.

The above description of the specific embodiments of the present invention is not intended to limit the scope of the invention, and those skilled in the art should understand that those skilled in the art can do without creative work on the basis of the technical solutions of the present invention. Various modifications or variations are still within the scope of the invention.

Claims (4)

A new use of Mfsd2a is in the preparation of products for the diagnosis of purulent meningitis. A biological product for use in the preparation of a diagnostic purulent meningitis product, characterized in that the biological product is capable of detecting the concentration of Mfsd2a. The biological product according to claim 2, wherein said biological product is for detecting the concentration of Mfsd2a in human cerebrospinal fluid. A method using fluorescent labeling, which uses Mfsd2a as a detection marker, and uses circulating cerebral vascular endothelial cells isolated from human peripheral blood by UEA I magnetic bead method to be used for experimental diagnosis of purulent meninges in vitro. inflammation.

Images