CN111474370B

CN111474370B - Method for detecting content of formyl peptide or formyl peptide receptor-1 ligand and application

Info

Publication number: CN111474370B
Application number: CN202010322743.6A
Authority: CN
Inventors: 李治国; 施福东; 李裕琳; 韩金蕊; 么阳
Original assignee: Tianjin Medical University General Hospital
Current assignee: Tianjin Medical University General Hospital
Priority date: 2020-04-22
Filing date: 2020-04-22
Publication date: 2021-04-06
Anticipated expiration: 2040-04-22
Also published as: CN111474370A

Abstract

The disclosure relates to methods and uses for detecting formyl peptide or formyl peptide receptor-1 ligand content. In particular, the disclosure relates to the application of a reagent for detecting FPR1 ligand in preparing a reagent or a kit for detecting acute brain injury condition or brain edema change, and a method for evaluating and predicting the brain edema change after acute brain injury such as cerebral hemorrhage. The application and the method can determine that the level of N-formyl phthalein or FPR1 ligand in peripheral blood is in positive correlation with the development of hematoma surrounding after cerebral hemorrhage, are used for evaluating and predicting the change of acute cerebral injury such as the change of the hematoma surrounding the cerebral hemorrhage after the cerebral hemorrhage and provide a biomarker basis for judging the evolution of the cerebral injury.

Description

Method for detecting content of formyl peptide or formyl peptide receptor-1 ligand and application

Technical Field

The present disclosure relates to the field of biomedicine. In particular to related reagents and technologies for detecting FPR1 ligand, such as N-formyl phthalein or FPR1 ligand content in blood, and the reagents and technologies are used for evaluating and predicting the change of the cerebral edema around acute brain injury such as hematoma after cerebral hemorrhage and providing biomarker basis for evaluating the evolution of the cerebral injury.

Background

Danger signaling molecules (DAMPs) are a class of endogenous molecules released by dead cells after tissue injury, activate immune responses after binding to cells expressing the corresponding ligands, and are key signals for activating tissue innate immune cells and mobilizing peripheral immune cells to the site of injury after tissue injury. N-formyl phthalides (NFPs) belong to mitochondria-derived DAMPs, and dead cells release the NFPs into extracellular matrix due to rupture of cell membranes and can permeate into a blood circulation system, and content change can be detected in blood by a certain technical means. NFPs are mainly recognized by formyl phthalein receptor-1 (FPR 1), and may induce immune inflammatory responses at the site of injury and throughout the body. Recent studies have shown that levels of NFPs can effectively identify the damaged state of tissue cells in vivo. In acute diseases such as systemic inflammatory response syndrome caused by acute lung injury, surgery, infection, etc., and sepsis, the levels of NFPs in plasma of patients are increasing within 24h after onset. However, no correlation has been reported between the concentration of NFPs and the volume of cerebral edema surrounding a hematoma in a patient with cerebral hemorrhage.

Ct (computed tomography), which is a common medical image examination technique, uses X-ray beams, gamma rays, ultrasonic waves, etc., the detector with extremely high sensitivity is used for scanning the sections of a certain part of a human body with a certain thickness one by one, then converting the signal into visible light, converting the visible light into an electric signal through photoelectric conversion, finally converting the electric signal into a digital signal through an analog/digital converter (analog/digital converter), arranging correlation coefficients obtained by calculating the information obtained by scanning into a digital matrix (digital/analog converter), converting each digit in the digital matrix into small blocks with different gray scales from black to white through a digital/analog converter (digital/analog converter), i.e. pixels, arranged in a matrix, i.e. constituting a CT image, for the examination of various diseases, which is characterized by fast scan time, clear image, etc. Through the use of such a reconstruction procedure of an image on a CT apparatus, it is possible to view the relationship of an organ and a lesion from different angles by reconstructing a slice image of a coronal plane and a sagittal plane.

The CT can be used for checking and diagnosing nervous system diseases, cardiovascular systems, breast diseases, abdominal organs, pelvic organs, bone and joint diseases, liver diseases and the like, wherein the diagnosis value of the CT on the nervous system diseases is high, and the CT is generally applied. Comprises the diagnosis of diseases such as craniocerebral trauma, intracranial tumors, abscesses and granulomas, traumatic hematomas, acute cerebrovascular diseases and the like. In the clinical diagnosis of acute cerebrovascular diseases such as cerebral hemorrhage, subarachnoid hemorrhage, cerebral infarction, cerebral aneurysm, arteriovenous malformation rupture hemorrhage and the like, images of the cross section, the coronary surface and other angles of the brain tissue of a patient can be clearly presented by utilizing CT scanning, and a more intuitive basis is provided for disease judgment. The cerebral hemorrhage refers to rupture and hemorrhage of cerebral parenchymal blood vessels, which is mostly caused by hypertension, cerebral arteriosclerosis, blood diseases and the like, and the bleeding parts are commonly seen in an inner sac-basal ganglia and a thalamus region, and then cerebellum and brainstem. The condition of acute hemorrhage and calcification of a patient can be clearly displayed through CT examination, and the application value in clinical diagnosis is higher.

However, at present, there is no method for evaluating the progression of cerebral edema after cerebral hemorrhage by examining the content of mitochondrial formyl peptide in blood.

Disclosure of Invention

Problems to be solved by the invention

The present disclosure provides new biomarker indications for cerebral edema progression and corresponding treatment decision assessment in patients with cerebral hemorrhage.

The present disclosure provides an application of a reagent for detecting FPR1 ligand in preparing a reagent or a kit for detecting acute brain injury condition or brain edema change and a method for detecting acute brain injury condition or brain edema change, which can determine the relationship between the edema volume around hematoma of a cerebral hemorrhage patient and the content of free mitochondrial N-formyl phthalein in blood.

Means for solving the problems

The technical scheme related to the disclosure is as follows.

(1) Use of a reagent for detecting a ligand of FPR1 in the manufacture of a reagent or kit for detecting an acute brain injury condition or a change in cerebral edema.

(2) The use of (1), wherein the FPR1 ligand is N-formyl phthalide; alternatively, the N-formyl peptide is selected from formylated peptides as set forth in SEQ ID NO: 1-3 itself or a protein comprising the sequence thereof.

(3) The use according to (1) or (2), wherein the detection is performed by:

obtaining a sample to be detected;

and detecting the content of the N-formyl phthalein or FPR1 ligand in the sample to be detected.

(4) The use of (3), wherein the sample to be tested is derived from plasma.

(5) A kit for detecting acute brain injury conditions or changes in brain edema, wherein the kit comprises reagents for detecting FPR1 ligand.

(6) The kit according to (5), wherein the FPR1 ligand is N-formyl peptide; alternatively, the N-formyl peptide is selected from formylated peptides as set forth in SEQ ID NO: 1-3 itself or a protein comprising the sequence thereof.

(7) The kit according to (5) or (6), wherein the reagent is selected from a polyclonal antibody or a monoclonal antibody.

(8) A method of detecting an acute brain injury condition or a change in brain edema, the method comprising the steps of:

obtaining a sample to be detected;

detecting the content of FPR1 ligand in the sample to be detected;

and judging the acute brain injury condition or the change of the cerebral edema of the testee by the content change of the FPR1 ligand.

(9) The method of (8), wherein said FPR1 ligand is an N-formyl peptide; alternatively, the N-formyl peptide is selected from formylated peptides as set forth in SEQ ID NO: 1-3 itself or a protein comprising the sequence thereof.

ADVANTAGEOUS EFFECTS OF INVENTION

In one aspect, the method provided by the present disclosure can accurately record the volume peak of edema around the hematoma at different time CT scan points.

In another embodiment, the present disclosure finds that the amount of FPR1 ligand in plasma is associated with patients with acute brain injury or cerebral hemorrhage.

In another embodiment, the level of FPR1 ligand in the patient's peripheral blood is found to be positively correlated with the progression of edema around the hematoma following cerebral hemorrhage by a best fit curve determined by regression analysis.

In a specific embodiment, the FPR1 ligand is a mitochondrial N-formyl peptide.

Drawings

Part a of figure 1 shows a schematic representation of the results of plasma N-formyl phthalein content (FPR1 ligand) ELISA quantification. Including 6h (n-50), 24h (n-80), 72h (n-36) and healthy control (n-47) after ICH patients develop disease. P < 0.01.

Section B of FIG. 1 shows the linear regression analysis of plasma N-formyl phthalein content 24h after onset of ICH patients versus PHE (PHE/hematoma). n is 80. Data are presented as mean ± standard deviation.

Detailed Description

Definition of

In the claims and/or the description of the present disclosure, the words "a" or "an" may mean "one," but may also mean "one or more," at least one, "and" one or more than one.

As used in the claims and specification, the terms "comprising," "having," "including," or "containing" are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Also, the terms "comprising," "having," "including," or "containing" are intended to be inclusive and mean that there may be additional, unrecited elements or method steps.

Although the disclosure supports the definition of the term "or" as merely an alternative as well as "and/or," the term "or" in the claims means "and/or" unless expressly indicated to be merely an alternative or a mutual exclusion between alternatives.

In the present disclosure, "multislice helical CT" (MSCT) refers to an imaging system capable of simultaneously obtaining more than 4 slices when an X-ray tube rotates 360 degrees during CT scanning. The multilayer spiral CT scanning principle is that an X-ray bulb tube detector rotates around a human body and is matched with the uniform motion of an examination bed, the detector receives X-rays penetrating through the human body, the X-rays are converted into electric signals, and image reconstruction processing is carried out through a data acquisition system. In the present disclosure, the multislice CT is referred to as a multislice CT dataset. Illustratively, the multi-slice helical CT dataset in the present disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24 slices, each 5mm thick.

By "peri-hematoma edema (PHE) volume in this disclosure is meant the volume that exists between the volume of the injury region and the volume of the hematoma. Exemplarily, PHE volume is the lesion region volume-hematoma volume.

"DAMPs" in this disclosure refers to injury-associated molecular patterns (DAMPs), which refer to endogenous molecules released by the death of the body's own cells, i.e., endogenous danger signals, derived from immune cells activated by damaged or necrotic tissue.

The present disclosure relates to the use of "N-formyl phthalides (NFPs)" in place of "N-formylated polypeptides", which are important chemotactic compounds that induce leukocyte chemotaxis by binding to their receptors, thereby enriching physiological functions at sites of inflammation and tissue injury.

In one embodiment of the disclosure, the N-formylated polypeptides detected are derived from NADH dehydrogenase (NCBI gene ID: 4535, 4536, 4537, 4538, 4540 and 4541) and human mitochondrially encoded protein cytochrome C oxidase (NCBI gene ID: 9997) itself and its protein degradants, respectively.

In one embodiment of the disclosure, the N-formylated polypeptide detected may be selected from formylated SEQ ID NOs: 1-3 or a protein comprising the sequence thereof:

MLKLIV (SEQ ID NO: 1); MMYALF (SEQ ID NO: 2) or MFADRW (SEQ ID NO: 3).

That is, in a particular embodiment, the polypeptide detected by the present disclosure is fMLKLIV, fmyalf, or fmmaadrw itself or a protein comprising the sequence thereof.

The 'Formyl Peptide Receptors (FPRs)' in the disclosure belong to the G-protein-coupled receptor (GPCR) superfamily, regulate and control a plurality of important physiological functions, are widely involved in the occurrence and development of various inflammatory diseases, and comprise three formyl peptide receptors, namely FPR1, FPR2 and FPR3 in a human body. Illustratively, the FPRs in this disclosure are FPR 1.

The Molecular biological methods used in the present disclosure can be referred to the corresponding methods described in publications such as "Current Protocols in Molecular Biology, Wiley publication", "Molecular Cloning, A Laboratory Manual, Cold spring harbor Laboratory publication", and the like.

Examples

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

All reagents used in the examples were commercially available unless otherwise noted.

Example 1: CT imaging and volume assessment of PHE and hematoma

Patients with cerebral hemorrhage were enrolled and subjected to cranial CT tomography using a fourth generation CT scanner (Somatom 64 or Somatom AS +, Siemens Healthcare, Erlangen) with each scan containing a multi-slice helical CT dataset (1-24 slices, 5mm thick). The lesion area and the hematoma are delineated on each slice of the cranial CT image, the lesion area volume and the hematoma volume are calculated using MRIcron software (christ roots's MRIcron, version 4AUG2014), and the peri-hematoma edema (PHE) volume is calculated using the formula: PHE volume-lesion area volume-hematoma volume. For more accurate assessment of PHE volume, we counted 24-hour CT scan images of cerebral hemorrhage and recorded PHE volume and hematoma volume of cerebral hemorrhage patients. We performed blood collection 24 hours after cerebral hemorrhage on these cerebral hemorrhage patients (see example 2).

Example 2: plasma mitochondrion N-formyl phthalein ELISA quantitative detection

Plasma mitochondrial N-formyl phthalein content was determined using a human methionine (fMET) ELISA kit according to the finishing instructions. Plasma samples were diluted 1:5 with sample diluent in the kit before addition to the ELISA plate.

2.1 sample Collection and storage

Plasma-sample is collected in a collecting pipe containing anticoagulant EDTA or heparin, and the sample is centrifuged for 1000g and 15min within 30min after being collected and is at 2-8 ℃. Supernatants were collected and tested immediately, or supernatant samples were stored in batches at-20 ℃ or-80 ℃ for later use. The samples were protected from repeated freeze thawing. Note that: the sample should be sufficiently centrifuged to avoid hemolysis or the presence of particles.

2.2 preparation work

All reagents were left at room temperature before use. If crystals precipitate in the concentrated buffer, it can be gently heated until completely dissolved.

Washing liquid- -diluted 1:20(48T)/1:30(96T) with distilled or deionized water in proportion

Standard-150 ul of standard dilutions were added sequentially to the tubes and equal amounts of standard were added to the tubes to produce a 2-fold dilution series (below). The previous liquid should be well mixed before each transfer. Undiluted standards were used as the highest standard.

Note that: if the sample yields a value above the maximum standard, the sample is diluted with a sample diluent and retested.

2.3 detection step

2.3.1 prepare all reagents before starting the assay. It is recommended that all standards and samples be added to the microplate in duplicate.

2.3.2 addition of standards: and setting a concentration gradient of a standard substance and detecting a standard curve. To the standard well, 50. mu.l of the diluted standard was added.

2.3.3 addition of sample: add 40. mu.l of sample dilution to the sample well. Then 10. mu.l of the sample was added and the blank wells were not loaded with any substance.

2.3.4 Elisa plates were sealed with plate lids and incubated at 37 ℃ for 45 minutes.

2.3.5 absorbing and removing liquid in the hole, adding washing liquid, repeating the absorbing and removing-washing process for 4 times, and washing for 5 times, wherein each time lasts for 1-3 minutes. And (3) washing process: the wells are filled with wash solution (250. mu.l) using a pipette or other device, and complete removal of the liquid from the wells after each wash is critical for good detection results. In the last 1 wash, the well plate was inverted after the liquid was aspirated off with a pipette and the liquid was blotted dry with a clean paper towel.

2.3.6 except for blank wells, 50. mu.l of HRP-coated detection antibody was added to each well. When detected, the antibody specifically binds to the polypeptide fmkliv, fmyalf or fMFADRW itself or a protein comprising the sequence thereof.

2.3.7 cover with plate lid and incubate for 30min at 37 ℃.

2.3.8 the blotting/washing process of step 2.3.5 was repeated for a total of 5 washes.

2.3.9 mu.l of the color developing solution A and 50. mu.l of the color developing solution B were added to each well. Mix gently and incubate at 37 ℃ for 15 minutes. And (4) avoiding light.

2.3.10 mu.l of stop buffer was added to each well. The color of the liquid in the well should change from blue to yellow. If the liquid in the well is green or the color change is not uniform, the Elisa plate can be gently tapped to ensure adequate mixing.

2.3.11 read the absorbance at 450nm (Optical sensitivity, O.D.) of the liquid in each well of the Elisa plate within 15 minutes.

2.4 data analysis

And (4) calculating a result: the average of each standard, control and sample well and its duplicate wells was calculated separately and the average zero standard o.d value was subtracted. The data was reduced using computer software with a four parameter logic fitting method to create a standard curve. Alternatively, the absorbance of each standard is plotted on the X-axis, the concentration of each standard is plotted on the Y-axis, and the optimal standard curve is constructed from the points on the graph. The data were linearized by plotting the log of the human formyl peptide concentration versus the log of the o.d. value and the best fit line was determined by regression analysis. This process will produce a suitable but less accurate fit of the data. The sample has been diluted and the concentration read from the standard curve needs to be multiplied by the dilution factor.

2.5 results

2.5.1 detection of mitochondrial-derived ligand-FPR 1 in patients with cerebral hemorrhage and its relationship to changes in cerebral edema

Cell death following tissue injury passively releases intracellular DAMPs into the extracellular space and into the peripheral circulation, thereby stimulating innate and peripheral immune systems within the tissue, causing an inflammatory response at the site of injury. After acute brain injury, dead brain cells release their endogenous molecules into the blood circulation (e.g., mitochondrial DNA, S-100 β). Since mitochondria contain a plurality of proteins containing formyl peptide sequences, mitochondrial proteins are released and degraded by in vivo enzymes into small fragments such as fmkliv, fmyalf or fmmaadrw.

We examined the level of mitochondria-derived N-formyl phthalein (an endogenous ligand to FPR1, wherein the N-formyl peptide comprises the polypeptide fmkliv, fmyalf or fMFADRW itself or a protein comprising the sequence thereof, in the plasma of patients with cerebral hemorrhage.

We found that N-formyl peptides (including the polypeptide fmkliv, fmmmyalf or fMFADRW itself or proteins comprising the sequence thereof) were maintained at baseline levels 6h after onset, followed by a peak at 24h and maintained until 72h post-hemorrhage (part a in fig. 1). In addition, we found that the level of mitochondrial N-formyl phthalein in the peripheral blood of patients was positively correlated with the progression of edema around the hematoma 24h after cerebral hemorrhage (r-0.4883, p <0.001) (part B in fig. 1).

The experimental results prove that the detection method and the detection target point can be used for evaluating and predicting the evolution of acute brain injury, such as the change of cerebral edema around hematoma after cerebral hemorrhage.

The above examples of the present disclosure are merely examples provided for clearly illustrating the present disclosure and are not intended to limit the embodiments of the present disclosure. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present disclosure should be included in the protection scope of the claims of the present disclosure.

Sequence listing

<110> general Hospital of Tianjin medical university

<120> method for detecting content of formyl peptide or formyl peptide receptor-1 ligand and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6

<212> PRT

<213> Homo sapiens

<400> 1

Met Leu Lys Leu Ile Val

1 5

<210> 2

<211> 6

<212> PRT

<213> Homo sapiens

<400> 2

Met Met Tyr Ala Leu Phe

1 5

<210> 3

<211> 6

<212> PRT

<213> Homo sapiens

<400> 3

Met Phe Ala Asp Arg Trp

1 5

Claims

1. The application of the reagent for detecting the FPR1 ligand in preparing a reagent or a kit for detecting the change of the cerebral edema around the hematoma after the cerebral hemorrhage;

the FPR1 ligand is a mitochondrial N-formyl peptide selected from formylated peptides as set forth in SEQ ID NO: 1-3 itself;

the mitochondrial N-formyl peptide is derived from plasma.

2. Use according to claim 1, wherein the detection is performed by:

obtaining a sample to be detected;

and detecting the content of the FPR1 ligand in the sample to be detected.