Novel protein biomarker for determining severity of muscle injury, and obtaining method therefor and use thereof

Abstract

A novel protein biomarker for determining the severity of muscle injury, and an obtaining method therefor and a use thereof. The novel protein biomarker comprises at least one of thirty-six proteins. The obtaining method for the novel protein biomarker specifically comprises: establishing experimental rat models with different severities of muscle injury; collecting the plasma of rats in different groups for proteomic detection and performing differential comparison to obtain candidate protein biomarkers; and using the blood of clinical patients with different severities of muscle injury to validate the candidate protein biomarkers and determining diagnostic reference ranges of the candidate protein biomarkers. The use of the proteomic biomarker is: using the novel protein biomarker to make a kit or using the novel protein biomarker for detection to determine the severity of muscle injury by means of mass spectrometry or any other methods. A new basis is provided for the determination of the severity of muscle injury, facilitating quantification of the severity of muscle injury.

Description

Novel protein markers for judging muscle injury degree and obtaining method and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of clinical medicine, more particularly to a group of novel protein markers for judging muscle injury degree. BACKGROUND

[0002] Orthopedic trauma is often accompanied by muscle soft tissue injury, and severe skeletal muscle injury usually leads to incomplete regeneration, resulting in dysfunction. In clinical practice, understanding the degree of skeletal muscle injury in patients with limb trauma is crucial for choosing treatment methods and evaluating prognosis.

[0003] At present, there is a lack of specific indicators for evaluating the severity of mechanical skeletal muscle injury. The existing classic blood protein markers related to muscle tissue decomposition and fiber injury include creatine kinase, lactate dehydrogenase, myoglobin, aldolase and aspartate aminotransferase, among which creatine kinase is the most commonly used serum marker for muscle injury. However, these established enzymes lack high diagnostic reliability, and they cannot effectively distinguish the severity of injury.

[0004] Therefore, it is urgent to provide a group of novel protein markers for judging muscle injury degree. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a group of novel protein markers for judging muscle injury degree and obtaining method and application thereof to solve the problems in the background.

[0006] To solve the above technical problems, the technical solutions adopted by the present application are as follows.

[0007] The novel protein markers for judging muscle injury degree are ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; At least one of them.

[0008] This invention also provides a method for obtaining a novel protein biomarker for determining the degree of muscle damage, comprising the following steps:

[0009] S1. Establish experimental rat models with different degrees of muscle damage: Mechanical compression injuries of different degrees were induced in the lower legs of rats using a self-made airbag cuff compression device;

[0010] S2. Three hours after the rats were injured, plasma was collected from the rat model by cardiac puncture. The expression profile of rat plasma proteins was detected by LC-MS / MS-4D-DIA quantitative proteomics technology. Candidate protein biomarkers were obtained through differential analysis.

[0011] S3. The concentrations of 36 candidate protein biomarkers in the blood of patients with different degrees of muscle injury were detected using the ELISA method.

[0012] To further optimize the technical solution, the experimental rat model in step S1 includes a control group, a mild injury group, and a severe injury group. The procedure for the control group rats in step S1 is as follows: anesthetize the rats and fix their right hind leg in a self-made, pressure-free airbag cuff. The procedure for the mild injury group rats is as follows: anesthetize the rats and fix their right hind leg in the self-made airbag cuff, applying pressure at 300 mmHg for 2 hours. The procedure for the severe injury group rats is as follows: anesthetize the rats and fix their right hind leg in the self-made airbag cuff, applying pressure at 300 mmHg for 6 hours. The tibialis anterior muscle of the three groups of rats is harvested 3 hours and 3 days after injury for hematoxylin and eosin staining to assess the degree of injury. Masson's trichrome staining is performed 28 days after injury to assess the degree of muscle fibrosis.

[0013] The present invention also provides the application of novel protein biomarkers for assessing the degree of muscle damage, using the novel protein biomarkers to prepare kits or for detection by mass spectrometry or any other method to assess the degree of muscle damage in subjects.

[0014] With the above technical scheme, the technical progress achieved by the present application is as follows.

[0015] The group of novel protein markers provided by the present application can be used to judge the muscle injury degree, help clinicians to formulate individualized treatment plans for patients, and improve the treatment effect and life quality of patients. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a schematic diagram of the method for establishing the experimental rat model with different muscle injury degrees in the present application;

[0017] Figure 2 is a typical hematoxylin and eosin (H&E) staining image of the tibialis anterior muscle of the control group, the mild injury group and the severe injury group at 3 hours and 3 days after injury in the present application;

[0018] Figure 3 is an analysis of the muscle injury severity based on the hematoxylin and eosin (H&E) staining results of the control group, the mild injury group and the severe injury group in the present application;

[0019] Figure 4 is a typical Masson trichrome staining image of the tibialis anterior muscle of the control group, the mild injury group and the severe injury group at 28 days after injury in the present application;

[0020] Figure 5 is an analysis of the fibrosis area based on the Masson trichrome staining of the control group, the mild injury group and the severe injury group at 28 days after injury in the present application. DETAILED DESCRIPTION

[0021] The present application will be further described in detail below with reference to specific embodiments.

[0022] A group of novel protein markers for judging the muscle injury degree, the novel protein markers are (fast skeletal muscle type troponin T3); (alpha-1, 4-glucan phosphorylase); ; (four half LIM domains 1); (PDZ and LIM domain 3); (phosphoglucomutase 1); (adenosine succinate synthetase isozyme 1); (hormone-sensitive lipase); (DNA topoisomerase I); (LIM and half-cysteine-rich domain 1); (glucose-6-phosphate isomerase); ; (triose phosphate isomerase); (chromosome condensation regulatory factor 1); (cytoplasmic glycerol-3-phosphate dehydrogenase); (Nicotinamide phosphoribosyltransferase) (Malate dehydrogenase) (Phosphoglycerate kinase 1) (Sugar phosphatase phosphatase)

[0023] (Fumarylacetoacetate hydrolase domain-containing protein 2A) (Exportin-2) (DNAJ heat shock protein family (Hsp40) member B4) (Elongation factor 1-gamma) (Phosphatidylethanolamine / phosphatidylcholine phosphatase 1) (Adenosine succinylate synthase) (5'-3' Exonuclease) (Small nuclear ribonucleoprotein D2) (Small nuclear ribonucleoprotein F) (Lactoylglutathione lyase) (Adenosine homocysteinase) (DNA (cytosine-5) methyltransferase) (Eukaryotic translation initiation factor 3 subunit B) (Melanocyte proliferation gene 1 exonuclease) (Small nuclear ribonucleoprotein D3) (Valine-tRNA ligase) (Alcohol dehydrogenase class-3) (Tumor necrosis factor receptor superfamily member 11B) (At least one of BPI fold-containing family A member 2).

[0024] A method for obtaining a novel protein marker for judging the degree of muscle injury, in combination with FIGS. 1-5, includes the following steps:

[0025] S1, Establishing experimental rat models with different degrees of muscle injury: As shown in Figure 1, different degrees of mechanical crush injury of rat calf were manufactured by self-made pneumatic cuff pressure device. The operation of the control group rats was as follows: the right hind leg of the rat was fixed in the self-made pneumatic cuff pressure device without pressure after anesthesia; the operation of the mild injury group rats was as follows: the right hind leg of the rat was fixed in the self-made pneumatic cuff pressure device and pressurized at 300 mmHg for 2 hours after anesthesia; the operation of the severe injury group rats was as follows: the right hind leg of the rat was fixed in the self-made pneumatic cuff pressure device and pressurized at 300 mmHg for 6 hours after anesthesia.

[0026] At 3 hours and 3 days after injury, the tibialis anterior muscle (TA) of the three groups of rats was taken for hematoxylin and eosin (H&E) staining to evaluate the degree of injury. The results showed that: H&E staining showed that in the control group, muscle fibers were normal and no inflammatory cells were observed; in the mild injury group, muscle fibers were slightly damaged, moderate interstitial edema, and accompanied by inflammatory cell infiltration; in the severe injury group, muscle fibers showed severe damage, accompanied by significant infiltration of inflammatory cells, and large areas of necrotic muscle fibers. The proportion of damaged fibers in the severe injury group was significantly higher than that in the mild injury group and the control group (as shown in Figure 3). At 28 days after injury, the muscle tissue of the mild injury group basically recovered to normal, while the severe injury group showed extensive fibrosis (as shown in Figures 4 and 5).

[0027] S2, At 3 hours after injury, the plasma of the control group, the mild injury group and the severe injury group rats was collected by heart puncture, and the LC-MS / MS-4D-DIA quantitative proteomics technology was used to detect the plasma protein expression profile of the three groups of rats, and a total of 2,797 proteins with at least one unique peptide were identified, FDR≤1%. Through differential analysis, when the P value is less than 0.05, the fold change is more than 1.5 is considered to be significantly up-regulated, and less than 1 / 1.5 is considered to be significantly down-regulated. Compared with the control group, 237 proteins (198 up-regulated and 39 down-regulated) in the mild injury group showed significant differences, and 541 proteins (412 up-regulated and 129 down-regulated) in the severe injury group showed significant differences. Compared with the mild injury group, 287 proteins (204 up-regulated and 83 down-regulated) in the severe injury group showed significant differences. Cross protein analysis was performed to find candidate new protein markers that can distinguish different degrees of muscle injury, and the relative quantification values of 34 proteins increased with the increase of the degree of muscle injury, while the relative quantification values of 2 proteins decreased with the increase of the degree of muscle injury.

[0028] S3, the concentration of 36 candidate protein markers in the plasma of patients with different degrees of muscle injury was detected by ELISA method. The test results are shown in Table 1 below:

[0029] Table 1

[0030] [Corrected according to Rule 26 15.08.2025]

[0031] The application of the new protein marker for judging the degree of muscle injury is: using the new protein marker to make a kit for detecting to judge the degree of muscle injury of the subject, or detecting the concentration of the new protein marker in the blood by mass spectrometry method or any other method to judge the degree of muscle injury of the subject.

Claims

1. A novel protein marker for judging the degree of muscle injury, characterized by: The novel protein marker is at least one of ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ​ 2. A method for obtaining a novel protein marker for judging the degree of muscle injury according to claim 1, characterized by, Specifically comprising the following steps: S1, establishing experimental rat models with different degrees of muscle injury: manufacturing different degrees of mechanical crush injury of rat lower leg by self-made air bag cuff pressure device; S2, collecting the plasma of rats in the experimental rat model by heart puncture after 3 hours of injury, detecting the protein expression profile of rats by LC-MS / MS-4D-DIA quantitative proteomics technology, and obtaining candidate protein markers through differential analysis; S3, detecting the concentration of 36 candidate protein markers in the blood of patients with different degrees of muscle injury in clinic by ELISA method.

3. The method of claim 2, wherein the method is characterized by: The experimental rat model in step S1 includes a control group, a mild injury group and a severe injury group; the operation of the control group rats in step S1 is to anesthetize and fix the right hind leg of the rat in the self-made air bag cuff pressure device without pressure; the operation of the mild injury group rats is to anesthetize and fix the right hind leg of the rat in the self-made air bag cuff pressure device under 300 mmHg pressure for 2 hours; the operation of the severe injury group rats is to anesthetize and fix the right hind leg of the rat in the self-made air bag cuff pressure device under 300 mmHg pressure for 6 hours; the tibialis anterior muscle of the rats in the three groups is obtained respectively at 3 hours and 3 days after injury for hematoxylin and eosin staining to evaluate the injury degree; Masson trichrome staining is performed at 28 days after injury to evaluate the degree of muscle fibrosis.

4. Use of novel protein markers for judging the extent of muscle damage, characterized in that: The new protein marker is used to make a kit or is detected by mass spectrometry to judge the degree of muscle injury of the subject.

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