Novel protein marker for judging muscle injury degree as well as obtaining method and application of novel protein marker

Abstract

The invention discloses a novel protein marker for judging muscle injury degree as well as an obtaining method and application of the novel protein marker. The novel protein marker comprises at least one of 36 proteins; the invention discloses a novel protein marker obtaining method. The method specifically comprises the following steps: establishing experimental rat models with different muscle injury degrees, collecting plasma of each group of rats, performing proteomics detection, comparing differences to obtain candidate protein markers, verifying the candidate protein markers by using blood of patients with different clinical muscle injury degrees, and determining a diagnosis reference interval range of the candidate protein markers; according to the application of the proteomic marker, the novel protein marker is used for preparing a kit or detecting and judging the muscle injury degree through a mass spectrum method or any other method. According to the method, a new basis is provided for judging the muscle injury degree, and the muscle injury degree can be quantified conveniently.

Description

Technical Field

[0001] The present invention relates to the technical field of clinical medicine, and more specifically to a group of novel protein markers for judging the degree of muscle damage. Background Art

[0002] Orthopedic trauma is often accompanied by muscle and soft tissue injuries, and severe skeletal muscle injury usually leads to incomplete regeneration, resulting in functional impairment. In clinical practice, understanding the extent of skeletal muscle injury in patients with limb trauma is crucial for selecting treatment methods and assessing prognosis.

[0003] Currently, there is a lack of specific indicators for assessing the severity of mechanical skeletal muscle injury. Existing classic blood protein markers associated with muscle tissue breakdown and fiber damage 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 are also unable to effectively distinguish the severity of injury.

[0004] Therefore, there is an urgent need to provide a group of new protein markers for determining the extent of muscle damage. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a group of novel protein markers for judging the degree of muscle damage and a method for obtaining and applying the same, so as to solve the problems in the background technology.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0007] A novel protein marker for judging the degree of muscle damage, the novel protein marker being Troponin T3, fast skeletal type; Alpha-1,4glucanphosphorylase; Four and ahalfLIM domains1; PDZ and LIM domain 3; Phosphoglucomutase 1; Adenylosuccinate synthetase isozyme1; Hormone-sensitive lipase; DNAtopoisomerase I; LIM and cysteine-rich domains1; Glucose-6-phosphate isomerase; Triosephosphate isomerase; Regulator of chromosome condensation 1; Glycerol-3-phosphate dehydrogenase [NAD (+)], cytoplasmic; Nicotinamide phosphoribosyltransferase; Malate dehydrogenase; Phosphoglycerate kinase 1; Sugarphosphate phosphatase; Fumarylacetoacetatehydrolase domain containing 2A; Exportin-2; DnaJ heat shockprotein family(Hsp40)member B4;

[0008] At least one of Adenosylhomocysteinase; DNA(cytosine-5)-methyltransferase; Eukaryotictranslation initiation factor 3subunit B; MYG1 exonuclease; Smallnuclearribonucleoprotein Sm D3; Valine--tRNAligase; Alcohol dehydrogenaseclass-3; Tumornecrosis factor receptor superfamily member 11B; BPI fold-containing familyAmember 2.

[0009] The present invention also provides a method for obtaining a novel protein marker for determining the degree of muscle damage, comprising the following steps:

[0010] S1. Establishing experimental rat models with different degrees of muscle injury: using a homemade air bag cuff pressurization device to create different degrees of mechanical compression injury in the rat calf;

[0011] S2. 3 hours after the rats were injured, the plasma of the rats in the experimental rat model was collected by cardiac puncture, and the expression profile of rat plasma proteins was detected by LC-MS / MS-4D-DIA quantitative proteomics technology. The candidate protein markers were obtained by differential analysis.

[0012] S3. The ELISA method was used to detect the concentrations of 36 candidate protein markers in the blood of patients with different degrees of muscle injury in clinical practice.

[0013] The technical scheme is further optimized, and the experimental rat model in step S1 includes a control group, a mild injury group and a severe injury group; the operation on the control group rats in step S1 is: anesthetizing and fixing the right hind calf of the rat in a pressure-free homemade air bag cuff pressurization device; the operation on the mild injury group rats is: anesthetizing and fixing the right hind calf of the rat in a homemade air bag cuff pressurization device and pressurizing at 300 mmHg for 2 hours; the operation on the severe injury group rats is: anesthetizing and fixing the right hind calf of the rat in a homemade air bag cuff pressurization device and pressurizing at 300 mmHg for 6 hours; the tibialis anterior muscles of the three groups of rats are obtained 3 hours and 3 days after injury, respectively, for hematoxylin and eosin staining to evaluate the degree of injury; Masson trichrome staining is performed 28 days after injury to evaluate the degree of muscle fibrosis.

[0014] The present invention also provides an application of a novel protein marker for determining the degree of muscle damage, and a kit is prepared using the novel protein marker or used for detection by mass spectrometry or any other method to determine the degree of muscle damage in a subject.

[0015] Due to the adoption of the above technical scheme, the technical progress achieved by the present invention is as follows.

[0016] The present invention provides a group of novel protein markers that can be used to determine the degree of muscle damage, which helps clinicians develop individualized treatment plans for patients and improve the treatment effects and quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the method for establishing experimental rat models with different degrees of muscle damage in the present invention;

[0018] Figure 2 Typical hematoxylin and eosin (H&E) staining images of the tibialis anterior muscle in the control group, mild injury group and severe injury group 3 hours and 3 days after injury in the present invention;

[0019] Figure 3 Analysis of the severity of muscle injury based on hematoxylin and eosin (H&E) staining results in the control group, mild injury group and severe injury group in the present invention;

[0020] Figure 4 The typical Masson trichrome staining images of the tibialis anterior muscle in the control group, mild injury group and severe injury group 28 days after injury in the present invention;

[0021] Figure 5 This is the fibrosis area analysis based on Masson's trichrome staining in the control group, mild injury group and severe injury group 28 days after injury in the present invention. DETAILED DESCRIPTION

[0022] The present invention will be further described in detail below with reference to specific embodiments.

[0023] A group of novel protein markers for judging the degree of muscle damage, wherein the novel protein markers are Troponin T3, fast skeletal type; Alpha-1,4glucan phosphorylase; Four and ahalf LIM domains 1; PDZ and LIMdomain 3; Phosphoglucomutase 1; Adenylosuccinate synthetase isozyme 1; Hormone-sensitive lipase; DNA topoisomerase I; LIM and cysteine-rich domains 1; Glucose-6-phosphate isomerase; Triosephosphate isomerase; Regulator of chromosome condensation 1; Glycerol-3-phosphate dehydrogenase [NAD(+)], cytoplasmic; Nicotinamidephosphoribosyltransferase; Malate dehydrogenase; Phosphoglycerate kinase 1; Sugarphosphate phosphatase; Fumarylacetoacetate hydrolase domain containing 2A; Exportin-2; DnaJ heat shock protein family (Hsp40) member B4; Elongation factor 1-gamma; Phosphoethanolamine / phosphocholine phosphatase 1 (phosphatidylethanolamine / phosphatidylcholine phosphatase 1);Adenylosuccinate lyase; 5'-3' exoribonuclease; Small nuclearribonucleoprotein Sm D2; Sm protein F; Lactoylglutathione lyase; Adenosylhomocysteinase; DNA(cytosine-5)-methyltransferase; Eukaryotic translation initiation factor 3 subunit B; MYG1 exonuclease; Small nuclearribonucleoprotein Sm D3; Valine--tRNA At least one of valine-tRNA ligase, alcohol dehydrogenase class-3, tumor necrosis factor receptor superfamily member 11B, and BPI fold-containing family A member 2.

[0024] A method for obtaining a new protein marker for judging the degree of muscle damage, combined with Figure 1-5 As shown, the following steps are included:

[0025] S1. Establishment of experimental rat models with different degrees of muscle injury: Figure 1 As shown, mechanical compression injuries of different degrees were made in the rat calf by a homemade air bag cuff pressurization device. The operation of the control group rats was as follows: the right hind calf of the rats was fixed in a homemade air bag cuff pressurization device without pressure; the operation of the mild injury group rats was as follows: the right hind calf of the rats was fixed in a homemade air bag cuff pressurization device under 300mmHg for 2h; the operation of the severe injury group rats was as follows: the right hind calf of the rats was fixed in a homemade air bag cuff pressurization device under 300mmHg for 6h.

[0026] Tibialis anterior (TA) muscles of the three groups of rats were obtained 3 hours and 3 days after injury for hematoxylin and eosin (H&E) staining to assess 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 found; in the mild injury group, muscle fibers were slightly damaged, with moderate interstitial edema, 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 (e.g. Figure 3 28 days after injury, the muscle tissue of the mild injury group basically returned to normal, while the severe injury group showed extensive fibrosis (e.g. Figure 4 , 5 ).

[0027] S2. Three hours after the rats were injured, the plasma of the control group, the mild injury group, and the severe injury group was collected by cardiac puncture. The plasma protein expression profiles of the three groups of rats were detected by LC-MS / MS-4D-DIA quantitative proteomics technology. A total of 2,797 proteins with at least one unique peptide were identified, with an FDR ≤ 1%. Through differential analysis, when the P value was less than 0.05, the fold change of more than 1.5 was considered to be significantly upregulated, and less than 1 / 1.5 was considered to be significantly downregulated. Compared with the control group, 237 proteins (198 upregulated and 39 downregulated) showed significant differences in the mild injury group, and 541 proteins (412 upregulated and 129 downregulated) showed significant differences in the severe injury group compared with the control group. Compared with the mild injury group, 287 proteins (204 upregulated and 83 downregulated) showed significant differences in the severe injury group. Cross-protein analysis was performed to identify candidate novel protein markers that could distinguish different degrees of muscle damage. The relative quantitative values ​​of 34 proteins increased with the increase of the degree of muscle damage, while the relative quantitative values ​​of 2 proteins decreased with the increase of the degree of muscle damage.

[0028] S3. ELISA method was used to detect the concentration of 36 candidate protein markers in the plasma of patients with different degrees of muscle damage in clinical practice. The test results were prepared as the following Table 1:

[0029] Table 1

[0030]

[0031]

[0032] The application of the new protein marker for determining the degree of muscle damage is: using the new protein marker to prepare a kit for detecting and determining the degree of muscle damage in a subject, or detecting the concentration of the new protein marker in the blood by mass spectrometry or any other method to determine the degree of muscle damage in the subject.

Claims

1. A new protein marker for determining the degree of muscle damage, characterized by: The novel protein markers are Troponin T3, fast skeletal type; Alpha-1,4 glucan phosphorylase; Four and a half LIM domains 1; PDZ and LIM domain 3; Phosphoglucomutase 1; Adenylosuccinate synthetase isozyme 1; Hormone-sensitive lipase; DNA topoisomerase I; LIM and cysteine-rich domains 1; Glucose-6-phosphate isomerase; Triosephosphate isomerase; Regulator of chromosome condensation 1; Glycerol-3-phosphate dehydrogenase [NAD(+)], cytoplasmic; Nicotinamide phosphoribosyltransferase; Malate dehydrogenase; Phosphoglycerate kinase 1; Sugar phosphate phosphatase; Fumarylacetoacetate hydrolase domain containing 2A; Exportin-2; DnaJ heat shock protein family (Hsp40) member B4; Elongation factor 1-gamma; Phosphoethanolamine / phosphocholine phosphatase 1; Adenylosuccinate lyase; 5'-3' exoribonuclease; Small nuclear ribonucleoprotein SmD2; Sm protein F; Lactoylglutathione lyase; At least one of Adenosylhomocysteinase; DNA(cytosine-5)-methyltransferase; Eukaryotictranslation initiation factor 3subunit B; MYG1 exonuclease; Smallnuclearribonucleoprotein Sm D3; Valine--tRNAligase; Alcohol dehydrogenaseclass-3; Tumornecrosis factor receptor superfamily member 11B; BPI fold-containing familyAmember 2.

2. A method for obtaining a novel protein marker for determining the degree of muscle damage as claimed in claim 1, characterized in that: The specific steps include: S1. Establishing experimental rat models with different degrees of muscle injury: using a homemade air bag cuff pressurization device to create different degrees of mechanical compression injury in the rat calf; S2. 3 hours after the rats were injured, the plasma of the rats in the experimental rat model was collected by cardiac puncture, and the expression profile of rat plasma proteins was detected by LC-MS / MS-4D-DIA quantitative proteomics technology. The candidate protein markers were obtained by differential analysis. S3. The ELISA method was used to detect the concentrations of 36 candidate protein markers in the blood of patients with different degrees of muscle injury in clinical practice.

3. The method for obtaining a novel protein marker for judging the degree of muscle damage according to claim 2, characterized in that: The experimental rat model in step S1 includes a control group, a mild injury group and a severe injury group; the operation on the control group rats in step S1 is: anesthetizing and fixing the right hind calf of the rat in a pressure-free homemade air bag cuff pressurizing device; the operation on the mild injury group rats is: anesthetizing and fixing the right hind calf of the rat in a homemade air bag cuff pressurizing device and pressurizing at 300 mmHg for 2 hours; the operation on the severe injury group rats is: anesthetizing and fixing the right hind calf of the rat in a homemade air bag cuff pressurizing device and pressurizing at 300 mmHg for 6 hours; the tibialis anterior muscles of the three groups of rats are obtained 3 hours and 3 days after injury, respectively, for hematoxylin and eosin staining to evaluate the degree of injury; Masson trichrome staining is performed 28 days after injury to evaluate the degree of muscle fibrosis.

4. Application of a novel protein marker for determining the degree of muscle damage, characterized in that: The degree of muscle damage in the subjects can be determined by making a kit using a novel protein marker or by using mass spectrometry for detection.

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