A marker, a kit and a drug for diagnosing, differentially diagnosing and evaluating the prognosis of stroke

Abstract

The present invention belongs to the field of stroke diagnosis and evaluation, and specifically relates to a marker, a kit and a drug for diagnosing, differentially diagnosing and prognostic evaluation of stroke. The present invention found that the expression of ninjurin1 in stroke patients was significantly higher than that in healthy controls, and the expression level of ninjurin1 was positively correlated with the severity of stroke. Based on this, a marker of stroke is provided, which can be used as a detection target molecule for stroke diagnosis and differential diagnosis reagents, and is applied to the diagnosis, efficacy observation and prognosis evaluation of stroke patients. It can improve the diagnosis and treatment level of stroke and is suitable for clinical promotion and application.

Description

Technical Field

[0001] The present invention belongs to the field of stroke diagnosis and evaluation, and specifically relates to a marker, a kit and a medicine for diagnosing, differentially diagnosing and prognostic evaluating stroke. Background Art

[0002] Stroke has a high morbidity and mortality rate. Among them, ischemic stroke (IS) accounts for about 87% of stroke cases, and hemorrhagic stroke (HS) accounts for 13% of stroke cases. Neuroimaging is the main diagnostic method for the detection and localization of stroke, and it is also the gold standard for distinguishing between hemorrhagic stroke and ischemic stroke patients. However, computed tomography or magnetic resonance imaging scans take a long time and are not recommended for patients with ischemic stroke, because timely thrombolytic intervention is crucial to the prognosis of ischemic stroke. In addition, computed tomography or magnetic resonance imaging also has disadvantages such as high examination costs, contrast agent nephrotoxicity, and radiation damage.

[0003] The detection of serum markers is one of the methods for diagnosing stroke. It has certain clinical application value for the diagnosis of stroke, observation of therapeutic efficacy, judgment of disease progression and monitoring of recurrence. It has the advantages of being simple and non-invasive, quantitative and objective measurement results, repeatable measurement for dynamic monitoring, and relatively low cost. However, the current clinical application of serum markers is relatively lacking. In addition, cerebrospinal fluid testing is widely used in stroke clinically, especially cerebrospinal fluid protein, chloride, glucose, etc. However, the biomarkers currently used in clinical practice still have certain limitations, with certain false positive and false negative rates, and have certain limitations for early diagnosis and differential diagnosis of stroke.

[0004] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the invention

[0005] The object of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a marker, a kit and a drug for diagnosing, differentially diagnosing and prognostic evaluation of stroke. Wherein, the marker is soluble nerve injury-induced protein 1 (ninjurin1). Ninjurin1 is significantly highly expressed in the serum and cerebrospinal fluid of stroke patients, and its content in serum and cerebrospinal fluid can be detected to provide a more reliable basis for the diagnosis, differential diagnosis and prognostic evaluation of stroke.

[0006] Ninjurin1 is a double-transmembrane molecule that exists in various tissues of the body. It is highly expressed in the damaged peripheral nervous system and plays an important role in angiogenesis. It can mediate the rupture of the plasma membrane during cell death. This protein has two hydrophobic transmembrane domains and an extracellular NH2-terminal (ENT) domain. Its extracellular NH2-terminal region can be cleaved by matrix metalloproteinase 9 (MMP9) and released into the blood circulation to form soluble ninjurin1.

[0007] To achieve the above object, the present invention provides a marker for diagnosis, differential diagnosis and prognosis evaluation of stroke, wherein the stroke marker is ninjurin1.

[0008] Preferably, the ninjurin1 is derived from serum and / or cerebrospinal fluid.

[0009] Preferably, the expression level of ninjurin1 is positively correlated with the severity of stroke.

[0010] Preferably, the expression of ninjurin1 in hemorrhagic stroke patients is higher than the expression of ninjurin1 in ischemic stroke patients.

[0011] Based on the same technical concept, the present invention further provides a kit for diagnosing, differentially diagnosing and prognostic evaluation of stroke, which includes a reagent capable of detecting the marker ninjurin1. For example, the marker can be used as one of the key components of a stroke diagnosis and differential diagnosis test kit by designing primer sequences, preparing monoclonal antibodies, etc., and the marker can be detected in clinical samples of stroke patients.

[0012] Preferably, the kit further comprises a mass spectrometry identification reagent, an antibody and / or an antigen-binding fragment thereof.

[0013] Preferably, the detection subject of the kit is human.

[0014] Based on the same technical concept, the present invention also provides a drug for treating stroke, which is a drug targeting ninjurin1 target and related pathways.

[0015] The beneficial effects of the present invention are:

[0016] The present invention provides a marker for stroke, which can be used as a detection target molecule for stroke diagnosis and differential diagnosis reagents, and is applied to the diagnosis, efficacy observation, and prognosis evaluation of stroke patients. It can improve the diagnosis and treatment level of stroke and is suitable for clinical promotion and application.

[0017] More profoundly, the present invention utilizes the characteristic of high expression of ninjurin1 in the serum and cerebrospinal fluid of stroke patients. Subsequently, according to the specific expression level of ninjurin1, the relationship between ninjurin1 and the severity of the patient's disease, prognosis and survival can be explored and new drugs can be provided. By combining specific clinical data, follow-up, establishment of mathematical models and other methods, a stroke prognosis model based on ninjurin1 is constructed, providing a new direction for the treatment and prognosis evaluation of stroke patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is a schematic diagram of the expression difference of ninjurin1 content in serum of hemorrhagic stroke (HS), ischemic stroke (IS) and normal controls.

[0020] Figure 2 This is a schematic diagram of the expression difference of ninjurin1 content in cerebrospinal fluid of hemorrhagic stroke (HS), ischemic stroke (IS) and normal controls.

[0021] Figure 3 is the ROC curve diagram of patient diagnosis (serum); where:

[0022] Figure 3 A in the figure is the ROC curve of serum ninjurin1 in the diagnosis of hemorrhagic stroke (HS) patients;

[0023] Figure 3 B in the figure is the ROC curve diagram of serum ninjurin1 in the diagnosis of ischemic stroke (IS) patients.

[0024] Figure 4 is the ROC curve of patient diagnosis (cerebrospinal fluid); where:

[0025] Figure 4 A in the figure is the ROC curve of cerebrospinal fluid ninjurin1 in the diagnosis of hemorrhagic stroke (HS) patients;

[0026] Figure 4 Figure B is the ROC curve of cerebrospinal fluid ninjurin1 in the diagnosis of ischemic stroke (IS) patients.

[0027] Figure 5This is the ROC curve of cerebrospinal fluid ninjurin1 in differential diagnosis of hemorrhagic stroke (HS) and ischemic stroke (IS) patients.

[0028] Figure 6 This is the result of correlation analysis of hemorrhagic stroke (HS); among them:

[0029] Figure 6 A in the figure is the correlation analysis result between cerebrospinal fluid ninjurin1 and hemorrhagic stroke (HS);

[0030] Figure 6 Figure B is the result of the correlation analysis between serum ninjurin1 and hemorrhagic stroke (HS).

[0031] Figure 7 This is the correlation analysis result diagram of ischemic stroke (IS); among them:

[0032] Figure 7 A in the figure is the correlation analysis result between cerebrospinal fluid ninjurin1 and ischemic stroke (IS);

[0033] Figure 7 B in the figure is the result of correlation analysis between serum ninjurin1 and ischemic stroke (IS).

[0034] Figure 8 The ROC curve for predicting the prognosis of hemorrhagic stroke (HS) is shown in Figure 2.

[0035] Figure 8 A in the figure is the ROC curve of serum ninjurin1 predicting the prognosis of hemorrhagic stroke (HS);

[0036] Figure 8 Figure B is the ROC curve of cerebrospinal fluid ninjurin1 in predicting the prognosis of hemorrhagic stroke (HS).

[0037] Fig. 9 It is the ROC curve for predicting the prognosis of ischemic stroke (IS); among them:

[0038] Fig. 9 A in the figure is the ROC curve of serum ninjurin1 predicting the prognosis of ischemic stroke (HS);

[0039] Fig. 9 Figure B is the ROC curve of cerebrospinal fluid ninjurin1 in predicting the prognosis of ischemic stroke (HS). DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention.

[0041] Example

[0042] 1. Detection method: ELISA was used to detect the levels of ninjurin1 in the serum and cerebrospinal fluid of stroke patients and normal healthy subjects.

[0043] 1. The clinical samples of this embodiment include peripheral blood (111 ischemic stroke patients, 93 hemorrhagic stroke patients), cerebrospinal fluid (37 ischemic stroke patients, 36 hemorrhagic stroke patients). The relevant samples were collected clinically, and the collection procedures of peripheral blood and cerebrospinal fluid samples were as follows:

[0044] 2.5-3.5 mL of fasting venous blood from the research subjects was placed in a coagulant tube and allowed to stand vertically at room temperature for 25-35 min. It was then centrifuged at 2500-3500 r / min for 5 min at room temperature. The upper layer of liquid was aspirated into a labeled EP tube and stored at -80°C for later use.

[0045] 1.5-2.5 mL of cerebrospinal fluid of the research subjects was placed in a coagulant tube and allowed to stand vertically at room temperature for 25-35 min. It was then centrifuged at 2500-3500 r / min for 5 min at room temperature. The upper layer of liquid was aspirated into a labeled EP tube and stored at -80°C for later use.

[0046] 2. ELISA test

[0047] After equilibration at room temperature for 30 minutes, take out the required strips from the ELISA kit, seal the remaining strips with a ziplock bag and put them back at 4°C; add 100 μL of samples or standards of different concentrations to the corresponding wells, add 100 μL of universal diluent to the blank wells, cover with a sealing film and incubate at 37°C for 1 hour; take out the ELISA plate, discard the liquid, do not wash, directly add 100 μL of biotinylated antibody working solution to each well, cover with a sealing film and incubate at 37°C for 1 hour; discard the liquid, add 300 μL of washing solution (1x) to each well, and incubate at 37°C for 1 hour. Leave for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat the washing process 3 times; add 100 μL of enzyme conjugate working solution to each well, cover with sealing film and incubate at 37°C for 30 minutes; discard the liquid and wash according to the step 4 method, wash the plate 5 times; add 90 μL of substrate (TMB) to each well, cover with sealing film, and incubate at 37°C in the dark for 15 minutes; take out the ELISA plate, directly add 50 μL of stop solution to each well, immediately measure the OD value of each well at a wavelength of 450 nm, and calculate the concentration of ninjurin1 in the serum of each group according to the standard curve.

[0048] 3. Statistical analysis of serum ninjurin1 detection results

[0049] Independent sample t test or Mann-Whitney test was used to compare the serum ninjurin1 levels of patients with different types of stroke and normal healthy subjects (the results are shown in Figure 1 and Figure 2 ROC curve analysis was used to evaluate the diagnostic and differential diagnostic value of serum and cerebrospinal fluid ninurin1 for stroke (the results are shown in Figure 3 , Figure 4 , Figure 5 , Figure 8 , Fig. 9 mRS score is used to evaluate the prognosis of stroke patients (the results are shown in Figure 6 , Figure 7 , Figure 8 , Fig. 9 The Spearman correlation test was used to analyze the correlation between serum and cerebrospinal fluid ninurin1 and the prognosis of hemorrhagic stroke and ischemic stroke (mRS score).

[0050] 4. Conclusion:

[0051] Depend on Figure 1 It can be seen that the serum ninurin1 concentration in the hemorrhagic stroke group [146.05 (148.95) pg / mL] was significantly higher than that in the ischemic stroke group [118.51 (99.74) pg / mL], and the serum ninurin1 levels in both the hemorrhagic stroke and ischemic stroke groups were significantly higher than those in the normal control group.

[0052] Depend on Figure 2It can be seen that the concentration of ninurin1 in the cerebrospinal fluid of the hemorrhagic stroke group [174.20 (265.89) pg / mL] was significantly higher than that of the ischemic stroke group [69.72 (84.71) pg / mL], and the ninurin1 in the cerebrospinal fluid of the hemorrhagic stroke and ischemic stroke groups were significantly higher than that of the non-stroke group.

[0053] Depend on Figure 3 It can be seen that:

[0054] Figure 3 A in the figure shows the ROC curve of serum ninurin1 for diagnosing hemorrhagic stroke, with an area under the curve (AUC) of 0.934.

[0055] Figure 3 Figure B shows the ROC curve of serum ninurin1 for diagnosing ischemic stroke, with an area under the curve of 0.903.

[0056] It can be seen that serum ninurin1 can be used as a diagnostic marker for hemorrhagic stroke and ischemic stroke.

[0057] Depend on Figure 4 It can be seen that:

[0058] Figure 4 A in the figure shows the ROC curve of cerebrospinal fluid ninurin1 for diagnosing hemorrhagic stroke, with an area under the curve (AUC) of 0.971.

[0059] Figure 4 Figure B shows the ROC curve of cerebrospinal fluid ninurin1 in diagnosing ischemic stroke, with an area under the curve of 0.941.

[0060] It can be seen that cerebrospinal fluid ninurin1 can be used as a diagnostic marker for hemorrhagic stroke and ischemic stroke.

[0061] Depend on Figure 5 It can be seen that the ROC curve of cerebrospinal fluid ninurin1 for differential diagnosis of hemorrhagic stroke and ischemic stroke has an area under the curve of 0.77. It can be seen that cerebrospinal fluid ninurin1 can be used as a biomarker for differential diagnosis of hemorrhagic stroke and ischemic stroke.

[0062] Depend on Figure 6 It can be seen that:

[0063] Figure 6 A in the figure shows: The correlation analysis between cerebrospinal fluid ninurin1 and mRS score in patients with hemorrhagic stroke showed that cerebrospinal fluid ninurin1 was moderately positively correlated with the prognosis score of hemorrhagic stroke, with a correlation coefficient of 0.407.

[0064] Figure 6B in the figure shows: The correlation analysis between serum ninurin1 and mRS score in patients with hemorrhagic stroke showed that serum ninurin1 was slightly positively correlated with the prognosis score of hemorrhagic stroke, with a correlation coefficient of 0.390.

[0065] It can be seen that cerebrospinal fluid and serum ninurin1 are strongly correlated with the prognosis of patients with hemorrhagic stroke.

[0066] Depend on Figure 7 It can be seen that:

[0067] Figure 7 A in the figure shows: The correlation analysis between cerebrospinal fluid ninurin1 and mRS score in patients with ischemic stroke showed that cerebrospinal fluid ninurin1 was moderately positively correlated with the prognosis score of ischemic stroke, with a correlation coefficient of 0.545.

[0068] Figure 7 B in the figure shows: The correlation analysis between serum ninurin1 and mRS score in patients with ischemic stroke showed that serum ninurin1 was moderately positively correlated with the prognosis score of ischemic stroke, with a correlation coefficient of 0.475.

[0069] It can be seen that cerebrospinal fluid and serum ninurin1 are strongly correlated with the prognosis of patients with ischemic stroke.

[0070] Depend on Figure 8 It can be seen that:

[0071] Figure 8 A in the figure shows that the area under the ROC curve (AUC) of serum ninurin1 in predicting the prognosis of patients with hemorrhagic stroke is 0.713.

[0072] Figure 8 B in the figure shows that the area under the ROC curve (AUC) of cerebrospinal fluid ninurin1 in predicting the prognosis of patients with hemorrhagic stroke is 0.838.

[0073] Therefore, cerebrospinal fluid ninurin1 has a better ability to predict the prognosis of patients with hemorrhagic stroke than serum ninurin1.

[0074] Depend on Fig. 9 It can be seen that:

[0075] Fig. 9 A in the figure shows that the area under the ROC curve (AUC) of serum ninurin1 in predicting the prognosis of patients with ischemic stroke is 0.762.

[0076] Fig. 9B in the figure shows that the area under the ROC curve (AUC) of cerebrospinal fluid ninurin1 in predicting the prognosis of ischemic stroke patients is 0.817.

[0077] Therefore, cerebrospinal fluid ninurin1 has a better ability to predict the prognosis of patients with ischemic stroke than serum ninurin1.

[0078] 5. Conclusion:

[0079] (i) The expression level of serum ninjurin1 in patients with hemorrhagic stroke and ischemic stroke was significantly higher than that in healthy controls.

[0080] (ii) The expression level of ninjurin1 in CSF of patients with hemorrhagic stroke and ischemic stroke was significantly higher than that of patients without stroke.

[0081] (iii) The expression levels of serum and cerebrospinal fluid ninjurin1 in patients with hemorrhagic stroke were significantly higher than those in patients with ischemic stroke.

[0082] (iv) Serum and cerebrospinal fluid ninjurin1 have strong diagnostic efficacy for stroke.

[0083] (v) Cerebrospinal fluid ninjurin1 has a strong ability to differentiate between patients with hemorrhagic stroke and patients with ischemic stroke.

[0084] (vi) The ability of cerebrospinal fluid ninjurin1 to predict the prognosis of hemorrhagic stroke is stronger than that of serum ninjurin1.

[0085] (vii) Cerebrospinal fluid ninjurin1 has a stronger ability to predict the prognosis of ischemic stroke than serum ninjurin1.

[0086] Therefore, ninjurin1, as a new marker for the diagnosis and differential diagnosis of stroke, can improve the sensitivity and specificity of stroke diagnosis, and can assess the severity and clinical prognosis of stroke patients. At the same time, targeting ninjurin1 targets and related pathways may provide new ideas for the preparation of stroke treatment drugs.

[0087] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. Use of a stroke marker in the preparation of a kit for differential diagnosis of hemorrhagic stroke and ischemic stroke, characterized in that: The stroke marker is ninjurin1; The expression of ninjurin1 in patients with hemorrhagic stroke was higher than that in patients with ischemic stroke.

2. The use according to claim 1, characterized in that: The ninjurin1 is derived from serum and / or cerebrospinal fluid.

3. The use according to claim 1, characterized in that: The expression level of ninjurin1 is positively correlated with the severity of stroke.

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