阅读说明：本技术 N-乙酰葡萄糖胺作为标志物在制备诊断缺血性脑中风试剂中的应用 (Application of N-acetylglucosamine as marker in preparation of ischemic cerebral apoplexy diagnosis reagent ) 是由 寇俊萍 马慧芬 李芳� 宫帅帅 张媛媛 余伯阳 于 2021-05-24 设计创作，主要内容包括：本发明公开了一种N-乙酰葡萄糖胺作为标志物在制备诊断缺血性脑中风试剂中的应用；本发明表明N-乙酰葡萄糖胺在缺血性脑中风和非缺血性脑中风对照者血清中的差异性表达,以N-乙酰葡萄糖胺作为标志物,对其血清中的代谢水平进行定量检测,同时,可将N-乙酰葡萄糖胺与调控酶N-乙酰葡萄糖胺激酶结合,减轻脑损伤。因此,N-乙酰葡萄糖胺代谢水平检测在缺血性脑中风的诊断中具有很高的临床应用价值,为缺血性脑中风的快速有效诊断提供了一种全新的途径。(The invention discloses an application of N-acetylglucosamine as a marker in preparing a reagent for diagnosing ischemic cerebral apoplexy; the invention shows that the N-acetylglucosamine is differentially expressed in the serum of ischemic cerebral apoplexy and non-ischemic cerebral apoplexy contrast persons, the N-acetylglucosamine is taken as a marker to quantitatively detect the metabolic level in the serum, and simultaneously, the N-acetylglucosamine can be combined with a regulatory enzyme N-acetylglucosamine kinase to reduce the brain injury. Therefore, the N-acetylglucosamine metabolic level detection has high clinical application value in the diagnosis of ischemic cerebral apoplexy, and provides a brand new way for the rapid and effective diagnosis of ischemic cerebral apoplexy.)

The application of N-acetylglucosamine as a marker in the preparation of a reagent for diagnosing ischemic cerebral stroke.

2. The use of claim 1, wherein the agent further comprises the regulatory enzyme N-acetylglucosamine kinase.

3. The use of claim 1, wherein the agent for diagnosing ischemic stroke comprises an agent for diagnosing ischemic stroke from migraine, epilepsy or stroke, including ischemic stroke and hemorrhagic stroke.

4. The use of claim 1, wherein the ischemic brain stroke is an early ischemic brain stroke.

5. A kit for diagnosing ischemic cerebral stroke, comprising N-acetylglucosamine as a marker, and auxiliary agents capable of being used for detecting the metabolic level of N-acetylglucosamine.

6. The kit of claim 5, further comprising N-acetylglucosamine kinase as a regulatory enzyme.

7. The kit according to claim 5, wherein the kit can use N-acetylglucosamine alone as a marker to complete early screening and differential diagnosis of ischemic cerebral stroke.

Technical Field

The invention relates to application of N-acetylglucosamine as a marker in preparation of a reagent for diagnosing ischemic cerebral apoplexy, belonging to the field of medical diagnosis.

Background

Stroke is a neurological deficit caused by acute focal injury to the central nervous system (i.e., brain, retina or spinal cord) due to vascular causes and can be broadly divided into two categories, ischemic and hemorrhagic stroke. Among them, ischemic stroke is ischemic infarction caused by arterial obstruction, which is a main type of stroke. At present, thrombolytic therapy is the most effective method for clinically treating acute ischemic stroke, but the treatment time window is extremely narrow, cerebral ischemia-reperfusion injury is easily caused, and the worsening degree of the disease is aggravated. Therefore, the timely diagnosis and effective treatment of ischemic stroke are receiving more and more attention from broad scholars.

Currently, clinical diagnosis of cerebral apoplexy mainly depends on the judgment of the clinician on the symptoms and signs of the patient and the examination result of brain imaging. However, other diseases such as migraine and seizure are similar to the clinical symptoms of stroke, and imaging of the brain, while a relatively straightforward way to diagnose stroke at present, has certain limitations such as the presence of metal implants or agitation in some patients that can affect the examination. Therefore, identifying and monitoring molecular biomarkers present in the natural course of ischemic stroke would aid in the diagnosis, prognosis and formulation of rational treatment strategies for stroke.

Metabolomics is an important component of system biology, where circulating small molecule compounds are the subject of study, and the changes in small molecule compounds under different physiological or pathological conditions are observed to reflect metabolic network activities that cause changes in these metabolites, such as drug or dietary intake and changes in the gut microbiota, and thus to yield basic information about the underlying biological state of the system under study. Therefore, the method is more and more applied to the fields of human epidemiology, drug effect evaluation, basic mechanism research and the like, and even can guide the personalized use of clinical drugs or predict the occurrence probability of diseases.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the defects and shortcomings of a clinical diagnosis means of ischemic cerebral apoplexy in the prior art, the invention provides application of N-acetylglucosamine as a marker in preparing a reagent for diagnosing ischemic cerebral apoplexy.

The technical scheme is as follows: the invention provides application of N-acetylglucosamine as a marker in preparing a reagent for diagnosing ischemic cerebral stroke.

The reagent for detecting the metabolism level of the N-acetylglucosamine is applied to the preparation of the reagent for diagnosing the ischemic cerebral apoplexy.

Further, the reagent also comprises a regulatory enzyme N-acetylglucosamine kinase.

Further, the agent for diagnosing ischemic cerebral stroke includes an agent for diagnosing ischemic cerebral stroke from migraine, epilepsy or cerebral stroke including ischemic cerebral stroke and hemorrhagic cerebral stroke; the ischemic cerebral apoplexy is early ischemic cerebral apoplexy.

The kit for diagnosing ischemic cerebral stroke of the present invention is characterized by comprising N-acetylglucosamine as a marker, and an auxiliary agent capable of being used for detecting the metabolic level of N-acetylglucosamine.

Further, N-acetylglucosamine kinase is included as a regulatory enzyme.

The kit can independently adopt N-acetylglucosamine as a marker to complete early screening and differential diagnosis of ischemic cerebral apoplexy.

The components of the test kit may be packaged in aqueous medium or in lyophilized form. Suitable containers in a kit typically include at least one vial, test tube, or the like, in which one component may be placed and suitably aliquoted. Where more than one component is present in the kit, the kit will also typically comprise a second, third or other additional container in which the additional components are separately disposed. However, different combinations of components may be contained in one vial. The kit of the invention will also typically include a container for holding the reactants, sealed for commercial sale. Such containers may include injection molded or blow molded plastic containers in which the desired vials may be retained.

The invention is based on the discovery of differential expression of N-acetylglucosamine in the serum of ischemic stroke and non-ischemic stroke control patients by the applicant, and the quantitative detection of the metabolic level in the serum by taking the N-acetylglucosamine as a marker can be used as a specific diagnostic index of ischemic stroke. In addition, applicants have also reduced brain damage by combining N-acetylglucosamine with the regulatory enzyme N-acetylglucosamine kinase.

Has the advantages that: compared with the prior art, the invention has the following remarkable advantages:

(1) the invention discloses a metabolic marker N-acetylglucosamine related to ischemic cerebral apoplexy, which can judge whether a subject suffers from ischemic cerebral apoplexy by detecting the level of the metabolite, has higher accuracy and specificity, and aims to realize early diagnosis of ischemic cerebral apoplexy, thus carrying out intervention treatment at the early stage of ischemic cerebral apoplexy and improving the treatment effect;

(2) the invention also confirms the function of the metabolite N-acetylglucosamine and the regulatory enzyme N-acetylglucosamine kinase in the ischemic cerebral apoplexy, the level of the metabolite N-acetylglucosamine in vivo is increased to promote the MCAO/R induced brain injury, the regulatory enzyme N-acetylglucosamine kinase is combined to provide a potential target for preventing and treating the ischemic cerebral apoplexy, the MCAO/R induced brain injury can be improved through the synergistic effect, and the volume of the cerebral infarction caused by the cerebral apoplexy is reduced to 20%.

Drawings

FIG. 1: the N-acetylglucosamine kinase NAGK carrier information constructed in the metabolite regulation enzyme function verification test;

FIG. 2: the level of N-acetylglucosamine in serum of stroke mice;

FIG. 3: the level of N-acetylglucosamine in the serum of patients with clinical cerebral stroke;

FIG. 4: receiver Operating Curve (ROC) plotted against the level of N-acetylglucosamine in serum of patients with clinical cerebral stroke;

FIG. 5: the effect of N-acetylglucosamine on the volume of cerebral infarction induced by MCAO/R;

FIG. 6: counting the cerebral infarction volume induced by MCAO/R by N-acetylglucosamine;

FIG. 7: the effect of N-acetylglucosamine on the extent of MCAO/R-induced brain tissue damage;

FIG. 8: the effect of N-acetylglucosamine on MCAO/R-induced neurological dysfunction;

FIG. 9: the effect of overexpression of N-acetylglucosamine kinase on the level of N-acetylglucosamine in serum;

FIG. 10: the effect of overexpressing N-acetylglucosamine kinase on the volume of MCAO/R-induced cerebral infarction;

FIG. 11: counting the cerebral infarction volume induced by MCAO/R by over-expressing N-acetylglucosamine kinase;

FIG. 12: the effect of overexpression of N-acetylglucosamine kinase on the extent of MCAO/R-induced brain tissue damage;

FIG. 13: effect of overexpression of N-acetylglucosamine kinase on MCAO/R-induced neurological dysfunction.

Detailed Description

The technical scheme of the invention is further explained by combining the attached drawings.

Materials, reagents and the like used in examples are commercially available unless otherwise specified.

The biological materials, reagents, kits and the like used in the invention can be obtained by conventional commercial purchase, and the experimental techniques are all routine operations in the field or operations according to the instruction of corresponding commodities.

Example 1

Targeted metabonomics validation:

1. preparation of middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model

Mice were injected intraperitoneally with 1% pentobarbital sodium, anesthetized, fixed in supine position, and incised at the midline of the neck. And separating and exposing the common carotid artery, the external carotid artery and the internal carotid artery on the right side, reserving ligatures at the proximal end of the common carotid artery in sequence, tying the external carotid artery at the distal end, and dissociating the distal end of the external carotid artery. A section of internal carotid artery is dissociated towards the deep part along the internal carotid artery, and a ligature is reserved at the proximal end of the external carotid artery. A small noninvasive vascular clamp is adopted to clamp and close a proximal common carotid artery and a distal internal carotid artery, a small opening is cut at the distal end of an external carotid artery by using a pair of microscissors, and a wire plug is inserted. Bifurcate through the common carotid artery and enter the internal carotid artery. The line segment is inserted until slight resistance is met, the depth is preferably 8-9mm beyond the bifurcation of the common carotid artery, and the head end of the line segment is inserted into the anterior cerebral artery by about 1 mm. After the insertion, the ligature previously placed in the external carotid artery is tied up. After MCAO for 60min, separating and exposing external carotid artery, loosening the fixing thread, removing the thread plug, loosening the carotid artery ligature to realize reperfusion, and conventionally suturing the neck wound. The temperature of the anus of the animal is kept at 37 ℃ in the operation process, and the animal is placed in a feeding box with a cleaning pad to freely drink and eat water after the operation.

2. Sample collection and processing

Mouse samples: collecting blood by eye ball picking method, standing whole blood at room temperature for 60min, centrifuging at 3500r/min for 10min, collecting supernatant, packaging, and storing at-70 deg.C.

Clinical samples: the serum samples of 272 subjects were collected by the Hospital of traditional Chinese medicine of Jiangsu province and stored at-70 ℃. Subjects were clinically diagnosed as healthy control group (Normal group) and Stroke group (Stroke group). Clinical sample information is shown in table 1:

TABLE 1 clinical sample information

Taking 50 mu L of frozen serum sample, thawing, adding 150 mu L of methanol containing an internal standard compound, uniformly mixing by vortex, centrifuging at 13000r/min at 4 ℃ for 10min, taking supernatant, centrifuging again, taking supernatant into a sample injection bottle, and placing the sample injection bottle at 4 ℃ for testing.

The chromatographic conditions are shown in table 2: the column temperature, autosampler and sample volume were set to 25 ℃, 4 ℃ and 5 μ L, respectively. Mobile phase A: 0.1% aqueous formic acid, mobile phase B: 0.1% formic acid acetonitrile. Gradient elution and flow rates are shown in the table below. Before each injection, the column was equilibrated for 5min at initial mobile phase conditions.

TABLE 2 gradient elution and flow Rate of chromatographic conditions

Mass spectrum conditions: detection is carried out in an electrospray ionization (ESI) negative ion mode, a signal acquisition selective reaction detection mode (MRM) is adopted, the GlcNAc fragmentation law is m/z 266.1 → 119.1, the capillary voltage, the nozzle voltage, the airflow, the atomizer pressure, the sheath gas temperature, the sheath gas flow rate, the collision energy and the fragmentation voltage are 3500V, 1500V, 7L/min, 50psi, 350 ℃, 12L/min, 9V and 55V respectively.

3. Results of targeting verification

As shown in FIG. 2, the metabolite N-acetylglucosamine (GlcNAc) was significantly elevated in the serum of MCAO/R mice; as shown in fig. 3, the metabolite N-acetylglucosamine (GlcNAc) is significantly elevated in the serum of patients with cerebral stroke; all the above results indicate that cerebral stroke is the main cause of a significant increase in the serum level of the metabolite N-acetylglucosamine (GlcNAc).

4. Plotting of ROC curves

Tools such as Receiver Operating Curve (ROC) analysis can be used to assess the performance of such tests, or to test the accuracy with which disease groups are distinguished from control groups. The ROC curve is a graphical representation of sensitivity and specificity spectra generated using sensitivity as the y-axis, 1-specificity as the x-axis, and various cut-offs. In the ROC curve, the true positive rate (sensitivity) is plotted as a function of FP rate (100-specificity) for different cut-off points. Each point on the ROC curve represents a sensitivity/specificity pair corresponding to a particular decision threshold. The ROC curve for the test with perfect discriminatory power (no overlap in the two distributions) passes through the upper left corner (sensitivity 100%, specificity 100%). Thus, qualitatively, the closer the graph is to the upper left corner, the higher the overall accuracy of the test. The area under the ROC curve (AUC) reflects the accuracy of the test and is shown in the lower left corner of the graph. As shown in FIG. 4, the area under the ROC curve is 0.9412, which indicates that GlcNAc can be used for preparing a reagent for clinically diagnosing cerebral stroke, and has higher accuracy and specificity.

Example 2

Functional verification of metabolites:

1. TTC dyeing

After MCAO/R modeling, all groups of mouse brain tissues are taken out, put into a refrigerator at the temperature of 70 ℃ below zero until the brain tissues are completely frozen, taken out, cut the frozen brain tissues into 5 coronary brain slices with the thickness of about 2mm on an ice box, quickly put the cut brain slices into 2 percent TTC solution, and pay attention to placing the brain slices towards one direction. Subsequently, incubation in an oven at 37 ℃ for 30min protected from light. The brain infarction volume is measured by taking a picture on a black paperboard and transmitting the picture into a computer and adopting Image J Image analysis software. The calculation formula is as follows:

infarct volume percentage right half cerebral infarct volume/total brain volume x 100%;

the larger the infarct volume, the more severe the stroke. In general, the volume of cerebral infarction caused by MCAO/R is 30-40%; as shown in fig. 5 and 6, administration of GlcNAc increased the volume of cerebral stroke-induced cerebral infarcts, up to 50%; as shown in fig. 10 and 11, the cerebral infarction volume induced by cerebral apoplexy can be significantly reduced by over-expressing NAGK, and the cerebral infarction volume is reduced to 20%, so that it can be seen that GlcNAc aggravates cerebral apoplexy, and the cerebral apoplexy can be improved by over-expressing NAGK.

2. H & E staining

Each group of mice was sacrificed after 24h of reperfusion, brains were taken, brain tissues were soaked in an EP tube containing 4% paraformaldehyde solution, and were sent to the pathology room of the drug safety evaluation center of Jiangsu province for detection.

3. Neurological scoring assay

After ischemia for 1h and reperfusion for 24h, neurobehavioral scoring of each group of mice was performed according to Longa5 score, and the criteria were as follows: 0 minute: no obvious nerve injury symptom; 1 minute: the left front paw cannot be completely straightened; and 2, dividing: when walking, the walking stick rotates to the left side; and 3, dividing: the walking stick inclines to the left side when walking; and 4, dividing: unconsciousness and inability to walk spontaneously. Thus, the higher the score the more severe the somatic neurological dysfunction is tested. Mean neurological scores of approximately 2.5 for mice after MCAO/R modeling and enhancement of stroke-induced neurological dysfunction following GlcNAc administration, as shown in figure 8; overexpression of NAGK improved brain stroke-induced neurological dysfunction, as shown in figure 13. The results indicate that GlcNAc can exacerbate stroke and overexpression of NAGK can improve stroke.

4. And (4) conclusion: function of metabolite GlcNAc in cerebral apoplexy

After exogenously administering the metabolite GlcNAc, the cerebral infarction volume induced by MCAO/R is increased, and the neurological dysfunction and the pathological damage of brain tissues are promoted, and the result shows that the increase of GlcNAc can aggravate the ischemic cerebral apoplexy.

Example 3

Metabolite regulatory enzyme functional validation

1. Experiment reagent and equipment

N-acetylglucosamine kinase (NAGK) overexpression adeno-associated virus (AAV-NAGK) and unloaded adenovirus (AAV-control) were provided by Jiman Biotech, Inc., Shanghai, and were tested: the titer of the GPAAV-CMV-M _ Nagk-EF1-ZsGreen1-WPRE is 1.21E +13 VG/mL; microsyringe (1 μ L); brain orientation apparatus (Stoelting, usa).

2. Synthesis of primers

Carrier information as in fig. 1; designing PCR amplification fragment primers, and introducing homologous sequences at the tail ends of a linearized cloning vector into the 5 ' ends of the primers, so that the 5 ' and 3 ' extreme sequences of amplification products are completely consistent with the two tail end sequences of the linearized cloning vector respectively, wherein the primer sequences are as follows:

3. brain in situ injection of adeno-associated virus

After the mouse is anesthetized by isoflurane, the head of the mouse is fixed on a brain positioning instrument by an ear rod, and simultaneously, the isoflurane is continuously given to the mouth and the nose of the mouse so as to ensure the anesthetic state of the mouse in the operation process, and the caliper rule is adjusted. The skin of the mouse head was cut open with an ophthalmic scissors, exposing the skull, and after iodophor disinfection the right brain synovium of the head was gently pulled open with a cotton swab. The micro syringe which absorbs 1 microlitre of the adeno-associated virus is fixed on the positioning instrument, and the position is adjusted so that the syringe can be inserted into the cerebral cortex. Adeno-associated virus (1 uL/5 min) was injected slowly, after the injection was completed, the syringe was pulled out gently, and the syringe hole was quickly sealed with bone wax. The suture was placed in a clean mouse cage after normal suturing and sterilization with iodophor, and the experiment was started 15 days later.

4. Grouping animals

AAV-control mice given 20 mice were randomized into 2 groups: sham (Sham, n ═ 10) and model (MCAO/R, n ═ 10); 20 mice given AAV-NAGK were randomized into 2 groups: sham (Sham, n ═ 10) and model (MCAO/R, n ═ 10).

5. Data statistics

All data were statistically analyzed using Graphpad Prism 5 software, expressed as mean + -SD, with Students't test for comparisons between groups, one-way analysis of variance when comparing three or more groups, and Dunnett's test for post-tests, P < 0.05 considered statistically significant. As shown in figure 2, the metabolite GlcNAc in the serum is obviously increased after MCAO/R modeling, and a significant difference is found after comparison. As shown in FIG. 3, the GlcNAc metabolite in the serum of the patients with cerebral apoplexy is obviously increased, and a significant difference is found after comparison. As shown in FIGS. 6 and 7, GlcNAc significantly increased the volume of MCAO/R-induced cerebral infarction and promoted neurological dysfunction, and significant differences were observed by comparison. As shown in FIG. 9, the increase of GlcNAc induced by MCAO/R can be obviously reduced by over-expressing NAGK, and a significant difference is found after comparison. As shown in FIGS. 11 and 13, the over-expression of NAGK can significantly reduce the cerebral infarction volume and the neurological dysfunction induced by MCAO/R, and significant differences are found after comparison.

6. And (4) conclusion: function of metabolite GlcNAc regulatory enzyme NAGK in cerebral apoplexy

As shown in figure 9, the metabolite GlcNAc after MCAO/R modeling is obviously increased, the high-level GlcNAc can promote brain injury induced by cerebral apoplexy and increase the cerebral infarction area, and the metabolite GlcNAc in vivo can be obviously reduced after NAGK is over-expressed, so that the cerebral infarction volume and brain tissue injury induced by MCAO/R are obviously reduced, and the neurological dysfunction is obviously improved, and the NAGK is a potential target for preventing and treating ischemic cerebral apoplexy.

The research discovers the metabolism marker N-acetylglucosamine related to the ischemic cerebral apoplexy for the first time, and whether a subject suffers from the ischemic cerebral apoplexy can be judged by detecting the level of the metabolism marker N-acetylglucosamine so as to realize the early diagnosis of the ischemic cerebral apoplexy, thus carrying out intervention treatment at the early stage of the ischemic cerebral apoplexy and improving the treatment effect. In addition, the research also confirms the functions of the metabolite GlcNAc and the metabolic enzyme NAGK thereof in ischemic cerebral apoplexy, the increase of the in-vivo level of the metabolite GlcNAc can promote the cerebral injury induced by MCAO/R, and the over-expression of the metabolic enzyme NAGK thereof can improve the cerebral injury induced by MCAO/R, thereby providing a potential target for preventing and treating the ischemic cerebral apoplexy.