CN110564841A - Application of cerebral ischemia related gene as biomarker for behavioral characteristic analysis of ischemic stroke - Google Patents
Application of cerebral ischemia related gene as biomarker for behavioral characteristic analysis of ischemic stroke Download PDFInfo
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Abstract
The invention discloses a preparation for assisting in diagnosing ischemic stroke or monitoring the treatment effect of ischemic stroke, which is a primer for amplifying at least one cerebral ischemia related gene P2RY12, NOS1, UCP2, MMP9 and VWf. The invention adopts CatWalk system, carries out gait analysis on MCAO mouse model at 7 days after modeling, collects genes related to cerebral ischemia from literature, reveals obvious correlation between gene expression related to cerebral ischemia and gait parameters, quantitatively and reliably evaluates the motor behavior of MCAO animal model according to the research result, has important biological significance for understanding the function of gene expression during early onset of ischemic stroke and long-term influence of gait variation, is helpful for researching disease progress of cerebral ischemia reperfusion, and provides new treatment direction for mouse model of ischemic stroke.
Description
Technical Field
the invention relates to the field of biological medicine, in particular to application of a cerebral ischemia related gene as a biomarker for behavioral characteristic analysis of ischemic stroke.
Background
cerebral stroke is the third most common death-causing disease in the world that can cause long-term complications such as type II diabetes; the annual global mortality rate for cerebral stroke is estimated to be 470 million.
Stroke patients have difficulty with memory, thinking, and partial paralysis and impaired motor ability. Global or focal cerebral ischemia associated with stroke can lead to death or permanent disability. The number of new and recurrent stroke cases that occur in the united states each year exceeds 70 million, while in china, 250 million new stroke patients are generated each year, of which 750 million survivors die 160 million, which places a serious economic burden on society.
Complex pathologies of ischemic stroke include bioenergy failure, acidosis, excitotoxicity, activation of nerve cells and glial cells, impairment of the blood-brain barrier (BBB) due to influx of immune cells and inflammatory reactions. Currently, intravenous recombinant tissue plasmin activator (rt-PA) is the only drug approved by the U.S. food and drug administration for thrombolytic treatment of acute ischemic stroke, minimizing nerve damage within 3 hours after tissue reperfusion following initiation of thrombolysis and occlusion of cerebral arteries, where the Middle Cerebral Artery (MCA) and its branches are severely affected during human cerebral ischemia, 70% of the infarcts occur. The prior art simulates human ischemic stroke to study the pathophysiology of the disease and designs an effective treatment strategy, and in the existing research technology, Middle Cerebral Artery Occlusion (MCAO) is one of the most commonly adopted methods which do not need craniotomy and can intensively occlude external carotid artery so as to reduce the cerebral blood flow velocity of a mouse animal model, the animal model constructed by the method is called MCAO animal model, the MCAO animal model is characterized by high-repeatability infarction and is essential for replication and research of human ischemic stroke and related cell death, inflammation and damage to BBB.
gait variation analysis allows simultaneous assessment of static (e.g., coordination, paw pressure, paw contact area) and dynamic (e.g., support phase time, swing phase, support phase proportion, stride, swing speed) parameters of motor performance in animal models of neurological diseases such as parkinson's disease, cerebral hemorrhage, sciatica, however, in mouse models of transient focal ischemic stroke, the description of gait changes is rare, thus limiting current understanding of pathophysiology of the disease, more comprehensive assessment of gait variability in mouse models of ischemic stroke is important for the study of dyskinesia caused by cerebral ischemia, and it is useful for developing targeted treatment regimens.
Disclosure of Invention
The invention aims to solve the technical problems that the research on the gait change of a mouse model of transient focal ischemic stroke (TIA) is less and a targeted treatment scheme for the disease is lacked in the prior art, and aims to provide a new treatment direction for the ischemic stroke by constructing an ischemic stroke animal model and researching the relevance between the gait change of the ischemic stroke animal model and an ischemia-related gene.
According to an aspect of the present invention, there is provided a kit for aiding diagnosis of ischemic stroke or monitoring therapeutic effect of ischemic stroke (ischemic stroke is subacute ischemic stroke), the kit comprising: the primers are used for amplifying at least one cerebral ischemia related gene P2RY12, NOS1, UCP2, MMP9 and VWf.
The present invention has been completed through the following research and analytical concepts:
First, this study blank was filled by examining 182 gait parameters observed in the mouse middle cerebral artery embolic cerebral ischemia (MCAO) model treated with MCAO on day 7 (the mouse model treated with MCAO on day 7 was selected to simulate subacute ischemic stroke) by automatic gait analysis using a computer-assisted catwalk system, of which 39 are significantly different from the sham group.
Then, the expression characteristics of cerebral ischemia related genes in the motor cortex of the mouse in the MCAO treated mouse middle cerebral artery embolism cerebral ischemia model on the 7 th day are analyzed through reverse transcription and real-time fluorescent quantitative PCR research.
Finally, the correlation of 39 gait parameters detected by the analysis of the pearson correlation coefficient and the expression of the genes related to the cerebral ischemia is found as follows: on day 7 of modeling, the model group showed more significant changes in mRNA expression levels of 39 gait parameters and 5 cerebral ischemia-related genes (p <0.005) compared to the control group.
The invention discloses a method for detecting the gait change of ischemic stroke, which comprises the following steps of (1) knowing that an ischemia related gene is related to ischemic stroke, but the correlation between the ischemia related gene and the gait change after the ischemic stroke is not clearly researched; or reduce the overall cost of clinical drug trials, reduce the duration of trials, and accelerate the drug development process.
The pearson correlation coefficient analysis was aimed at determining the linear relationship between the mRNA expression levels of the 8 ischemia-associated genes and the gait variability of the MCAO surgery-treated animals at day 7. The research of the invention finds that 5 of 8 ischemia-related genes (P2RY12, NOS1, UCP2, MMP9 and VWf) are obviously related to 26 of 182 gait defects (a related coefficient r is more than 0.7 or less than-0.7) in the MCAO operation treatment group.
The genes related to cerebral ischemia are related to the occurrence and development of cerebral ischemia, but the correlation between the genes related to cerebral ischemia and the behavior of ischemic stroke has not been discussed widely. Only MMP9, P2RY12, NOS1, and VWf of the 8 ischemia-related genes analyzed in this study were differentially regulated, showing strong correlation with 26 gait parameters that were significantly altered in the MCAO surgical treatment group. This suggests that not all of the cerebral ischemia-related genes are correlated with ischemic stroke behavior, but only some of the cerebral ischemia-related genes that are pathophysiologically correlated. The invention opens up a new research idea, finds the relevance between the cerebral ischemia related gene and the ischemic stroke ethology through research, and provides a new diagnosis and treatment scheme for the ischemic stroke.
Specifically, the kit can amplify target genes (P2RY12, NOS1, UCP2, MMP9 and VWf) in a tissue sample of a patient with ischemic stroke through primers for expanding cerebral ischemia related genes P2RY12, NOS1, UCP2, MMP9 and VWf, and can assist in diagnosing gait changes of the patient with ischemic stroke according to the relation between the ischemia related genes and the change of the post-ischemic stroke state, or can also be used for monitoring disease progression and nerve function recovery after treatment.
In some specific embodiments, the primer sequence is as set forth in SEQ ID NO: 1-10, the primers can specifically expand cerebral ischemia-related genes P2RY12, NOS1, UCP2, MMP9 and VWf.
In some more specific embodiments, the kit can be a test kit, for example, a kit for aiding in the diagnosis of ischemic stroke or monitoring the efficacy of treatment of ischemic stroke (ischemic stroke is subacute ischemic stroke), in addition to a kit comprising SEQ ID NO: 1-10, and can also comprise buffer solution, enzyme and other related reagents.
Specifically, the buffer and the enzyme may be those used in conventional real-time fluorescent quantitative PCR.
According to another aspect of the present invention, the present invention also provides the use of a cerebral ischemia-related gene as a biomarker of post-ischemic stroke change for non-diagnostic purposes.
The research of the invention discovers that in the preparation of the mouse middle cerebral artery embolism cerebral ischemia model, the age, sex and weight of animals, the inserting length, diameter and coating of a embolism wire are beneficial to the repeatability of the occurrence of an infarction focus and cause the obvious change of the degree and distribution of ischemic injury, thereby causing the obvious change of gait parameters of the mouse middle cerebral artery embolism cerebral ischemia model animals, so that the prepared middle cerebral artery embolism cerebral ischemia model is determined in advance, and the expression quantity of ischemia related genes of a cerebral motor cortex of the mouse middle cerebral artery embolism cerebral ischemia model can be researched on the basis of the model.
According to another aspect of the present invention, the present invention also provides the use of a cerebral ischemia-related gene as a biomarker of gait changes in a non-diagnostic purpose cerebral middle artery embolization cerebral ischemia model mouse; wherein the mouse is C57BL/6J mouse, and the preparation of the mouse middle cerebral artery embolism cerebral ischemia Model (MCAO) comprises the following steps:
(1) selecting male C57BL/6 mice with the weight of 25-28 g;
(2) Inserting the embolism line into the internal carotid artery by 0.8-1.5 cm to form an ischemia phenomenon until resistance is felt;
(3) the embolic wire was carefully withdrawn 1 hour after ischemia.
In addition, the embolism line is withdrawn after 1 hour of ischemia, so that the influence on blood flow can be avoided.
In some specific embodiments, the gait changes are obtained by analyzing gait parameters, and the gait analysis results show that the two gait parameters, namely the maximum contact area average pressure of the left anterior paw and the average pressure of the complete left anterior paw footprint, have the highest correlation degree with the expression of the cerebral ischemia related genes, wherein the correlation coefficients of the maximum contact area average pressure of the left anterior paw and the P2RY12, NOS1, MMP9 and VWf are respectively 0.76, -0.73 and-0.74; the correlation coefficients of the average pressure of the full left anterior paw footprint with P2RY12, NOS1, MMP9, and VWf were 0.72, 0.92, -0.85, -0.73, respectively. Thus, in some embodiments, the gait parameters in the above application are the left anterior maximum contact mean pressure and the mean pressure of a full left anterior foot footprint.
In some embodiments, the middle cerebral artery embolization ischemia Model (MCAO) mice herein are used to mimic ischemic stroke, particularly where ischemic stroke is subacute.
the cerebral ischemia related gene which is obviously related to gait change in the mouse model of subacute ischemic stroke is used as a biomarker to assist diagnosis and monitoring of disease progression and nerve function recovery after treatment intervention, and the application is specifically shown as follows: determining the gait behavior of the patient with subacute ischemic stroke by monitoring the expression level of the cerebral ischemia related gene of the patient with subacute ischemic stroke, so as to analyze the gait of the patient; and finally, determining the disease progression and nerve function recovery condition of the ischemic stroke after treatment and intervention according to the gait analysis result of the patient.
The cerebral ischemia related gene which is obviously related to the gait change in the mouse model of the subacute ischemic stroke is used as a biomarker for reducing the total cost of clinical drug experiments, shortening the duration of the experiments and accelerating the drug development process, and the application is specifically shown as follows: 1. the method comprises the steps of developing a clinical medicine for a mouse model of subacute ischemic stroke, 2, applying the clinical medicine to the mouse model of subacute ischemic stroke, and monitoring gait behaviors of the mouse model of subacute ischemic stroke at different periods after the clinical medicine is taken by measuring the expression quantity of cerebral ischemia related genes of the mouse model of subacute ischemic stroke, so as to analyze the gait of a model mouse, and finally determining the curative effect of the clinical medicine according to the gait analysis result of the model mouse.
the two specific applications, namely determining the disease progression and the neurological function recovery condition of ischemic stroke after therapeutic intervention according to the gait analysis result of a patient and determining the curative effect of a researched clinical drug according to the gait analysis result of a model mouse, belong to the auxiliary diagnosis of the focal state of ischemic stroke by detecting the expression quantity of cerebral ischemia related genes.
The present invention also studied mRNA of cerebral motor cortex of MCAO surgically treated animals, and the results showed that mRNA expression levels of MMP9 and VWf were significantly down-regulated by 37.8% and 31.2%, respectively, in the MCAO surgically treated group compared to the sham operated control group. In contrast, the mRNA expression levels of P2RY12 and NOS1 were increased by 79.9% and 55.4%, respectively, in the MCAO surgical treatment group. These results indicate that the mRNA expression levels of 4 ischemia-related genes MMP9, VWf, P2RY12, and NOS1 were significantly changed in the motor cortex of mice treated with MCAO surgery on day 7, suggesting that these four genes may be involved in the regulation of motor function in animals with ischemic stroke.
According to another aspect of the invention, there is also provided SEQ ID NO: 1-10 in the preparation of an auxiliary diagnostic reagent for ischemic stroke.
In some specific embodiments, the secondary diagnosis of ischemic stroke is: the method is used for assisting in determining the disease progression and the neurological function recovery condition of the ischemic stroke patient by detecting the expression quantity of the cerebral ischemia related gene of the ischemic stroke patient.
More specifically, i.e. as SEQ ID NO: 1-10 as a primer, amplifying the expression level of the cerebral ischemia related gene in the body of the cerebral ischemic stroke patient, and judging the motor ability of the cerebral ischemic stroke patient according to the expression level.
In some specific embodiments, the cerebral ischemia-related gene is selected from at least one of P2RY12, NOS1, MMP9, and VWf.
Specifically, the above criterion for judging and judging the motor ability of the cerebral ischemic stroke patient is as follows: when the detected cerebral ischemia-related gene is MMP9 or VWf, the expression of MMP9 or VWf of a cerebral ischemic stroke patient is down-regulated, namely the motor ability of the cerebral ischemic stroke patient is reduced; when the detected cerebral ischemia related gene is P2RY12 or NOS1, the expression of P2RY12 or NOS1 of a cerebral ischemic stroke patient is up-regulated, namely the motor ability of the cerebral ischemic stroke patient is reduced.
In summary, the invention firstly quantitatively evaluates the gait defect of the mouse on the 7 th day after MCAO treatment, and discovers the strong correlation between the gait variability of the mouse treated by MCAO on the 7 th day of modeling and the expression spectrum of ischemia-related genes, thereby providing a method for quantitatively and reliably evaluating the movement performance of the MCAO model. The research has important biological significance for understanding the functional role of gene expression in the early onset of ischemic stroke and the long-term influence of gait variation, is beneficial to researching the disease progression of cerebral ischemia reperfusion, and provides a new treatment direction for a mouse model of ischemic stroke.
Drawings
Fig. 1 is an exploratory data analysis of gait parameters, calculated by qlucore omics explorer 3.0, PCA scatter of day 7 MCAO surgical treatment group (n-10) and sham surgical control group (n-20);
Fig. 2 is a hierarchical cluster analysis of gait parameters, which was calculated by qlucore omics explorer 3.0 to obtain a cluster analysis of 39 gait parameters between the MCAO surgery treatment group (n-10) and the sham surgery control group (n-20) on day 7;
Fig. 3 shows the expression of defect-associated genes, each box plot showing the relative mRNA expression of AQP4, GPX1, MMP9, P2RY12, S1000B, UCP2, NOS1 and VWf in the cerebral cortex of the MCAO surgery treatment group (n 10) and the sham surgery control group (n 20), where P <0.05, + represents the mean value of each group, the box represents a 25% to 75% percentage value, the horizontal line in the middle of the box indicates the median, the whisker line goes down to the minimum and up to the maximum (minimum to maximum), the grid line on the Y axis is set to 1, and the data of each group are obtained as 4 means ± standard error;
FIG. 4 is a correlation analysis between the expression levels of cerebral ischemia-related genes and gait changes, and the right heat map shows that the Pearson's correlation coefficient of the group correlated between 26 gait parameters and the expression levels of mRNA of 8 cerebral ischemia-related genes is greater than 0.7 or less than-0.7, and the left graph is a relationship network created using Cytoscape in which the shades of blue located on the outer side of the circle represent the number of links between 26 gait parameters and gene expression;
FIG. 5 is a typical pathway functional annotation and disease enrichment analysis of ischemia-associated genes with IPA and the use of Cytoscape to generate a relationship net, with red intensity indicating the percentage of overlap of the first 10 typical pathways with associated disease and gene expression disorders.
Detailed Description
Animal for experiment
Adult C57BL/6J mice were obtained from the laboratory animal service center, university of Chinese, hong Kong. All Animal handling and Experimental procedures followed The "Experimental Animal usage rules of Chinese university of Hong Kong-attached Table 7" (CUHK Regulations on Use of Experimental Animals-Schedule 7), "International ethical guidelines for Biomedical Research Involving Animals" (The International guidelines for Biomedical Research investigating Animals), "Hong Kong laboratory Animal Care and Use Practice protocols" (Hong Kong Code of Practice for Care and Use of experiments for Experimental Animals) and "The Animal health guide of Chinese university of Hong Kong" (The CUHK guide for Experimental Care Use).
30 adult (8 to 10 weeks) male C57BL/6 mice, weighing 25-28g, were used in this study. The mice are placed in an environment with a suitable temperature (22 +/-1 ℃) and humidity of 45% -55%, and light-dark alternation is carried out for 12 hours respectively, and the mice are fed according to the standard of a laboratory and are fed with water freely. In order to make all animals available for the experiment, all animals were cultured daily one week prior to the experiment as described above and all animal handling and experimental procedures were approved by the ethical committee on animal experiments of chinese university in hong kong.
Second, MCAO mouse model establishment
MCAO models were constructed according to the chinese et al literature reports and surgery was performed on MCAO model mice to obtain MCAO surgery treated group (n ═ 10) and sham operated control group (n ═ 20).
the MCAO model mouse operation method comprises the following steps: mice were anesthetized by intraperitoneal injection with 20% amobarbital (1ml/kg) and fixed on an operating table in the supine position to keep breathing unobstructed. The neck was wiped with an alcohol cotton ball, the incision was made in the center of the neck, the submandibular gland was isolated bluntly, the left common carotid artery was isolated from the microscope downstream, and the vessel was clamped closed with a microangioclip. Dissociating the left external carotid artery, cauterizing and ligating the superior thyroid artery by using an electrocoagulation pen, ligating the distal end of the external carotid artery by using a 6-0 suture, tying two loose knots between the bifurcation of the left common carotid artery and the ligature of the previous external carotid artery, and clamping the intersection of the left common carotid artery and the carotid artery of the left neck by using a micro vascular clamp. A small opening is cut between two loose knots at the proximal section of the left external carotid artery and a distal ligature by using microscopic ophthalmic scissors, the MCAO ligature is inserted downwards to the common carotid artery, the two loose knots are tied tightly, a micro vascular clamp at the bifurcation of the common carotid artery is loosened, and the insertion end of the embolism ligature is positioned at the bifurcation of the left common carotid artery. The left external carotid artery is cut off below the ligation part at the far end of the external carotid artery and above the inserted embolism thread, and the external carotid artery is pulled to the outer lower part to be in the same straight line with the internal carotid artery. The plug wire was inserted about lcm into the internal carotid until resistance was felt. After 1 hour of ischemia, the embolic thread was carefully removed, the silk thread at the external carotid artery was tied, and the vascular clamp at the left common carotid artery was removed after 1 minute, and the skin was sutured. In the sham-operated control group, the left external carotid artery was cut off without inserting a plug thread, and then the left common carotid artery was clamped for l hours, and the vascular clamp was withdrawn and the skin was sutured. After operation, the mice are placed in an incubator at 30-33 ℃, after the mice are awake, the neurobehavioral changes of the mice are observed, and then the mice are placed in a feeding box with a cleaning pad, and the mice are kept at room temperature of 20-23 ℃ for free drinking and eating.
Third, data analysis
Heatmaps that enable hierarchical cluster analysis and PCA were calculated and visualized using qlucore omics explorer 3.0. The gait of mice from the day 7 MCAO surgery treatment group (n ═ 10) and sham control group (n ═ 20) was calculated using R-package version 3.0.1(R statistical basis, vienna, austria) of the (R, Y) function, and the pearson correlation coefficients of the 182 gait parameters obtained were analyzed. All data are expressed as mean ± Standard Error (SEM) of the mean.
the t-test was used to statistically evaluate the mRNA expression levels in the cerebral motor cortex of mice in the day 7 MCAO surgery-treated group (n ═ 10) and the sham-operated control group (n ═ 20), and a P value of ≦ 0.05 was considered to be statistically significant. The qPCR results were analyzed using Prism 7forWindows (american glapard).
Example 1
1. Catwalk gait and posture analysis
Gait and posture analysis employed a video-based Catwalk system (Vaginine, Netherlands, Nodastic information technology). The system consists of a long glass plate, a patent footprint lighting technology and a high-speed camera. Only when any part of the mouse's body is in contact with the glass plate, green light is internally reflected and refracted. The signal produced by the refracted light is converted to a digital image by the camera and transmitted to a computer for analysis by the Catwalk XT 10.5.505 software.
One week prior to gait analysis experiments, mice were placed on glass plates and allowed to walk freely at least 6 times a day until all mice could pass through the walkway without interruption, as is customary for culturing mice. Only the trajectory of the straight walking direction in which the mouse was not interrupted or hesitated was recorded. The camera gain is set to 20 and the detection threshold is set to 0.2. The settings recorded the gait of mice passing through the observation zone for only 0.5-5s, with a maximum velocity change across the entire observation zone of 60%. At least 4 recordings were made per mouse. For each mouse, 5 acquisition traces containing at least 5 complete walking cycles were analyzed by 2 experimenters. The analysis trace values for each mouse were averaged and used for data processing. Gait measurements of mice in the day 7 MCAO surgery-treated group (n ═ 10) and the sham-operated control group (n ═ 20) were normalized to those of mice in the day 0 MCAO surgery-treated group (n ═ 10) and the sham-operated control group (n ═ 20).
As a result: mice in the MCAO surgery treated group (n ═ 10) showed gait deficits in the CatWalk gait analysis.
The CatWalk gait analysis system was used to study the gait of day 7 (i.e. brain ischemia subacute) mice, and a total of 182 gait parameters were examined comprehensively and quantitatively in order to assess the motor function performance of MCAO surgery treated group (n 10) mice. Pattern recognition was performed using Principal Component Analysis (PCA) and MCAO surgery-treated (n ═ 10) mice and sham-operated control (n ═ 20) mice were compared at day 7 using Qlucore.
As shown in figure 1, PCA produced two different clusters (p <0.05) between the two groups, indicating a significant difference in gait between MCAO surgery treated (n ═ 10) and sham operated control (n ═ 20) mice.
Next, unsupervised hierarchical clustering analysis was performed using Qlucore to plot changes in gait in MCAO surgery-treated (n ═ 10) mice and sham-operated control (n ═ 20) mice on day 7. The tree heatmap in fig. 2 shows the entire data matrix for both groups. Here, 39 gait parameters (see table 1) were significantly changed (p <0.005), and 29 of the gait parameters in the sham-operated control group (n ═ 20) showed an upward trend (p <0.005, gait parameters numbered 1 to 29 in table 1) and 10 of the gait parameters showed a downward trend (p <0.005, gait parameters numbered 30 to 39 in table 1) compared to the MCAO-operation-treated group (n ═ 10).
Three gait parameters, mean pressure intensity (mean pressure intensity), maximum pressure intensity (maximum pressure intensity), and maximum pressure intensity at maximum limb contact (maximum pressure four-limbic) of MCAO surgery treated mice were significantly decreased (p <0.005) compared to sham control (n ═ 20). In addition, similar to the results of the sham-operated control group (N ═ 20), the results of the maximum contact area and the maximum contact length of the paw mark (maximum contact area and length of the left and right hind paws) of the MCAO-operated treated group (N ═ 10) mice showed a significant decrease (p < 0.005).
TABLE 1
Note: RF in table 1 is right front paw; RH is the right hind paw; LF is the left front paw; LH is the left hind paw.
In contrast, the MCAO surgery treated group (n 10) mice had a significantly smaller base of hind limbs (bases of support) than the sham control group (n 20) (95% CI: -0.56 to-0.01, p < 0.005). Interestingly, the mice in the MCAO surgery treated group (n ═ 10) had significantly greater forelimb support cardinalities (bases of support beta front limbs) than the sham operated control group (n ═ 20) (95% CI: -0.19 to-0.17, p < 0.005). Similarly, the relative average time (the relative average left and right stroke duration) for left hind paw swing in MCAO surgery treated group (n ═ 10) was significantly higher than that in sham surgery control group (n ═ 20) (95% CI: -0.01 to 0.02, p < 0.005). In addition, the standing time of the right hind paw (the time of the right hand paw) of MCAO surgery treated (n ═ 10) mice was significantly increased (95% CI: -0.01 to 0.02, p <0.005) compared to the standing time of sham-operated control (n ═ 20) mice.
A significant difference in limb coordination was detected between the MCAO surgery treated group (n-10) and the sham operated control group (n-20). MCAO surgery treated (n ═ 10) mice placed the right hindpaw faster when the right forepaw was standing (95% CI: 3.77 to 8.57, p <0.005) compared to sham control (n ═ 20). The placement times of the right hind paw of MCAO surgery treated (n ═ 10) and sham control (n ═ 20) mice were statistically significantly different during swing when the right anterior paw was in a standing position (95% CI: -8.98 to-3.71, p < 0.005).
Similarly, during swing, mice in the MCAO surgical treatment group (n ═ 10) had their left hind paw placed in a standing position, with their right forepaw placed in delay (95% CI: 0.02 to 0.15, p < 0.005). No significant difference was observed in the measurements of left anterior paw placement when right anterior paw was standing, right anterior paw placement when left anterior paw was standing, right posterior paw placement when left posterior paw was standing, or left posterior paw placement when right posterior paw was standing in the MCAO surgical treatment group (n ═ 10) and sham surgical control group (n ═ 20). These results indicate significant changes in gait variability between MCAO surgery-treated (n-10) and sham-operated control (n-20) mice at day 7.
2. qRT-PCR of cerebral cortex of MCAO operation treatment group (n ═ 10) and sham operation control group (n ═ 20) mice
(1) Cerebral motor cortex separation
After completing the CatWalk gait analysis on day 7 post-surgery, all mice were sacrificed using deep anesthesia. The cerebral cortex of MCAO surgery treated (n ═ 10) and sham control (n ═ 20) mice were removed on ice: for each mouse, the bottom blade of a small scissors was inserted into the foramen magnum to cut the median line of the brain and the skull on both sides was peeled to expose the brain. The skull was gently inverted and the tissue was sloughed off the skull. Four points (ML + -2 mm, AP +2.5mm, ML + -1mm and AP-1mm) were used for accurate localization of the motor cortex.
(2) qRT-PCR experiment
The gait impairment was more severe in the day 7 MCAO surgery treated group (n 10) mice compared to the sham control group (n 20), which suggested that the mid-cerebral artery embolism affected the motor cortex controlling motor behavior. Based on the above results, the cerebral ischemia-related genes were searched from the PubMed database and qPCR experiments were performed to verify the expression of the motor cortex mRNA in the MCAO surgery treatment group (n ═ 10).
Frozen motor cortex tissues (about 50mg each) of mice from the MCAO surgery treated group (n ═ 10) and sham surgery control group (n ═ 20) were homogenized and resuspended in 1ml of Trizol reagent (invitrogen, usa, CAT 15596-026) to extract total RNA, according to the following experimental procedure:
The RNA concentration was measured using a NanoDrop spectrophotometer (Thermo Scientific, Mass.) in the United states. Reverse transcription reactions were performed using a high capacity reverse transcriptase kit (applied biosystems, Calif., USA) and 1. mu.g of RNA sample. The first strand of cDNA was synthesized by the following thermocycling conditions: incubating at 25 deg.C for 10min, annealing/extending at 37 deg.C for 120min, and denaturing at 85 deg.C for 5 min.
Quantitative real-time PCR (RT-PCR) was performed using the QuantStaudio 7Flex real-time fluorescent quantitative PCR system using SYBR Master Premix Ex Taq as a reagent according to the manufacturer's (applied biosystems, Calif., USA) instructions. The thermal cycling conditions were: relative gene expression levels in the brains of mice in the MCAO-treated group (n-10) and sham-operated control group (n-20) were calculated by the 2-delta-CT method using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control, after denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s (40 cycles), annealing/elongation at 60 ℃ for 34 s.
on the NCBI website, pubmed retrieves "braiin" and "ischemia" and retrieves 8 genes associated with cerebral ischemia: aquaporin 4(AQP4), glutathione peroxidase 1(GPX1), matrix metalloproteinase 9(MMP9), purinergic receptor P2y12(P2RY12), S100 calcium binding protein B (S100B), uncoupling protein 2(UCP2), nitric oxide synthase 1(NOS1), and von willebrand factor (VWf).
The 8 genes related to cerebral ischemia were selected as target genes, primers for RT-PCR reaction are shown in Table 2, and reaction systems for RT-PCR for the respective genes related to cerebral ischemia are shown in Table 3.
TABLE 2
TABLE 3
Results of mRNA analysis: as shown in fig. 3, the mRNA expression levels of MMPs 9 and VWf were significantly down-regulated by 37.8% (95% CI: 0.9331-0.07688, P ═ 0.0278) and 31.2% (95% CI: 0.5248-0.01021, P ═ 0.044), respectively, in the MCAO surgical treatment group (n ═ 10) compared to the sham-operated control group (n ═ 20). In contrast, the mRNA expression levels of P2RY12 and NOS1 in the MCAO surgical treatment group (n ═ 10) were increased by 79.9% (95% CI: 0.1309-1.399, P ═ 0.026) and 55.4% (95% CI: 0.05855-1.221, P ═ 0.036), respectively. These results indicate that mRNA expression levels of 4 cerebral ischemia-related genes MMP9, VWf, P2RY12, and NOS1 were significantly changed in the motor cortex of mice treated with MCAO surgery on day 7, suggesting that these four genes may be involved in the regulation of motor function after ischemic stroke.
Pearson correlation analysis was aimed at determining the linear relationship between mRNA expression levels of 8 cerebral ischemia-related genes and gait variability at day 7 in MCAO surgery-treated mice. As shown in fig. 4, 5 of the 8 cerebral ischemia-related genes (P2RY12, NOS1, UCP2, MMP9, and VWf) were significantly correlated with 26 of the 182 gait defects in the MCAO surgical treatment group (correlation coefficient r) being greater than 0.7 or less than-0.7. For example, there are 12 gait parameters positively correlated with P2RY12 gene expression, including total surface area of the left hind paw (r ═ 0.92), and 1 gait parameter negatively correlated with P2RY12 gene expression, including the base of struts between hind limbs (r ═ 0.80); 17 gait parameters were positively correlated with NOS1 gene expression, including maximum pressure at maximum contact of the left anterior paw (r ═ 0.94), and 3 gait parameters were negatively correlated with NOS1 gene expression, including the temporal relationship between the positions of the right posterior paw and the right anterior paw in one stride period (r ═ 0.89).
In addition, only 1 gait parameter (i.e., maximum pressure at maximum contact of right anterior paw) is positively correlated with UCP2 gene expression (r ═ 0.71), and only 1 gait parameter (i.e., base of anterior support, base of superior left the front strips) is negatively correlated with UCP2 gene expression (r ═ 0.74); 7 gait parameters are inversely related to MMP9 gene expression, including the mean pressure across the left hind paw (r ═ 0.92). No gait parameters are positively correlated with MMP9 gene expression; the 3 gait parameters are positively correlated with VWf gene expression, including the placing time relationship of the right hind paw and the right forepaw in a stride period (r ═ 0.91), and the 8 gait parameters are negatively correlated with VWf gene expression, including the placing time relationship of the right forepaw and the right hind paw in a stride period (temp dispersion) between the right forepaw and the right forepaw in a stride period (r ═ 0.90).
As seen in the right panel of fig. 4, gait measurements of the left anterior paw maximum contact area average pressure (maximum intensity at maximum contact of the left front paw, P9) and the average pressure of the intact left anterior paw footprint (maximum intensity of the complete left front paw, P10) show the highest degree of correlation with mRNA expression of the four genes-P2 RY12 (r-0.76), NOS1 (r-0.76), MMP9 (r-0.73) and VWf (r-0.74), indicating a close relationship between these two gait parameters and the expression of the cerebral ischemia-related genes (see fig. 4, right panel). The mRNA expression levels of these several cerebral ischemia-related genes were strongly correlated with the CatWalk gait measurement observed in the MCAO surgical treatment group (n ═ 10) at day 7.
(3) IPA analysis
Typical pathway and disease-enrichment analysis of 8 cerebral ischemia-related genes using the original pathway analysis (IPA, Qiagen) showed that they span several biological pathways, including amyotrophic lateral sclerosis signaling, the HIF-1 pathway, citrulline-nitrate oxide circulation, and neuroinflammatory signaling pathways, which are widely associated with a range of diseases (e.g., cardiovascular, neurological, inflammatory) (see figure 5). Interestingly, only MMP9, P2RY12, NOS1 and VWf were differentially regulated in the disease model, showing a strong correlation with 26 gait parameters that were significantly altered in the MCAO surgical treatment group.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Sequence listing
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Claims (10)
1. a kit for aiding in the diagnosis of ischemic stroke or monitoring the effectiveness of a treatment for ischemic stroke, the kit comprising: the primers are used for amplifying at least one cerebral ischemia related gene P2RY12, NOS1, UCP2, MMP9 and VWf.
2. The kit for assisting in diagnosing ischemic stroke or monitoring the therapeutic effect of ischemic stroke according to claim 1, wherein the primer sequence is as set forth in SEQ ID NO: 1 to 10.
3. The application of cerebral ischemia related gene as non-diagnostic biomarker of gait change after ischemic stroke.
4. The application of cerebral ischemia related gene as non-diagnosis purpose cerebral artery embolism cerebral ischemia model mouse gait change biomarker; the mouse is a C57BL/6J mouse, and the preparation of the mouse cerebral middle artery embolism cerebral ischemia model comprises the following treatment:
(1) Selecting male C57BL/6 mice with the weight of 25-28 g;
(2) Inserting the embolism line into the internal carotid artery by 0.8-1.5 cm to form an ischemia phenomenon until resistance is felt;
(3) The embolic wire was carefully withdrawn 1 hour after ischemia.
5. Use according to claim 3 or 4, wherein the gait changes are obtained by analyzing gait parameters, the gait parameters being the left anterior paw maximum contact mean pressure and the mean pressure of a full left anterior paw footprint.
6, SEQ ID NO: 1-10 in the preparation of an auxiliary diagnostic reagent for ischemic stroke.
7. Use according to claim 6, characterized in that the auxiliary diagnosis of ischemic stroke is: the method is used for assisting in determining the disease progression and the neurological function recovery condition of the ischemic stroke patient by detecting the expression quantity of the cerebral ischemia related gene of the ischemic stroke patient.
8. The use of claim 7, wherein the cerebral ischemia-related gene is selected from at least one of P2RY12, NOS1, MMP9 and VWf.
9. The use of claim 8, wherein the expression of MMP9 or VWf is down-regulated in patients with cerebral ischemic stroke when the cerebral ischemia-related gene is MMP9 or VWf.
10. The use of claim 8, wherein when the cerebral ischemia-related gene is P2RY12 or NOS1, the expression of P2RY12 or NOS1 in a patient with cerebral ischemic stroke is up-regulated.
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