CN111621558A - Application of blood brain barrier damage degree-related serum exosome miR-410-3p and detection method thereof - Google Patents
Application of blood brain barrier damage degree-related serum exosome miR-410-3p and detection method thereof Download PDFInfo
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Abstract
The invention provides a serum exosome miR-410-3p related to the blood brain barrier damage degree after cerebral ischemia, and the sequence of the serum exosome miR-410-3p is AATATAACACAGATGGCCTGT. The serum exosome miR-410-3p can be used as a biomarker for evaluating the degree of blood brain barrier destruction after cerebral ischemia. The method for detecting the expression level of the serum exosome miR-410-3p provided by the invention comprises the following steps: (1) establishing a mouse middle cerebral artery embolism model, performing ischemia reperfusion, and collecting serum; (2) separating exosomes in serum; (3) and detecting the expression level of miR-410-3p in the serum exosomes at different stages of cerebral ischemia-reperfusion injury and recovery by using a real-time fluorescence quantitative polymerase chain reaction technology. The technical scheme of the invention proves that the expression trend of miR-410-3p in the serum exosome is basically consistent with the BBB injury and repair process, and the serum exosome miR-410-3p can be used as a biomarker for judging cerebral ischemic injury.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to application of a serum exosome miR-410-3p related to the degree of blood brain barrier destruction after cerebral ischemia and a detection method of an expression level of the serum exosome miR-410-3 p.
Background
Global Burden of Disease (GBD) data in 2016 showed that stroke is the second cause of death and disability worldwide, with the highest incidence in east asia (Gorelick, 2019). With the increase of the average life of population in China and the aging of society, the burden of stroke diseases in China has a explosive growth situation in the last 30 years, and the number of stroke patients over 40 years old is over 1200 ten thousand (Chinese stroke prevention and treatment report 2018). The stroke brings great physical and psychological pains to individuals and causes heavy economic burden to families and society. Currently, the effective methods for treating stroke clinically are intravenous thrombolysis and mechanical embolectomy, but only a small fraction (3.4% -5.2%) of patients are effectively treated due to the narrow therapeutic time window and the limited use of many factors (Donnan, Fisher, Macleod, & Davis, 2008).
The blood-brain barrier (BBB) is a tightly regulated interface between the peripheral circulation and the Central Nervous System (CNS), limiting the invasion of toxins and pathogens, protecting delicate nervous tissue, maintaining brain microenvironment homeostasis, and ensuring proper functioning of the nervous system by regulating the influx and efflux of ions, oxygen, and nutrients between blood and brain tissue (Pardridge, 2003). After stroke occurs, the permeability of the blood-brain barrier is increased, the integrity is destroyed, and a large amount of blood components enter ischemic tissues, thereby causing inflammation and edema. The extent of blood brain barrier disruption in patients with acute stroke is closely related to the prognosis of the patient (Jiang et al, 2018) and also positively related to the risk and severity of intracranial hemorrhage (ICH) following acute intravascular treatment (Ren-u et al, 2015).
Currently, there are two main types of methods for detecting the integrity of BBB: in vitro detection methods and in vivo imaging methods (Domi, Honarrvar, & Kassner, 2019). Commonly used in vitro detection methods include: evans Blue (EB) semi-quantitative analysis, immunohistochemistry and electron microscopy. EB staining is the most commonly used semi-quantitative method for evaluating BBB integrity. However, the operation process of the method is complicated and is easily interfered by human factors. The permeability of BBB can be semi-quantitatively evaluated by immunohistochemical method for detecting serum albumin and immunoglobulin IgG in blood, and morphological observation can be performed. The electron microscope can observe the change of the BBB ultrastructure, which is considered as a 'gold standard' for detecting BBB damage, but the electron microscope detection has the defects of no quantitative analysis, time and labor consumption, poor sensitivity and the like. The 3 detection methods all sacrifice experimental animals and cannot be continuously and dynamically studied.
The in vitro detection method for clinically evaluating the integrity of the BBB of the stroke patient is mainly based on biochemical indexes of cerebrospinal fluid and blood. Because of the presence of the BBB, blood and many substances in cerebrospinal fluid are greatly different, and when the BBB is destroyed, the contents of cerebrospinal fluid or blood substances change, and the magnitude of the change is related to the destruction degree of the BBB. Thus, the function and permeability of the BBB can be assessed by measuring the content of these substances in body fluids. However, such methods are indirect and the sensitivity of such indicators is different. Common methods are: 1. cerebrospinal fluid/serum albumin ratio (BBB index = cerebrospinal fluid/serum albumin) is a common and well established method of assessing blood brain barrier permeability, but it is invasive (requires puncture); in addition, since it is greatly affected by the flow of cerebrospinal fluid, the blood brain barrier permeability cannot be reliably reflected. 2. Myelin basic protein, the extent of BBB destruction was assessed by measuring myelin basic protein in blood.
The imaging method for evaluating the permeability of BBB in vivo mainly comprises the following steps: positron emission computed tomography (PET), single-photon emission computed tomography (SPECT), cranial CT perfusion (CTP), MR-PWI, and the like. PET and SPECT combined with molecular biology achieve accurate quantitative assessment of BBB permeability, but are difficult to be routine detection methods due to their radioactivity and expensive price. CTP has the characteristics of non-invasiveness, low cost, easy operation and the like, and quantitatively analyzes BBB damage to a certain extent, but has the defects of small coverage area, large radiation dose and the like. MR-PWI receives more and more attention in the BBB permeability in the field of quantitative analysis in vivo due to its advantages of non-invasiveness, non-radiativity, easy operability, repeatability, high time resolution and the like. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is a newer technology that can measure BBB leakage at all levels, including levels not seen in conventional imaging (Cramer, Simonsen, Frederiksen, Rostrup, & Larsson, 2014). However, the analysis of the DCE-MRI results is complicated and depends not only on the acquisition of image data but also on the application of pharmacokinetic models. However, the number of models available for DCE-MRI is large, and various models have different assumption conditions, which brings difficulties for comparing different technologies and methods and standardizing the technologies and methods. In general, the in-vitro imaging detection method can well make up the defects of the in-vitro detection method, and can perform noninvasive quantitative analysis on BBB destruction, so as to be beneficial to continuous dynamic research and subsequent curative effect evaluation. The application of the method is mainly limited by the cost of instruments and detection, and in addition, the methods have certain damage and potential risks to the detected objects.
Exosomes are small vesicles of diameter between 30-150 nm, originally found in sheep reticulocyte culture supernatant (Johnstone, Adam, Hammond, Orr, & Turbide, 1987). Recent studies have found that almost all cells can release exosomes (Thery, Amigorena, Raposo, & Clayton, 2006). In addition to playing an important role in cell-cell communication, these vesicles, which carry components such as host cell proteins and nucleic acids, play a role (Gyorgy, Hung, Breakefield, & Leonard, 2015; Lai & Breakefield, 2012), and their components also reflect the state of the host cell (Garcia, Otoria-Ovido, Gonzalez-King, Diez-Juan, & Sepulveda,2015), which are ideal targets for biomarker research (Galazka, Mycko, Selmaj, Raine, & Selmaj,2017; Momen-Heravi et al, 2015; Skog et al, 2008).
Mirnas are a class of single-stranded RNAs of predominantly 22 nucleotides in length that regulate gene expression by targeting the 3' UTR of the mRNA to degrade the mRNA or inhibit its translation (Bartel, 2004). There are 1,915 and 2,588 (miRbase, Release 21) mirnas that have been found in humans and mice, respectively, some of which have certain tissue specificity. Exosomes are the predominant form of body fluid mirnas. These host cell-derived mirnas have high stability due to protection of the exosome vesicle structure. Mirnas in serum exosomes are important subjects for current clinical marker studies (Creemers, Tijsen, & Pinto, 2012).
Disclosure of Invention
The technical problem to be solved by the invention is to provide the application of the serum exosome miR-410-3p related to the blood brain barrier damage degree after cerebral ischemia and the detection method of the expression level thereof, and prove that the serum exosome miR-410-3p can be used as a biomarker for evaluating the blood brain barrier damage degree after cerebral ischemia.
In order to solve the technical problems, the embodiment of the invention provides a serum exosome miR-410-3p related to the degree of blood brain barrier destruction after cerebral ischemia, and the sequence of the serum exosome miR-410-3p is AATATAACACAGATGGCCTGT.
The invention also provides application of the serum exosome miR-410-3p related to the blood brain barrier damage degree after cerebral ischemia, and the application is used for evaluating a biomarker of the blood brain barrier damage degree after cerebral ischemia.
The invention also provides a method for detecting the expression level of the blood-brain barrier damage degree related blood-brain barrier miR-410-3p of the blood serum exosome after cerebral ischemia, which comprises the following steps:
(1) establishing a mouse middle cerebral artery embolism model, performing ischemia reperfusion, and collecting serum;
(2) separating exosomes in serum;
(3) the real-time fluorescence quantitative polymerase chain reaction technology is used for detecting the expression level of miR-410-3p in the serum exosomes at different recovery stages and cerebral ischemia-reperfusion injury.
Wherein, the specific steps of the step (1) are as follows:
(1-1) 10 weeks old male C57BL/6 mice, 4% isoflurane induced anesthesia, 2% isoflurane maintenance; separating the left common carotid artery under a stereomicroscope, respectively tying a dead knot at the proximal end of the CCA, tying a loose knot at the distal end, cutting a cut between the two knots, inserting a thread plug at the cut with the depth of 10mm, fastening the loose knot, and taking out the thread plug for reperfusion after 60min of ischemia; sham group used the same surgical procedure but did not cut the vessel;
(1-2) detecting the volume of infarction caused by ischemia by TTC staining, anesthetizing and killing a mouse, quickly dissecting and taking out brain tissue, and performing coronary section by using a mould with the thickness of 2 mm; sections were incubated in 1% TCC dye at 37 ℃ for 30min and fixed with 4% paraformaldehyde; scanning and recording, and carrying out Image analysis by using Image J software;
infarct ratio = area of contralateral half-ipsilateral non-infarcted area/contralateral half-brain area 100%;
(1-3) after the mouse dies in an excessive anesthesia, collecting blood from the left ventricle, standing the collected whole blood at 37 ℃ for 30min, standing at 4 ℃ for 1h, centrifuging at 3000 g for 10min to separate serum, wherein the normal serum is amber, discarding the sample with obvious hemolysis, carefully transferring the serum into a new EP tube, centrifuging at 4 ℃ and 20000 g for 10min, transferring the supernatant into a new EP tube, marking, and storing at-80 ℃.
Wherein, the specific steps of the step (2) are as follows:
(2-1) separating exosomes in serum by using an ExoQuck kit, namely melting the serum stored at the temperature of-80 ℃ on ice, centrifuging at the temperature of 21000g for 15min at the temperature of 4 ℃, transferring the supernatant into a new EP tube, adding 1/4 volumes of ExoQuick solution, slightly reversing and uniformly mixing, incubating at the temperature of 4 ℃ for 2h, centrifuging at the temperature of 1500g for 30min, removing the supernatant, and keeping precipitates for later use;
(2-2) detection of the particle size and the quantity of the serum exosomes: analyzing the number and the particle size distribution of exosomes in a sample by using Nanoparticle-packing analysis (NTA); resuspend the pellet with 30 μ l particle-free PBS and dilute 1000 times before testing;
(2-3) observing the form of the serum exosome by using a lens electron microscope: preparing an electron microscope observation sample, resuspending the precipitate with 30 mul PBS, and adding equivalent 4% w/v paraformaldehyde for fixation; dripping 10 mu l of the fixed exosome solution onto a Formvar/carbon-coated nickel net, air-drying for 20min, washing for several times by PBS, fixing for 5min by 1% v/v glutaraldehyde, washing for several times by pure water, dyeing for 5min by 4% w/v uranium acetate, embedding by mixed liquid of uranium acetate and methyl cellulose, absorbing the redundant liquid by filter paper, air-drying for later use, and observing the form and size of exosome by a transmission electron microscope;
(2-4) Westen blot detection of exosome markers: extracting exosome protein by using RIPA lysate, determining protein concentration by using BCA, separating protein by 12% SDS-PAGE, wherein the sample amount of each channel is 40 microgram of total protein, transferring the protein onto a PVDF membrane by a wet transfer method, sealing 5% skimmed milk powder at room temperature for 1h, respectively adding CD63, CD9 and CD81, sealing overnight at 4 ℃, washing 3 times by TBST, adding HRP-labeled 2 antibody, incubating at room temperature for 1h, enhancing the color development by a chemiluminescence method, and recording by using a film.
Wherein, the specific steps of the step (3) are as follows:
and detecting the expression level of miR-410-3p in the serum exosome by adopting a miRNA qRT-PCR kit based on a poly-A tailing method and a real-time quantitative PCR instrument.
The technical scheme of the invention has the following beneficial effects:
the technical scheme of the invention proves that the expression trend of miR-410-3p in the serum exosome is basically consistent with the BBB injury and repair process, and the serum exosome miR-410-3p can be used as a biomarker for judging cerebral ischemic injury.
Drawings
FIG. 1 is a schematic diagram showing the comparison of infarct sizes of a model group and a pseudo-surgical group in the present invention;
FIG. 2 is a graph showing the results of the isolation and identification of the serum exosomes of the present invention;
FIG. 3 is a comparison graph of the extent of blood brain barrier disruption measured by Evans blue at different times after cerebral ischemia-reperfusion according to the present invention;
FIG. 4 is a graph showing the dynamic changes of serum exosomes during ischemia-reperfusion injury and recovery in the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.
The invention provides a serum exosome miR-410-3p related to the blood brain barrier damage degree after cerebral ischemia, and the sequence of the serum exosome miR-410-3p is AATATAACACAGATGGCCTGT.
The application of the serum exosome miR-410-3p provided by the invention can be used for evaluating biomarkers of blood brain barrier destruction degree after cerebral ischemia.
The invention also provides a method for detecting the expression level of the blood-brain barrier damage degree related blood-brain barrier miR-410-3p of the blood serum exosome after cerebral ischemia, which comprises the following steps:
(1) establishing a mouse middle cerebral artery embolism model, performing ischemia reperfusion, and collecting serum;
wherein, the specific steps of the step (1) are as follows:
(1-1) 10 weeks old male C57BL/6 mice, 4% isoflurane induced anesthesia, 2% isoflurane maintenance; separating the left common carotid artery under a stereomicroscope, respectively tying a dead knot at the proximal end of the CCA, tying a loose knot at the distal end, cutting a cut between the two knots, inserting a thread plug at the cut with the depth of 10mm, fastening the loose knot, and taking out the thread plug for reperfusion after 60min of ischemia; sham group used the same surgical procedure but did not cut the vessel;
(1-2) detecting the volume of infarction caused by ischemia by TTC staining, anesthetizing and killing a mouse, quickly dissecting and taking out brain tissue, and performing coronary section by using a mould with the thickness of 2 mm; sections were incubated in 1% TCC dye at 37 ℃ for 30min and fixed with 4% paraformaldehyde; scanning and recording, and carrying out Image analysis by using Image J software;
infarct ratio = area of contralateral half-ipsilateral non-infarcted area/contralateral half-brain area 100%;
(1-3) after the mouse dies in an excessive anesthesia, collecting blood from the left ventricle, standing the collected whole blood at 37 ℃ for 30min, standing at 4 ℃ for 1h, centrifuging at 3000 g for 10min to separate serum, wherein the normal serum is amber, discarding the sample with obvious hemolysis, carefully transferring the serum into a new EP tube, centrifuging at 4 ℃ and 20000 g for 10min, transferring the supernatant into a new EP tube, marking, and storing at-80 ℃.
(2) The exosome in the serum is separated, and the specific steps are as follows:
(2-1) thawing serum stored at-80 ℃ on ice, centrifuging at 21000g and 4 ℃ for 15min, transferring the supernatant to a new EP tube, adding 1/4 volumes of ExoQuick solution, slightly reversing and uniformly mixing, incubating at 4 ℃ for 2h, centrifuging at 1500g for 30min, removing the supernatant, and keeping the precipitate for later use; separating exosomes in serum by using an Exoquick kit;
(2-2) analysis of particle size and concentration of exosomes: the number and size distribution of the particles were analyzed using Nanoparticle-tracking analysis (NTA); resuspend the pellet with 30 μ l particle-free PBS and dilute 1000 times before testing;
(2-3) observing the form of the exosome by using a lens electron microscope: preparing an electron microscope observation sample, resuspending the precipitate with 30ul PBS, and adding equivalent 4% w/v paraformaldehyde for fixation; dripping 10ul of the fixed exosome solution onto a Formvar/carbon-coated nickel net, air-drying for 20min, washing with PBS for several times, fixing for 5min with 1% v/v glutaraldehyde, washing with pure water for several times, dyeing for 5min with 4% w/v uranium acetate, embedding with mixed liquid of uranium acetate and methyl cellulose, absorbing the redundant liquid with filter paper, air-drying for later use, and observing the form and size of exosome by a transmission electron microscope;
(2-4) Westen blot detection of exosome markers: extracting exosome protein by using RIPA lysate, determining the protein concentration by using BCA, separating the protein by 12% SDS-PAGE, wherein the sample amount of each channel is 40 microgram of total protein, transferring the protein onto a PVDF membrane by a wet transfer method, sealing 5% skimmed milk powder at room temperature for 1h, respectively adding CD63, CD9 and CD81, sealing overnight at 4 ℃, washing 3 times by TBST, adding HRP-labeled 2 antibody (1: 1000), incubating at room temperature for 1h, enhancing the color development by a chemiluminescence method, and recording by using a film; NTA analysis shows that the particle sizes of the separated exosomes are not uniform and are mainly distributed near 100nm, which indicates that the serum exosomes are successfully separated.
(3) Detecting the expression level of miR-410-3p in the serum exosomes at different stages of cerebral ischemia-reperfusion injury and recovery by using a real-time fluorescent quantitative Polymerase Chain Reaction (PCR) technology.
Wherein, the specific steps of the step (3) are as follows:
and detecting the expression level of miR-410-3p in the serum exosome by adopting a miRNA qRT-PCR kit based on a poly-A tailing method and a real-time quantitative PCR instrument.
The change trend of miR-410-3p in serum exosomes after cerebral ischemia is consistent with BBB injury and recovery processes by combining with the specific embodiment, and the injury degree of BBB can be evaluated by detecting the expression condition of miR-410-3p in serum exosomes as the injury of BBB begins to increase and the recovery of BBB decreases.
Firstly, establishing a mouse Middle Cerebral Artery Occlusion (MCAO) ischemia reperfusion model
Male C57BL/6 mice, 10 weeks old, were anesthetized with 4% isoflurane induction and maintained with 2% isoflurane. The left Common Carotid Artery (CCA) was isolated under a stereomicroscope and a dead knot was tied proximal to the CCA and a loose knot was tied distal to the CCA, respectively. Then, a cut is cut between the two knots, a thread plug is inserted into the cut with the depth of about 10mm, the slipknot is fastened, and the blood is ischemic for 60min and then is perfused to form a model group (MCAO). The Sham group (Sham group) used the same procedure but did not cut the vessels.
TTC staining was used to detect the volume of infarct caused by ischemia, mice were sacrificed under anesthesia, brain tissue was rapidly dissected and removed, and coronal sections were performed using a mold with a thickness of 2 mm. Sections were incubated in 1% TCC dye at 37 ℃ for 30min and fixed with 4% paraformaldehyde. Scan record, Image J software for Image analysis. Infarct ratio = area of contralateral half-ipsilateral non-infarcted area/contralateral half-brain area 100%. As shown in FIG. 1, P < 0.001, wherein FIG. 1A shows TTC staining pattern, normal tissue was stained red and infarct tissue was stained white, and FIG. 1B shows statistical results of infarct volume in mice, and the infarct volume in MCAO group was 51.24% + -7.89%.
Second, separation and identification of serum exosomes
2.1, thawing the serum stored at minus 80 ℃ on ice, and centrifuging for 15min at 21000g and 4 ℃; the supernatant was transferred to a new EP tube, and 1/4 volumes of ExoQuick solution were added and mixed by gentle inversion, incubated at 4 ℃ for 2h, centrifuged at 1500g for 30min, the supernatant was removed and the pellet was kept for further use. Exosomes were isolated from serum using the Exoquick kit (System Biosciences inc., mountain view, CA, USA) from SBI corporation.
2.2, analyzing the particle size and concentration of exosome: the number of particles and the size distribution were analyzed by Nanoparticle-tracking analysis (NTA), and the pellet was resuspended in 30. mu.l of particle-free PBS and diluted 1000-fold before testing.
2.3, observing the form of the exosome by a transmission electron microscope: electron microscopy samples were prepared as described by Thery (Thery et al, 2006) et al, resuspended in 30ul PBS and the pellet, fixed with an equal volume of 4% w/v paraformaldehyde, 10ul of the fixed exosome solution was dropped onto a Formvar/carbon-coated nickel mesh and air dried for 20 min. After washing with PBS several times, fixing with 1% v/v glutaraldehyde for 5min, washing with pure water several times, dyeing with 4% w/v uranium acetate for 5min, embedding with mixed liquid of uranium acetate and methyl cellulose, blotting excess liquid with filter paper and air drying for use. The morphology and size of the exosomes were observed by transmission electron microscopy (JEM-2100 JEOL, Tokyo, Japan).
2.4 Westen blot detection of exosome markers: exosome proteins were extracted using RIPA (Pierce, USA) lysates and protein concentrations were determined by BCA (Pierce, USA). Proteins were separated by 12% SDS-PAGE, and the amount of sample per lane was 40 μ g total protein. The proteins were transferred to PVDF membrane by wet transfer, blocked with 5% skimmed milk for 1h at room temperature, separately added with CD9 (Abcam, USA) and CD81 (Abcam, USA) antibodies (1: 1000), blocked overnight at 4 deg.C, washed 3 times with TBST, added with HRP-labeled 2-antibody (1: 1000), incubated for 1h at room temperature, Enhanced Chemiluminescence (ECL) (Pierce, USA), and recorded on film.
Because the particle size of exosome is extremely small, the traditional ultracentrifugation method is time-consuming and labor-consuming and has low separation efficiency, and an exotick method (Thery et al, 2006) for precipitating exosome in solution by using a high-molecular polymer is adopted. The serum exosomes obtained by this method in this experiment were approximately round, partially concave, with particle size below 100nm (fig. 2A and 2D), and expressed marker molecules for exosomes such as CD 9-and CD81 (fig. 2C). SDS-PAGE and Coomassie blue staining showed that mouse serum and exosome-depleted serum had similar protein expression profiles, but differed greatly from those of extracellular vesicles (FIG. 1B). Further NTA analysis showed that the size of the isolated exosomes was not uniform, mainly distributed around 100nm (fig. 2E), indicating successful isolation of human serum exosomes. Fig. 2 is a graph showing the results of separation and identification of serum exosomes, in which fig. 2A is a mixture of ExoQuick and mouse serum and centrifuged to obtain a vesicle-containing pellet, fig. 2B is SDS-PAGE of proteins in mouse serum, exosome-depleted serum and exosomes, fig. 2C is Western blot detection of exosome marker proteins CD9 and CD81, fig. 2D is a TEM electron micrograph of ExoQuick pellet, fig. 2E is a particle size distribution and concentration of NTA detection particles, and fig. 2F is a Bioanalyzer analysis of length distribution and concentration of RNA in serum exosomes.
III, Evans blue staining for detecting integrity of BBB in ischemia reperfusion at different time
Mice were subjected to ischemia reperfusion for 0.5d, 1d, 3d and 7d, and then injected with 2% Evans blue solution intravenously in orbit at a dose of 4mL/kg, and then subjected to isoflurane anesthesia after 2h injection. The heart was perfused with PBS to drain the circulating system of Evans blue dye until the outflow was colorless, and the brain was removed by cutting the head and photographed.
As shown in fig. 3, evans blue staining results showed no significant leakage of BBB at 12h of ischemia reperfusion, increasing gradually at 1d, with the most leakage at 3d and decreasing at 7 d.
Fourthly, serum exosome RNA extraction and real-time quantitative PCR detection
Serum exosomes are extracted by a trizol method, Cel-mir-39 is added as an external reference in the extraction process, and an ultra-micro ultraviolet spectrophotometer is used for quantitative determination and purity determination. MiDETECT A Track based on poly-A tailing method is adoptedTMThe miRNA qRT-PCR kit (Rugby, Guangzhou Sharp) and the CFX 96 PCR instrument (Biorad) detect the miR-410-3 p.
FIG. 4 is a graph showing the dynamic changes of miR-410-3p in serum exosomes during ischemia-reperfusion injury and recovery in the present invention.
miR-410-3p in mouse serum exosomes subjected to ischemia-reperfusion for 12h begins to increase, the ischemia-reperfusion reaches a peak value in 3 days, is remarkably reduced in 7 days, and is reduced to a level close to a normal level in 14 days. The expression trend is basically consistent with BBB damage and repair processes, so that the expression trend can be used as a biomarker reflecting BBB damage.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (6)
1. A serum exosome miR-410-3p related to the degree of blood brain barrier destruction after cerebral ischemia, wherein the sequence of the serum exosome miR-410-3p is AATATAACACAGATGGCCTGT.
2. The application of a serum exosome miR-410-3p related to the blood brain barrier damage degree after cerebral ischemia is characterized in that the application is used as a biomarker for evaluating the blood brain barrier damage degree after cerebral ischemia.
3. A method for detecting the expression level of a serum exosome miR-410-3p related to the degree of blood brain barrier destruction after cerebral ischemia is characterized by comprising the following steps:
(1) establishing a mouse middle cerebral artery embolism model, performing ischemia reperfusion, and collecting serum;
(2) separating exosomes in serum;
(3) and detecting the expression level of miR-410-3p in the serum exosomes at different stages of cerebral ischemia-reperfusion injury and recovery by using a real-time fluorescence quantitative polymerase chain reaction technology.
4. The method for detecting the expression level of the serum exosome miR-410-3p related to the degree of blood brain barrier destruction after cerebral ischemia according to claim 3, wherein the specific steps in the step (1) are as follows:
(1-1) 10 weeks old male C57BL/6 mice, 4% isoflurane induced anesthesia, 2% isoflurane maintenance; separating the left common carotid artery under a stereomicroscope, respectively tying a dead knot at the proximal end of the CCA, tying a loose knot at the distal end, cutting a cut between the two knots, inserting a thread plug at the cut with the depth of 10mm, fastening the loose knot, and taking out the thread plug for reperfusion after 60min of ischemia; sham group used the same surgical procedure but did not cut the vessel;
(1-2) detecting the volume of infarction caused by ischemia by TTC staining, anesthetizing and killing a mouse, quickly dissecting and taking out brain tissue, and performing coronary section by using a mould with the thickness of 2 mm; sections were incubated in 1% TCC dye at 37 ℃ for 30min and fixed with 4% paraformaldehyde; scanning and recording, and carrying out Image analysis by using Image J software;
infarct ratio = area of contralateral half-ipsilateral non-infarcted area/contralateral half-brain area 100%;
(1-3) after the mouse dies in an excessive anesthesia, collecting blood from the left ventricle, standing the collected whole blood at 37 ℃ for 30min, standing at 4 ℃ for 1h, centrifuging at 3000 g for 10min to separate serum, wherein the normal serum is amber, discarding the sample with obvious hemolysis, carefully transferring the serum into a new EP tube, centrifuging at 4 ℃ and 20000 g for 10min, transferring the supernatant into a new EP tube, marking, and storing at-80 ℃.
5. The method for detecting the expression level of the serum exosome miR-410-3p related to the degree of blood brain barrier destruction after cerebral ischemia according to claim 3, wherein the specific steps in the step (2) are as follows:
(2-1) separating exosomes in serum by using an ExoQuck kit, namely melting the serum stored at the temperature of-80 ℃ on ice, centrifuging at the temperature of 21000g for 15min at the temperature of 4 ℃, transferring the supernatant into a new EP tube, adding 1/4 volumes of ExoQuick solution, slightly reversing and uniformly mixing, incubating at the temperature of 4 ℃ for 2h, centrifuging at the temperature of 1500g for 30min, removing the supernatant, and keeping precipitates for later use;
(2-2) detection of the particle size and the quantity of the serum exosomes: analyzing the number and the particle size distribution of exosomes in a sample by adopting Nanoparticle-packing analysis; resuspend the pellet with 30 μ l particle-free PBS and dilute 1000 times before testing;
(2-3) observing the form of the serum exosome by using a lens electron microscope: preparing an electron microscope observation sample, resuspending the precipitate with 30 mul PBS, and adding equivalent 4% w/v paraformaldehyde for fixation; dripping 10 mu l of the fixed exosome solution onto a Formvar/carbon-coated nickel net, air-drying for 20min, washing for several times by PBS, fixing for 5min by 1% v/v glutaraldehyde, washing for several times by pure water, dyeing for 5min by 4% w/v uranium acetate, embedding by mixed liquid of uranium acetate and methyl cellulose, absorbing the redundant liquid by filter paper, air-drying for later use, and observing the form and size of exosome by a transmission electron microscope;
(2-4) Westen blot detection of exosome markers: extracting exosome protein by using RIPA lysate, determining protein concentration by using BCA, separating protein by 12% SDS-PAGE, wherein the sample amount of each channel is 40 microgram of total protein, transferring the protein onto a PVDF membrane by a wet transfer method, sealing 5% skimmed milk powder at room temperature for 1h, respectively adding CD63, CD9 and CD81, sealing overnight at 4 ℃, washing 3 times by TBST, adding HRP-labeled 2 antibody, incubating at room temperature for 1h, enhancing the color development by a chemiluminescence method, and recording by using a film.
6. The method for detecting the expression level of the serum exosome miR-410-3p related to the degree of blood brain barrier destruction after cerebral ischemia according to claim 3, wherein the specific steps in the step (3) are as follows:
and detecting the expression level of miR-410-3p in the serum exosome by adopting a miRNA qRT-PCR kit based on a poly-A tailing method and a real-time quantitative PCR instrument.
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