CN107287199A - Applications of the MicroRNA 29a 5p in treatment acute cerebral ischemia and cerebral ischemia re-pouring injured medicine is prepared - Google Patents
Applications of the MicroRNA 29a 5p in treatment acute cerebral ischemia and cerebral ischemia re-pouring injured medicine is prepared Download PDFInfo
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Abstract
The invention discloses a kind of applications of miR 29a 5p in treatment acute cerebral ischemia and cerebral ischemia re-pouring injured medicine is prepared, the miR 29a 5p have such as SEQ ID No:1 and SEQ ID No:Base sequence shown in 2.Present invention discover that miR 29a 5p are the potential target spots of acute ischemic stroke neuroprotection; up-regulation miR 29a 5p are to improve a kind of neuroprotection new strategy of acute cerebral ischemia revascularization therapeutic effect; the present invention treats acute cerebral ischemia and cerebral ischemia re-pouring injured there is provided new method for miR 29a 5p, the innovative means of reperfusion injury after again leading to as treatment acute ischemic stroke intravascular intervention.
Description
Technical field
It is the invention belongs to beyond body nucleic acid detection technique field, specially microRNA 29a-5p and its anxious preparing treatment
Application in property cerebral ischemia and cerebral ischemia re-pouring injured medicine.
Background technology
Cerebral apoplexy is to cause one of main causes of death in world wide, is also the master for causing long-lasting nerve dysfunction
Want reason.Cerebral apoplexy becomes increasingly conspicuous to the harmfulness of Chinese population health, and China human mortality health field is faced with palsy and brought
Serious challenge.During the examination of China national palsy investigates newest result of study discovery 2002 to 2013 years, 40 years old people of China
The stroke prevalence rate and the incidence of disease of group is in rapid growth, and there is trend (Guan T, Ma J, the Li M, et of rejuvenation
al.Rapid transitions in the epidemiology of stroke and its risk factors in
China from 2002to 2013.Neurology.2017May 31.[Epub ahead of print])。
It is acute ischemic stroke to be injected intravenously tissue plasminogen activator (tPA) and carry out medicine thromboembolism treatment
First-line treatment, but therapeutic time window limits to, recanalization rate is relatively low, and benefited patient crowd is less.Most middle severe soldier
Middle patient loses the therapy apparatus meeting of early stage revascularization, so that the state of an illness enters one because tPA thrombolysis is avoided or tPA thrombolysis is invalid
Step is aggravated, and serious cerebral infarction is occurred, is caused death or handicap.Carrying out intervention using endovascular technology takes bolt to treat
It is an important advance in Treatment of Cerebral Stroke field in recent years, multiple association of country such as the current U.S., Europe and China are all with most
It is high-grade to recommend intravascular intervention support to take bolt technology to be applied to controlling for acute ischemic stroke caused by intracranial vessel occlusion
Treat.
Intravascular intervention takes bolt treatment to significantly improve the recanalization rate of intracranial vessel occlusion, but obtains and be satisfied with blood vessel
Logical patients with acute ischemic stroke is not that can finally obtain good clinical therapeutic efficacy again.Closed from the cerebrovascular
Plug intracranials hemorrhage conversion to caused by after the time delay and reperfusion as treatment of revascularization and ischemical reperfusion injury is acute
Ischemic Stroke intervention takes the major reason of " invalid revascularization " after bolt treatment.In recent years, increasing sufficient evidence table
Clear intravascular intervention takes bolt to lead to a kind of effective treatment method that technology is acute ischemic stroke again.
Reperfusion injury and conversion of intracranialing hemorrhage further have aggravated brain damage caused by after revascularization treatment, cause portion
Divide the apoplexy patient clinical prognosis of revascularization poor.Intravascular intervention support takes bolt to lead to treatment again and the big blood vessel of encephalic occlusion is produced
Quickly lead to again, cause the vescular bed moment Reperfu- sion of ischemic, but machinery causes ischemical reperfusion injury and cranium after leading to treatment again
The mechanism of internal haemorrhage conversion is not yet illustrated.Research finds commenting for blood marker before treatment, neuroimaging and prediction scoring
Estimating helps to formulate the decision-making that intervention takes bolt to lead to treatment again, also can determine whether the risk of conversion of being intracranialed hemorrhage after revascularization treatment.
But, it there is no reliable method prediction intervention to take the generation of reperfusion injury and conversion of intracranialing hemorrhage after bolt treatment at present.In addition,
Although by lasting research for many years, effectively treatment cerebral ischemia clinically still being lacked at present and mitigates ischemical reperfusion injury
Nerve protection medicine.The generation of reperfusion injury and conversion of intracranialing hemorrhage after bolt treatment is taken by carrying out and illustrating intravascular intervention
Mechanism, early warning and prophylactico-therapeutic measures, will be expected to improve the revascularization therapeutic effect of acute ischemic stroke.
MicroRNAs is a single-stranded microRNA of class endogenous non-coding, is about 20-24 nucleotides, by with target base
Because of the non-translational region complementary pairing of mRNA molecules, degraded mRNA or suppression mRNA translations, so as to participate in the table after target gene transcription
Up to regulation and control.One microRNA generally can adjust multiple target gene functions, and a gene may also be adjusted by multiple microRNA.
The regulating networks of interaction composition complexity between MicroRNA and protein coding gene.
Research finds that microRNAs participates in Ischemic Stroke and cerebral ischemia re-pouring injured occurrence and development.Star glue
Cell plastid is a kind of cell type most abundant in brain, and it plays important in terms of normal nervous system normal function is maintained
Effect, including maintain ion and acid-base balance, regulation nerve conduction and maintain neuronal energy deposit (Clarke LE,
Barres BA.Emerging roles of astrocytes in neural circuit development.Nat Rev
Neurosci.2013；14(5):311-321.).
Research in the past finds that microRNA-29 is main and expressed in astroglia.MicroRNA-29 families by
Tri- member compositions of microRNA-29a, microRNA-29b and microRNA-29c, and cluster be distributed in two it is different
Gene loci, microRNA-29a/b-1 is located at No. 6 chromosomes of mouse, No. 13 chromosomes of rat, No. 7 chromosomes of the mankind respectively,
MicroRNA-29c/b-2 is located at mouse, No. 1 chromosome of the mankind, No. 4 chromosomes of rat respectively.Research finds miR-29a/b-1
Expression is lowered in Alzheimer's disease patient's brain tissue, miR-29b and miR-29c are in people and mouse Huntington's disease brain group
Knit middle lower and express (Karnati HK, Panigrahi MK, Gutti RK, et al.miRNAs:Key players in
neurodegenerative disorders and epilepsy.J Alzheimers Dis.2015；48(3):563-
580.).Suppress the expression of microRNA-29 in Mice brain tissues, neurological dysfunction and Neuronal cell death will be caused
(Roshan R,Shridhar S,Sarangdhar MA,et al.Brain-specific knockdown of miR-
29results in neuronal cell death and ataxia in mice.RNA.2014；20(8):1287-
1297.)
In Cerebral Ischemia-reperfusion in Mice damage model, up-regulation microRNA-29a-3p is by targeting in rush apoptosis
Bcl2 family genes change, and are played a protective role to cerebral ischemia re-pouring injured.In astrocytes cell experiment,
MicroRNA-29a simulants alleviate the damage that ischemic stress be to astroglia, suppress microRNA-29a and result in star
Shape glial cell death (Ouyang YB, Xu L, Lu Y, et al.Astrocyte-enriched miR-29a targets
PUMA and reduces neuronal vulnerability to forebrain ischemia.Glia.2013；61
(11):1784-1794.).Up-regulation microRNA-29a-3p can reduce the star that sugar deprives rear Hippocampal CA 1 and dentate fascia region
Shape spongiocyte damages (Stary CM, Sun X, Ouyang Y, et al.miR-29a differentially regulates
cell survival in astrocytes from cornu ammonis 1and dentate gyrus by
targeting VDAC1.Mitochondrion.2016；30:248-254.).Research finds cell caused by arachidonic acid
The missing of glutathione causes nerve cell death to be caused by the miR-29b relied on by 12- LOXs is lacked.It is exogenous to mend
Filling miR-29b simulants has neuroprotection, and miR-29b inhibitor has aggravated cell death.(Khanna S,Rink
C,Ghoorkhanian R,et al.Loss of miR-29b following acute ischemic stroke
contributes to neural cell death and infarct size.J Cereb Blood Flow
Metab.2013；33(8):1197-1206.).
Existing patent report prompting microRNA-29a-5p turns in tumor in digestive tract such as colorectal cancer early detection and at a distance
Application in shifting, and drug development values of the microRNA-29a-3p in the migration and invasion and attack for suppressing glioma cell.But
It is that there is no patent report on microRNA-29a-5p in acute cerebral ischemia and cerebral ischemia re-pouring injured medicine so far
Using.
The content of the invention
Based on conventional research and report, in order to overcome the defect of above-mentioned prior art, the invention provides one kind
MicroRNA (miR), i.e. miR-29a-5p, and it is in treatment acute cerebral ischemia and cerebral ischemia re-pouring injured medicine is prepared
Application.
In order to realize foregoing invention purpose, this invention takes following technical scheme：
A kind of miR-29a-5p, the miR-29a-5p have such as SEQ ID No:1 and SEQ ID No:Base shown in 2
Sequence.
Treatment acute cerebral ischemia and cerebral ischemia re-pouring injured medicine are being prepared present invention also offers above-mentioned miR-29a-5p
Application in thing.
The present invention is in acute cerebral ischemia and the brain tissue and blood of cerebral ischemia re-pouring animal model based on miR-29a-5p
Downward expression in liquid, and miR-29a-5p target genes nadph oxidase (NOX) 4 and Caspase-3 are in above-mentioned animal model brain
Up-regulated expression in tissue and make, compared with prior art, the invention has the advantages that：
1st, present invention demonstrates the direct target gene that NOX4 is miR-29a-5p；
2nd, in rat pattern of ischemia reperfusion, intravenous administration miR-29a-5p activators after intravascular machinery leads to again,
Inhibit the downward of miR-29a-5p in rat cerebral tissue to express, alleviate the early stage after acute cerebral ischemia and cerebral ischemia re-pouring
Encephaledema formation and blood-brain barrier disruption, reduce cerebral infarct volume, reduce the death rate after cerebral ischemia and ischemia-reperfusion
And neurological dysfunction；
3rd, exogenous miR-29a-5p activators of giving inhibit acute cerebral ischemia and brain after ischemia reperfusion NOX4
With Caspase-3 genes and the up-regulated expression of protein level, and nerve is played by regulating and controlling oxidative stress and Apoptosis Mechanism
Protective effect；
4th, miR-29a-5p is the potential target spot of acute ischemic stroke neuroprotection, and up-regulation miR-29a-5p is to improve anxious
Property cerebral ischemia revascularization therapeutic effect a kind of neuroprotection new strategy, the present invention for miR-29a-5p treat acute cerebral ischemia
New method is provided with cerebral ischemia re-pouring injured, will turn into after treatment acute ischemic stroke intravascular intervention leads to again and fill again
Note the innovative means of damage.
Brief description of the drawings
Fig. 1 is influence result figures of the miR-29a-5p in test example 1 to 3 ' UTR NOX4 uciferase activities；
Fig. 2 is injection miR-29a-5p activators reduction acute cerebral ischemia in rats encephaledema content in the medium sized vein of test example 2；
Fig. 3 is that injection miR-29a-5p activators are broken to the blood-brain barrier of acute cerebral ischemia in rats in the medium sized vein of test example 3
Bad improvement result；
Fig. 4 is injection miR-29a-5p activators reduction acute cerebral ischemia in rats cerebral infarct volume in the medium sized vein of test example 4；
Fig. 5 is miR-29a-5p in test example 5 in rat blood (A) and ischemic side peri-infarct region brain tissue (B)
Expression figure；
Fig. 6 is qRT-PCR and Western blot analyzing rat ischemic sides peri-infarct region brain tissue in test example 6
NOX4mRNA and albumen expression；
Fig. 7 is qRT-PCR and Western blot analyzing rat ischemic sides peri-infarct region brain tissue in test example 6
Caspase-3mRNA and albumen expression；
Immuning fluorescent dyeing analysis NOX4 is in ischemic side peri-infarct region brain tissue neuron and star in Fig. 8 test examples 7
Expression in spongiocyte；
Fig. 9 is the situ hybridization analysis microRNA-29a-5p of test example 7 in ischemic side peri-infarct region brain tissue star
Quantitative expression in spongiocyte；
Figure 10 is the expression of DHE fluorescence probes dyeing ischemic side peri-infarct region brain tissue superoxide anion in test example 7
Difference；
Figure 11 is the table of DCFH-DA fluorescence probes dyeing ischemic side peri-infarct region brain tissue hydrogen peroxide in test example 7
Up to difference；
Figure 12 be test example 7 in TUNEL/NeuN or GFAP staining analysis ischemic sides peri-infarct region brain tissue neuron and
Astrocyte apoptosis degree；
Figure 13 is the injection miR-29a-5p activator reduction acute cerebral ischemia in rats death rates (A) in the medium sized vein of test example 8
With the nervous function state (B) of rat after improvement ischemia-reperfusion.
Embodiment
Described in detail below in conjunction with the drawings and specific embodiments in the present invention, following examples unless otherwise specified, institute
Commercially available, to use method to be well known to those skilled in the art conventional practices are derived from using raw material.
In embodiment all data using the softwares of SPSS 20.0 carry out statistical analysis, all measurement datas with mean ±
Standard deviationRepresent, whether be in normal distribution using single sample KS test and judges measurement data；Normal distribution data is used
One-way analysis of variance is examined, and Non-Gaussian Distribution data is analyzed using Mann-Whitney non-parametric tests.Enumeration data is adopted
With Fisher is accurate or Chi-square Test.P value thinks that difference has statistical significance less than 0.05.
The miR-29a-5p activator sequences of embodiment 1
Sequence is as shown in the table.
Relation between the miR-29a-5p of test example 1 and NOX4
Using the relation between Dual-Luciferase active technologies clear and definite NOX4 and miR-29a-5p.
MiR-29a-5p simulants (mimics) (Shanghai Ji Ma pharmaceutical technologies Technology Co., Ltd.) are respectively with carrying fluorescence
Wild type and saltant type NOX4-3 ' UTR (Shanghai and first Bioisystech Co., Ltd) common transfection HEK 293T cells of plain enzyme,
Observe influences of the miR-29a-5p to NOX4 luciferase reporter genes activity.
1st, microRNA and plasmid co-transfection cell
24h before transfection, 293T cells are inoculated on 96 orifice plates, set 6 multiple holes.On the transfection same day, prepare DNA (NOX4)
And transfection reagent, per boring ratio, example is 0.2 μ g Flucs carriers (Firefly), 0.01 μ g sea pansy fireflies during transfected plasmids
Light element zymophore (Renilla), 0.25 μ l transfection reagents.Prepare miR-29a-5p simulants and transfection reagent dilution, miR-
The final concentration of 100nM of 29a-5p simulants, transfection reagent is per the μ L of hole 0.25.By the DNA diluted (NOX4) and miR-29a-
5p simulants are mixed with transfection reagent respectively, and normal temperature stands 20 minutes.Culture medium is discarded per hole, is added respectively per hole cell sample
25 μ L DNA (NOX4) transfection cocktails and 25 μ L miR-29a-5p simulants.After transfection 6 hours, fresh complete culture is changed
Base.
2nd, double reporter gene detections
The operating procedure detection luciferase element activity provided according to the plain detection kit of double luciferases of Promega companies,
Transfect after 48h, PBS is washed 2 times.Cell cracking is carried out with PLB (Passive Lysis Buffer).10 μ L cell pyrolysis liquids are added
Enter white 96 hole elisa Plates, 50 μ L luciferase assay reagents II (Luciferase Assay mixed in advance are added per hole
Reagent II, LAR II), it is quick to mix, stand readings after 2s.Then 50 μ L Stop＆Glo are added in the sample of every hole
Reagent, after quick mixing, is put into luminometer, after static 2 seconds, measurement data, parallel statistical analysis.
3rd, miR-29a-5p regulates and controls direct target gene NOX4 determination
MiR-29a-5p simulants transfect HEK 293T cells jointly with luciferase NOX4-3 ' UTR, with wild type
NOX4-3 ' UTR groups are compared, and miR-29a-5p simulant group uciferase activity ratios are remarkably decreased (P<0.001).It is prominent with two
Modification NOX4-3 ' UTR groups are compared, and miR-29a-5p simulant group uciferase activity ratios are remarkably decreased (P<0.001；P<
0.01), miR-29a-5p simulants weaken (result such as Fig. 1 to the rejection ability of saltant type NOX4-3 ' UTR uciferase activities
It is shown).
These results indicate that NOX4 is miR-29a-5p direct target gene.
Tests below example 2~7 with drag and experiment packet by being carried out.
1st, set up acute cerebral ischemia and intravascular machinery leads to cerebral Ischemia re-perfusion model again
Using the method for the inaccessible arteria cerebri media (MCA) of line brush generally acknowledged both at home and abroad, MCA permanent occlusions are set up
(pMCAO) acute cerebral ischemia model of 24 hours, and 2 hours recession Outlet bolt machinery of MCA occlusions lead to 22 hours (tMCAO) again
Acute cerebral ischemia machinery lead to cerebral Ischemia re-perfusion model again.Supplied using laser Doppler flowmetry measurement rat homonymy MCA
Blood region leads to front and rear cerebral blood flow (CBF) again.After rat anesthesia success, take and open left side 5mm to hand over by 2mm and center line after rat bregma
Point, cuts the region scalp, and laser Doppler flowmetry detects the basic cerebral blood flow (CBF) before the occlusion of the region.When bolt line is inserted about
After 18-20mm, inaccessible side brain blood flow value declines more than 70% compared with basic value, shows MCA occlusion successes；Extract after bolt line, close
Plug side brain blood flow value reaches more than the 50% of basic value, shows that the success of infarct area blood flow is led to again.Line bolt is pulled out again to lead to after 22h
Rats with bilateral cerebral hemisphere is taken to be tested.
The rat of following several situations, including the monitoring of (1) laser-Doppler occur for rejecting, and MCA is inaccessible and leads to rear blood flow again
Amount declines or recovery extent is not reaching to standard；(2) materials confirm that encephalic there occurs subarachnoid hemorrhage.
2nd, experiment packet
Rat is randomly divided into 5 groups, i.e. sham-operation group, pMCAO groups, tMCAO groups, tMCAO+ intravenous injection microRNA skies
White control group (after leading to again at once), tMCAO+ intravenous injection miR-29a-5p activators (after leading to again at once) group.Pharmaceutical intervention
Group MCA machinery again lead to after at once, according to 50nmol/kg dosage by tail vein inject miR-29a-5p activators,
MicroRNA blank control groups inject Isodose microRNA placebos in tail vein at once after MCA machineries lead to again.
3rd, in cerebral ischemia 24 hours, materials carry out following detect.
Influence of the miR-29a-5p activators of test example 2 to acute cerebral ischemia in rats early stage encephaledema
Rat MCA occlusions ischemic 24 hours, collects cerebral ischemia and the whole cerebral hemisphere tissue of non-ischemic, inserts respectively
The masking foil weighed is weighed, and is weight in wet base；It is then placed in 100 DEG C of baking box 24 hours, weighs for the second time, is dry weight.Calculate brain
Water content judges early stage degree of cerebral edema, brain water content (%)=(weight in wet base-dry weight)/weight in wet base].
Compared with tMCAO groups, injection miR-29a-5p activator groups significantly reduce brain water content (P after tMCAO leads to again<
0.05).Illustrate that miR-29a-5p activators reduce acute cerebral ischemia in rats early stage encephaledema (result is as shown in Figure 2).
Influence of the miR-29a-5p activators of test example 3 to blood-brain barrier permeability
Injection 2% Evans blue (Evans Blue) liquid in 21 hours (putting to death first 3 hours) tail veins of cerebral ischemia, before execution
Through 4 DEG C of physiological saline of intra-cardial perfusion up to limpid, a cerebral hemisphere tissue is quickly rounded, -20 DEG C of freezings cut 2mm thickness hats
6, shape face brain piece.All brain piece photograph, judge the Evans blue dye of cerebral hemisphere peri-infarct region and infraction core space tissue
Situation.Spectrophotometer quantitative analysis brain tissue Evans Blue ooze out index, specify the change of blood-brain barrier permeability.
Normal rat brain tissue is taken, standard curve is made.Weigh EB reagents be dissolved in PBS solution configuration it is final concentration of
160ug/ml solution.Supernatant is taken after brain tissue homogenate, centrifugation.Take 200 respectively, 125,125,125,125,125,125,
125uL adds 50ul EB solution (160ug/ml) as the 1st pipe, using coubling dilution, from the 1st pipe in 1-8 EP pipes
Take 125uL to be added to the 2nd pipe, take 125uL to be added to the 3rd pipe from the 2nd pipe, the rest may be inferred, and work 7 is managed altogether and blank tube 1 is managed, and its is dense
Degree is respectively 8,4,2,1,0.5,0.25,0.125 and 0ug/mL.Then (volume ratio 1 is diluted with 75% ethanol:3), in wavelength
OD value (OD) is determined under the conditions of 620nm, calibration curve equation is calculated.
The brain piece of ischemic side and non-ischemic side cerebral hemisphere is collected respectively, is weighed, and adds 50% trichloroacetic acid solution
4ml is homogenized, and is centrifuged 30 minutes with 10000rpm rotating speeds, is taken supernatant with 75% ethanol 1:3 dilution proportions, 620nm spectrophotometrics
The lower detection absorbance of meter.Absorbance is substituted into calibration curve equation, obtained per the EB contents in the cerebral hemisphere brain tissue of side, with
Represented (ug/g) per Borneo camphor tissue content.EB seepage discharges are calculated, EB oozes out index=ischemic side brain tissue EB contents/non-ischemic
Side brain tissue EB contents.
Rat ischemia side cerebral hemisphere Evans blue seepage discharges, are found compared with tMCAO groups, and tMCAO is injected after leading to again
MiR-29a-5p activator groups significantly suppress the rise (P of blood-brain barrier permeability<0.05) (result is as shown in Figure 3).Explanation
MiR-29a-5p activators improve blood-brain barrier disruption after acute cerebral ischemia.
Influence of the miR-29a-5p activators of test example 4 to acute cerebral ischemia in rats cerebral infarct volume
Rat MCA occlusions ischemic 24 hours, materials leave and take whole cerebral hemisphere, and -20 DEG C freeze 30 minutes, according to coronal-plane
Cut 6 2mm thickness brain pieces.Add in 2%TTC liquid, dyed 15-30 minutes in lucifuge, 37 DEG C of water baths, it is every to shake within 5-10 minutes
Shake once, it is in level dyeing to make brain tissue.Then 4% paraformaldehyde continues fixed brain tissue 24-48 hours, takes pictures.Using
The image analysis software network analysis cerebral infarction degree of Image Pro Plus 6.0, i.e. cerebral infarct volume (%)=(contralateral brain
Hemisphere volume-non-the cerebral infarct volume in focus side)/contralateral hemispheres volume.
Compared with tMCAO groups, injection miR-29a-5p activator groups significantly reduce infarct volume (P after tMCAO leads to again<
0.01) (result is as shown in Figure 4).Illustrate that miR-29a-5p activators reduce acute cerebral ischemia in rats cerebral infarct volume.
Expression of the miR-29a-5p activators of test example 5 on miR-29a-5p in acute cerebral ischemia in rats brain tissue influences
Rat MCA occlusions ischemic 24 hours, the chloraldurate deep anaesthesia of excess injection 10%.Using xiphoid-process as mark, " V " type
The wall of the chest is cut off, exposure heart most goes out to insert vein blood taking needle, other end insertion PAXgene blood rna collections by force in apex beat
Pipe collects whole blood.After the completion of blood collection, the physiological saline about 200ml of precooling is irrigated through atrium dextrum, until perfusion liquid is limpid.It is disconnected
Head is put to death after rat, quickly takes full brain, is removed olfactory bulb, cerebellum and brain stem, is left and taken whole cerebral hemisphere.Respectively collect cerebral ischemia and
Non- ischemic brain hemisphere peri-infarct region tissue, is detected for fluorescence quantitative RT-RCR and Western blot.
Whole blood RNA is extracted, miR-29a-5p expression is detected using TaqMan stem-loop RT-PCR.
1st, miR-29a detection of expression in blood
750 μ l TRI REAGENT BD blood rnas extracts reagents and 20 μ l glacial acetic acid, hand are added in 200 μ l blood samples
Dynamic acutely vibration body extracts whole blood total serum IgE, miR-29a-5p is detected using TaqMan stem-loop RT-PCR to mixing
Expression.After the RNA synthesis cDNA of sample, Realtime PCR reaction systems are respectively configured in all cDNA samples,
The progress miRNA PCR amplifications and interpretation of result on ViiA 7Real-time PCR System, miR-29a-5p and U6's
Primer sequence (is shown in Table 1).All samples are repeated 3 times, as a result using comparison loop threshold value (CT) standard measure, and computational methods are Δ Δ
Ct methods.
The real-time quantitative PCR of table 1 uses list of primers
QRT-PCR detections are found, compared with sham-operation group, and tMCAO is organized after leading to again and significantly reduced whole blood miR-29a-
5p expression (P<0.01).Compared with tMCAO groups, injection miR-29a-5p activator groups are significantly increased after tMCAO leads to again
MiR-29a-5p expression (P<0.05).(result is as shown in Figure 5A).Illustrate that machinery reduces acute cerebral ischemia in rats after leading to again
MiR-29a-5p is expressed in whole blood.
2nd, peri-infarct region brain tissue miR-29a detection of expression
QRT-PCR detects peri-infarct region brain tissue, finds compared with sham-operation group, and tMCAO is significantly reduced after leading to again
MiR-29a-5p expresses (P<0.001)；Compared with tMCAO groups, injection miR-29a-5p activator groups are notable after tMCAO leads to again
Add miR-29a-5p expression (P<0.001).(result is as shown in Figure 5 B).
The miR-29a-5p activators of test example 6 are to acute cerebral ischemia in rats brain tissue target gene NOX4, Caspase-3
MRNA and protein expression influence
24 hours after cerebral ischemia, ischemic side peri-infarct region is taken to organize.Extract the RNA of ischemic side peri-infarct region tissue
And total protein, 5 groups of rat ischemia side cerebral hemisphere tissue target genes are detected using qRT-PCR and Western blot methods
NOX4, Caspase-3 mRNA and protein expression.Primary antibody is respectively NOX4 (1:1000)、Caspase-3(1:1000) and
β-actin(1:1000).Secondary antibody is the secondary antibody (1 that HRP is marked:1000).The light that Image J softwares determine target protein band is close
Angle value, is corrected with β-actin albumen as internal reference.
1st, brain tissue prediction target gene NOX4 detection of expression
After the total serum IgE for extracting brain tissue, NOX4, Caspase- are detected using SYBR green real-time RT-PCR
3mRNA expression.After the RNA synthesis cDNA of sample, Realtime PCR reactants are respectively configured in all cDNA samples
System, microRNA and mRNA PCR amplifications and interpretation of result is carried out on ViiA 7Real-time PCR System, is used
Primer Premier V5.0Software design NOX4 and Caspase-3 primers (being shown in Table 1).All samples are repeated 3 times, knot
Fruit uses comparison loop threshold value (CT) standard measure, and computational methods are Δ Δ Ct methods.
QRT-PCR and Western blot detect peri-infarct region brain tissue, find compared with tMCAO groups, tMCAO leads to again
Injection miR-29a-5p activator groups significantly suppress up-regulated expression (mRNA, the P of NOX4mRNA and albumen afterwards<0.05) (egg
In vain, P<0.001).Compared with pMCAO groups, injection miR-29a-5p activator groups significantly suppress after tMCAO leads to again
NOX4mRNA and albumen up-regulated expression (mRNA, P<0.001) (albumen, P<0.001) (result is as shown in Figure 6).
2nd, Caspase-3 detection of expression
QRT-PCR and Western blot detect peri-infarct region brain tissue, find compared with sham-operation group, tMCAO groups
Up-regulated expression (mRNA, the P of Caspase-3mRNA and albumen are significantly raised<0.05；P<0.001).It was found that with tMCAO groups
Compare, injection miR-29a-5p activator groups significantly suppress the up-regulated expression (P of Caspase-3 albumen after tMCAO leads to again<
0.001).Compared with pMCAO groups, injection miR-29a-5p activator groups significantly suppress Caspase-3 eggs after tMCAO leads to again
White up-regulated expression (P<0.001) (result is as shown in Figure 7).
NOX4, miR-29a-5p expression and active oxygen are expressed, carefully in the neuron of test example 7 and astroglia
Born of the same parents' apoptosis situation
Through irrigating 4 DEG C of physiological saline in rat heart until after limpid, the paraformaldehyde 200ml of Reperfu- sion 4% is to fix brain
Tissue, leaves and takes in whole cerebral hemisphere, 4% paraformaldehyde solution and is dehydrated respectively with 20% and 30% sucrose solution after immersion 24h,
Section judges substantially bleeding, OCT embedding liquid nitrogen flash freezers, -80 DEG C of preservations, for immunofluorescence dyeing, In situ hybridization,
Quantitation active oxygen and Apoptosis degree analyzing.
1st, immunofluorescence double staining is dyed, distribution and expression feelings of the quantitative analysis NOX4 in neuron and astroglia
Condition
Materials leave and take whole cerebral hemisphere, and -20 DEG C are freezed 30 minutes, and 6 2mm thickness brain pieces are cut according to coronal-plane.Brain
Liquid nitrogen flash freezer after OCT embeddings is organized, middle 2 brain pieces is chosen, makes 7 μ m thick frozen sections.The alcohol of frozen section 95% is consolidated
Fixed 5 minutes, distilled water aquation 10 minutes, PBST is washed 3 times, and 5 minutes every time, citric acid was repaired liquid hot high pressure and repaired 5 minutes, from
So cooling is repaired.Section 5%BSA is closed 1 hour, and premix is added dropwise is used for double labelled staining primary antibody, and (14 is small for 4 DEG C of overnight incubations
When more than).Immunofluorescence primary antibody is NOX4, NeuN and GFAP, and dilution factor is 1:200.
Rewarming 45 minutes under normal temperature, PBST is washed 2 times, and 5 minutes every time, PBS was washed 5 minutes.The donkey of premix is added dropwise in lucifuge
Anti- (the dilution factors 1 of mouse Alexa Fluo 488:250) with (dilution factors 1 of donkey anti-rabbit Alexa Fluo 594:250) fluorescence secondary antibody, 37
DEG C lucifuge is incubated 1 hour, and PBST is washed 2 times, and 5 minutes every time, PBS was washed 5 minutes.DAPI (dilution factors 1 are added dropwise:10) born of the same parents are redyed
Core 10 minutes.Anti- fluorescent quenching mountant mounting.Fluorescence microscopy Microscopic observation destination protein fluorescence in other neurovascular cells is determined
Position is simultaneously taken a picture, the excitation wavelength 488nm of Alexa Fluo594 excitation wavelengths 594nm, Alexa Fluo 488.
Each sample chooses 3 400 × high power fields, takes pictures, and judges NOX4 in ischemic side peri-infarct region and infraction core
The expression of heart district tissue, and the distribution in neuron and astroglia.Calculate respectively NOX4 in neuron and
Degree, i.e. NOX and neuron or astroglia positive coexpression area/neuron or star are expressed in astroglia
The ratio of spongiocyte positive expression area.
It was found that compared with tMCAO groups and pMCAO groups, intravenous injection miR-29a-5p activators show after tMCAO leads to again
Write the up-regulated expression (P for inhibiting NOX4 albumen in the neuron of rat peri-infarct region<0.001；P<0.001) (result such as Fig. 8 A
It is shown).
It was found that compared with tMCAO groups and pMCAO groups, intravenous injection miR-29a-5p activators show after tMCAO leads to again
Write the up-regulated expression (P for inhibiting NOX4 albumen in the astroglia of rat peri-infarct region<0.001；P<0.01) (result is such as
Shown in Fig. 8 B).
2nd, In situ hybridization, distribution and expression situations of the quantitative analysis miR-29a-5p in astroglia
Liquid nitrogen flash freezer after brain tissue OCT embeddings, chooses middle 2 brain pieces, makes 7 μ m thick frozen sections.95% ethanol
Fix 10 minutes；Distilled water aquation 10 minutes；Citric acid repairs liquid Pressure method 5 minutes, natural cooling reparation；30%H2O2+ pure
Methanol (1:50) mix, room temperature treatment 30 minutes, distillation water washing 5 minute 3 times；Exposure mRNA nucleic acid fragments：It is added dropwise in section
The pepsin (1ml3% citric acids add 2 drop concentrated type pepsins, mix) of 3% citric acid diluted fresh, room temperature digestion 5-
120 seconds, in situ hybridization was washed 5 minutes 3 times with PBS, distillation washing 5 minutes；1% paraformaldehyde, room temperature fixes 10 minutes, distilled water
Washing 5 minutes 3 times；Prehybridization solution is added dropwise, 38-42 DEG C of insulating box is incubated 2-4 hours, draws surplus liquid, do not wash；Plus hybridization
Liquid, by situ hybridization privacy protection membrane cover in section, 38-42 DEG C of incubation hybridized overnight of insulating box；Post-hybridization washing：Take lid off
Slide, 2*SSC is washed 15 minutes；Confining liquid is added dropwise, 37 DEG C are incubated 30 minutes；The anti-digoxin of biotinylation mouse is added dropwise：Room temperature is incubated
Educate 2 hours；In situ hybridization is washed 5 minutes 4 times with PBS；SABC-FITC is added dropwise：Take 1ul SABC-FITC plus in situ hybridization PBS
100ul, 37 DEG C are incubated 30 minutes, and in situ hybridization is washed 5 minutes 3 times with PBS；5%BSA is closed 1 hour；Primary antibody, 4 DEG C of incubations is added dropwise
Overnight (more than 14 hours).Primary antibody is GFAP, and dilution factor is 1:200；Rewarming 45 minutes under normal temperature, PBST washings 2 times, every time
5 minutes, PBS was washed 5 minutes.Fluorescence secondary antibody is added dropwise in lucifuge, and 37 DEG C of lucifuges are incubated 1 hour, PBST washings 2 times, 5 minutes every time,
PBS is washed 5 minutes；DAPI (dilution factors 1 are added dropwise:10) karyon is redyed 10 minutes；Anti- fluorescent quenching mountant mounting；Image
The image analysis software quantitative analysis positive expression areas of Pro Plus 6.0.Hybridization in situ technique judges miR-29a-5p in ischemic
The expression of side peri-infarct region tissue.The double mark dyeing detection rat cerebral tissue infractions of miR-29a-5p/GFAP in situ hybridizations
MiR-29a-5p expression in peripheral region astroglia.
It was found that compared with tMCAO groups, injection miR-29a-5p activator groups significantly increase star glue after tMCAO leads to again
MiR-29a-5p expression (P in cell plastid<0.05).Compared with pMCAO groups, injection miR-29a-5p excitements after tMCAO leads to again
Agent group significantly increases miR-29a-5p in astroglia and expresses (P<0.05) (result is as shown in Figure 9).
3rd, quantitation active oxygen
Liquid nitrogen flash freezer after brain tissue OCT embeddings, chooses middle brain piece, makes 7 μ m thick frozen sections.Frozen section
95% alcohol fixation 5 minutes, distilled water aquation 10 minutes, PBST is washed 3 times, 5 minutes every time, adds final concentration of 10 μm of ol/L
37 DEG C of lucifuges of DHE probes or DCFH-DA probes be incubated 25 minutes, be placed in fluorescence microscopy Microscopic observation ischemic side cerebral hemisphere group
Knit middle fluorescence localization and take pictures, parameter is excitation wavelength 518nm, wavelength of transmitted light 610nm.DHE and DCFH-DA fluorescence probes
The expression of dyeing detection peri-infarct region brain tissue superoxide anion and hydrogen peroxide.Using the softwares of Image Pro Plus 6.0
Measure superoxide anion and hydrogen peroxide OD value in brain tissue, analysis active oxygen expression degree.
It was found that compared with tMCAO groups, injection miR-29a-5p activator groups significantly suppress active oxygen after tMCAO leads to again
Up-regulated expression (superoxide anion, P<0.01) (result is as shown in Figure 10).Compared with tMCAO groups, tMCAO is injected after leading to again
MiR-29a-5p activator groups significantly suppress up-regulated expression (hydrogen peroxide, P of active oxygen<0.001) (result such as Figure 11 institutes
Show).
4th, TUNEL kits are incubated, and the ratio of apoptotic cell in measurement neuron and astroglia, analysis cell withers
Die degree
Liquid nitrogen flash freezer after brain tissue OCT embeddings, chooses middle 2 brain pieces, makes 7 μ m thick frozen sections.Frozen section
95% alcohol fixation 5 minutes, distilled water aquation 10 minutes, PBS is washed 3 times, and 5 minutes every time, citric acid repaired liquid high pressure hot repair
It is multiple 5 minutes, natural cooling reparation.PBS is washed 3 times, and 5 minutes every time, 20 μ g/ml Proteinase K Solutions were incubated at room temperature 10 minutes,
PBS wash 3 times, 5 minutes every time, then level pad be incubated 30 minutes, and then with premix containing deionized water, 5 ×
37 DEG C of the TdT incubation buffers of level pad, BrightRed mark mixtures and TdT enzymes are incubated 60 minutes, PBST washings,
Premix is added dropwise is used for double labelled staining primary antibody, and 4 DEG C are incubated overnight (more than 14 hours).Immunofluorescence primary antibody be NeuN or
GFAP, dilution factor is 1:200.Rewarming 45 minutes under normal temperature, PBST is washed 2 times, and 5 minutes every time, PBS was washed 5 minutes.Lucifuge
Drip anti-mouse Alexa Fluo488 (dilution factors 1:250) fluorescence secondary antibody, 37 DEG C of lucifuges are incubated 1 hour, and PBST washs 2 times, and every time 5
Minute, PBS is washed 5 minutes.DAPI (dilution factors 1 are added dropwise:10) karyon is redyed 10 minutes, anti-fluorescent quenching mountant mounting.Put
Fluorescence localization and take pictures, seen under the conditions of excitation wavelength 620nm in the cerebral hemisphere tissue of fluorescence microscopy Microscopic observation ischemic side
Examine the DAPI that blueness is observed under the conditions of the red fluorescence of apoptosis tissue, 420nm.Measured using Image Pro Plus6.0 softwares
Neuron Apoptosis ratio in brain tissue, analyzes apoptosis degree.TUNEL/NeuN or the dyeing of astroglia Double immunofluorescence
Detect peri-infarct region neuron and astrocyte apoptosis degree.
It was found that compared with tMCAO groups, injection miR-29a-5p activator groups significantly suppress neuron after tMCAO leads to again
Apoptosis degree (P<0.05).Compared with pMCAO groups, injection miR-29a-5p activator groups are significantly inhibited after tMCAO leads to again
Apoptosis degree (the P of neuron<0.05) (result is as illustrated in fig. 12).
It was found that compared with tMCAO groups, injection miR-29a-5p activator groups significantly suppress star glue after tMCAO leads to again
Apoptosis degree (the P of cell plastid<0.01).Compared with pMCAO groups, injection miR-29a-5p activator groups show after tMCAO leads to again
Write the apoptosis degree (P for inhibiting astroglia<0.001) (result is as shown in Figure 12 B).
Influence of the miR-29a-5p activators of test example 8 to rat function damage
Rat respectively in MCA occlusions 2 hours, lead to again after lead at once and again after 22 hours (ischemic 24 hours), using Longa
Point system checks the degree of nervous function damage, is divided into 0-4 points.0 point：Normally, impassivity defective symptom；1 point：Slight nerve work(
Energy defect, fore paw is unable to full extension；2 points：Moderate neurologic impairment, turn-takes during walking to hemiplegia side；3 points：Severe neurological
Functional impairment, rolls during walking to hemiplegia；4 points：It spontaneous can not walk, lose consciously.
Compared with tMCAO groups, injection miR-29a-5p activator groups are significantly reduced after ischemia-reperfusion after tMCAO leads to again
The death rate (P<0.05).Compared with pMCAO groups, injection miR-29a-5p activator groups significantly reduce scarce after tMCAO leads to again
The death rate (P after blood Reperfu- sion<0.05).
Compared with tMCAO groups, injection miR-29a-5p activator groups are significantly improved after ischemia-reperfusion after tMCAO leads to again
Nervous function state (the P of rat<0.001).Compared with pMCAO groups, injection miR-29a-5p activator groups show after tMCAO leads to again
Work improves the nervous function state (P of rat after ischemia-reperfusion<0.001) (result is as shown in figure 13).
Embodiment described above only expresses the several embodiments of the present invention, and it describes more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Sequence table
<110>No.1 Hospital Affiliated to Zhongshan Univ.
<120>Applications of the MicroRNA 29a-5p in treatment acute cerebral ischemia and cerebral ischemia re-pouring injured medicine is prepared
The ＞ 2 of ＜ 130
The ＞ PatentIn version 3.1 of ＜ 170
The ＞ 1 of ＜ 210
The ＞ 22 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ artificial sequences of ＜ 213
The ＞ 1 of ＜ 400
acugauuucu uuugguguuc ag 22
The ＞ 2 of ＜ 210
The ＞ 22 of ＜ 211
The ＞ DNA of ＜ 212
The ＞ artificial sequences of ＜ 213
The ＞ 2 of ＜ 400
gaacaccaaa agaaaucagu uu 22
Claims (2)
1. a kind of miR-29a-5p, it is characterised in that it has such as SEQ ID No:1 and SEQ ID No:Base sequence shown in 2
Row.
Applications of the 2.miR-29a-5p in treatment acute cerebral ischemia and cerebral ischemia re-pouring injured medicine is prepared, its feature exists
In the miR-29a-5p has such as SEQ ID No:1 and SEQ ID No:Base sequence shown in 2.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109006662A (en) * | 2018-07-02 | 2018-12-18 | 中山大学附属第医院 | Acute cerebral ischemia machinery is intracranialed hemorrhage transformation model and its microRNA screening technique and application after leading to again |
| CN110806485A (en) * | 2019-11-12 | 2020-02-18 | 广州医科大学附属第二医院 | Colloidal fiber acidic protein antibody detection kit and application thereof |
| WO2020073935A1 (en) * | 2018-10-11 | 2020-04-16 | 伽蓝（集团）股份有限公司 | Method for screening active material using skin exogenous aging target, and active material for improving skin exogenous aging |
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| WO2013057513A2 (en) * | 2011-10-19 | 2013-04-25 | Nhs Blood & Transplant | Therapeutic compositions and uses |
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| WO2013057513A2 (en) * | 2011-10-19 | 2013-04-25 | Nhs Blood & Transplant | Therapeutic compositions and uses |
| WO2016156865A1 (en) * | 2015-04-02 | 2016-10-06 | The University Of Bristol | Exosomes |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109006662A (en) * | 2018-07-02 | 2018-12-18 | 中山大学附属第医院 | Acute cerebral ischemia machinery is intracranialed hemorrhage transformation model and its microRNA screening technique and application after leading to again |
| WO2020073935A1 (en) * | 2018-10-11 | 2020-04-16 | 伽蓝（集团）股份有限公司 | Method for screening active material using skin exogenous aging target, and active material for improving skin exogenous aging |
| CN110806485A (en) * | 2019-11-12 | 2020-02-18 | 广州医科大学附属第二医院 | Colloidal fiber acidic protein antibody detection kit and application thereof |
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