CN105648073A - Ischemic stroke screening kit and application thereof - Google Patents

Abstract

The invention discloses an application of miRNA-98 to preparation of an ischemic stroke screening kit and discloses that miRNA-98 affects a PAFR (platelet activating factor receptor) and the signal transduction process related to downstream inflammations so as to play roles in stokes through targeted negative regulation of expression of the PAFR. A new action mechanism is provided for the application of the miRNA-98 to diagnosis of ischemic strokes.

Description

A kind of ischemic cerebral apoplexy kit for screening and application thereof

Technical field

The invention belongs to technical field of gene detection, be specifically related to a kind of ischemic cerebral apoplexy kit for screening and application thereof.

Background technology

RNA is one of most important material base in organism, and it constitutes the framework of life together with DNA and protein. But for a long time, RNA had once been regarded as merely " transition " between DNA and protein, and it obtains the order of oneself from DNA, then hereditary information is changed into protein there. But, a series of research shows, these microRNAs in fact handle many cell functions, and it can react on DNA by the combination of complementary series, thus the expression of closedown or regulator gene. The RNA family of a recently discovered class non-protein coding, microRNA (miRNA, i.e. Microrna), it is possible to carry out controlling gene expression by being combined or regulate the protein translation process of specific mRNA with specific mRNA. In current human genome, confirmed miRNA has kind more than 1,000, it is possible to participate in the expression of the regulation and control mankind 30% protein coding gene.

MicroRNA is referred to as miRNA, and normal length is 19��25 nucleotide, is widely present in various animals and plants even unicellular eukaryote, is a class little molecule single stranded RNA of high conservative on evolving. MiRNA is positioned at gene intron region, and the genetic transcription encoding miRNA in nucleus becomes pri-microRNA (pri-miRNA). Pri-miRNA, under the effect of a kind of DroshaRNase, is cut into about 70 length of nucleotides, has the miRNA precursor (pre-miRNA) of loop-stem structure. Under the effect of the caryoplasm that pre-miRNA relies at Ran-GTP/cytoplasm albumen Exportin5, transport kytoplasm in core. Under the effect of Dicer enzyme, pre-miRNA is cut into the double-strand miRNA:miRNA of 21��25 length of nucleotides. Double-strand miRNA:miRNA untwists subsequently, and ripe miRNA enters in endochylema RISC (silencing complex of RNA induction) and plays gene silencing function. 3 ' ends noncoding region (3 ' UTR) of the messenger RNA (mRNA) of miRNA and downstream specific gene produce base pair complementarity, cause the degraded of this mRNA or suppress it to translate, and cause protein expression failure. The gene in these downstreams is called the target gene of miRNA by us, they are generally of important biological function in cell, miRNA exercises important function by regulating the expression of target gene in cell, and the growth of embryo, growth promoter, cell propagation, break up closely related. Scientist is it has proven convenient that the nucleotides sequence of miRNA is listed between different plant species high conservative, and mutation rate is extremely low, and its expression has gene cluster collection phenomenon, space-time and tissue specificity. MiRNA determines process and the multiformity of a series of important vital movement such as cell differentiation, fetal development at the express spectra of post-transcriptional level modulin plasmagene.It has been found that miRNA is not only the important regulating and controlling factor of cell proliferation, differentiation and apoptosis, the miRNA of more than 50% is positioned at tumor-related gene group region, including LOH district, chromosome amplification district and fragile site etc. Identified, miRNA express spectra in multiple hematological system tumor and solid tumor changes, and this discloses disease mechanism for us from the angle of RNA regulation and control and provides fundamental basis, and provides new gene target and molecular marker for medical diagnosis on disease and treatment.

In human genomic sequence, miR-98 is a kind of MicroRNA, is positioned at Xp11.22. The sequence of miR-98 is 5 '-UGAGGUAGUAAGUUGUAUUGUU-3 ' (GeneBank:GeneID:407054). MiR-98 is high conservative on evolving, and there is homology between different plant species.

Showing according to the data that World Health Organization (WHO) (WHO) announces, apoplexy is one of major disease that the whole world threatens human health, is the primary the third-largest fatal disease disabled and be only second to cancer and cardiovascular disease at present. Epidemiology shows, stroke onset is the trend of rejuvenation and cumulative year after year. Apoplexy is broadly divided into ischemic and hemorrhagic apoplexy according to the difference of the cause of disease, and wherein cerebral infarction accounts for 87%. Research discloses and affect that apoplexy occurs, the principle pathological mechanism that develops includes energy metabolism impairment, neuronic excessive depolarization, Excitotoxicity, ionic homeostasis is unbalance, inflammation and dysimmunity, apoptosis and autophagy etc. Control apoplexy case fatality rate and to improve life quality be a Medical research difficult problem urgently to be resolved hurrily. The approval of current U.S. FDA is for treating the only tissue type plasminogen activator (tPA) of apoplexy, the therapeutic time window strict due to it and easily bring out the complication such as cerebral hemorrhage and significantly limit the extensive use of its clinic, the medicament research and development of apoplexy is still shouldered heavy responsibilities. Along with going deep into of genetic engineering research, utilization genetic engineering is developed medicine and is shown keen interest by scientist.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of ischemic cerebral apoplexy diagnostic kit.

Technical scheme: ischemic cerebral apoplexy kit for screening, carries out stem-loopreal-timePCR and detects miR-98 level,

RT primer: SEQIDNO:9:CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAACAATAC;

PCR primer:

Forward primer sequence SEQIDNO:1:5 '-GGCGCAGTAAGTTGTATTGTT-3 ';

Reverse primer sequences SEQIDNO:2:5 '-GTGCAGGGTCCGAGGT-3 '. The primer sequence of PCR.

The application in preparing brain soldier's type screening agent of the above-mentioned ischemic cerebral apoplexy kit for screening.

A kind of ischemic cerebral apoplexy examination primer, including following primer:

RT primer: SEQIDNO:9:CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAACAATAC;

PCR primer:

Forward primer sequence SEQIDNO:7:5 '-GGCGCAGTAAGTTGTATTGTT-3 ';

Reverse primer sequences SEQIDNO:8:5 '-GTGCAGGGTCCGAGGT-3 '.

The application in preparing brain soldier's type screening agent of the above-mentioned ischemic cerebral apoplexy examination primer.

In the clinical practice of the present invention, design primer, detect the expression of miR-98 in patients serum by real-timePCR method. When MiR-98 expression reduces at least up to 50%, point out acute ischemic cerebral apoplexy. The expression of the microRNA sequence that detection is ripe, that conventional is stem-looprealtime-PCR.Experimental principle is illustrated in fig. 11 shown below.

It is the gene that miRNA-98 targeting regulates that the present invention discloses platelet activating factor receptor (plateletactivatingfactor, PAFR). PAFR is the specific receptor of platelet activating factor (plateletactivatingfactor, PAF). PAF is a kind of and the closely-related endogeneous activity phosphoric acid medium of arachidonic acid metabolic, produced by the Various Tissues and cell (such as macrophage, endotheliocyte, platelet, polymorphonuclear cell) that participate in allergy and inflammatory reaction, act on PAFR and participate in pathophysiological process widely. PAF can cause brain injury by following mechanism after extremely discharging and be combined with PAFR: (1) increases neurocyte calcium ion concentration, upsets cell membrane function, neuron is caused coup injury; (2) promote platelet aggregation and activation, discharge vaso-active substance, cause blood vessel embolism etc., increase the weight of cerebral edema; (3) chemotactic and activation inflammatory cell, promote the release of inflammatory factor, start and increase the weight of inflammatory damage etc. A large amount of zooperies and clinical trial show, Plasma in Patients with Acute Cerebral Infarction and in Cerebral Infarction Model murine brain PAF content significantly raise. Above-mentioned three kinds of mechanism prompting PAF participate in the pathological process that development occurs of cerebral infarction, and closely related with lapsing to of ischemic cerebrovascular.

Present invention is disclosed miRNA-98 to be combined with the promoter region of PAFR, affect PAFR expression, in ischemic cerebral apoplexy patients serum, miRNA-98 sharply declines, cause PAFR expression raise, the abnormal release of PAF in ischemic cerebral apoplexy patients serum, and be combined with PAFR and further result in brain injury. Therefore, being related closely of the expression of miR-98 and ischemic cerebral apoplexy, if the expression of detection miR-98 lower than normal expression level 50% and more than. As in figure 2 it is shown, miR-98 level is 0.89 in our actually detected value normal healthy controls serum, and in patients with cerebral apoplexy serum, miR-98 level is 0.19, is only the 21% of normal healthy controls; In zoological specimens, in stroke groups animal serum, miR-98 is only sham operated rats 53%, and ischemic penumbra miR-98 is sham operated rats 28%, sets time separation accordingly. Then can determine that detected person belongs to ischemic cerebral apoplexy. The expression detection kit of miR-98 can be completely used for brain soldier crowd is carried out in detection examination.

Beneficial effect: by the miR-98 checking to PAFR gene target, to the detection of miRNA-98 expression in patients with acute hemorrhagic cerebral apoplexy serum, cerebral infarction model mouse serum and penumbral brain tissue, and to the detection that PAFR in PAF in patients with cerebral apoplexy serum and cerebral infarction model mouse brain expresses, miR-98 effect in cerebral infarction diagnoses is described.

Accompanying drawing explanation

Fig. 1 hsa-miR-98-5p is on the PAFR impact expressed.

The content (A) of miR-98 in Fig. 2 patients with cerebral apoplexy and normal healthy controls crowd's serum; The expression of cerebral infarction rat blood serum (B) and penumbra region (C) miR-98.

The level of platelet activating factor in Fig. 3 patients with cerebral apoplexy and normal healthy controls crowd's serum.

The expression of Fig. 4 cerebral infarction rat penumbra region platelet activating factor receptor.

The expression of Fig. 5 cerebral infarction rat penumbra region p65.

The expression of Fig. 6 cerebral infarction rat penumbra region p-IKK �� albumen.

The expression of Fig. 7 cerebral infarction rat penumbra region p-p38 albumen.

The expression of Fig. 8 cerebral infarction rat penumbra region p-JNK albumen.

The expression of Fig. 9 cerebral infarction rat penumbra region Bcl-2 (A) and Bax (B) albumen.

The expression of Figure 10 cerebral infarction rat penumbra region cleavedcaspase-3 albumen.

Figure 11 stem-looprealtime-PCR schematic diagram.

Figure 12 miRNA-98 and PAFR promoter region pairing schematic diagram.

Detailed description of the invention

According to following embodiment, it is possible to be more fully understood that the present invention. But, as it will be easily appreciated by one skilled in the art that the content described by embodiment is merely to illustrate the present invention, and should without the present invention described in detail in restriction claims.

In embodiment end indicate actual conditions experimental technique, be substantially all write according to Sambrook, J et al. " Molecular Cloning: A Laboratory guide (the 3rd edition) " (MolecularCloning:ALaboratoryManual, 3^rdEd. yellow training hall etc. are translated, Science Press .2002.8) described in condition and method or according to material supplier it is proposed that condition and method carry out, other technology being not described in is the standard method known corresponding to those skilled in the art.

The material of the present invention: microorganism, cell line, plasmid or other expression vectors mentioned in the application and culture medium all have supply of commodities or can for public's gained with other approach; they are only for example; it not unique to the present invention, can replace with other instrument being suitable for and biomaterial respectively.

Embodiment 1: prediction miRNA-98 acts on the target site of PAFR, the interaction of application luciferase reporter gene detection miRNA-98 and PAFR promoter region.

(1) shown in pairing signal Figure 12;

(2) design primer

PTAFR-miR98-5pF (SEQIDNO:1): XhoI:cacaactcgagTCATTTCCTGTGTACCGGGC;

PTAFR-miR98-5pR (SEQIDNO:2): BamHI:aaggatccTAAGGGACCTGCAAAGCCTG;

PTAFR-miR98-5pMR (SEQIDNO:3): CTCCATCCTTTAACCTCATAGGTAATGACCCTAACTCCATCGAAATTCAGTGCCTG GT;

PTAFR-miR98-5pMF (SEQIDNO:4): GATGGAGTTAGGGTCATTACCTATGAGGTTAAAGGATGGAGATGGGATTGTTATAC GCC;

PCR primer size 442bp.

(3) pLUC-PTAFR Reporter gene vector is built:

Contained 3 ' UTR sequence following (SEQIDNO:5):

TCATTTCCTGTGTACCGGGCAAACACCGGATTGCTGATTTTGAGAAATGCCTCTCGATGGACCTGTAACCTGCTGGAGTCTGGGATGGTAGCTGTGGGCTGGACTTGGCTGATGGGATGACCCGGTGGCTAGTGCAGCATCACACAAGCCTGGTTCAAGTCTTGGCTGTGTCATTTCCTGCTGAGGGACTAGGGTCATTATGGGATTGTTATACGCCACTAATGTTGAGGCAGACACCTCTTGGCAGGGTGACTGCTCATCTTAGACCCTCCCCTTTTCTGCGAATTTGGGCCCCTTGATCCTCTGATGGGAGCTGAAAGGATGAGAGGTGGGCATCTAGATTTAGGGAGGCTGTTCAGGCTTTGCAGGTCCCTTA��

(4) pLUC-PTAFRMut Reporter gene vector is built:

Contained 3 ' UTR sequence following (SEQIDNO:6):

TCATTTCCTGTGTACCGGGCAAACACCGGATTGCTGATTTTGAGAAATGCCTCTCGATGGACCTGTAACCTGCTGGAGTCTGGGATGGTAGCTGTGGGCTGGACTTGGCTGATGGGATGACCCGGTGGCTAGTGCAGCATCACACAAGCCTGGTTCAAGTCTTGGCTGTGTCATTTCCTGCTGAGGGACTAGGGTCATTATGGGATTGTTATACGCCACTAATGTTGAGGCAGACACCTCTTGGCAGGGTGACTGCTCATCTTAGACCCTCCCCTTTTCTGCGAATTTGGGCCCCTTGATCCTCTGATGGGAGCTGAAAGGATGAGAGGTGGGCATCTAGATTTAGGGAGGCTGTT

(note: " _ _ _ _ " is primer sequence position,For miRNA action site)

(5) miR-98 target gene prediction and checking test.

Detection method: by hsa-miR-98-5pmimics (with control for comparison) respectively with containing prediction target gene PTAFR-3'UTR or the luciferase reporter vector transient cotransfection 293T cell of its mutant, cell lysis after 48 hours, Dual-Luciferase activity (Promega is detected with test kit, Cat#E1980), checking miRNA suppresses candidate targets translation skill.

Testing result: hsa-miR-98-5p is on the PTAFRWT&Mut impact expressed such as table 1, Fig. 1.

Interpretation of result: owing to hsa-miR-98-5pmimics has significant difference (p value is 0.001) with the luciferase reporter gene cotransfection checking test containing PTAFR gene 3'UTR fragment relative to negative control (mimiccontrol tests with the luciferase reporter gene cotransfection containing PTAFR gene 3'UTR fragment), fluorescence ratio is the 65.8% of negative control.Gene expression dose containing PTAFR-3'UTR predicting function site is had inhibitory action by hsa-miR-98-5pmimics, but after predicting function site mutation, inhibitory action substantially weakens. Therefore, hsa-miR-98-5p works likely via PAFR.

Table 1 by hsa-miR-98-5pmimics respectively with the luciferase reporter vector transient cotransfection 293T cell 48 hours containing prediction target gene PTAFR-3'UTR or its mutant after Dual-Luciferase activity

Embodiment 2: the detection that patients with cerebral apoplexy serum, ischemic Stroke Models animal serum and penumbra region miRNA-98 express

Detection method: Real-timePCR

Testing result: as shown in Fig. 2 A��C.

Interpretation of result: compared with normal healthy controls group, in patients with cerebral apoplexy serum, miRNA-98 significantly reduces; Compared with sham operated rats animal, the expression of cerebral infarction animal serum and penumbra region miRNA-98 significantly reduces.

Embodiment 4: the detection of platelet activating factor in patients with cerebral apoplexy serum.

Detection method: application ELISA measures PAF content in test kit detection patients serum.

Result measures: as shown in Figure 3.

Interpretation of result: compared with normal healthy controls group, in patients with cerebral apoplexy serum, PAF raises 3.4 times, up to 505.7 �� 93.6pg/ml.

Embodiment 5: the detection that in cerebral infarction animal model penumbral brain tissue, PAFR expresses.

Method: application westernblotting method detection PAFR protein expression.

Result: as shown in Figure 4.

Interpretation of result: compared with sham operated rats animal, apoplexy animal ischemic penumbra PAFR expresses notable rising.

The detection that embodiment 6:PAFR downstream signaling pathway is expressed.

Method: the expression of the albumen such as application westernblotting method detection p65, pIKK ��, p-p38MAPK, p-JNK, Bcl-2, Bax and cleavedcaspase-3.

Result: result is as shown in Figure 5-10.

Interpretation of result: compared with sham operated rats animal, p65, pIKK ��, p-p38MAPK, p-JNK, cleavedcaspase-3, Bax all significantly raise, and Bcl-2 expresses and significantly reduces.

Statistical analysis in the present invention adopts SPSS11.0 software (Release11.0, SPSSInc.) to carry out data process, and data representation is with means standard deviation. Carrying out t inspection, result has significant with P < 0.05 for difference.

Embodiment 7: luciferase reporter gene checking is analyzed.

Build gene 3 '-UTR luciferase reporter gene plasmid extraction:

Cell is cultivated: HEK293T cell with containing 10%FBS, the DMEM culture medium of 1% dual anti-(100 �� g/ml streptomycins, 100M/ml penicillin), 37 DEG C, 5%CO2 incubator is cultivated. In day before transfection, an appropriate number of cell is seeded in Tissue Culture Plate, every hole adds without antibiotic culture medium, enable cell density during transfection to reach 50%.

1.DNA-FugenHD mixes:

By every 96 hole 0.2 �� g plasmid DNA, the consumption of 0.3 �� LFugeneHD, three multiple holes of one group of experiment, PCR pipe is sequentially added into 0.45 �� gmiRNAmimics, miRNANC as comparison. 0.15 �� gsensor reporter gene, 30 �� LOpti-MEM cultivate dilution plasmids, are eventually adding 0.9 �� LFugeneHD, and rear chamber is gentle and quiet puts 15min in mixing.

2. transfection:

DNA-FugeneHD mixture is taken 10 �� L and uniformly drips in every porocyte, every three Kong Weiyi experimental grouies, it is placed in 37 DEG C, 5%CO2 incubator continues to cultivate.

3. luciferase assays:

After having transfected 48h, LuciferaseReporterAssaySystem (purchased from Promega company) is adopted to detect.

(1) 5 �� cell pyrolysis liquid (5 �� lysisbuffer) distilled water is diluted to 1 �� working solution, absorb the culture medium transfected in each hole, 80 �� L, the 1 �� cell pyrolysis liquid diluted is added in each hole, it is placed on decolorization swinging table and shakes 1h, collect the cell pyrolysis liquid in each hole, 12,000rpm, centrifugal 1min precipitated impurities.

(2) take above-mentioned cell pyrolysis liquid in the opaque 96 each holes of orifice plate, be sequentially added into LUC Photinus pyralis LUC Photinus pyralis FL and renilla luciferase substrate to specifications, by the multi-functional microplate reader detection of TecanInfiniteF200/M200 type.

Embodiment 8: target gene is cloned.

(1) 3 '-UTR design of primers

Obtaining gene 3 ' untranslated region sequence from ncbi database, be designed to pcr amplification primer, primer sequence is primer sequence is laboratory report Green mark part.

Pcr amplification reaction is carried out as template, it is thus achieved that 3 '-UTR genetic fragments using genomic DNA.

Reaction system is as follows:

PCR condition: 94 DEG C, 5min; 94 DEG C, 30sec; 60 DEG C, 30sec (miRNA)/60 DEG C, 3min (UTR); 72 DEG C, 40sec; Circulating 35 times, last 72 DEG C extend 5min.

(2) agarose gel electrophoresis separates and glue recovery fragment

1. through 1.5% or 1% agarose gel electrophoresis separation PCR primer

2. cut the blob of viscose containing PCR primer (volume about 100 �� L) to be placed in 1.5mlEP pipe, add 500 �� L sol solutionses, 55 DEG C of colloidal sol 10min, period reverse mixing 2-3 time;

3. adsorption column on mixed liquor, 12,000rpm, centrifugal 1min, repeated centrifugation once abandons waste liquid afterwards;

4. DNA, 12,000rpm on rinsing adsorption column, centrifugal 1min, abandon waste liquid, repeat rinsing once, abandons after waste liquid 12,000rpm, and centrifugal 2min removes residual rinse liquid;

5. drying at room temperature adsorption column 5min, changes to adsorption column in new 1.5mlEP pipe, takes appropriate TE buffer and collects PCR primer. Stand 2min, 12,000rpm, centrifugal 1min.

(3) 3 '-UTR fragments and pLUC carrier double digestion

3 hours rear electrophoresis of 37 degree of enzyme action reclaim.

(4) coupled reaction:

PLUC and 3 with identical restriction enzyme site by after enzyme action purification '-UTRPCR fragment, it is about the ratio mixing of 1:3 with mole ratio, adds 1 �� L10 �� connection buffer, 1 �� LT4DNA ligase, adding aseptic double-distilled water to system volume is 10 �� L

Linked system is as follows:

It is transformed into competent cell TOP10 after 22 DEG C of connection 2h.

(5) recombinant plasmid transformed escherichia coli:

1. take the competent cell 100 �� L of fresh preparation, add 10 �� L and connect product, after mixing, ice bath 20min;

2. 42 DEG C of heat shock 60s, at once ice bath 2min;

3. 800 �� LLB fluid mediums are added, 37 DEG C of gentle jolting recovery cell 45min;

4. taking appropriate converted product and coat on the LB flat board of Amp+, 37 DEG C of incubators are inverted and are cultivated 16h, observe colony growth situation.

(6) screening of recombinant bacterial strain and qualification

Expression vector double digestion system:

Detect in 1.5% agarose gel electrophoresis after enzyme action 3-4h at 37 DEG C.

Recombiant plasmid order-checking is identified: identifying that correct clone's bacterium solution send order-checking through bacterium colony PCR and plasmid PCR, the sequence information of checking recombinant clone Insert Fragment, the correct plasmid containing aim sequence is named with gene name.

Embodiment 9: prepared by focal brain ischemia-reperfusion injury model.

By rats by intraperitoneal injection chloral hydrate (10%, 1ml/100g) anaesthetize, skin of neck sterilization medisection, separate common carotid artery (CCA) and external carotid artery (ECA), by ECA distal end ligation, detachment, ECA stump is made to dissociate, standby at the proximal part threading of stump, ligation CCA, cutting off by stump place at ECA, insert line bolt to internal carotid artery (ICA), the degree of depth is about 2cm, then fix ECA stump and line bolt, pull out Outlet bolt after ischemia 90min again and carry out blood flow and fill again.

Embodiment 10: real-time quantitative PCR

1, cDNA synthesis

(1) RT mixed reaction solution is prepared:

RT primer (SEQIDNO:9): SLRT-miR-98-5p:5 '-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAACAATAC-3 '

(2) RT reaction is carried out in PCR amplification instrument: 16 DEG C of 30min; 42 DEG C of 40min; 85 DEG C of 5min. Reaction places it in stand-by on ice or-20 DEG C of preservations after terminating.

2, real-time quantitative PCR

(1) configuration RealtimePCR reaction system. System configurations is as follows:

Forward primer F (SEQIDNO:7): 5 '-GGCGCAGTAAGTTGTATTGTT-3 ';

Reverse primer R (SEQIDNO:8): 5 '-GTGCAGGGTCCGAGGT-3 ';

(2) in the enterprising performing PCR reaction of Real-timePCR instrument:

U6 and all of index are all undertaken by following procedure: 95 DEG C, 10min; 40 PCR cycle (95 DEG C, 10 seconds; 60 DEG C, 60 seconds (collection fluorescence).

In order to set up the melting curve of PCR primer, after amplified reaction terminates, by (95 DEG C, 10 seconds; 60 DEG C, 60 seconds; 95 DEG C, 15 seconds); And it is heated slowly to 99 DEG C (it is 0.05 DEG C/sec that instrument carries out RampRate automatically) from 60 DEG C.

3, result and calculating: the purpose miRNA of each sample and internal reference (U6) carry out RealtimePCR reaction respectively. Data acquisition is with 2^{-����CT}Method is analyzed.

Embodiment 11: detected by Western blot measures.

1. extract total protein of cell.

2.SDS-PAGE electrophoresis:

(1) clean, glass plate, potsherd are installed.

(2) preparation perfusion separation gel, 37 DEG C stand 25min.

(3) preparation perfusion concentration glue, room temperature stands 20min.

(4) adding protein sample and molecular weight marker, each period, albumen applied sample amount was 40 �� g.

(5) constant voltage electrophoresis (concentration glue 80V, separation gel 160V).

3. half dry type transferring film:

(1) after electrophoresis terminates, wear glove, cut glue (corner cut labelling) according to destination protein molecular weight ranges with reference to molecular weight marker, cut formed objects nitrocellulose filter (corner cut labelling) and filter paper 2.

(2) transferring film buffer soaks glue, film and filter paper 10min.

(3) tiling from bottom to up on transferring film instrument: filter paper, film, gel, filter paper, catches up with bubble removing, especially notes prohibiting between film and gel staying bubble. Dry surplus liquid, 0.8mA �� membrane area (cm2) electric current transferring film 1.5 hours.

(4) immunoblotting

A. transferring film terminates, and whether inspection marker has turned on film (or Ponceaux dyeing) to judge transferring film effect. After TBST cleans film, 5% defatted milk powder (TBST preparation) 4 DEG C is closed overnight.

B. primary antibodie Caspase-3/7 (activated form) monoclonal antibody (1:500 and 1:500) is added, room temperature shake 2h.

C.TBST rinses 3 times, each 10min.

D. add two anti-(1:200), room temperature shake 1h.

E.TBST rinses 3 times, each 10min.

(5) ECL detection

A.AB liquid mixes, and after film does not drip, tiles on it.

B. after reaction 5min, according to fluorescence intensity darkroom tabletting 5s��30min.

C. develop 15��30s, washing, fixing 1��3min.

D. picture scanning and quantitative analysis.

Embodiment 12: immunohistofluorescence stain.

Rat irrigates normal saline and 4% paraformaldehyde of 100ml respectively through left ventricle, and broken end takes brain, is placed in 4% paraformaldehyde fixing, and 4 DEG C overnight. Cerebral tissue after conventional serial dehydration, paraffin embedding, in the upper serial section (5 ��m) of microtome (Leica, Germany), collect forebrain (containing tricorn) brain sheet, take one for testing every six. Through dimethylbenzene, graded ethanol dewaxing aquation, PBS washes 3 times, and PBS is washed 3 times, each 2min.Section is immersed in 0.01M, PH6.0 citrate buffer, power-off after microwave-oven-heating extremely boiling, cooling. Dropping primary antibodie (the anti-GFAP polyclonal antibody of rabbit), 4 DEG C of overnight incubation. 0.02MPBS washes 3 each 2min. Add fluorescence two to resist: Alexa488 goat anti-mouse IgG (1:800, Invitrogen), Alexa488 rabbit anti goat igg (1:800, Invitrogen) incubated at room 2 hours, wet box keeps in Dark Place.

Embodiment 13: cell counting.

Randomly select the section 6 of the immunofluorescence dyeing of every rat, and under the microscope every section is randomly selected 6 visuals field, it is analyzed calculating the cell of NeuN, GFAP and Iba1 stained positive in each visual field by ImageProPlus5.0 software, with mm²Represent for unit.

Claims (4)

1. an ischemic cerebral apoplexy kit for screening, it is characterised in that include following primer:

RT primer: SEQIDNO:9: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAACAATAC;

PCR primer:

Forward primer sequence SEQIDNO:7: 5 '-GGCGCAGTAAGTTGTATTGTT-3 ';

Reverse primer sequences SEQIDNO:8: 5 '-GTGCAGGGTCCGAGGT-3 '.

2. the application in preparing brain soldier's type screening agent of the ischemic cerebral apoplexy diagnostic kit described in claim 1.

3. an ischemic cerebral apoplexy examination primer, it is characterised in that include following primer:

RT primer: SEQIDNO:9:CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAACAATAC;

PCR primer:

Forward primer sequence SEQIDNO:7:5 '-GGCGCAGTAAGTTGTATTGTT-3 ';

Reverse primer sequences SEQIDNO:8:5 '-GTGCAGGGTCCGAGGT-3 '.

4. the application in preparing brain soldier's type screening agent of the primer described in claim 3.