CN103784974B - Application of interferon regulatory factor 8 (IRF8) in cerebral apoplexy disease - Google Patents

Abstract

The invention discloses function and application of an IRF8 gene in a cerebral apoplexy disease, belonging to the field of the function and the application of a gene. According to the invention, IRF8 gene knockout mice and neuron-specific IRF8 transgenic mice are taken as experimental subjects, a brain middle artery ischemia reperfusion model is adopted, and the results show that the cerebral infarction volume of the IRF8 gene knockout mice is obviously increased and the neurological function is obviously worsened as well in comparison with that of the wild C57 mice, the infarction volume of the IRF8 transgenic mice is obviously reduced, and the neurological function obviously gets better. The invention discloses the function of the IRF8 gene in the cerebral apoplexy disease, which mainly means that the IRF8 gene has an effect of protecting the function of a nervous system, especially an effect that the IRF8 gene can protect the cerebral apoplexy disease. According to the abovementioned function of the IRF8, the invention provides the application of the IRF8 in preparation of a drug for treating the cerebral apoplexy disease.

Description

Interferon regulatory factor 8 (IRF8) application in apoplexy disease

Technical field

The invention belongs to function and the application of gene, particularly a kind of interferon regulatory factor 8(interferon regulatory factor 8, IRF8) application in apoplexy disease.

Background technology

Cerebral infarction is the whole world mainly lethal disease disabled at present, current tissue plasminogen activator (tissue-typeplasminogenactivator, tPA) fibrinolytic is still the primary treatments for the treatment of ischemic cerebrovascular, but adjoint ischemia-reperfusion can increase the weight of ischemic neurological cell injury further.Research shows, neuro-protective strategy can improve brain function in the long period after cerebral ischemia, reduces neuronal cell loss.Apoptosis is one of fundamental mechanism of cell death in cerebrum ischemia/refilling process, but its regulatory mechanism is illustrated not yet completely.Therefore, the molecular mechanism of neuronal apoptosis existence during research cerebrum ischemia/Reperfu-sion, will contribute to for neuro-protective provides new therapeutic strategy and method.

Cerebrum ischemia reperfusion injury can cause multiple difference but overlapped signal path, modulating apoptosis in platelets or death.During neuron focal cerebral ischemia, infarction core space overwhelming majority cell all necroses (necrosis), feature be energy supply reduce sharply cause cellular edema, organelle breaks and the irreversible death of cell.Infarction core periphery Low perfusion cerebral tissue is called " cerebral ischemic penumbra ", and because it still has metabolic activity, if cerebral blood perfusion improves, this region cytoactive still can be recovered.Therefore save cerebral ischemic penumbra, for Post stroke treatment, there is important value.Although 1996 Nian Qi tissue plasminogen activators (tPA) namely ratify to be used for cerebral infarction treatment, it remains Bureau of Drugs Supervision of the U.S. (FDA) and uniquely audits the thrombolytic drug passed through up to now.Because extend bleeding risk in time to increase, the therapeutic time window of tPA is only 4.5 hours; Consider ischemic and hemorrhagic apoplexy in early days iconography not easily differentiate, delay further patient accept tPA treatment chance.The ischemic cerebral stroke patients being only less than 5% at present uses tPA thromboembolism treatment.In addition, if research finds the long-time severe ischemic anoxia of cerebral tissue, even if recover cerebral blood flow in the later stage still can cause irreversible damage to cerebral tissue, therefore current still in the urgent need to the therapeutic strategy of research for pathophysiologic event caused by hypoxic-ischemic and (or) Reperfu-sion.Since the nineties in 20th century, the therapeutic strategy of research neuroprotective and cerebral tissue is the focus of Treatment of Cerebral Stroke always, and these strategies not only can extend the therapeutic time window of tPA, also can alleviate the brain tissue impairment of ischemia-reperfusion induction.Multiple nerve protection medicine achieves stem-winding result in zoopery; but after entering apoplexy 3 phase clinical experiment; overwhelming majority medicine does not all attain the results expected; one of its primary failed reason is that most of known Neuroprotective Mechanisms acts within Post stroke 4-6 hour; and be difficult in clinical practice so of short duration be in time window, implement treatment, therefore illustrate further to promote in longer a period of time after apoplexy occurs or protection brain tissue impairment molecular mechanism for the effective Treatment of Stroke target spot of research or strategy significant.

Interferon regulatory factor (interferon regulatory factor, IRF) family has had now found that 10 members, and it consists of IRF1 ~ IRF10.Existing research prompting, IRF family member take part in biological process widely, relates generally to the natural immunity and the acquired immune response, and regulating cell growth and existence, apoptosis and propagation, participate in hemopoietic, antitumor formation etc.IRF8 is the other associated proteins of common recognition (interferon consensus sequence-binding protein, ICSBP) of interferon, is a member of IRF family, as a transcription factor, and can transcriptional control DNA thus play a role.IRF8 is found to be in myelocyte and lymphocyte first, there are some researches show that IRF8 plays central role in immunomodulating and myeloid differentiation.Multinomial research shows, IRF8 may participate in multiple sclerosis, central nervous system's inflammatory demyelinating disease, the diseases such as peripheral nerve injury.

Summary of the invention

For solving defect and the deficiency of above-mentioned prior art, primary and foremost purpose of the present invention is to provide the application of a kind of IRF8 in preparation treatment nervous system disease agent.

Another object of the present invention is to provide the application of a kind of IRF8 in preparation treatment apoplexy disease medicament.

Object of the present invention is achieved through the following technical solutions:

The present invention with IRF8 knock out mice and Neuron-specific IRF8 transgenic mice for experimental subject, by Cell transplantation model, result shows to contrast with wild type C57 mice, IRF8 knock out mice head infarction obviously increases the weight of, function of nervous system also obviously worsens, the Infarction volume of Neuron-specific IRF8 transgenic mice is then suppressed, and function of nervous system also takes a turn for the better.This prompting IRF8 gene has the effect of neuroprotective systemic-function, can protect the nerve injury that cerebral ischemia causes, for the new drug and New Policy studying control cerebral ischemia provides theoretical foundation and Clinical Basis.

The function of IRF8 gene in apoplexy disease, be mainly reflected in the effect that IRF8 gene has neuroprotective systemic-function, particularly IRF8 gene can protect the effect of Imaging in Patients with Cerebral Ischemia Disease.

For the above-mentioned functions of IRF8 gene, IRF8 is provided to apply in the medicine of preparation treatment nervous system disease.

Treat a medicine for nervous system disease, comprise IRF8.

For the above-mentioned functions of IRF8 gene, IRF8 is provided to apply in preparation treatment apoplexy disease medicament.

Treat a medicine for apoplexy disease, comprise IRF8.

Achievement in research of the present invention shows, in the damage that artery ischemia Reperfu-sion causes in the brain of IRF8-KO mice, mice Infarction volume obviously increases the weight of, and function of nervous system obviously worsens, and neuronal apoptosis also obviously increases.Prove that IRF8 gene has important protective effect in apoplexy disease model.

The present invention has following advantage and effect relative to prior art:

1. the present invention finds the New function of IRF8 gene, and namely IRF8 gene can protect the effect of apoplexy disease.

2. the effect of IRF8 in protection apoplexy disease, for the preparation for the treatment of apoplexy disease medicament.

Accompanying drawing explanation

Fig. 1 is structure and the qualification result figure of nerve-specific IRF8 transgenic mice

A is the design of graphics of nerve-specific IRF8 transgenic mice;

B is the qualification result figure of nerve-specific IRF8 transgenic mice;

Fig. 2 is the TTC coloration result figure of WT and IRF8-KO mice.

A is TTC coloration result figure;

B is cerebral infarction volume statistics block diagram;

C is Neuroscore statistics block diagram;

Fig. 3 is the TTC coloration result figure of IRF8-TG and NTG mice.

A is TTC coloration result figure;

B is cerebral infarction volume statistics block diagram;

C is Neuroscore statistics block diagram;

Fig. 4 is the cerebral tissue infarction surrounding zone neuronal apoptosis situation measurement result figure of WT and IRF8-KO mice.

Figure A is Fluoro Jade B detection display figure and statistical result figure;

Figure B is TUNEL apoptosis figure and statistical result figure;

Fig. 5 is the cerebral tissue infarction surrounding zone neuronal apoptosis situation measurement result figure of IRF8-TG and NTG mice.

Figure A is Fluoro Jade B detection display figure and statistical result figure;

Figure B is TUNEL apoptosis figure and statistical result figure;

Detailed description of the invention

Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.

Animal for research and raising

Laboratory animal: select 11-12 age in week, body weight is at 25-30g; background is the wild-type mice (WT of male C57BL/6 strain; purchased from Beijing China Fukang bio tech ltd), IRF8 knock out mice (IRF8-KO; C57BL/6J background; purchased from EMMA; article No. EM:02414), nontransgenic mice (NTG, Neuron-specific Cre transgenic mice (CaMKII α-Cre; Purchased from Jackson Laboratory, Stock No. 005359)) and nerve-specific IRF8 transgenic mice (IRF8-TG, obtained by IRF8-flox transgenic mice and CaMKII α-Cre mouse hybrid, IRF8-flox transgenic mice is built by this laboratory oneself, and the building process of IRF8-flox transgenic mice as described hereinafter).

Feeding environment: all experiment mices are all raised in angiocardiopathy institute of Wuhan University SPF level Experimental Animal Center.Mice special feed is provided by Chinese military medicine academy of science animal center.Rearing conditions: room temperature is between 22-24 DEG C, and humidity is between 40-70%, and it is 12h that light and shade replaces lighting hours, freely drinks water and ingests.

The structure of [embodiment 1] nerve-specific IRF8 transgenic mice

IRF8-flox transgenic mice builds information:

Transgene carrier builds information: with forward primer, i.e. 5 '-CCAGATTACGCTGATTGTGACCGGAACGGCGGGCG-3 ' (SEQ ID NO. 1); Downstream primer, 5 '-AGGGAAGATCTTGATTTAGACGGTGATCTGTTGAT-3 ' (SEQ ID NO. 2), amplification mice IRF8 full-length gene (NCBI, Gene ID:15900, NM_008320.3), cDNA is inserted pCAG-CAT-LacZ carrier, this carrier comprises the β-actin gene (CAG of a cmv enhancer and a chicken, chicken β-actin gene) promoter, and be connected to chloramphenicol acetyl transferasegene (CAT, chloramphenicol acetyltransferase), loxP site is positioned at CAT both sides.The expression of neurocyte IRF8 is obtained (Figure 1A) by CAG promoters driven.IRF8-floxed mouse: the pCAG-IRF8-CAT-LacZ carrier of structure is configured to fertilized embryo (C57BL/6J background) by microinjection, obtains IRF8-floxed transgenic mice.Neuronal specificity IRF8 transgenic mice is obtained by IRF8-flox mice and the breeding of CaMKII α-Cre mouse hybrid.

Transgenic mice gets genomic DNA by cutting tail, use PCR qualification, PCR identifies that primer information is: detect forward primer 5 '-CCAGATTACGCTGATTGTGACCGGAACGGCGGGCG-3 ' (SEQ ID NO. 3), detects reverse primer 5 '-AGGGAAGATCTTGATTTAGACGGTGATCTGTTGAT-3 ' (SEQ ID NO. 4).Expression by IRF8 albumen in the different transgenic mouse head of western blotting (Western Blot) experimental identification: extract different transgenic mouse cerebral tissue albumen, by polyacrylamide gel electrophoresis (SDS-PAGE), checking IRF8 process LAN (Figure 1B)

We construct a few strain nerve-specific IRF8 transgenic mice (IRF8-TG).In order to reflect the change of IRF8 under pathological and physiological condition, we have selected IRF8-TG5 mice, and Western Blot and quantitative analysis display, in its cerebral tissue, IRF8 expression is about normal structure 10.5 times.

[embodiment 2] mouse brain Infarction Model (I/R) obtains

1. laboratory animal grouping: male C57BL/6 strain wild-type mice, IRF8 knock out mice and head specificity IRF8 transgenic mice and nontransgenic mice, sets up Cerebral Infarction Model (I/R) by Cell transplantation.Be divided into 8 groups at random, often organize 10 mices: C57BL/6 strain wild-type mice sham operated rats (WT SHAM) and I/R art group (WT I/R), IRF8 knock out mice sham operated rats (KO SHAM) and I/R art group (KO I/R), nontransgenic mice sham operated rats (NTG SHAM) and I/R art group (NTG I/R), neuronal specificity IRF8 transgenic mice sham operated rats (TG SHAM) and I/R art group (TG I/R).

2. line brush cerebral infarction I/R performs the operation and adopts mouse brain medium-sized artery ischemia-reperfusion (middle cerebral artery Ischemia Reperfusion) model manipulation flow process:

(1) capture mice, use 3% isoflurane anesthesia mice, 8% sodium sulfide sloughs the Mus hair of cervical region, and calvarium Mus hair operating scissors is cut rapidly, 3% povidone iodine sterilization cervical region and calvarium skin 2 times, and 75% ethanol takes off iodine 1 time;

(2) at the calvarium position cross sections of mice, expose skull, peel off the connective tissue of skull surface with tweezers gently.The fibre-optical probe biogum of laser Doppler flowmetry is fixed on bregma rear 2mm, the position of left side 5mm;

(3) lain on the back by mice fixing, neck median line otch, along sternocleidomastoid inner edge separating muscle and fascia, is separated left common carotid (CCA), external carotid artery (ECA) and internal carotid artery (ICA).Prick at ECA distal end 8-0 toe-in, ECA proximal part place hanging wire is for subsequent use.Temporarily press from both sides with arteriole folder and close ICA, CCA; Then in the middle of the ligation of ECA distal end and proximal part hanging wire, an osculum is cut, line bolt is sent to CCA by clip, and the hanging wire of ECA proximal part is made a call to a slip-knot at clip, elasticity can free in and out with line bolt but the sense that slightly rubs is advisable, loose ICA bulldog clamp again, line bolt is sent into ICA, from vascular bifurcation place, calculates distance, when insertion depth stops to the decline power that is hampered of blood flow at about 9-11mm.At this moment will fasten gently around ECA proximal part place slip-knot and fasten line, whole process must maintain the anus temperature of mice at 37 ± 0.5 DEG C;

(4) enter cerebrovascular to blood flow from line bolt to decline the timing the power stopping time that is hampered, ECA proximal part place slip-knot is first unclamped after 45min, line bolt is extracted, and ECA proximal part place slip-knot is tightened, unclamp rapidly CCA place bulldog clamp, and by the ligation of ECA proximal part (Sham group takes out Outlet bolt when entering cerebrovascular from line bolt and declining be hampered power to blood flow).Note observing restoration of blood flow situation, select blood flow to decline more than 75%, the mice that restoration of blood flow reaches more than 70% includes experiment in;

(5) sew up mice cervical region and skin of head, and to sterilize wound with povidone iodine.After operation terminates, be placed on by mice in incubator, case temperature maintains 28 DEG C, and feedwater and feedstuff are to drawing materials.

[embodiment 3] Cerebral Infarction Model (I/R) mouse brain Infarction volume measures

The evaluation index of the cerebral ischemia/reperfusion injury order of severity mainly comprises infarction of brain volume and Neuroscore, these indexs all with the positive correlation of the ischemia/reperfusion injury order of severity.

(1) respectively 24h, 72h carry out function of nervous system and shape for learning scoring before drawing materials after surgery;

(9 points of systems) is improved one's methods based on Berderson Neuroscore:

0 point: the symptom that impassivity is impaired;

1 point: when carrying tail, offside forelimb is curled, or can not arrive Ipsilateral forelimb completely;

2 points: offside shoulder adduction when carrying tail;

3 points: horizontal sliding: when promoting to offside, resistance declines;

4 points: can be spontaneous to all directions motion, but only to turn to offside when de-tail;

5 points: turn-take during autonomic movement or only to turn;

6 points: without autonomic movement, the only motion when stimulating;

7 points: without autonomic movement, also without motion during stimulation;

8 points: the death relevant with cerebral ischemia.

(2) capture mice, lumbar injection 3% pentobarbital sodium anesthetized mice, cuts off mice thoracic cavity, breaks heart blood-letting;

(3) skin of neck after volume fraction 75% alcohol disinfecting, cut off rear skin of neck, expose head and cervical region, neck marrow is cut off from cervical vertebra, be separated the rear musculi colli of removing, eye scissors longitudinally cuts off the outer skull of brain stem cerebellum, with stricture of vagina tooth pincers strip off skull, be separated the cerebral dura mater of brain surface, avoid cerebral dura mater to scratch cerebral tissue.When getting brain from oblongata, careful separation basis cranii tissue, avoids damaged brain;

(4) cerebral tissue taken off is put into the culture dish rinse that PBS is housed, blot PBS with gauze, cerebral tissue is put into 1mm mouse brain mould, be placed in-20 DEG C of refrigerators frozen (being no more than 4h);

(5) cerebral tissue 2,3, 5-Triphenyltertrazoliumchloride (2,3,5-Triphenyltetrazolium chloricej, TTC) dyeing: take out cerebral tissue from-20 DEG C of refrigerators, be cut into 1mm slab immediately, comprise bregma front and cut 4, rear cuts 3, cuts 7 altogether.Section is placed in immediately the serum bottle of 10mL2% TTC solution, 37 DEG C of constant-temperature incubation 10min.Frequently stir section, even tissue is dyeed.In cerise after normal cerebral tissue's dyeing, and infarcted region is pale asphyxia;

(6) cerebral tissue is fixed: the cerebral tissue in beaker and solution are together proceeded to and carry out in the cup of labelling, discard TTC solution, fix brain tissue slice with 10% neutral formalin solution, take pictures and use IPP software analysis after 24h;

(7) cerebral infarction volume computing: Infarction volume %=(contralateral hemispheres volume-non-Infarction volume in infarction side)/(contralateral hemispheres volume × 2) × 100%;

Total Infarction volume is respective 7 brain sheet result data sums.

TTC coloration result as shown in Figure 2, through I/R ischemia 45min Reperfu-sion after 24 hours IRF8-KO mice Infarction volume comparatively wild-type mice increase; And this deterioration acts on I/R within postoperative 72 hours, still continues, and Neuroscore all increases the weight of at I/R for postoperative 24 hours, 72 hours.

As shown in Figure 3, through I/R ischemia 45min Reperfu-sion after 24 hours IRF8-TG mice Infarction volume comparatively wild-type mice obviously alleviate; And this protective effect still continues at I/R for postoperative 72 hours, and Neuroscore all alleviates at I/R for postoperative 24 hours and 72 hours.

Embodiment 4. cerebral tissue infarction surrounding zone neuronal apoptosis situation measures

1. cerebral tissue frozen section preparation

1) experiment mice presses the anesthesia of 50mg/kg dosage lumbar injection pentobarbital sodium;

2) open breast and expose heart, with injection needle puncture as left ventricle, cut off right atrium simultaneously;

3) 0.1mol/l PBS(pH7.4 is used) after 100mmHg pressure perfusion loses color to liver, with 4% paraformaldehyde perfusion 15min;

4) open cranium and take out mouse brain rapidly, after room temperature 4% paraformaldehyde, fix 6-8h;

5) excise the olfactory bulb of cerebral tissue and cerebellum, then prolong median line and brain is divided into first latter two part, fix 15min again with previous fixative;

6) be immersed in the phosphate buffer containing 30% sucrose subsequently, 4 DEG C of refrigerators sink to the bottom and spend the night;

7) after 30% sucrose mixes by 1:1 with OCT, appropriate in embedding frame, the tissue of back is taken out, soak a little while in this embedding frame after gauze sucks liquid, be transferred to again and first added in another embedding frame of 2 OCT, the position of adjustment tissue, makes it just in time be positioned at the center of embedding frame;

8) will contain the embedding frame of tissue, move in dry ice, make it be in horizontal position, slightly after a little while, continue to add OCT, certain height is organized in submergence as far as possible, after OCT solidifies, is stored in the refrigerator of-80 DEG C;

9) frozen section of 5 μm is cut by the standardization program of freezing microtome for subsequent use.

2.TUNEL test kit staining examine apoptosis.

With TUNEL test kit staining examine apoptosis.(TUNEL test kit: ApopTag Plus In Situ Apoptosis Fluorescein Detection Kit (S7111, Chemicon)):

1) ice is cut the paraformaldehyde that tissue slice is placed in (pH 7.4) 1%, room temperature fixes hydrolysis 10 minutes;

2) PBS washes twice, each 5 min;

3) be placed in the ethanol of pre-cooling: acetic acid (2:1) solution ,-20 DEG C are soaked 5 minutes, remove surplus liquid, but note not dry;

4) PBS washes twice, each 5 min;

5) filter paper carefully sucks surplus liquid, presses 75 μ L/5 cm immediately in section ²directly add level pad, incubated at room 1-5 min;

6) filter paper carefully sucks surplus liquid, presses 55 μ l/5 cm immediately in section ²directly add TdT enzyme reaction solution, be placed in lucifuge moisture preservation box and act on 1 h(negative control and add not containing the reactant liquor of TdT enzyme);

7) section is placed in termination/lavation buffer solution, shakes 15 sec gently, incubated at room 10 min; Now prepare appropriate anti digoxin antibody, be preheated to room temperature, note lucifuge;

8) PBS washes three times, each 1 min;

9) filter paper carefully sucks surplus liquid, directly in section, presses 65 μ L/5 cm ²add anti digoxin antibody, under room temperature, in the wet box of lucifuge insulation, act on 1 h;

10) PBS washes four times, each 2 min;

11) SlowFade Gold antifade reagent with DAPI(Invitrogen, S36939) mounting;

12) viewed under fluoroscopy, takes pictures.Preserve if need, 4 DEG C of preservations in dark wet box.At fluorescence microscopy Microscopic observation, take pictures, counting Apoptotic neuron cell.(if needing to preserve, 4 DEG C of preservations in dark wet box)

3. FJB(Fluoro Jade B) dyeing

1) ice is cut tissue slice and dry 1 hour in an oven;

2) 1% NaOH+80% dehydrated alcohol 5min;

3) 70% dehydrated alcohol 2min;

4）dd H ₂O 2min；

5) Flouro jade B diluent (AG310, Millipore, Billerica, MA), room temperature lucifuge 20min;

6) dd H ₂o 1min washes 3 times;

7) sheet 5-10min is dried in an oven;

8) dimethylbenzene process 2-3min;

9) mounting, takes pictures.

Cerebral tissue infarction surrounding zone neuronal apoptosis situation measurement result is shown in Fig. 4, Fig. 5.Fig. 4 is IRF8-KO mice and wild-type mice I/R postoperative 24 hours cerebral tissue infarction surrounding zone neuronal apoptosis situations.Fluoro Jade B dyes (A) and TUNEL dyeing (B) detects apoptosis, and result display IRF8-KO mice neuronal cells apoptosis rate all increases than wild-type mice, and prompting IRF8 is dead relevant to during neuronal cell ischemia/reperfusion further.Same Fig. 5 is IRF8-TG mice and NTG mice I/R postoperative 24 hours cerebral tissue infarction surrounding zone neuronal apoptosis situations, and Fluoro Jade B dyes (A) and TUNEL dyes, and (B) result display IRF8-TG mice neuronal cells apoptosis rate all reduces than NTG mice.These results show, promote that IRF8 expresses and can improve brain tissue ischemia/reperfusion injury, and may be closely related with neuronal apoptosis.

Our achievement in research shows, in the damage that artery ischemia Reperfu-sion causes in the brain of IRF8 KO mice, after we find that IRF8 knocks out, mice Infarction volume significantly increases, and function of nervous system obviously worsens, and neuronal apoptosis is showed increased also.Prove that IRF8 gene has important protective effect in apoplexy disease model.

Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.

SEQUENCE LISTING

 

<110> Wuhan University

<120> interferon regulatory factor 8(IRF8) application in apoplexy disease

<130> 1

<160> 4

<170> PatentIn version 3.3

 

<210> 1

<211> 35

<212> DNA

<213> Artificial

<223> IRF8 forward primer

<400> 1

ccagattacg ctgattgtga ccggaacggc gggcg 35

 

<210> 2

<211> 35

<212> DNA

<213> Artificial

 

<223> IRF8 downstream primer

<400> 2

agggaagatc ttgatttaga cggtgatctg ttgat 35

 

<210> 3

<211> 35

<212> DNA

<213> Artificial

<223> detects forward primer

<400> 3

ccagattacg ctgattgtga ccggaacggc gggcg 35

 

<210> 4

<211> 35

<212> DNA

<213> Artificial

<223> detects reverse primer

<400> 4

agggaagatc ttgatttaga cggtgatctg ttgat 35

Claims (1)

1. interferon regulatory factor 8 or the application of its gene in preparation treatment apoplexy disease medicament.