CN104083754A - Function and application of type-II oncostatin-M receptor (OSMR) in cerebral apoplexy diseases - Google Patents

Abstract

The invention discloses the function and application of type II oncostatin M receptor (OSMR) in cerebral apoplexy, and belongs to the field of gene function and application. The present invention uses OSMR gene knockout mice and nerve-specific OSMR transgenic mice as experimental objects, and simulates stroke disease models through middle cerebral artery ischemia-reperfusion injury. The results show that the volume of cerebral infarction in OSMR knockout mice is significantly increased, The neurological function deteriorated significantly, and there were more apoptotic neurons, and the JAK2/STAT3 signaling pathway was significantly down-regulated; while the infarct volume of the OSMR transgenic mice was significantly reduced, the neurological function was significantly improved, the apoptotic neurons were also reduced, and the JAK2/STAT3 signaling pathway was significantly down-regulated. STAT3 signaling pathway was significantly up-regulated. Therefore, the OSMR gene can be used as a drug target for screening drugs for the prevention, mitigation and/treatment of stroke; the OSMR gene can be used as a target gene in gene therapy for the prevention, mitigation and/treatment of stroke in design and preparation Drugs and/or biological reagents provide an effective new way for the treatment of stroke.

Description

The function and application of II type oncostatinM receptor (OSMR) in apoplexy disease

Technical field

The invention belongs to function and the application of gene, the particularly application of a kind of II type oncostatinM receptor (OSMR) in treatment apoplexy disease specifically applied in the medicine of prevention, alleviation and/or treatment apoplexy disease.

Background technology

Apoplexy can be divided into two large classes substantially: cerebral infarction, accounts for (70～80) %; Hemorrhagic apoplexy, accounts for (20～30) %.Cerebral infarction is to have blocked brain trunk or its branch due to thrombosis or embolus, permanent or the temporary cerebral tissue hypoxic-ischemic damage causing, global the fourth-largest lethal factor and second largest disability-causing factor at present, after it, in several minutes to a few hours, there is irreversible brain injury, patient's life and health is caused to serious harm.

Apoplexy causes cerebral tissue blood to supply and energy supply reduces, and induces a series of overlapped molecular mechanisms finally to cause brain cell death: to comprise excitotoxicity, Ca ²⁺ion overload, oxidative stress, inflammation, apoptosis and the depolarization of infarction surrounding zone.These events do not continue several minutes, a few hours or a couple of days not etc. after cerebral ischemia re-pouring, and neuronal cell, glial cell and blood vessel component etc. are produced to damage in various degree.Neuronal cell is the important ingredient of central nervous system, but its high metabolic rate reduced hypoxic-ischemic environment tolerance, is therefore more vulnerable to damage compared with other neural blood vessel element components.Research shows, neuro-protective strategy can improve brain function after cerebral ischemia in the long period, reduces neuronal cell loss.

Most research thinks, recovers cerebral tissue blood supply and be the effective method for the treatment of cerebral ischemia.Although 1996 Nian Qi tissue plasminogen activators (tPA) ratify for cerebral infarction treatment, it remains the thrombolytic drug that the unique examination & verification of Bureau of Drugs Supervision of the U.S. (FDA) is passed through up to now.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 has been obtained stem-winding result in zoopery; but enter after 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 acted on after apoplexy in 4-6 hour; and in clinical practice, be difficult to implement treatment in so of short duration time window, after therefore further illustrating apoplexy and occurring, in longer a period of time, promote or the molecular mechanism of protection brain tissue impairment significant for the effective Treatment of Stroke target spot of research or strategy.

OSMR is the receptor of oncostatinM (OSM), is a kind of cytokine with various biological activity.OSM belongs to interleukin IL-6 family, can inhibition tumor cell.OSM is as the multi-functional cell regulating factor of one; can act on various kinds of cell; there is various biological activity; the potential function of energy regulator gene activity, cell survival, propagation and differentiation; show unique biologic activity at aspects such as inflammatory reaction, hemoposieis, liver development and immune systems, there is important biological action; Existing studies confirm that OSM be a kind of can be in vivo, the neuroprotective factor (the Oncostatin M is a neuroprotective cytokine that inhibits excitotoxic injury in vitro and in vivo of outer inhibition excitatory neuron damage; 2006; Weiss TW et al.); (Oncostatin M:foe or friend shields in nervous system; 2003, Pelletier JP et al.).OSM has the receptor of two types, be respectively I type and II type OSM receptor (OSMR), I type OSMR is identical with leukaemia inhibitory factor (LIF) receptor (LIFR), the heterodimer being made up of the combination subunit of gp130 and a LIF; The heterodimer (progress of oncostatinM, 2008, Hu Zhiqing) that II type OSMR is made up of 1 gp130 and 1 OSMR β subunit (OSMR β), OSMR β subunit has expression on neuron smooth muscle.Cerebral ischemia re-pouring brain injury can discharge a large amount of inflammatory cytokines, as interleukin 1 (interleuk in-1, IL-1), interleukin-6 (interleuk in-6, IL-6), tumor necrosis factor-alpha (tumor necrosis factor-, TNF-α) etc., their (Determental and beneficial effects of injury-induced in flammation and cytokine expression in the nervous system that plays an important role in ischemia-reperfusion neural cell injury, 2002, Stroll G et al).The cytokine of various releases must be by cytokine receptor superfamily intracellular signal transduction performance effect because of, therefore, OSMR probably improves brain function after cerebral ischemia, reduces in neuronal cell loss and is playing the part of key player.In view of brain and the mankind's life closely related, cerebral infarction is again to threaten human health, threaten one of reason of human life, how research OSMR improves brain function in cerebral infarction disease, reduce neuronal cell loss, for abundant exploitation, it has more importantly meaning in the clinical practice aspect apoplexy.

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 determine the expression of II type oncostatinM receptor (OSMR) and the mutual relation of cerebral infarction associated diseases, provides a kind of II type OSMR for preventing, alleviate and/or treat the new purposes of medicine of cerebral infarction associated diseases.

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

The present invention is taking OSMR knock out mice and nerve-specific OSMR transgenic mice as experimental subject, by Cell transplantation damage model, result shows to contrast with wild type control mice, OSMR knock out mice cerebral infarction volume obviously increases, function of nervous system obviously worsens, the neuronal cell of apoptosis is also more, and the activation of JAK2/STAT3 signal path is obviously lowered; And nerve-specific OSMR transgenic mice Infarction volume obviously reduces, function of nervous system is clearly better, and the neuronal cell quantity of apoptosis significantly reduces, and the activation of JAK2/STAT3 signal path is obviously raised.This prompting OSMR has the effect that can significantly improve apoplexy disease.For the above-mentioned functions of OSMR, OSMR can be used for preparation prevention, alleviate and/medicine for the treatment of apoplexy disease.

The inventor studies have shown that: in apoplexy model, thereby OSMR promotes to activate JAK2/STAT3 signal path neuroprotective system, particularly improves the effect of apoplexy disease.

For the above-mentioned functions of OSMR, the application of a kind of OSMR is provided, major embodiment is that OSMR applies in the medicine of preparation prevention, alleviation and/or treatment apoplexy disease.

Prevention, alleviation and/medicine for the treatment of nervous system disease, comprise OSMR.

Prevention, alleviation and/medicine for the treatment of apoplexy disease, comprise OSMR.

As a kind of prevention, alleviation and/medicine for the treatment of nervous system disease or apoplexy disease, OSMR albumen can be applied to clinical from two aspects.An aspect is can make its label that enters cell (as TAT) by OSMR albumen being added to some; or build and can cross the carrier (as defective adenoviral or retrovirus retrovirus) of expressing OSMR albumen; then utilize conventional process for preparing medicine; adopt medicinal excipient or carrier to be made into injection; then be expelled in cerebrovascular; can improve the level of OSMR in brain neuroblastoma cell, promote to activate JAK2/STAT3 signal path, brain ischemia is produced to protective effect.Another aspect is the new function that the OSMR albumen that confirms according to the present invention has, new compound is screened, selection can inducing cell in the OSMR compound of expressing as medicine, also can play the effect of protection cerebral ischemia.

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

(1) the present invention finds the new function of OSMR gene, and OSMR can improve the effect of apoplexy disease.

(2) the present invention is directed to OSMR in the effect that improves apoplexy disease, is the drug provision basis of preparation treatment apoplexy, can be used for the medicine of preparation prevention, alleviation and/or treatment apoplexy.

Brief description of the drawings

Fig. 1 is that OSMR nerve-specific transgenic mice builds schematic diagram.

Fig. 2 is the postoperative TTC dyeing of WT and OSMR-KO mice I/R, Infarction volume statistics and function of nervous system's scoring cartogram, and result shows that OSMR knocks out remarkable deterioration cerebral infarction and function of nervous system (*: p < 0.05vs WT I/R group).

A is the postoperative TTC coloration result of I/R figure;

B is the postoperative large cerebral infarction volume statistics block diagram of I/R;

C is the postoperative function of nervous system of I/R scoring statistics block diagram.

Fig. 3 is the postoperative TTC dyeing of NTG and OSMR-TG mice I/R, Infarction volume statistics and the scoring statistical result figure of function of nervous system; result shows that OSMR crosses expression and significantly suppresses cerebral infarction, neuroprotective function (*: p < 0.05vs NTG I/R group).

A is the postoperative TTC coloration result of I/R figure;

B is the postoperative large cerebral infarction volume statistics block diagram of I/R;

C is the postoperative function of nervous system of I/R scoring statistics block diagram.

Fig. 4 is the postoperative cerebral tissue infarction surrounding zone neuronal cell apoptosis situation of TUNEL staining examine WT and OSMR-KO mice I/R, and result shows that OSMR knocks out the apoptosis of remarkable promotion neuronal cell.

Fig. 5 is the postoperative cerebral tissue infarction surrounding zone neuronal cell apoptosis situation of TUNEL staining examine NTG and OSMR-TG mice I/R, and result shows that OSMR crosses the apoptosis of expressing remarkable inhibitory neuron cell.

Fig. 6 is that western blotting detects WT and postoperative JAK2/STAT3 signaling pathway protein expression figure and the quantitative statistics block diagram of OSMR-KO mice I/R, and result shows that OSMR knocks out the phosphorylation activation level that can suppress JAK2 and STAT3; Wherein " p-" represents the kinases (JAK2 and STAT3) of phosphorylation, " T-" represents total (phosphorylation with unphosphorylated) kinases (JAK2 and STAT3), GAPDH is as internal reference (*: p < 0.05vs WT Sham group, #:p < 0.05vs WT I/R group).

Fig. 7 is that western blotting detects NTG and postoperative JAK2/STAT3 signaling pathway protein expression figure and the quantitative statistics block diagram of OSMR-TG mice I/R, and result shows that OSMR crosses expression and can promote the phosphorylation activation level of JAK2 and STAT3; Wherein " p-" represents the kinases (JAK2 and STAT3) of phosphorylation, " T-" represents total (phosphorylation with unphosphorylated) kinases (JAK2 and STAT3), GAPDH is as internal reference (*: p < 0.05vs NTG Sham group, #:p < 0.05vs NTG I/R group).

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 to this.

Animal for research and raising

Laboratory animal: select 11-12 age in week, male, body weight is 25-30g mice, wild-type mice (the WT of background C57BL/6, purchased from Fukang bio tech ltd of China, Beijing, the certification of fitness number: 949431), OSMR knock out mice (OSMR-KO, C57BL/6J background, buy from RIKEN BRC company (BRC article No.: RBRC02711)), nerve-specific OSMR transgenic mice (OSMR-TG, by OSMR-flox mice and CaMKII α-Cre (purchased from Jackson Laboratory, Stock No. 005359) mice hybridization obtains, OSMR-flox mice is built by the Li Hongliang of angiocardiopathy institute of Wuhan University professor laboratory, the building process of OSMR-flox mice is as described hereinafter) and non-transgenic mice (NTG, littermate control non-transgenic mice).

Feeding environment: all experiment mices are all raised in the SPF of angiocardiopathy institute of Wuhan University level Experimental Animal Center.Mice special feed is provided by Chinese military medicine academy of science animal center.Raising condition: 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 OSMR transgenic mice

Transgene carrier builds information: with forward primer, i.e. 5 '-GTGATTAATTAAGCCACCATGGCTTTCTCTGTGGTCCT-3 ', downstream primer, 5 '-GTGATTAATTAATTACTGTTCACCTTGGCCTA-3 ', amplification mice OSMR full-length gene (NCBI, Gene ID:18414, XM_006519974.1), cDNA is inserted to pCAG-CAT-LacZ carrier, β-actin gene (CAG that this carrier comprises 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 OSMR obtains (Fig. 1) by CAG promoters driven.The pCAG-OSMR-CAT-LacZ carrier of structure, by the microinjection embryo's (C57BL/6J background) that is configured to be fertilized, is obtained to OSMR-floxed mice.Nerve-specific OSMR transgenic mice obtains by OSMR-flox mice and CaMKII α-Cre mice outbreeding.

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

1. laboratory animal grouping: wild-type mice, OSMR knock out mice, nerve-specific OSMR transgenic mice and non-transgenic mice, set up Cerebral Infarction Model (I/R) and sham operated rats (Sham) by Cell transplantation.Be divided at random 8 groups: wild-type mice sham operated rats (WT Sham), OSMR knock out mice sham operated rats (KO Sham), wild-type mice I/R operation group (WT I/R), OSMR knock out mice I/R operation group (KO I/R), non-transgenic mice sham operated rats (NTG Sham) and I/R operation group (NTG I/R), Neuron-specific OSMR transgenic mice sham operated rats (TG Sham) and I/R operation group (TG I/R).

2. Cerebral Infarction Model (I/R) operation adopts MCAO(middle cerebral artery occlusion, middle cerebral artery occlusion) model manipulation flow process:

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

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

(3) mice is lain on the back fixing, neck median line otch, along sternocleidomastoid inner edge separating muscle and fascia, separates left carotid (CCA), external carotid artery (ECA) and internal carotid artery (ICA).Close ICA, CCA with the temporary transient folder of arteriole folder, in the ligation of ECA distal end with cut an osculum, line bolt is sent into ICA by clip, when line bolt penetration depth stops to the blood flow decline power that is hampered in 9-11mm left and right, whole process must maintain the anus temperature of mice at 37 ± 0.5 DEG C;

(4) enter cerebrovascular from line bolt and in the time that blood flow decline is hampered power, start timing, after 45min, line bolt is extracted, and by the ligation of ECA proximal part, unclamp rapidly CCA place bulldog clamp (Sham group enter from line bolt cerebrovascular to blood flow decline take out Outlet bolt while being hampered power).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% is included experiment in;

(5) sew up mice cervical region and skin of head, and with the povidone iodine wound of sterilizing.After operation finishes, mice is placed in incubator, case temperature maintains 28 DEG C, and feedwater and feedstuff are drawn materials 6 hours, 24h, 72h respectively.

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

(1) 24h after operation respectively, carries out function of nervous system and behavioristics's scoring before 72h draws materials;

Based on Berderson function of nervous system scoring improve one's methods (9 points of systems):

0 point: the symptom that impassivity is impaired;

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

2 points: while carrying tail, in offside shoulder, receive;

3 points: horizontal sliding: while promotion to offside, resistance declines;

4 points: can be spontaneous to all directions motions, but only turn to offside in the time of de-tail;

5 points: when autonomic movement, turn-take or only to turning;

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

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

8 points: the death relevant with cerebral ischemia.

(2) capture mice, lumbar injection 3% pentobarbital sodium anesthetized mice, gets brain;

(3) cerebral tissue taking off is put into the culture dish rinse that PBS is housed, blotted PBS with gauze, cerebral tissue is put into 1mm mouse brain mould, be placed in-20 DEG C of refrigerators frozen;

(4) 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 immediately 1mm slab, cut altogether 7.Section is placed in to 10ml 2% TTC solution, 37 DEG C of constant-temperature incubation 10min immediately.After normal cerebral tissue's dyeing, be cerise, and infarcted region is pale asphyxia;

(5) with the fixing brain tissue slice of 10% neutral formalin solution, substantially take pictures;

(6) cerebral infarction volume calculates (Image-Pro Plus 6.0 softwares): Infarction volume %=(not Infarction volume of offside cerebral hemisphere volume-infarction side)/(offside cerebral hemisphere volume × 2) × 100%;

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

TTC is fat-soluble photaesthesia complex, it is the proton acceptor of pyridine-nucleoside structure enzyme system in respiratory chain, take on a red color, and in ischemic tissue, dehydrogenase activity declines with dehydrogenase reaction in normal structure, can not react, therefore can not change and be pale asphyxia.

The evaluation index of the cerebral ischemia reperfusion injury order of severity mainly comprises the scoring of infarction of brain volume and function of nervous system, these indexs all with ischemical reperfusion injury order of severity positive correlation.The TTC coloration result of OSMR knock out mice as shown in Figure 2 A, pours into after 24 hours OSMR-KO mice Infarction volume (Fig. 2 B) through I/R ischemia 45min obvious more serious than wild-type mice again; And this deterioration acts on I/R and still continues for postoperative 72 hours, and function of nervous system's scoring (Fig. 2 C) all increases at I/R for postoperative 24 hours, 72 hours, illustrate that function of nervous system worsens serious, the disappearance that shows OSMR gene can worsen infarction of brain and the nervous system of the apoplexy mice that ischemical reperfusion injury causes.Cross the TTC coloration result (Fig. 3) of expressing mice by OSMR, we find that OSMR crosses and express the Infarction volume of mice and obviously reduce, function of nervous system is clearly better, and illustrates that OSMR can significantly improve infarction and the nervous system of the apoplexy that cerebrum ischemia reperfusion injury causes.

[embodiment 4] cerebral tissue infarction surrounding zone neuronal cell apoptosis situation is measured

1. cerebral tissue frozen section preparation

1) experiment mice is pressed the anesthesia of 50mg/kg dosage lumbar injection pentobarbital sodium for 24 hours after surgery;

2) open breast and expose heart, puncture into left ventricle with injection needle, cut off right auricle;

3) use 0.1mol/L PBS(pH7.4) after 100mmHg pressure perfusion loses color to liver, with 4% paraformaldehyde perfusion 15 minutes;

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

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

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

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

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

9) cut the frozen section of 5um with 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 to tissue slice and is placed in the paraformaldehyde of (pH 7.4) 1%, the fixing hydrolysis of room temperature 10 minutes;

2) PBS washes twice, each 5 minutes;

3) be placed in the ethanol of pre-cooling: acetic acid (2:1) solution ,-20 DEG C are soaked 5 minutes, remove unnecessary liquid, but note dry;

4) PBS washes twice, each 5 minutes;

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

6) filter paper sucks unnecessary liquid, presses immediately 55 μ L/5cm in section ²directly add TdT enzyme reaction solution, be placed in lucifuge moisture preservation box effect 1 hour (negative control adds the not reactant liquor containing TdT enzyme);

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

8) PBS washes three times, each 1 minute;

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

10) PBS washes four times, each 2 minutes;

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

12) at fluorescence microscopy Microscopic observation counting Apoptotic neuron cell.

By detecting OSMR-KO mice and postoperative 24 hours cerebral tissue infarction surrounding zone neuronal cell apoptosis situation (see figure 4)s of wild-type mice I/R, the neuronal cell apoptosis quantity of finding OSMR-KO group mice is obviously many than WT group mice, and this shows that OSMR gene delection increases the apoptosis quantity of neuronal cell.The cerebral tissue infarction surrounding zone neuronal cell apoptosis quantity of OSMR transgenic mice significantly reduces (see figure 5), this show OSMR cross expression can neuroprotective unit's cell.

[embodiment 5] western blotting (Western blot) detects JAK2/STAT3 signal path.

1. cerebral tissue sample preparation

Experiment mice is pressed the anesthesia of 50mg/kg dosage lumbar injection pentobarbital sodium for 6 hours after surgery;

Open breast and expose heart, puncture as left ventricle with injection needle, on right auricle, do a little otch, so that drain;

With 0.55 number sword-shaped needle, connect disposable transfusion device, insert left ventricle, perfusion ice PBS buffer (about 20mL), until flow out limpid perfusate in right auricle, stops perfusion;

Open cranium and take out rapidly mouse brain, the cerebral tissue taking off is first rinsed and washed with ice PBS, put into again the culture dish of ice, take out olfactory bulb and cerebellum with blade rapidly, again by left and right brain separately, the whole operation of each removal 1mm(before and after left brain to be carried out on ice chest), remaining tissue is packed into the EP pipe of carrying out labelling, be placed in liquid nitrogen container, be finally placed in-80 DEG C of Refrigerator stores.

2. protein extraction

Prepare lysate by formula (1mL lysate 720 μ LRIPA, 20 μ LPMSF, 100 μ LComplete, 100 μ LPhosstop, 50 μ LNaF, 10uLNa3VO4).

In-80 DEG C of required samples of taking-up, put into dry ice.Each EP pipe is put into 3-4 steel ball, puts into dry ice pre-cooling.Cut required sample with eye scissors and put into corresponding EP pipe, weigh and record the weight of each sample, add lysate in sample.

After-80 DEG C of pre-cooling grinding pot 5-8min, sample symmetry is positioned in grinding pot, puts into beveller.Abrasive parameters is set to 30Hz/S, 90s.

After grinding finishes, take out steel ball and be placed in dehydrated alcohol, sample is placed 10min on ice.

Ultrasonic degradation instrument lysed sample 5KHz/ time, each 1s, interval 1s, repeats 10-15 time.For preventing bubble, when cracking, probe gos deep under liquid level 2/3.After ultrasonic completing, place 10min on ice.

It is centrifugal that sample is put into the centrifuge of 4 DEG C of pre-coolings.Parameter is set to 4 DEG C, 12000rpm, 30min.

Accurately draw supernatant quantitative, record the cracking volume of each sample.

3. protein quantification (BCA Protein Assay Kit is quantitative)

Preparation liquid: press A liquid: B liquid=50:1 prepares working solution, the volume of required A liquid is (N+6) * 400 μ L.The volume of B liquid is A liquid 1/50.N is detected sample number.A liquid and B mix, and low temperature is placed for subsequent use.

Prepare detected sample: the sample of extraction is taken out in the ddH2O that 4 μ L join 116 μ L and shakes and mix.

Prepare standard substance, in 500 μ LEP pipes, standard substance are diluted

Draw the standard substance or the sample 25 μ L that have diluted and join in 96 orifice plates, every duplicate samples is vertically done 2 multiple holes.Add the working solution of 200 μ L.

In 37 DEG C of incubators, hatch 30min.

Take out 96 orifice plates and be placed in microplate reader and detect absorbance (wavelength 562nm), and calculate sample concentration (microplate reader is from tape program) according to standard curve.

Add 10 × DTT of respective amount according to surveyed concentration, total protein is calibrated to same concentration by lysate and 4 × LB.

By after protein sample mix homogeneously in 12000rpm/min wink from, subpackage sample, 72 DEG C of water-bath 10min degeneration (every 2-3min stirs once), is placed in-80 DEG C and saves backup.

4. Protein Detection

Protein sample is loaded in SDS-PAGE glue well successively to beginning constant voltage 120V electrophoresis after point sample completes by the applied sample amount of 50ug/ sample.

Clamping plate left and right is spread out, and negative pole to the right.One side of black is negative pole, and white is anodal.Both sides are respectively spread and are used in advance transferring film buffer (3.03g Trisbase, 14.4g glycine, 200mL methanol, 800mL distilled water) to soak that 1-2 opens sponge and 4-5 opens filter paper.

After electrophoresis completes, take out the gel in gel slab, with transferring film liquid wetting gel, pvdf membrane is covered on it, cut the place of breach and should align with of macromolecule Marker jiao, folder train wheel bridge.

Clamping plate are put into transferring film groove, and the negative pole (black side) of transferring film groove should be put together with the negative pole of clamping plate (black side), fills transferring film liquid to flood gel.

Transferring film groove switches on power, and the about 0.2A of constant current starts electrophoretic transfer, and starting voltage should be advisable at 100 ~ 130V, shifts 1.5h.

After transfer finishes, there is the pvdf membrane of albumen to be placed in preprepared TBS by turning, the transferring film liquid washing away on film is placed on 5% the middle sealing of defatted milk powder confining liquid (5g defatted milk powder is dissolved in the TBS of 100mL), slowly shake on decolorization swinging table, room temperature sealing 2-4h.

Sealing finishes rear with TBST washing pvdf membrane 3 times, each 5min, and sealing machine is enclosed pvdf membrane in hybridization bag, adds appropriate primary antibodie, seals after catching up with most foaming, and hybridization bag is put into slowly shake on 4 DEG C of shaking tables, spends the night.

Pvdf membrane is taken out with TBST washing 3 times, each 5 minutes.Reclaim primary antibodie.

Film is put into the corresponding two anti-two anti-diluents (5% defatted milk powder: 5g defatted milk powder is dissolved in 100mLTBST) that are added with, and lucifuge is hatched 1h.In the anti-diluent of 8ml bis-, add two of 0.6uL to resist.

Two anti-hatching after end with TBST washing 3 times, each 5min.Film is placed in to Odyssey fluorescence detector, testing goal band.Preserve and analysis result.

Utilize western blotting to detect the variation of JAK2/STAT3 signal path, the results are shown in Figure 6.Protein expression and quantitative analysis results show, phosphorylation JAK2(p-JAK2) and pSTAT3 (p-STAT3) will be higher than Sham group at the I/R expressing quantity of postoperative 6 hours, illustrate that, after I/R model, JAK2/STAT3 signal path is activated; The OSMR-KO group p-JAK2 of mice and the expressing quantity of p-STAT3 will be lower than WT groups, respectively organize total JAK2 and STAT3 expressing quantity without significant difference.This shows after OSMR gene knockout, JAK2/STAT3 signal path downward in mouse brain tissue.OSMR transgenic mice is after I/R, and the expressing quantity of p-JAK2 and p-STAT3 will be organized (see figure 7) higher than NTG, illustrates that OSMR can pass through to raise the protein expression level of p-JAK2 and p-STAT3, thereby reduces cerebral infarction, neuroprotective function.

Our achievement in research shows, damage in the apoplexy disease model causing at Cell transplantation, the Infarction volume of mice after OSMR gene knockout significantly increases, function of nervous system obviously worsens, the neuronal cell of apoptosis is also more, and the activation of JAK2/STAT3 signal path is obviously suppressed; And the Infarction volume of OSMR transgenic mice significantly reduces, function of nervous system is clearly better, and the neuronal cell of apoptosis also reduces, and the activation of JAK2/STAT3 signal path is more remarkable.These presentation of results OSMR can improve apoplexy by the activation that promotes JAK2/STAT3 signal path, neuroprotective system, and OSMR has important protective effect in apoplexy disease model.

Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.

SEQUENCE LISTING

<110> Wuhan University

The function and application of <120> II type oncostatinM receptor (OSMR) in apoplexy disease

<130> 1

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 38

<212> DNA

<213> Artificial

<220>

<223> OSMR forward primer

<400> 1

gtgattaatt aagccaccat ggctttctct gtggtcct 38

<210> 2

<211> 32

<212> DNA

<213> Artificial

<220>

<223> OSMR downstream primer

<400> 2

gtgattaatt aattactgtt caccttggcc ta 32

Claims (4)

1. Type II Oncostatin M Receptor (OSMR) is used in the preparation of drugs for the prevention, alleviation and/or treatment of nervous system diseases. 2. A drug for preventing, alleviating and/or treating nervous system diseases, characterized in that it contains type II Oncostatin M receptor (OSMR). 3. Type II Oncostatin M Receptor (OSMR) is used in the preparation of drugs for the prevention, alleviation and/or treatment of stroke. 4. A drug for preventing, alleviating and/or treating cerebral apoplexy, characterized in that it contains type II Oncostatin M receptor (OSMR).