CN107583034A - Application of the growth and differentiation factor 11 in ischemia apoplexy disease medicament is prepared - Google Patents

Abstract

The present invention relates to application of the growth and differentiation factor 11 in ischemia apoplexy disease medicament is prepared.The present invention is in Focal Cerebral Ischemia Reperfusion model caused by intraluminal middle cerebral artery occlusion in rats occlusion, it have studied treatment and prevention effect of the growth and differentiation factor (GDF11) to cerebral ischemia, it was found that GDF11, which has, reduces cortical ischemia volume, improve the function of recovering animal sports ability, and then the mechanism of action that GDF11 treats and prevents to cerebral ischemia is found, and this process is realized by activating Smad2/3 signal paths.GDF11 can be designed as to targeted drug and carry out prophylactic treatment cerebral arterial thrombosis, there are potential huge applications to be worth the treatment of cerebral arterial thrombosis clinical prevention, and the biological function for further research GDF11 is laid a good foundation.

Description

Application of the growth and differentiation factor 11 in ischemia apoplexy disease medicament is prepared

Technical field

The present invention relates to growth and differentiation factor 11 (Growth differentiation factor 11, GDF11) to make Application in standby ischemia apoplexy disease medicament, belongs to ischemia apoplexy technical field of pharmaceuticals.

Background technology

Headstroke is one of clinical common disease, has the characteristics of incidence of disease, fatal rate and disability rate are high, headstroke The origin cause of formation has two kinds, and one kind is due to that rupture of blood vessel in brain causes hemorrhagic cerebral apoplexy caused by bleeding, accounts for 20%；Another kind is due to Thrombus obstruction cerebral artery cause intracerebral hypoxic-ischemic caused by ischemia apoplexy, account for 80%.After focal cerebral ischemia, ischemic core Heart Regional Blood Flow drastically declines, neuron mortality, is irreversible after the necrosis in this region.But Ischemic core domain edge, referred to as ischemic penumbra, it is considered to be the potential region saved.Treatment ischemia apoplexy at present Conventional medicine has three major types, be respectively by promoting the thromboembolism treatment medicine of revascularization resurgent, by reducing cellular damage, Improve the neuroprotective agent of brain blood flow, and Chinese herbal medicine treatment.But due to many medicines, there is curative effect is weak, side effect The problems such as more, the medicine for finding highly effective and safe are still shouldered heavy responsibilities.

In recent years by parabiosis model find young mice blood and old blood mutually blended circulation when, can So that function declines such as reducing muscular atrophy, heart failure, slow in reacting, cognitive ability all caused by aging Relax and even return to previous level.Played a role by the GDF11 that numerous studies are the discovery that in blood, GDF11 is in blood In expression be to be reduced with the increase at animal age, into aged mice body injection restructuring GDF11 albumen effect, with It is directly injected into that the effect of young mice blood is the same into aged mice body, both can significantly improve the myocardial function of mouse, Avoid heart failure；In addition, find when the GDF11 contents in aged mice body return to identical water in young mice body Usually, the structure of the skeletal muscle relevant with aging and physical function damage are repaired, and strengthen the strength of aged mice and resistance to Power；Research can strengthen the blood circulation of aged mice brain it has also been found that recombinating both GDF11 albumen and young mice blood, swash The nerve stem cell proliferation of subependymal region living, promotes the existence of new neuron, and finally improve age-related brain work( It can recover.

GDF11 be transforming growth factor β (Transforming growth factor beta, TGF-β) family into Member, TGF-β family include many protein moleculars for planting and influenceing body functions and such as growing, make a variation and be immunized, wherein flesh generation suppression Albumen myostatin and GDF11 homology are up to 90%, and actuating signal path is identical.GDF11 intracerebral expression more Extensively, mainly in hypothalamus, hippocampus and Purkinje cell group, it by combining the receptors of activin II on cell membrane, and then I receptor ALK4, ALK5 is recruited, activation downstream Smad2, Smad3 signal path plays a role.

The content of the invention

In view of the deficiencies of the prior art, the present invention provides a kind of (Growth of growth and differentiation factor 11 Differentiation factor 11, GDF11) application in ischemia apoplexy disease medicament is prepared, available for preparing ICVD medicine.

Technical solution of the present invention is as follows：

A kind of applications of growth and differentiation factor GDF11 in ischemia apoplexy disease medicament is prevented and treated.

According to currently preferred, the ischemia apoplexy disease is cerebral apoplexy.

It is injection according to currently preferred, described pharmaceutical dosage form.

Beneficial effect

1st, inventor is had found by studying, after cerebral ischemia re-pouring 24h, rat layer ischemic penumbra GDF11 expression It is horizontal significantly to rise；After being overexpressed slow virus pretreated rats 12 days with GDF11, cerebral ischemia re-pouring model is established, discovery can Rat cerebral ischemia volume is substantially reduced, reduces neurological deficit scoring.Its mechanism be by promote SVZ brain areas cell breed, Strengthen the regeneration of peripheral vessels and suppress penumbra region Apoptosis mode, reduce damage caused by cerebral ischemia re-pouring；In brain To intracerebroventricular injection vitro recombination GDF11 albumen after ischemia-reperfusion 24h, rat cerebral ischemia volume can be effectively reduced, reduces god Scored through functional defect.Its mechanism is shielded by way of suppressing penumbra region Apoptosis；GDF11 is pre- in cerebral ischemia Effect in anti-and treatment is realized by regulating and controlling Smad2/3 signal paths.This illustrates GDF11 and ischemia apoplexy is damaged The preventive and therapeutic action of wound.

2nd, this application discloses GDF11 prevention and treatment ischemia apoplexy damage in effect and different effect machines System, for ischemia apoplexy clinical prevention and treat and provide new targeted drug, it was demonstrated that GDF11 carries in ischemia apoplexy Potential huge applications in preceding prevention and clinical treatment.

Brief description of the drawings

GDF11 immunohistochemical staining figures after 2 hours Figure 1A, rat ischemia Reperfu- sion 24h, wherein：45 μm of scale；

GDF11 positive cell dyeings count column diagram after 2 hours Figure 1B, rat ischemia Reperfu- sion 24h, wherein：The μ of scale 45 m；

When 2 hours Fig. 1 C, rat cerebral ischemia Reperfu- sion different time points, the horizontal changes of cortex penumbra region GDF11mRNA Statistical chart；

Fig. 2A, MCAO rats with left intracerebroventricular external source recombinate GDF11 albumen, the brain section photo of TCC dyeing；In figure, Arrangement represents the direction of brain from front to back, scale in order from left to right for section：10mm

Fig. 2 B, MCAO rats with left intracerebroventriculars external source restructuring GDF11 albumen, ischemic area volume statistical chart；

Before and after Fig. 3, MCAO rats with left intracerebroventricular external source restructuring GDF11 albumen, the statistical chart of Bederson scorings；

Fig. 4 A, MCAO rats with left intracerebroventriculars external source restructuring GDF11 albumen, ischemic cortex caspase-3 SABCs Colored graph, wherein：45 μm of scale；

Fig. 4 B, MCAO rats with left intracerebroventriculars external source restructuring GDF11 albumen, ischemic cortex caspase-3 positive cells The statistical chart of number；

Fig. 5 A, MCAO rats with left intracerebroventriculars external source restructuring GDF11 albumen, ischemic cortex TUNEL colored graphs, wherein： 45 μm of scale；；

Fig. 5 B, MCAO rats with left intracerebroventriculars external source restructuring GDF11 albumen, ischemic cortex TUNEL dyeing statistical charts；

Fig. 6 A, MCAO rats with left intracerebroventriculars external source restructuring GDF11 albumen, SVZ areas BrdU-nestin is immunized dual glimmering Light tissue staining figure；

Fig. 6 B, MCAO rats with left intracerebroventriculars external source restructuring GDF11 albumen, SVZ areas Ki67 immunofluorescence tissue stainings Figure；

Fig. 6 C, MCAO rats with left intracerebroventriculars external source restructuring GDF11 albumen, SVZ areas BrdU positive cell statistical charts；

Fig. 6 D, MCAO rats with left intracerebroventriculars external source restructuring GDF11 albumen, SVZ areas Ki67 positive cell statistical charts；

Fig. 7 A, MCAO rats with left intracerebroventriculars external source restructuring GDF11 albumen, CD31 immunohistochemistries around SVZ areas Colored graph；

Fig. 7 B, MCAO rats with left intracerebroventriculars external source recombinate GDF11 albumen, and CD31 positive cells count around SVZ areas Figure；

Fig. 8 A, Rat Right intracerebroventricular injection GDF11 are overexpressed virus and MCAO models are established after 12 days, and TCC dyeing displays lack Blood volume；In figure, arrangement represents the direction of brain from front to back, scale in order from left to right for section：10mm

Fig. 8 B, Rat Right intracerebroventricular injection GDF11 are overexpressed virus and MCAO models, ischemic area volume system are established after 12 days Meter figure；

Fig. 9, Rat Right intracerebroventricular injection GDF11 are overexpressed virus and MCAO models, Bederson scoring systems are established after 12 days Meter figure；

Figure 10 A, Rat Right intracerebroventricular injection GDF11 are overexpressed virus and establish MCAO models, ischemic cortex after 12 days Caspase-3 immunohistochemical staining figures, wherein：45 μm of scale；

Figure 10 B, Rat Right intracerebroventricular injection GDF11 are overexpressed virus and establish MCAO models after 12 days, ischemic cortex Caspase-3 positive cell statistical charts；

Figure 11 A, Rat Right intracerebroventricular injection GDF11 are overexpressed virus and establish MCAO models, ischemic cortex TUNEL after 12 days Colored graph, wherein：45 μm of scale；

Figure 11 B, Rat Right intracerebroventricular injection GDF11 are overexpressed virus and establish MCAO models, ischemic cortex TUNEL after 12 days Positive cell statistical chart；

Figure 12 A, Rat Right intracerebroventricular injection GDF11 are overexpressed virus and establish MCAO models, SVZ areas BrdU- after 12 days Double fluorescent tissue staining figure is immunized in nestin, wherein：45 μm of scale；

Figure 12 B, Rat Right intracerebroventricular injection GDF11 are overexpressed virus and MCAO models are established after 12 days, and SVZ areas BrdU is positive Cell counts figure；

Figure 13 A, Rat Right intracerebroventricular injection GDF11 are overexpressed virus and establish MCAO models after 12 days, CD31 around SVZ areas Immunohistochemical staining figure, wherein：45 μm of scale；

Figure 13 B, Rat Right intracerebroventricular injection GDF11 are overexpressed virus and establish MCAO models after 12 days, CD31 around SVZ areas Positive cell statistical chart；

Figure 14 A, Western Blot give external source restructuring GDF11 albumen/pre- place of GDF11 overexpression viruses after showing ischemic After reason, cortex penumbra region p-Smad2/3 expression result of variations；

Figure 14 B, Western Blot give external source restructuring GDF11 albumen, cortex penumbra region p-Smad2/3 after showing ischemic The statistical chart of relative amount after internal reference standardizes；

After Figure 14 C, Western Blot show that GDF11 is overexpressed virus pretreatment, cortex penumbra region p-Smad2/3 warp Cross the statistical chart of relative amount after internal reference standardizes.

Embodiment

Technical scheme is further elaborated with reference to Figure of description and embodiment, but the present invention is protected Protect scope not limited to this.

The present invention, by the method for intracerebral cortex microinjection, have studied in Focal Ischemia-Reperfusion in Rats model Treatment and prevention that GDF11 damages to ischemia apoplexy effects, simultaneously, it was found that GDF11 is treating and preventing ischemic Different mechanism of action in headstroke damage.So as to facilitate the present invention.

Embodiment 1:Therapeutic action of the GDF11 foreign recombinant proteins in cerebral ischemia is given after ischemic

1. establish line brush Focal Ischemia-Reperfusion in Rats model (Middle cerebral artery Occlusion, MCAO model)：

Take 300 ± 5g of body weight Sprague-Dawley rats (purchased from Beijing company of dimension tonneau China), intraperitoneal injection 10% Chloraldurate (0.3~0.4ml/100g body weight) after, lie on the back and be fixed on operating table, along neck median incision, blunt separation Hypodermis, separation, which connects, pricks left common carotid (CCA), and separates internal carotid (ICA) and external carotid artery upwards along CCA (ECA) crotch, ECA near-ends are ligatured.An osculum is cut in arteria carotis communis infall, a diameter of 0.25mm bolts line is inserted from osculum Enter ICA about 20.5 ± 0.5mm, meet light resistance, show that bolt line through arteria cerebri media initiating terminal to near-end, will move in brain Arteries and veins initiating terminal blocks, and connects and pricks internal carotid for line.Sew up the incision, put the end of a thread in external, bolt line is evacuated in neck during Reperfu- sion and moved Arteries and veins initial part.Ischemia Time is 2h, and the Reperfu- sion time is 24h, every group of each 3~5 animal.Sham-operated control group is not inserted Line, remaining operation are identical with experimental group.

2. detect cortex penumbra region GDF11 expression：

Ischemic 2 hours, after Reperfu- sion 24h, perfusion, take full brain to make frozen section, compared with rats in sham-operated group, pass through The method of immunohistochemical staining finds notable compared with sham-operation group in cortex penumbra region, the quantity of GDF11 positive cells Increase (sham-operation group：20.64±1.57；MCAO groups：29.78±2.03；Figure 1A and Figure 1B).In addition, take successfully the big of modeling Mouse, ischemic 2 hours, take brain to be cut into slices after irrigating 2,6,12,24,48 hours respectively, TTC dyes are carried out in 37 DEG C of incubator Color, cortical ischemia scope is judged according to coloration result, the tissue of adjacent brain piece cortex penumbra region is drawn materials, extracted total RNA carries out fluorescence real-time quantitative PCR, and result of study finds that compared with sham-operation group (1 ± 0) GDF11 mRNA level in-site is small 2 When without significant change (0.93 ± 0.78), have within 6 hours, 12 hours significantly rise (1.69 ± 0.27；1.68 ± 0.21), and 24 Hour reaches highest (2.20 ± 0.22), and normal level (1.23 ± 0.33 is gradually restored to after 48 hours；Fig. 1 C)

3. intracerebral microinjection：

After the chloraldurate of SD rats (purchased from Beijing company of dimension tonneau China) intraperitoneal injection 10%, mouse brain is fixed on brain and stood On body position indicator, calvarium portion skin is cut, exposure skull, using skull face lambda as body surface symbol, is taken out with micro sample adding appliance PBS and 2ul (0.625ng/ μ l) GDF11 are taken according to literature method (J Cereb Blood Flow Metab, 1999,19 (12): 1329-1335) inject ventriculus sinister cerebri (coordinate：Front and rear (AP), -0.9mm；Left and right (L), ± 1.5mm；Carry on the back abdomen (V), -3.6mm), MCAO models are made after 24 hours.

4.TCC dyeing shows infarcted region：

Experimental animal broken end is taken into brain after ischemia-reperfusion 24h, the thick coronal sections of 2mm is prepared from front to back, is placed in and contains 37 DEG C of constant-temperature incubation 30min, Nao Pianzhong cortical infarction areas are not in 2%TCC (triphenyltetrazolium chloride) normal saline solution Dye and white is presented, undamaged brain tissue takes on a red color.4% paraformaldehyde is fixed 24h and taken pictures.Using digital medical graphical analysis The area and infarct size of every brain piece of system measurement, and the volume and Infarction volume of every brain piece are calculated, PBS control group cortex Ischemic volume ratio be 10.45 ± 1.99%, GDF11 pretreated group cortical ischemia volume ratios substantially reduce to 3.59 ± 1.75% (P<0.05 Fig. 2A, B).These results prompting GDF11 has protective effect to cerebral ischemia.

5. neurological deficit scores：

According to the 5 of Longa and Bederon points of methods of marking processed, scored after ischemic 2h Reperfu- sions 24h.Its mark that scores Standard is：0 point, impassivity injury symptoms；1 point, it is impossible to full extension operation offside forelimb；2 points, turn-taked to offside；3 points, to right Roll oblique；4 points, it is impossible to spontaneous walking, the loss of consciousness.0 point and 4 points of animal is given it up.Take the successful animal of modeling random It is divided into two groups of MCAO1 and MCAO2, respectively before and after statistics injection external source restructuring GDF11 albumen, two groups of animals of MCAO1 and MCAO2 Neurological deficit scoring score value.As a result show, before given after ischemia, the god for the two groups of animals of MCAO1 and MCAO2 being randomly assigned There is no difference (MCAO1 groups through functional defect scoring score value：1.88±0.35；MCAO2 groups：1.83 ± 0.21, Fig. 3). Restructuring GDF11 albumen is given after cerebral ischemia so that rat functional defect scoring score value significantly reduces (MCAO1+PBS groups： 1.75±0.16；MCAO2+GDF11 groups：1.13±0.13；P<0.05, Fig. 3).As a result restructuring GDF11 eggs are given after prompting ischemic Neurological deficit scoring, recovered part nervous function can be significantly reduced in vain.

6. cerebral cortex apoptotic cell detects：

After making ischemia-reperfusion injury model, after 2 hours Reperfu- sion 24h of ischemic, rat is subjected to cardiac perfusion, then breaked end Brain is taken, is fixed overnight with 4%PFA, graded sucrose solutions sedimentation, series frost is carried out with frozen section embedding medium investing tissue and cuts Piece, take frozen section to carry out fluorescent immunohistochemistry, as a result find compared with control group, injection restructuring GDF11 albumen The positive cell number of the cortex penumbra region Neuron Apoptosis GAP-associated protein GAP (caspase-3) of cerebral ischemia-reperfusion significantly reduces in group (control groups：52.41±5.33；GDF11 protein groups：29.14±3.82；Fig. 4 A, B).For further the result, hair A person of good sense takes two groups of cerebral ischemia-reperfusion piece to carry out TUNEL dyeing, and experimental group and the visible different degrees of TUNEL of control group are positive thin Born of the same parents are distributed, and have chromatin condensation, karyopycnosis, in the obvious Apoptosis feature (Fig. 5 A) such as brown color or yellowish-brown.With just Normal control group is compared, and the cell quantity of apoptosis is remarkably decreased (control groups in the rat brain of injection restructuring GDF11 albumen： 12.62 ± 1.32%；GDF11 protein groups：5.44 ± 0.65%；Fig. 5 A, B).Result above illustrates to recombinate GDF11 albumen pair The neuron of cerebral ischemia-reperfusion penumbra region has protective effect.

The horizontal detection of 7.SVZ brain areas propagation：

Rat line brush Focal Cerebral Ischemia Reperfusion model is established, 2 hours Reperfu- sions of ischemic are after 24 hours, according to Longa and Bederon 5 points of methods of marking screening successful rats of modeling processed, are randomly divided into two groups, respectively to its ventriculus sinister cerebri Vitro recombination GDF11 albumen and PBS are injected, cardiac perfusion takes brain after three days, BrdU (50mg/ is injected intraperitoneally within 2 hours before perfusion Kg), immunohistochemical staining is carried out after frozen section, is as a result found compared with control group, ventriculus sinister cerebri injection restructuring GDF11 albumen Rat significantly reduce (control groups in the SVZ brain area BrdU positive cell quantities of cerebral ischemia-reperfusion：138.64±6.56；GDF11 Protein groups：58.67±10.78；Fig. 6 A and C), the quantity of another cell proliferation markers Ki67 positive cells also significantly drops It is low.Illustrate that recombinating GDF11 albumen inhibits horizontal (the control groups of the propagation of SVZ brain area nerve cells：223.36±16.01； GDF11 protein groups：52.64±7.08；Fig. 6 B and D).The cerebral ischemia-reperfusion piece for injecting restructuring GDF11 albumen and PBS is taken to carry out respectively Dyeing, immunohistochemistry double staining is carried out using cell proliferation markers BrdU and neural stem cell marker nestin, As a result it is also BrdU positive cells to show nestin positive cells, prompts restructuring GDF11 albumen to inhibit SVZ brain area nerve cords thin The propagation (Fig. 6 A) of born of the same parents.

8.SVZ brain area peripheral vesselses regeneration level detects：

Establish rat line brush focal cerebral ischemia in rats, after ischemic 2h Reperfu- sions 24h, the restructuring of intracerebroventricular injection external source GDF11 albumen, cardiac perfusion is carried out after 3 days, then takes brain to cook frozen section, is exempted from by revascularization label CD31 Epidemic disease histochemical staining finds that in rat brain after the horizontal rises of GDF11, subependymal region peripheral vessels generative capacity is without significant changes (control groups：30.58±3.04；GDF11 protein groups：30.85±2.50；Fig. 7 A, 7B).This explanation restructuring GDF11 albumen pair Subependymal region peripheral vessels, which generates, has no influence, or the μ l of injection volume 2 of restructuring GDF11 albumen (0.625ng/ μ l) inadequate, Obvious action can not be produced to the angiogenesis around subependymal region.

Embodiment 2:It is overexpressed GDF11 and pre-processes the prevention effect in cerebral ischemia

1.GDF11 is overexpressed virus efficacy detection：

In order to express GDF11 state in a kind of long-acting height of intracerebral structure, its prevention effect to cerebral ischemia is studied, Inventor constructs GDF11 overexpression plasmids and is packaged into the slow virus with green fluorescent protein (GFP) first, and by internal And the overexpression efficiency of experiment in vitro detection virus.293 cell recovery cultures are taken, after cell density reaches 80% or so respectively The negative control virus and GDF11 for adding 5 μ g are overexpressed virus, do fluorescence real-time quantitative PCR after 48 hours, as a result show GDF11 is overexpressed virus and improves 6 times of (control groups in mRNA level in-site than Empty virus group：1±0；GDF11 overexpression groups： 6.08±0.14).The alternative SD rats for taking health of inventor are divided into two groups (n=3), give intracerebroventricular injection negative control respectively Each 2 μ l of virus are overexpressed with GDF11, treats that it expresses 14 days cardiac perfusions afterwards, takes brain to cook frozen section, it is considerable under the microscope Observe obvious green fluorescence.Prove that GDF11 is overexpressed virus formulation success.

2. being overexpressed TCC dyeing after GDF11 pretreatments shows infarcted region：

Slow virus and each 2 μ l of negative control virus are overexpressed to lateral ventricle of rat brain injection GDF11, treats expressing viral 12 days Afterwards, line brush Focal Cerebral Ischemia Reperfusion model is established, 2 hours Reperfu- sions of ischemic, by rat heart perfusion, take brain after 3 days Frozen section is done, carries out TTC dyeing (Fig. 8 A).As a result show that rat and the injection of injection GDF11 overexpression slow virus are negative right Compared according to the rat of virus, ischemic scope is remarkably decreased (control groups：11.34±2.22；GDF11 overexpression groups：3.44± 1.41；Fig. 8 A and B).Result above is consistent with the result of injection restructuring GDF11 albumen, prompts to be overexpressed GDF11 pretreatments to brain Ischemic injuries have protective effect.

3. neurological deficit scores after being overexpressed GDF11 pretreatments：

Virus and each 2 μ l of negative control virus are overexpressed to lateral ventricle of rat brain injection GDF11, after expressing viral 12 days, Line brush Focal Cerebral Ischemia Reperfusion model is established, after 3 days, the nervous function for recording rat lacks 2 hours Reperfu- sions of ischemic Score value is fallen into, evaluation is overexpressed influence of the GDF11 pretreatments to rats with cerebral ischemia nervous function.As a result show that injection GDF11 crosses table The rat functional defect scoring score value of da virus is significantly reduced (control groups：1.63±0.18；GDF11 is overexpressed Group：1.13±0.13；Fig. 9).Prompting, which is overexpressed GDF11 pretreatments, there is protection to make nervous function caused by cerebral ischemia With.

4. cerebral cortex apoptotic cell detects after being overexpressed GDF11 pretreatments：

The SD rats for choosing 250~300g carry out intracerebroventricular injection GDF11 overexpressions virus and each 2 μ of negative control virus L, expressing viral establish line brush Focal Cerebral Ischemia Reperfusion model after 12 days, and 2 hours Reperfu- sions of ischemic carry out the heart after 3 days Dirty perfusion, take brain to cook frozen section, then carry out immunohistochemical staining, as a result find compared with control group, intracerebroventricular injection GDF11 is overexpressed the positive of the viral rat in the cortex penumbra region Neuron Apoptosis GAP-associated protein GAP (Caspase-3) of cerebral ischemia-reperfusion Cell number significantly reduces (control groups：45.08±4.08；GDF11 overexpression groups：34.08±1.42；Figure 10 A and B). After TUNEL coloration results show that GDF11 continuation is overexpressed, the TUNEL positive cell apoptosis quantity of cortex penumbra region is notable Decline (control groups：55.23±1.81；GDF11 overexpression groups：38.29±2.07；Figure 11 A and B).Result above with note It is consistent to penetrate GDF11 albumen results, further demonstrate GDF11 has protective effect to the neuron in cerebral ischemia-reperfusion.

5. it is overexpressed the horizontal detection of SVZ brain areas propagation after GDF11 pretreatments：

Naoliqing capsule injection GDF11 is carried out to rat and is overexpressed virus and each 2 μ l of negative control virus, expressing viral 12 Establish line brush cerebral ischemic model after it, 2 hours Reperfu- sions of ischemic 3 days, carry out after BrdU is injected intraperitoneally 2 hours, carrying out heart Perfusion takes brain to cook frozen section, then does immunohistochemical staining, as a result finds that injection GDF11 is overexpressed the rat of virus in ischemic The SVZ brain area BrdU positive cell numbers of brain significantly raise (control groups：101.75±6.40；GDF11 overexpression groups： 192.33±14.38；Figure 12 A and B).BrdU and neural stem cell marker nestin is subjected to immunohistochemical double-labeled dyeing, knot Fruit confirms that GDF11 is overexpressed the propagation for remarkably promoting SVZ NSCs.

6. SVZ brain area peripheral vesselses regeneration level detects after being overexpressed GDF11 pretreatments：

Intracerebroventricular injection GDF11 is carried out to rat and is overexpressed virus and negative control virus after 12 days, establishes line brush office Stove cerebral ischemic model, ischemic 2h Reperfu- sions 3 days, after carrying out cardiac perfusion, take brain to make frozen section, carry out SABC dye Color, as a result find that GDF11 is overexpressed pretreatment and subependymal region vascular endothelial cell label CD31 expression can be caused significantly to increase Add (control groups：40.34±5.91；GDF11 overexpression groups：151.1±26.31；Figure 13 A and B), this prompting GDF11 locates in advance Reason has significant facilitation to the regeneration of MCAO rat SVZ brain areas peripheral vessels.

Embodiment 3:GDF11 is treated in cerebral ischemia and the molecular mechanism of prevention effect

The cortical ischemia Penumbra tissue of materials cerebral ischemia animal respectively, extraction albumen are Western Blot, as a result found After having injected external source restructuring GDF11 albumen, intracerebral pSmad2/3 (control groups：1±0.09；GDF11 protein groups：2.0± 0.24；Figure 14 A and B) expression dramatically increase.GDF11 albumen is recombinated it can be seen from the result by regulating and controlling Smad2/3 ginsengs With the therapeutic process of cerebral ischemia.

Cortical ischemia penumbra region histone is extracted, is detected through Western Blot, as a result finds that having injected GDF11 crosses table After da virus, pSmad2/3 (control groups in rat brain：1±0.66；GDF11 overexpression groups：1.57±0.10；Figure 14 A and C amount) dramatically increases.GDF11 is overexpressed the guarantor that cerebral ischemia is participated in by regulating and controlling Smad2/3 it can be seen from the result Shield acts on.

Claims (3)

1. A kind of 1. applications of growth and differentiation factor GDF11 in ischemia apoplexy disease medicament is prevented and treated.
2. 2. application as claimed in claim 1, it is characterised in that the ischemia apoplexy disease is cerebral apoplexy.
3. 3. application as claimed in claim 1, it is characterised in that described pharmaceutical dosage form is injection.