CN100381168C - Use of glucoprotein for pharmaceuticals - Google Patents
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- CN100381168C CN100381168C CNB2004100910342A CN200410091034A CN100381168C CN 100381168 C CN100381168 C CN 100381168C CN B2004100910342 A CNB2004100910342 A CN B2004100910342A CN 200410091034 A CN200410091034 A CN 200410091034A CN 100381168 C CN100381168 C CN 100381168C
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Abstract
The present invention discloses a new pharmaceutic application of glucoprotein, namely an application of fetuin in the preparation of medicine for curing ischemic brain injury which can be cerebral thrombosis, cerebral infarction, cerebral hemorrhage, etc.
Description
Invention field
The present invention relates to a kind of pharmaceutic usage of glycoprotein, particularly myosin (fetuin, α
2-HS glycoprotein, molecular weight 45kda--65 kda) pharmaceutic usage.
Background technology
Myosin (fetuin, α
2-HS glycoprotein, molecular weight 45kda--65 kda) be a kind of by the synthetic glycoprotein of liver, extensively be present in mammal (comprising people, cattle, Mus etc.) period of embryo's body, be height with blood plasma, cerebrospinal fluid, liver and bone content.The myosin of cattle (fetuin) contains intimate 30% sugar, contains the terminal relative less C-stub areas with of 2 N-.
Although myosin (fetuin) is found in nineteen forty-four, its function and purposes are still not fully aware of.Bibliographical information, myosin (fetuin) can increase cell growth rate and improve protein expression.Because the activity of its Profilin hydrolytic enzyme in cell culture is so can strengthen the extension of intercellular contact and cell.At present, myosin (fetuin) is mainly used in cell culture, particularly can increase cell growth and duplicate in the serum-free cell culture.
Summary of the invention
The objective of the invention is to disclose a kind of pharmaceutic usage of glycoprotein, particularly myosin (fetuin, α
2-HS glycoprotein, molecular weight 45kda--65 kda) pharmaceutic usage promptly is used for preparation treatment acute ischemic brain injury medicine in pharmaceutical field.
The preparation method of myosin preparation of the present invention has multiple, is wherein a kind of (doing raw material with hyclone is example) below:
Hyclone adds the 0.03-0.05M zinc acetate of 2-3 times of volume, regulate pH value to 6-6.8 with 28.5%-35% ethanol, placed 12-16 hour down for-5 ℃, discard precipitation after centrifugal, collect supernatant, adding 1.0-2.0M barium acetate and 95% ethanol and obtaining final concentration is 0.02 or the 0.04M barium ions; 25%-45% ethanol is regulated pH value to 6 or 6.7 and placed 2 hours, and is centrifugal, discards precipitation, collects supernatant, adds 95% ethanol and make concentration be 40% or 50% ethanol placement 12-16 hour, and centrifugal, precipitation (obtains myosin, FETUIN).
According to the drug formulation process of routine, myosin (fetuin) can be prepared into the pharmaceutical dosage form that is suitable for clinically.For example, injectable powder, aqueous injection, infusion solution, subcutaneous administration preparation and other medicine-releasing system.
Hyclone adds the 0.05M zinc acetate of 3 times of volumes, and 35%7 alcohol are regulated pH value to 6 and time placed 12-16 hours, discards precipitation after centrifugal, collects supernatant, and adding 1.5M barium acetate and 95% ethanol and obtaining final concentration is 0.04M barium ions (Ba++); 30% ethanol is regulated pH value to 6 and placed 2 hours, and is centrifugal, discards precipitation, collect supernatant, add 95%7 alcohol and make concentration be 50% ethanol placement 12-16 hour, centrifugal, precipitation (myosin, FETUIN), abandoning supernatant obtains myosin solution with the dissolving of 3% mannitol water for injection; Aseptic filtration repeatedly, packing, lyophilization promptly gets myosin (FETUIN) injectable powder.
Injectable powder to myosin of the present invention carries out pharmacodynamic study.The result shows that medicine of the present invention can significantly dwindle the infarct size of acute ischemic brain injury, obviously suppresses the expression of macrophage and the activatory microgliacyte and the tumor necrosis factor (TNF-α) of ischemic area; And can obviously improve the behavior of focal cerebral ischemia rat model nervous symptoms, significantly reduce intracellular adhesion molecule-1 (ICAM-1) and ET-1 content in the rat infarction local brain tissue, increase the VIII factor related antigen VIII:Ag. gross area and pipe density simultaneously.Illustrate that myosin (fetuin) can resist the inflammatory reaction of ischemic brain injury; the antagonism local vascular shrinks and impels new vessels to grow; obviously dwindle the ischemic brain infarction area and significantly protect brain cell; thereby treatment acute ischemic brain injury comprises the such class of the cerebral apoplexy serious brain disorders relevant with the acute cerebral ischemia pathological changes such as cerebral thrombosis, brain Geng plug, cerebral hemorrhage.
Following experimental example is used to further specify the present invention.
Experimental example 1: myosin (fetuin) is to the influence of brain focal cerebral ischemia infarct size
1, animal grouping: 88 of SD male rats, body weight 250 grams-350 grams are divided into 10 groups, 8 every group at random.Wherein 1 group, 6 groups is model control group, and all the other each groups are the treatment group of various dose and different dosing time.
2, operating procedure: myosin (fetuin) is (1ml volume) behind physiological saline solution, injects in the rat body (seeing Table 1) respectively at 15 minutes, 30 minutes veins of ischemia.
Table 1 myosin (fetuin) is to the influence of brain focal cerebral ischemia infarct size
Cause the focal cerebral ischemia in rats model after 24 hours, animal is put to death in anesthesia, takes out brain and fixes and be cut into the thick brain sheet of 2mm, and the TTC (2,3,5-hiphenyltetrazoliam chloride) of immersion 2% was hatched under 37 ℃ 30 minutes.Cerebral tissue after the dyeing is behind photo, and (Photoshop Adobe system) calculates cerebral infarct size by computer image analysis.
3, experimental result (seeing accompanying drawing 1): myosin (fetuin) significantly dwindles rat ischemia cerebrum block volume.A). gave various dose myosin (fetuin) by vein in 15 minutes behind the ischemia.Collect the capable TTC dyeing of brain after 24 hours.* P＜0.05, * P＜0.01 (comparing) with the 0mg/kg group.B). 15 minutes and 30 minutes give 50mg/kg myosin (fetuin) by vein behind the ischemia.Collect the capable TTC dyeing of brain after 24 hours.* P＜0.01 (organizing relatively) with 0mg/kg.
This shows that myosin (fetuin) can obviously dwindle and significantly dwindle the focal cerebral infarct size (Fig. 1) that the intraluminal middle cerebral artery occlusion in rats ischemia causes.Experiment shows that only 5mg/kg myosin (fetuin) can obviously reduce the ischemic infarct area, and treatment group rat cerebral infarction area is less than 1/2 of model control group rat infarct size.The ischemia resisting damaging action presents tangible dose-effect relationship.From the 5mg/kg body weight, increase and enhancing evident in efficacy with dosage; When consumption reached the 500mg/kg body weight, focus of infarct only was 1/10 of a model control group.Result of study shows,, myosin (fetuin) significantly dwindles the cerebral ischemia infarct size.Prompting myosin (fetuin) can be treated acute cerebral ischemia and be comprised cerebral apoplexy disease illness such as cerebral thrombosis, cerebral infarction, cerebral hemorrhage, obviously alleviates damage and the death of brain cell under the acute ischemia sexual state.
Experimental example 2: the influence that myosin (fetuin) is expressed the neural microglia and the macrophage of rat acute ischemic brain injury zone activated
1, animal grouping: 12 of male SD rats, body weight 250 grams-350 grams, be divided into 2 groups at random: 1 group is myosin (fetuin) treatment group, and 1 group is model control group, 6 every group.
2, operating procedure: myosin (fetuin) is through physiological saline solution (1ml volume), and intravenous injection in 15 minutes is gone in the treatment group rat body (50mg/kg) behind ischemia, and model group is only injected the equivalent normal saline.Anesthetized animal after 24 hours pours into rat (about 350ml) with 4% paraformaldehyde (pH 7.4) through left ventricle.Take out brain, be cut into 2mm thickness brain sheet, under 4 ℃, fix 12 hours, put into 30% sucrose solution 12 hours (4 ℃) with same fixed liquid.Frozen section (10 μ m).Show the variation of microgliacyte and macrophage with ED1 monoclonal antibody (1: 2000) through immunohistochemical method dyeing at damage field.
3, experimental result (seeing accompanying drawing 2): myosin (Fetuin) suppresses the expression intraluminal middle cerebral artery occlusion in rats of focus zone macrophage to be blocked back 15 minutes, vein give myosin (fetuin, 50mg/kg).Anesthetized animal after 24 hours.4% paraformaldehyde (pH 7.4) heart perfusion.Collect brain.Fix 12 hours after under the 4 degree conditions.Placed 12 hours in 30% sucrose.Frozen section.With ED1 monoclonal antibody row immunohistochemical staining.A, contrast (normal brain); B, myosin (fetuin, 50mg/kg) treatment back cerebral ischemia brain; C does not treat the cerebral ischemia brain.
This shows, myosin (fetuin) can obviously suppress the expression of ischemic region macrophage and activated microglia, macrophage and activated microglia quantity are obviously reduced, and prompting myosin (fetuin) is expressed relevant to the therapeutical effect of acute cerebral ischemia damage with the inflammatory response cell of its inhibition damage field.
Experimental example 3: the influence that myosin (fetuin) is expressed rat ischemia brain injury zone tumor necrosis factor-alpha (TNF-α)
1, animal grouping: with experimental example two;
2, operating procedure: myosin (fetuin) is through physiological saline solution (1ml volume), and 15 minutes angular veins inject in the treatment group rat body (50mg/kg) behind ischemia, and model group is only injected the equivalent normal saline.Anesthetized animal after 24 hours pours into tremulous pulse (about 300ml) with normal saline through left ventricle, takes out brain, is cut into 2mm brain sheet, puts and collects ischemia damaged tissue on ice, adds the lysis buffer, makes homogenate, puts on ice 30 minutes, centrifugal (4 ℃, 2000g).Collect supernatant, measure protein content,, be transferred to cellulose nitrate film, detect the TNF-alpha content according to the Western hybridization step with the anti-TNF-Alpha antibodies (1: 1000) of exempting from of polyclone through the SDS-page electrophoresis.
3, experimental result (seeing accompanying drawing 3): TNF-alpha expression obviously raise (TNF-α is a kind of and the closely-related cytokine of inflammation, because of obviously increasing in inflammatory reaction tissue is caused damage) at the ischemic injuries position.After myosin (fetuin) treatment, TNF-α obviously reduces in the expression at cerebrum ischemia position, inflammatory cell when it suppresses cerebral ischemia is described in the expression of areas of inflammation and discharge inflammatory factor, thereby protection ischemia brain exempts from the damage that inflammation causes.
Experimental example 4: myosin (fetuin) is to the ethological influence of focal cerebral ischemic model rat nervous symptoms
1, the foundation of model
The making of model, according to kolzulni in 1985 in Japanese apoplexy meeting reported first carry out reversibility middle cerebral artery occlusion model method rat with nylon wire.Concrete grammar is: adopt 4～No. 0 nylon monofilament lines or nylon fishing line, head end is heated into spheroidal, and the about 0.3mm of diameter dries standby after silication.Postanesthetic rat is got the fixed bit of lying on the back.The cervical region median incision separates and exposes a side common carotid artery (CCA) and the inside and outside tremulous pulse of neck, at the inside and outside aortic bifurcation of neck place's ligation external carotid artery, at proximal part ligation common carotid artery, and presets a ligature at its far-end.Cut an osculum at the about 3mm of common carotid artery ligation place far-end place, the nylon wire that with diameter is 0.165～0.185mm imports or inserts through ECA from otch, when ICA goes into the intracranial furcation, it is the blood flow of inflow middle cerebral artery capable of blocking (MCA), about 16～the 18mm of inlet wire length, afterwards with common carotid artery together with nylon wire ligation, skin suture then, promptly cause this side middle cerebral artery occlusion, cause focal cerebral ischemia or cerebral infarction.The person that makes the re-perfusion model if desired, then not ligation common carotid artery after inserting nylon wire to the scheduled time, then can be drawn back nylon wire, and plug wire is extracted the blood flow that can recover middle cerebral artery (MCA), realizes perfusion again.In order to strengthen blocking action better, what have burns till sphere to the nylon wire head end, or is coated with one deck silicone elastomer (length 5mm, diameter 0.25mm) at the end of a thread far-end, is convenient to the end of a thread swelling tremulous pulse, can reach the total blockage middle cerebral artery.Sham operated rats is except that not carrying out the line bolt blocks the same model group of surplus operating procedure.All experimental group animals are observed animal pattern neurological deficit symptom in 1-2h behind PMCAO clear-headed (the about 30min of postoperative):
(1), can not fully stretch left front pawl when static, turn-take to the left during walking or topple over;
(2), when animal climbs the plate test, always fall to the left;
(3), carry tail when test, receive in the left fore, head bends towards upper left;
(4), function of nervous system's scoring is at the 1-3 branch.
In addition, get the visible diameter 3～4mm of rat whole brain ischemic injuries district in dissection, be magneta colour, normal district is white.With the ischemic focus center is that section is done a crown section, and the ischemic injuries district degree of depth can be observed the cerebral infarction kitchen range and be positioned at the cortex of temporal lobe district at 2～3mm in experiment, and consistent with the cerebral blood supply zone of MCA domination, the modelling that this experiment is described is successful.
2. the evaluation of model
Before after modeling postoperative 1h and treatment, promptly putting to death respectively evaluation once by the method for Bederson etc. and improved, the modeling animal is carried out behavioristics's scoring.Specifically:
Carry the about chi of Mus tail built on stilts, observe forelimb flexing situation.Stretch to ground as two forelimb symmetries, be designated as 0 fen; As the flexing that shoulder flexing, elbow flexing, shoulder inward turning or existing shoulder elbow appear in the offside forelimb of performing the operation has inward turning person again, is designated as 1 fen.
Animal is placed on the level and smooth ground, push away both shoulders respectively, check resistance to side shifting.As bilateral resistance equity and be designated as 0 fen effectively; As resistance descender when the operation offside promotes, be designated as 1 fen.
Animal two forelimbs are put on the wire netting, observed the muscular tension of two forelimbs.Bilateral muscular tension equity and strong person are 0 minute; Operation offside muscle of anterior limb tension force descender is designated as 1 fen.
Carry the about chi of Mus tail built on stilts, animal has ceaselessly to operation offside revolver, is designated as 1 fen.According to above standard scoring, full marks are 4 minutes, and mark is high more, and the behavior disorder of animal is serious more.
3. the nervous symptoms behavior change is analyzed
All experimental group animals are giving Drug therapy end back of the present invention (1,2,3 day), observe animal pattern, model control animal neurological deficit symptom:
(1), can not fully stretch left front pawl when static, turn-take to the left during walking or topple over;
(2), when animal climbs firm and hard testing, always fall to the left;
(3), carry tail when test, receive in the left fore, head bends towards upper left;
(4), function of nervous system's scoring is at the 1-3 branch.
4. experimental result:
Relatively, the results are shown in Table 2 between each treated animal neurobehavioral detected value group.
Table 2 myosin (fetuin) is to the influence of MCAO rat nervous symptoms
Annotate: compare with while segment model group: ※ p＜0.05, ※ ※ p＜0.01; Compare before and after the treatment between group: ★ p＜0.05, ★ ★ p＜0.01.
Above result finds out, each organizes rat nervous symptoms behavior during 1h after modeling all has positive reaction in evaluation, before the treatment with model group and the medicine group comparison course of treatment (p＞0.05); Treatment back 24H group and 48H group there was no significant difference (p＞0.05), 72H group model group and medicine group relatively have significant difference p＜0.05, illustrate that this medicine nervous symptoms that damage causes to rat cerebral ischemia has clear improvement and therapeutical effect.
Experimental example 5: myosin (fetuin) is to infarction local brain tissue form and ICAM-1, ET-1, the influence of VIII factor related antigen
1, modeling and animal grouping: with experimental example four
2, each treated animal is after modeling, give Drug therapy of the present invention according to different course treatment 24H, 48H, 72H vein respectively, when finish the course of treatment each treated animal is anaesthetized again, fix with 4% paraformaldehyde (4 ℃) heart perfusion at official hour point, broken end is opened cranium and is got brain rapidly then, on ice pan be to do the thick crown section of about 4mm before and after the starting point from brain medium-sized artery infarcted region center, fix 30 hours in the neutral formalin fixative of input 10%, carry out paraffin respectively and cut HE dyeing and Nissl dyeing.
The HE staining procedure:
(1), section dewaxes dimethylbenzene I, II each 15 minutes;
(2), the gradient alcohol wash each 1 minute, 100%I, II, 95%I, II, 90%I, II, 80%, 70%, 60%, 50% each 1 minute;
(3), the tap water flushing is 5 minutes;
(4), Harris Lignum Sappan uniformly dyeing is 10 minutes;
(5), the tap water flushing is 5 minutes;
(6), the differentiation of 1% hydrochloride alcohol is 1-2 minute;
(7), the flowing water flushing is 1 hour;
(8), dyed 2 minutes in Yihong;
(9), the flowing water flushing is 5 minutes;
(10), gradient alcohol dehydration each 1 minute, each 1 minute fan of 50%-100%II dries up;
(11), dimethylbenzene I, II are transparent each 15 minutes;
(12), DPX natural gum mounting is preserved.
The Nissl staining procedure:
(1), paraffin section de-waxing is to water;
(2), 1% Toluidine blue staining, 45 ℃ of dye vats of heating, 30-40 minute dyeing;
(3), the 95%I ethanol differentiation several seconds, the 95%II ethanol differentiation several seconds, must how observe differentiation down by mirror, endochylema dyeing, karyon does not dye; (4) 100% ethanol I, II dehydration, dimethylbenzene is transparent, and DPX natural gum mounting is preserved.The blue particle that is dispersed in, karyon dye-free appear in the kytoplasm.
3, light microscopic is observed the morphological change of infraction local brain tissue Hippocampus CA3 district neurocyte down.
(1). SABC detects ICAM-1, ET-1, VIII factor related antigen
Method: adopt immunohistochemical two step method to detect ischemia side cerebral tissue ICAM-1, ET-1, VIII factor related antigen.Concrete steps are as follows:
1. the dewaxing, the aquation tissue slice;
2. 3% hydrogen peroxide was hatched 10 minutes, with the blocking-up endogenous peroxydase;
3. according to applied one anti-specific (special) requirements, tissue slice is carried out pretreatment;
4. it is anti-to drip ICAM-1 one respectively, and 4 ℃ are spent the night, PBS flushing, 2 minutes * 3;
5. increase reagent E nVision, hatched 20 minutes for 37 ℃, PBS flushing, 2 minutes * 3;
6. use the colour developing of DAB solution, adopt the DAKO developer;
7. distilled water flushing, redye, dehydration, mounting.
(2). image quantitative analysis:
Respectively 40 *, 100 *, under the 400 * times mirror, every section is in 5 visuals field of picked at random, ischemia side Hippocampus CA3 district, carry out quantitative analysis with the colored case history graphical analysis management system of multi-functional true (CMIAS2001A type), measure the gross area, the integration luminosity of ischemia side Hippocampus CA3 district positive cell yellowish-brown reactant respectively, reach the gross area of this district's microvascular endothelial positive cell yellowish-brown reactant, analyze the expression of ICAM-1, ET-1, VIII factor related antigen respectively.
(3). statistical procedures
Use the SPSS11.0 statistical software and carry out statistical procedures.Measurement data is checked with t or t`, and enumeration data is checked with x2, and ranked data carry out statistical procedures with the rank test or the Ridit method of inspection.
4, experimental result
(1). myosin (fetuin) is to the influence of focal cerebral ischemic model rat ischemia half blanking bar cell ICAM-1 protein expression
The gross area that table 3 laboratory animal ischemia side cerebral tissue half dark space cell ICAM-1 expresses
Compare with while segment model group: * p＜0.05, * * p＜0.01; Compare with sham operated rats: ▲ p＜0.05, ▲ ▲ p＜0.01.
As can be seen from Table 3, model group and sham operated rats be the positive cell number showed increased of each different course relatively, and utmost point significant difference (P＜0.01) is arranged, and the success of line bolt method focal cerebral ischemia model is described; And with medicine group and the model group comparison course of treatment, medicine 24H group increases than model 24H group positive cell number, medicine 48H group, the 72H group is organized than model 48H respectively, 72H group positive cell number obviously reduces, medicine 48H group relatively has utmost point significant difference (P＜0.01) with model 48H group, and medicine 72H group relatively has significant difference (p＜0.05) with model 72H group.
The integration luminosity that table 4 laboratory animal ischemia side half dark space positive cell ICAM-1 expresses
Compare with while segment model group: ※ p＜0.05, ※ ※ p＜0.01; Compare with sham operated rats: ▲ p＜0.05, ▲ ▲ p＜0.01.
As can be seen from Table 4, model group and sham operated rats be the positive cell number showed increased of each different course relatively, and utmost point significant difference (P＜0.01) is arranged, and the success of line bolt method focal cerebral ischemia model is described; Medicine 24 hours, 48 hours, 72 hours groups respectively with model 24 hours, 48 hours, group relatively had utmost point significant difference (P＜0.01) in 72 hours.
ICAM-1 is one of part of LFA; Be distributed widely in hemopoietic and non-hematopoietic cell, and activated T cell, mononuclear cell etc. are all expressed high-caliber ICAM-1.When inflammation, the ICAM-1 of vascular endothelial cell peaks, and some inflammatory cytokine such as interleukin-11 (IL-l), interferon г (IFN-г), tumor necrosis factor a (TNFa) etc. can stimulate vascular endothelial cell that the expression of ICAM-1 is raised rapidly.ICAM-l and m RNA thereof confirm in many ischemia-reperfusion animal models at the cerebrovascular endothelial cell up-regulated during cerebral ischemia, use the ICAM-1 monoclonal antibody and also can obviously alleviate the damage of ischemia-reperfusion murine brain.Show in the ICAM-1 early stage inflammation damnification mechanism of cerebral ischemia that leukocyte mediates after cerebral ischemia and play a significant role.This experimental result was with documents and materials were consistent in the past, ICAM-1 expresses obviously and raises during the Cerebral Ischemia damage, prompting ICAM-1 sticks early stage mediated leucocytes of cerebral ischemia and vascular endothelial cell, and leukocyte is swum out of blood vessel to cerebral ischemia district and caused cerebral tissue acute inflammation pathological lesion.Myosin (fetuin) can suppress the rising of cerebral ischemia area I CAM-1, reduces inflammatory cell to the moving about of ischemic tissue, thereby alleviates the organizing inflammatory reaction of cerebral ischemia zone and protect cerebral tissue to exempt from infringement.
(2). myosin (fetuin) is to the influence of cerebrovascular endothelium ET-1 positive expression
The gross area that table 5 laboratory animal ischemia side cerebral tissue half dark space cell ET-1 expresses
Compare with while segment model group: * p＜0.05, * * p＜0.01; Compare with sham operated rats: ▲ p＜0.05, ▲ ▲ P＜0.01.
As can be seen from Table 5, model group and sham operated rats be the positive cell number showed increased of each different course relatively, and utmost point significant difference P＜0.01 is arranged, and the success of line bolt method focal cerebral ischemia model is described; And with the course of treatment medicine group and model group relatively, 24 hours groups of 24 hours groups of medicine and model are there was no significant difference (p＞0.05) relatively, medicine 48 hours, the expression of 72 hours group ET-1 is starkly lower than model group (P＜0.01).
The optical density that each treated animal ischemia side half dark space positive cell ET-1 of table 6 expresses
Compare with while segment model group: * p＜0.05, * * p＜0.01; Compare with sham operated rats: ▲ p＜0.05, ▲ ▲ P＜0.01.
As can be seen from Table 6, model group and sham operated rats be the positive cell number showed increased of each different course relatively, and utmost point significant difference (P＜0.01) is arranged, and the success of line bolt method focal cerebral ischemia model is described; And with medicine group and the model group comparison course of treatment, medicine 24 hours, 48 hours groups and model 24 hours, 48 hours groups be there was no significant difference (p＞0.05) relatively, and 72 hours groups of 72 hours groups of medicine and the model relatively optical density expressed of ET-1 are starkly lower than model group (P＜0.01).Medicine group and model group compare, and 5 days groups of medicine also obviously reduce (P＜0.01) than 5 days groups of model positive cell number.
ET is the excretory a kind of vaso-active substance with strong vasoconstrictive effect of vascular endothelial cell, ET-1 directly overflows from impaired vascular endothelial cell, neurocyte and neurogliocyte behind the brain injury, the a large amount of releases of ET-1 and vascular endothelial injury make cerebrovascular shrink the brain energy metabolism obstacle.This medicine obviously reduces the expression of ischemic region ET-1, makes local vascular dilation and helps keeping the energy metabolism of organizing of ischemic area, thereby constituted the another effective mechanism of brain protection.
(3). myosin (fetuin) is to the influence of focal cerebral ischemic model rat brain microvascular endothelial VIIIR:Ag
The gross area that each treated animal ischemia side cerebral tissue half dark space cell VIIIR:Ag of table 7 expresses
Compare with while segment model group: * p＜0.05, * * p＜0.01.
As can be seen from Table 7, model group and sham operated rats be the positive cell number showed increased of each different course relatively, and utmost point significant difference P＜0.01 is arranged, and the success of line bolt method focal cerebral ischemia model is described; And with medicine group and the model group comparison course of treatment, 24 hours groups of 24 hours groups and models of medicine compare there was no significant difference (p＞0.05), medicine 48 hours, 72 hours groups respectively with model 48 hours, 72 hours groups relatively VIIIR:Ag are expressed and are obviously increased (P＜0.05).
The optical density that each treated animal ischemia side half dark space positive cell VIIIR:Ag of table 8 expresses
Compare with while segment model group: * p＜0.05, * * p＜0.01.
As can be seen from Table 8, with medicine group and the model group comparison course of treatment, 24 hours groups of medicine 24 hours group and model are there was no significant difference (p＞0.05) relatively, medicine 48 hours, and 48 hours, 72 hours groups of 72 hours groups and the model relatively optical density expressed of VIIIR:Ag also obviously increase (p＜0.05).
The complex that blood coagulation factor VIII is made up of blood coagulating protein and Factor IX associated protein (VIIIR) has antigenicity, claims Factor IX related antigen (VIIIR:Ag) again.It is synthetic that it mainly contains endotheliocyte, is distributed in the Weikel-palabe corpusculum of vascular endothelial cell, also is distributed in the platelet membrane.Every pathological changes that vascular endothelial cell infringement is arranged, as apoplexy etc., the VIIIR:Ag level increases in all visible blood plasma.And only on new vessels, express with the VIIIR:Ag that the SABC method shows.Therefore, can not only measure the relative amount of VIIIR:Ag in the hemorrhagic focus, also can be used as the labelling of new vessels simultaneously.This result of the test shows that 24 hours model group and medicine group do not have significant difference, and 48 hours, group VIIIR:Ag expressed then apparently higher than model group in 72 hours, illustrate that this medicine can promote ischemic area cerebral microvascular new life, improve and recover the blood supply of ischemic area and impel the recovery as early as possible of cerebral ischemic injury.
Description of drawings:
Fig. 1: myosin (fetuin) significantly dwindles rat ischemia cerebrum block volume.
Fig. 2: myosin (Fetuin) suppresses the expression of focus zone macrophage: A and is contrast (normal brain); B is myosin (50mg/kg) treatment back cerebral ischemia brain; C is not for treating the cerebral ischemia brain.
Fig. 3: myosin (Fetuin) suppresses the expression of focus zone tumor necrosis factor (TNF-a): 1 is contrast (normal brain); 2 is myosin (50mg/kg) treatment back cerebral ischemia brain; 3 for not treating the cerebral ischemia brain.
Embodiment 1:
Hyclone adds the 0.05M zinc acetate of 3 times of volumes, and 35%7 alcohol are regulated pH value to 6 and time placed 12-16 hours, discards precipitation after centrifugal, collects supernatant, and adding 1.5M barium acetate and 95% ethanol and obtaining final concentration is 0.04M barium ions (Ba++); 30% ethanol is regulated pH value to 6 and placed 2 hours, and is centrifugal, discards precipitation, collect supernatant, add 95% ethanol and make concentration be 50% ethanol placement 12-16 hour, centrifugal, precipitation (myosin, FETUIN), abandoning supernatant obtains myosin solution with the dissolving of 3% mannitol water for injection; Aseptic filtration repeatedly, packing, lyophilization promptly gets myosin (FETUIN) injectable powder.Be used for the treatment of the acute ischemic brain injury, comprise cerebral apoplexy diseases such as cerebral thrombosis, cerebral embolism, cerebral hemorrhage
Embodiment 2:
Hyclone adds the 0.03M zinc acetate of 2 times of volumes, and 28.5% ethanol is regulated pH value to 6.4 and time placed 12-16 hour, discards precipitation after centrifugal, collects supernatant, and adding 1.0M barium acetate and 95% ethanol and obtaining final concentration is 0.02M barium ions (Ba++); 25% ethanol is regulated pH value to 6.7 and placed 2 hours, and is centrifugal, discards precipitation, collects supernatant, adds 95% ethanol and make concentration be 40% ethanol placement 12-16 hour, and centrifugal, precipitation obtains myosin, makes granule according to conventional method.Be used for the treatment of the acute ischemic brain injury, comprise cerebral apoplexy diseases such as cerebral thrombosis, cerebral embolism, cerebral hemorrhage.
Claims (5)
1. the application of myosin in preparation treatment acute ischemic brain injury medicine.
2. application as claimed in claim 1 is characterized in that said treatment acute ischemic brain injury is the infarct size of dwindling the acute ischemic brain injury.
3. application as claimed in claim 1 is characterized in that said treatment acute ischemic brain injury is to suppress the macrophage of ischemic area and the expression of activatory microgliacyte or tumor necrosis factor.
4. application as claimed in claim 1 is characterized in that said treatment acute ischemic brain injury is to improve the behavior of focal cerebral ischemia nervous symptoms.
5. application as claimed in claim 1 is characterized in that said treatment acute ischemic brain injury is the inflammatory reaction of resisting ischemic brain injury, the protection brain cell.
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Non-Patent Citations (1)
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|---|
| 从新生小牛血清分离提取胎球蛋白. 王贤辅等.重庆医科大学学报，第17卷第4期. 1992 * |
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