CN1753686A - Methods, compositions and kits for promoting recovery from damage to the central nervous system - Google Patents

Abstract

The present application relates to methods, kits and compositions for improving a subject's recovery from CNS injury. In certain aspects, methods of the invention comprise administering to a subject cells and a neural stimulant. Recovery may be manifest by improvements in sensorimotor or cognitive abilities, e.g., improved limb movement and control or improved speech capability. In certain embodiments, subject methods can be used as part of a treatment for damage resulting from ischemia, hypoxia or trauma.

Description

Be used for promoting the method, compositions and the test kit that recover from central nervous system injury

The application requires the priority of the U.S. Provisional Application 60/149561 of application on August 18th, 1999, and this application is incorporated herein in full

1. background technology

Because central nervous system (CNS) cell only has limited repair ability, so CNS is under attack especially easily, causes part cell death or damage.As a result, the CNS disorder usually can cause the weak of patient's cognition and sensorimotor function, and produces irreversible degeneration to a great extent.Cause the disease of nerve cell death and damage to have a lot, from the degenerative imbalance, for example alzheimer disease is to the ischemic outbreak, and for example apoplexy does not wait to wound.

Worldwide, central nervous system (CNS) damage is to cause dead and disabled major reason.For example, in U.S.'s apoplexy is to cause dead and disabled the third-largest reason, estimate that sickness rate is annual 700,000 example (Furie et al. (1998) " CerebrovascularDisease " in The Atlas of Clinical Neurology, R.N.Rosenberg, Ed.Current Medicine:Philadelphia).2/3 paralytic can be survived after apoplexy in 1 year, on average can survive 7 years, and present like this have 4,800 in the U.S., survivor after the apoplexy more than 000.With regard to medical fee with and the wage of loss with regard to, apoplexy expends America's economy and reach more than 30,000,000,000 dollars every year.

After a few hours, almost nothing can prevent the coup injury of lacking of proper care and bringing to CNS because of CNS.For example, apoplexy treatment is carried out in back 6 hours in outbreak usually.According to the position that damage takes place in brain, side paralysis may take place in the patient, may lose the ability of speaking and seeing thing, and the possibility difficulty in walking, and all symptoms.Although may recover not exclusively, according to size and position and other factors of damage, the recovery of these functions is very common.

Because the cerebral tissue of damage can not be regenerated, recovery can only come from the remaining that part of brain that was not subjected to damage, remaining brain itself again machineization (reorganize) or wiring again (rewires) in case compensation because of some function of damage forfeiture.In fact, the research among the animal and human provides competent evidence to show the described machineization again of brain function after the apoplexy.Specifically, residue neuron in the complete hemisphere of damaged hemisphere and opposition side all can be grown new prominent (aixs cylinder and dendron) and be formed new being connected (synapse), they have and help recover (Kawamata et al. (1997) Proc.Natl.Acad.Sci.USA, 94:8179-8184; Jones et al. (1994) J.Neurosci., 14:2140-2152; Stroemer et al. (1998) Stroke, 29:2381-2395; Cramer et al. (1997) Stroke, 28:2518-2527).

For example, apoplexy treatment concentrates in the preceding several hrs after apoplexy the restriction to degree of injury.Apoplexy is normally because artery occlusion, thereby causes brain to stop up, and causes the brain cell death by the tremulous pulse supply.The treatment of apoplexy at present mainly concentrates on the treatment (controlling blood pressure, blood fat, heart disease etc.) that prevents artery occlusion, and in case prevents in the treatment of brain injury after stopping up.Back one class treatment comprises " thrombolytic medicine " (" clot bomb " be tPA for example) with broken tremulous pulse clot, and design is used for protecting cerebral tissue to avoid " nerve protection medicine " of risk of stroke.Described thrombolytic and nerve protection medicine must be used in a few hours just after apoplectic seizure and can be effective.

After cell death and damage take place, have only method seldom can promote that the patient recovers at present.Method used in the method for damage after date treatment early apoplexy and early stage a few hours is just different on mechanics.Promote the treatment that recovers to concentrate on usually and encourage neure growth to be connected (rewiring) again with nerve.

Directly use the neurotrophy growth factor for brain and can improve spontaneous functional rehabilitation (Kawamata et al. (1997) Proc.Natl.Acad.Sci.USA, the 94:8179-8184 that in the animal model of apoplexy, takes place; Kawamata et al. (1996) J.Cereb.BloodFlow Metab., 16:542-547; Kawamata et al. (1999) Exp.Neurol.158:89-96; Alps et al., U.S.Patent No.5,733,871, Fisher etal. (1995) J.Cereb.Blood Flow Metab., 15:953-959; Jiang etal. (1996) J.Neurol.Sci., 139:173-179).For example, basic fibroblast growth factor (bFGF) is a kind of support neuronal survival and the ramose protein of axon.When using bFGF after the apoplexy same day or a couple of days, animal recovers rapidly and return to better degree (opposite with the apoplexy side) in the sensorimotor function test of damage limb.This recovery is not because the reduction of source cerebral lesion degree.In fact, data show that this improvement of recovery situation may be because in remaining complete cerebral tissue, the increase that new neuron sprouts and synapse forms causes.As if described reshaping occur in damaged and the intac hemisphere.Other recover mechanism may comprise the IC neuronic endogenous neural stem cell that can be divided into subsequently, substitutes some neuron because of the apoplexy loss.

The another kind of effective ways that are used for the apoplexy recovery comprise the use neural stem cell.These are already present pluripotent cells in developmental and ripe mammal brain, give suitable stimulation, just can be divided into cerebral neuron and/or neurogliocyte.Some research worker have successfully been separated from Mus and human brain and have been cloned described neural stem cell system (Snyder et al. (1997) Proc.Natl.Acad.Sci.USA, 94:11663-11668; Gage etal. (1995) Proc.Natl.Acad.Sci.USA, 92:11879-11883; Kuhnet al. (1997) J.Neurosci., 17:5820-5829; McKay et al., U.S.Patent No.5,270,191; Johe, K., United States Patent (USP) 5,753,506; Carpenter, M., United States Patent (USP) 5,968,829; Weiss et al., United States Patent (USP) 5,750,376).In the time of in described stem cell being imported again growth or sophisticated brain, they can divide, move, grow prominent, therefore present neural phenotypes, comprise the expression by the neurotransmitter and the somatomedin of neuron normal process.Therefore, it is favourable using aspect two neural stem cell to be used for that apoplexy recovers at least: the dead regional cell that (1) partly makes apoplexy cause by described stem cell is bred again and is established again neural the binding; (2) the required important neurotransmitter of secretion brain is connected (rewire) with somatomedin so that produce nerve again after apoplexy.Promote existing (Park etc., (1999) J.Neurotrauma 16:675-687 of describing of the brain effort that recovery is done from damage of animal with neural stem cell; Park etc. (1995) Soc.Neurosci.Abs.21:2027; Stroemer etc. (1999) Soc.Neuroscience Abs.25:1310).Also described in animal (Borlongan etc. (1993) Int.J.Devl.Neuroscience11:555-568) and people (Kokaia etc. (1998) Eur.J.Neurosci., 10:2026-36) the middle work of being made of the deutero-cell line of lopsided cancerous cell.

The method that existing promotion recovers from the CNS damage only can recovered part function of nervous system.In the patient who suffers from weak property neurological handicap, the improvement of the increase of function has remarkable influence to quality of life.Because the limited of a large amount of patients and existing method arranged, therefore, be badly in need of other and improved method recover from damage to promote nervous system.The given Therapeutic Method of the present invention can recover from the CNS damage to a greater extent than present existing other known treatment methods.

2. summary of the invention

One aspect of the present invention relates to and is used for improving the method for patient from CNS damage or infringement recovery.In one aspect, the present invention includes preferred stem cell and be enough to improve patient's sensorimotor or cognitive competence, the Dexedrine of limb motion that for example improves and control or the ability quantity of speaking improved to individual dosed cells.

On the other hand, the invention provides the test kit that is used for the treatment of the CNS infringement.In specific embodiments, test kit of the present invention comprises stem cell and Dexedrine.In other embodiments, test kit of the present invention contains a kind of Dexedrine and the device that is used for containing from the individuality acquisition stem cell sample.In preferred embodiments, described test kit contains a kind of polypeptide growth factor and more preferably has 30% homogeneity at least with one of SEQ.ID.Nos.1-3 polypeptide but the best polypeptide that has 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% homogeneity at least.

On the other hand, the invention provides the pharmaceutical preparation that contains stem cell, Dexedrine and one or more pharmaceutically acceptable reagent.

In preferred embodiments, being used for stem cell of the present invention is to produce brain cell, for example the cell of neuron, oligodendroglia or astroglia.In particularly preferred embodiments, stem cell is neural stem cell, hematopoietic stem cell, the deutero-cell of lopsided cancerous cell or embryonic stem cell.In other embodiment preferred, from individuality, obtain stem cell, and randomly can before using, cultivate or enrichment external.

In other embodiments, can be by promoting the factor with one or more propagation of coding, for example the gene of vmyc, SV40T antigen, polyoma virus large T antigen, neu oncogene or the transfection of ras oncogene induce stem cell of the present invention at in-vitro multiplication.In preferred embodiments, described gene promotes propagation at external strong expression, but then seldom expresses after described cell enters the central nervous system, thereby described cell can not bred rapidly in vivo.

In further embodiment, described Dexedrine is a polypeptide growth factor.Preferred polypeptide growth factor comprises the polypeptide that is selected from following peptide family: fibroblast growth family member, neurotrophin family member, insulin like growth factor family, ciliary nerve nutrition growth factor family member; EGF family member, TGF 'beta ' family member, leukaemia inhibitory factor (LIF); Oncostatin M, interleukin 11; Interleukin-6; The member of platelet-derived growth factor family and VEGF family member.Be envisaged in the specific embodiment, various combinations of factors can be used.Preferred polypeptide contains the polypeptide of sequence that at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% homogeneity is arranged with the arbitrary aminoacid sequence of SEQ ID Nos.1-3.

In other embodiments, described Dexedrine is the active regulator of neurotransmitter (for example agonist or an antagonist).In preferred embodiments, described Dexedrine is for example for example amitriptyline or its combination of fluoxetine, amfetamine, methylphenidate, tricyclic antidepressants of antidepressant.In another embodiment, described Dexedrine is a for example tretinoin of neuron differentiation accelerator.In another embodiment, described Dexedrine is for example netrin, semaphorin, neuropilin or ephrin of so-called guide molecule.In other embodiments, described Dexedrine can be to stride cranium magnetic to stimulate (transcranial magneticstimulation).

On the other hand, the present invention includes the bioactive compound of co-administered cell and non-Dexedrine.Preferred bioactive compound comprises for example immunophilin (for example cyclosporin, FK506 and Thalidomide) and antibiotic tetracycline for example of immunosuppressant.

The various technology that are used to use cell of the present invention and Dexedrine have been imagined.Not needing in an identical manner or dosed cells and Dexedrine simultaneously, is preferable but can eclipsed mode use them with its effect.In preferred embodiments, after damage takes place, finished at least 6,10,12 or 24 hours and use.

3. accompanying drawing summary

Fig. 1 is data of making the embodiment 1 of diagram form.

Figure 1A is described in the result of forelimb placement test in the rat apoplexy model.With rat separately with bFGF, separately with stem cell, with bFGF and stem cell or handle with control vector separately.

Figure 1B explanation hind leg in the rat apoplexy model is placed the result of test.

The result of Fig. 1 C explanation health rotation (bodyswing) test in the rat apoplexy model.

The result of Fig. 1 D explanation spontaneous limb service test in the rat apoplexy model.

Fig. 2 is data of making the embodiment 2 of diagram form.

Fig. 2 A is described in the result of forelimb placement test in the rat apoplexy model.With rat separately with bFGF, separately with stem cell, with bFGF and stem cell or handle with control vector separately.

Fig. 2 B explanation hind leg in the rat apoplexy model is placed the result of test.

The result of Fig. 2 C explanation health rotation test in the rat apoplexy model.

The result of Fig. 2 D explanation spontaneous limb service test in the rat apoplexy model.

Fig. 3 is described in the result that rat apoplexy model median claw stretches out test.

Fig. 4 represents the aminoacid sequence of bFGF version (SEQ.ID.Nos.1-3).

4. detailed description of the present invention

4.1 definition

Term used herein " brain cell " refers to the cell that brain is contained, comprises neuron, astroglia, mesoglia and microglia cell. Many concrete cell types belong to kind separately. For example neuron comprises dopaminergic, cholinergic and paddy ammonia serotonergic neuron, and mentioned only is several examples.

" bioactive compound " comprises the compound with required effect when using when in the context of the invention. Bioactive compound comprises many Dexedrines (seeing below) and many compounds that is not considered to Dexedrine but also has required effect. For example immunodepressant such as immunophilin (for example, FK506) can show and suppress the transplanted cells rejection and neuroprotective activity double action (Bavetta etc., (1999) Exp.Neurol.158:382-393) is provided. Antibiotic, particularly Tetracyclines can suppress possible infection, and the effect useful to nerve cell (Yrjanheikki etc. (1998) PNAS 95:15769-74).

" cell culture " refers to it no matter is the cell of that growing, that breaking up or static any composition generically. Cell culture can present by various forms. For example, " suspension culture " refers to that cell wherein is suspended in the culture in the appropriate media. " continuous flow type culture " refers to that cultured cell is to keep Growth of Cells and vigor in the continuous flow type fresh culture. " continuously expansion " is to cultivate the method that makes Growth of Cells by continuous flow type.

Term used herein " central nervous system " (CNS) refers to comprise any component of the central nervous system of brain and spinal cord, cell and ECM and liquid.

With regard to giving individual dosed cells and Dexedrine or bioactive compound, this paper uses " co-administered ". Term " co-administered " does not also mean that and cell and Dexedrine must be used simultaneously. Can use at different time, with different time interval and different modes the component of administering drug combinations. But described administration should be overlapping on result for the treatment of.

Term " culture medium " is well known in the art, and is often referred to any material or the goods of cultivating for living cells.

Term used herein " growth conditioning agent " refers to the molecule regulating brain cell or have the development of stem cells of the ability that becomes brain cell.

This paper " focus cerebral ischemia " of use with regard to central nervous system refer to because giving an artery occlusion of brain or spinal cord supply blood, the dead and disease that produces of the cell element that causes this artery institute feed region.

" general cerebral ischemia " is that the blood flow that flows to whole brain reduces, and usually for example caused by heartbeat all standing or hypotension.In general cerebral ischemia, easy especially ischemic cell is dead easily or sustain damage, and causes the damage fragment that distributes around brain.This is different from the damage that takes place in the focus cerebral ischemia.

" guide molecule " is the protein that a class is found in extracellular matrix usually, and its function is meant that guided cell or cell prominent (aixs cylinder) are positioned to bring into play suitable function desired position.Example is semaphorins, netrins, neuropilins and ephrins.(Perris etc. (2000) Mech.Dev.95:3-21; Wilkinson (2000) Int.Rev.Cytol.196:177-244; (1999) Curr.Biol.9:R201-4 such as Van Vactor).

" hematopoietic stem cell " used herein is except that the potential daughter cell such as can producing (HSCs), also can produce the stem cell of the cell of at least a main hematopoietic lineage.Three kinds of main hemocyte pedigrees comprise lymph sample pedigree, for example B cell and T cell, myelocyte sample pedigree, for example mononuclear cell, granulocyte and megalokaryocyte, and cell born of the same parents class pedigree, for example Red blood corpuscle.Specific HSCs can produce many other cell types that comprise brain cell." pluripotency " HSCs is the HSCs that can produce at least three kinds of main hematopoietic lineages.

" homology " and " homogeneity " respectively refers to two sequence similarities between peptide sequence, and homogeneity will be carried out stricter comparison.By relatively determining homology and homogeneity for the position in each sequence of relatively arranging.When the position in being compared sequence is occupied by the same amino acid residue, can think that then described polypeptide is identical in this position; When the equivalent site when occupying, is thought then that described molecule is homologous in this position by same aminoacid (for example identical) or similar aminoacid (for example space and/or electronic property are similar).The percentage ratio of homology between sequences or homogeneity is the common coupling of two sequences or the function of homology positional number.The peptide sequence of " incoherent " or " nonhomologous " sequence and biologically active polypeptide of the present invention has the homogeneity below 40%, although preferably be lower than 25% homogeneity.

Instruct the insufficient any environment of blood supply that causes to tissue with term " ischemia outbreak ".The cerebral ischemia outbreak results from cerebripetal blood supply deficiency.Spinal cord also is central nervous system's a part, and is responsive equally to reduce the ischemia that produces because of blood flow.The ischemia outbreak can cause because of the contraction or the obstruction of blood vessel, as what take place under thrombosis or embolus situation.Perhaps, ischemia outbreak can result from any type of injured cardiac function, comprises the heartbeat all standing.

Term " Dexedrine " refer to affect the nerves function or active therapeutant.Described therapeutant is polypeptide growth factor normally, for example neurotrophin or fibroblast growth factor.Described therapeutant also is included in activated guide molecule and non-peptide molecule in the brain, for example neurotransmitter, neurotransmitter antagonists or agonist, and grow regulator." Dexedrine " can also be the factor that influences the same signal pathway that is influenced with the above-mentioned listed factor.The chemical substance of for example available activation bFGF receptor signal conduction is as Dexedrine." Dexedrine " also can comprise other chemistry or the Electromagnetic Treatment (for example striding cranium magnetic stimulates) of the molecule generation that changes affect the nerves function or activity.

(NSC) describe cell derived from the tissue of the central nervous system or the nervous system of growing with " neural stem cell ", they can produce at least a following basic neural pedigree: neuron, oligodendroglia and astroglia.In addition, neural stem cell must be able to produce the new NSCs with similar potential." pluripotency NSCs is the NSCs that can produce all above-mentioned neural pedigrees and be equal to the cell of potentiality of development.

" neuronal function " is often referred to neural all functions, for example sensorimotor function and cognitive function.

" neuroepithelial stem cell " is the stem cell population that separates from the neuroepithelium tissue of fetus.According to used herein, described cell can be considered to the subclass of neural stem cell." neuroepithelial cell " tends to pluripotency.

" neurotransmitter " is the micromolecule that works that discharges the aixs cylinder in synapse.The neurotransmitter of illustrative comprises catecholamine (for example epinephrine, norepinephrine and dopamine), 5-hydroxy tryptamine, acetylcholine, glutamate, Glu and GABA.

" patient " or " individuality " to be treated with described method is mammal, comprises the people.

" protein " used herein and " polypeptide " refer to any amino acid residue chain, do not consider length or post translational modification (for example glycosylation or phosphorylation)." biologically active polypeptide " used herein refers to have as the active polypeptide of Dexedrine.Example is polypeptide growth factor and guide molecule." biologically active polypeptide " also comprises the active fragment and the analog of the biologically active polypeptide of one or more biological functions with described factor.

" polypeptide growth factor " normally in the cell of at least a type, excretory polypeptide or its active fragment of stimulating cellular growth or cell prominent (for example aixs cylinder, dendron etc.) growth.

Used " active fragment " refers to have any part of at least a active polypeptide of full-length polypeptide when mentioning biologically active polypeptide.Many polypeptide have several different activity, but also preferably use the active fragment that a kind of activity or these active subclass are only arranged.Described active fragment will produce at least 20%, and preferably at least 50%, more preferably at least 70%, and the activity of the described full-length polypeptide of the best at least 90% (comprising up to 100%).Example is bFGF, and it can be a multiform, and observed molecular weight is 17.8,22.5,23.1 and 24.2kDa; All these forms is biologically active and can be used for the present invention all.

Be used alternatingly term " recombiant protein ", " heterologous protein " and " foreign protein " in this manual, and refer to the polypeptide that produces by recombinant DNA technology, wherein the dna molecular of coding said polypeptide is inserted in the suitable expression construct, then with described construct transformed host cell to produce heterologous protein.Promptly express described polypeptide from heterologous nucleic acids.

" apoplexy " is to lose suddenly because of the function that cerebripetal blood supply causes unusually.Apoplexy has the order of severity of varying level, from " temporary transient ischemia outbreak " or " TIA " (impermanent deformity) to " the non-progressing stroke of part " (continue but do not have catastrophic damage), to " complete apoplexy " (permanent, catastrophic neurological handicap).Ischemia (blood flow reduce or stop) and to block (primary cellular defect in the zone of ischemia and death) be the pathological process that causes in the apoplexy of neurological handicap." ischemic stroke " is that the obstruction of blood vessel by the supply brain causes.The main subspecies class of ischemic stroke is embolic stroke, embolus apoplexy and lacuna apoplexy." hemorrhagic apoplexy " is to be caused by the breaking of blood vessel of supplying brain.The main subspecies class of hemorrhagic apoplexy is subarachnoid hemorrhage (SAH) and ICH (ICH).

With regard to the inventive method, for example cell of " treatment effective dose " or Dexedrine refer to when using as the part of required dosage scheme, can cause the quantity of the treatment (in preparation) that neuronal function improves according to clinical acceptable standard.

" striding cranium magnetic stimulates " (TMS) is with the close head (electric current in the TMS coil can have the 8000A height) of coil, is the method that stimulating neuronal is come in the magnetic field between 2 to 4T by producing common field intensity momently.TMS often comprises the boost pulse with different pulses and time delay.Known TMS can raise the monoamine in the brain.

4.2 general introduction

The present invention's part is based on following wonderful discovery, and promptly co-administered cell and Dexedrine all can promote to recover from the CNS infringement than any independent treatment better.Aspect specific, the invention provides improved method, compositions and test kit that the cerebral tissue that is used to stimulate damaged recovers, no matter and damage is partial or general.In preferred embodiments, the present invention relates to from ischemia, hypoxgia and wound, recover.Aspect specific, method of the present invention comprises co-administered stem cell and Dexedrine, for example polypeptide growth factor or other molecules.Therapeutic alliance can produce greatly than any independent treatment and recover.The prospect of this method has been described in a research recently, in described research, polypeptide growth factor BDNF has been used to improve the recovery (Chen etc., 2000, Neuropharmacology 39:711-716) in the rat apoplexy model with medullary cell.The effect that weakens of CNS damage is even recovers to improve constantly the bigger improvement that also can cause patient's quality of life.

The inventive method can be widely used in treatment CNS damage.Thus, the inventive method can be used for, but is not limited to treat because the brain that ischemia, hypoxgia, wound, neurodegenerative disease, infectious disease, cancer, autoimmune disease and metabolism disorder cause and the treatment of spinal cord injury.Disorderly example comprises brain injury, Alzheimer, hungtington's chorea, Creutzfeld-Jakob disease, parkinson after apoplexy, head trauma, spinal trauma, hypotension, the breathing (arrested breathing) that stops, heartbeat all standing, Rey ' s syndrome, cerebral thrombosis, thromboembolism, cerebral hemorrhage, the cerebral tumor, encephalomyelitis, hydroencephalitis, operation and the operation, hardens and the amyotrophic side sclerosis more.

Thrombosis, embolus and general hypotension are the most common inducements of cerebral ischaemia outbreak.Other inducements of cerebral ischaemia comprise hypertension, hypertensive cerebral cerebrovascular disease, aneurysm rupture, hemangioma, blood dyscrasia, heart failure, heartbeat all standing, cardiogenic shock, septic shock, head trauma, spinal cord injuries receptor, epilepsy, tumor is hemorrhage or other are lost blood.With regard to wound, wound may relate to tissue injury, for example scratch, otch, dampen, stab, compressing etc., for example can produce from exterior object and head, neck or any position of vertebra or its appendicular contact.Other forms of traumatic damage can produce the narrow or compressing (for example obstruction of normal brain activity spinal fluid or functional disorder or Vitreous humour generation, renewal or volume-adjustment, perhaps following or intracranial hematoma or edema of dura mater) of the CNS tissue that causes from the inappropriate accumulation because of liquid.Equally, traumatic narrow or compressing is owing to there are a large amount of abnormal structures, for example shifts or primary tumor.

In some cases, the damage that is caused by above-mentioned disease mainly is positioned at an IC zone, focus ischemia for example, certain wound and parkinson.In other cases, damage may be more extensive or be distributed in the zones of different of brain, for example hypoxgia and general ischemia and Creutzfeld-Jakob disease.Because known specific cells of the present invention can move at the brain internal freedom, and, therefore expect that the inventive method and compositions will be used in the recovery of promotion from general and local brain injury owing to somatomedin can be provided so that make all cerebral tissue all can obtain described somatomedin.

Generally, therapeutic scheme of the present invention relates to the patient who suffers from the CNS damage and uses Dexedrine and stem cell.In preferred embodiments, the CNS damage is caused by ischemia, hypoxgia or wound.Treatment can comprise from the patient and obtain cell that randomly enrichment treatment cell is used described cell to described patient then.In this way, the patient is without any need for foreign cell, thereby advantageously avoided the immunoreation to described cell.

With regard to administering mode, timing administration and dosage, finish therapeutic scheme of the present invention from the negative consequence of central nervous system injury so that the patient can be recovered quickly; For example patient's motor skill (for example posture, balance, grasping or gait), cognitive skill, speak and/or consciousness (comprising visual capacity, the sense of taste, olfactory sensation and somesthetic sensibility) can improve because of treatment of the present invention.

Although without wishing to be held to specific mechanism of action, it is believed that method of the present invention is sprouted by stimulating neuronal and new synapse formation promotes to recover from the CNS damage.Under the situation of apoplexy, all treatments at present all concentrate on infraction minimizing and pre-antisitic defect basically.Therefore, the present invention relates to treat the unconventional and new method of CNS damage.

4.3 Dexedrine and other biological active factors

Dexedrine of the present invention comprises affect the nerves function or active therapeutant, chemical substance or other materials.Described therapeutant is biologically active polypeptide normally, but also can be that non-peptide molecule and naturopathy method are for example striden the stimulation of cranium magnetic.

In one group of embodiment preferred, described Dexedrine is a polypeptide growth factor.Pharmaceutically suitable carrier administration of described polypeptide growth factor also can be used with blended or unmixing cell.Described polypeptide growth factor can be the member in fibroblast growth factor (FGF) family; Neurotrophin family; Insulin-like growth factor (IGF) family; Ciliary neurotrophic factor (CNTF) family; EGF family; The TGF 'beta ' family; PDGF family; VEGF family; Leukaemia inhibitory factor (LIF) family; Interleukin (for example IL-11, IL-6, IL-1); Perhaps Oncostatin. (for example oncostatin M).The feature and the illustrative member of these families have been provided hereinafter with in the table 2.In preferred embodiments, described polypeptide factor is the human polypeptides factor.

At least 15 kinds of different factors are contained in FGF family, and they are high conservatives between the mammal kind, although each family member has the difference (homogeneity of 30-70% usually) of height to each other.FGFs is excretory protein, they all by one by the basic tertiary structure of forming with 12 folding beta chains of β-SANYE.But most of family member has mitogenesis and heparin-binding also to all kinds cell.FGF family example comprises: basic FGF (bFGF, FGF-2), acid FGF (aFGF, FGF-1), FGF-3 (int-2), FGF-4 (hst/kFGF), FGF-5, FGF-6, FGF-7 (KGF), FGF-8 (AIGF) and FGF-9 (GAF).(Stauber etc., (2000) PNAS 97:49-54; Wong etc. (1998) J.Biol.Chem.273:18617-18622; Szebenyi etc. (1999) Int.Rev.Cytol.185:45-106).

The neurotrophin family member comprises the excretory factor that several are relevant, and they mainly bring into play its effect to nervous system.Neurotrophin is produced as precursor protein matter usually, then it is carried out height processing and obtains mature form.Ripe neurotrophin carries one group of 6 cysteine, and wherein 1-4 (promptly the 1st and the 4th cysteine form disulfide bond) is individual, 2-5 is individual with 3-6 is individual forms relevant with disulfide bond.Usually the neurotrophin family member is by a surface of being made up of 3 antiparallels, and dimerization is along this surface generation.The illustrative member of neurotrophin family is: nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), neurotrophin 4/5 (NT4/5) neurotrophin 6.(Lewin etc. (1996) Annu.Rev.Neuroscience 19:289-317).

Insulin like growth factor family comprises having and the sequence of insulin and the excretory protein of structural similarity, and molecular weight is generally 5-10kDa.Can in blood flow, find these factors, relevant with one of 6 igf binding protein matter usually.The illustrative member of this family comprises IGF-1 and IGF-2.Known IGF-1 and-2 can promote to recover from the various attack to CNS.(Daughaday etc. (1989) Endocr.Rev.10:68-91; Rajaram etc. (1997) Endocr.Rev.18:801-831; Jones etc. (1995) Endocr.Rev.16:3-34).

Epidermal growth factor family is the extended familys of a relevant excreted factor.The EGF family member has at least 30% sequence homology and at this proteinic C-terminal one group of 6 conserved cysteine residue is arranged.Most of described albumen also contains an EGF-spline structure territory, and this is a domain that has fully been characterized and is present in many non-EGF family member albumen.Usually obtain the EGF family member from bigger precursor processing.The illustrative member of EGF family comprises EGF, TGF α, HB-EGF (heparin-bounding EGF), amphiregulin, β tunicin, cowpox somatomedin and neu differentiation factor.(Aviezer etc. (1994) 91:12173-12177; Higashyama etc. (1992) J.Biol.Chem.267:6205-6212; Pelles etc. (1992) Cell69:205-216).

In many organisms and cell type, the TGF beta superfamily is the important molecule relevant with growth with cell-cellular signal transduction of a class.The member of this family at first as have amino terminal signal sequence and variable size prodomain be synthesized (Kingsley, D.M. (1994) Genes Dev.8:133-146) than the larger precursor molecule.Then described precursor cracking is discharged 110-140 amino acid whose ripe C-terminal fragment.Active signal conduction portion is formed (Massague, J. (1990) Annu.Rev.Cell Biol.6:597-641) by exclusive OR with-dimeric C-terminal fragment.The activity form of this molecule and its acceptor interaction then, the receptor of this family molecule is formed (Massague, J.et al. (1992) Cell 69:1067-1070 by two kinds of closely not corresponding transmembrane serine/threonine kinases that are called I type and II receptor; Miyazono, K.A.et al.EMBO are J.10:1091-1101).TGF β directly combines with the II receptor, restores the I receptor then and by phosphorylation it is modified.The I receptor conducts signal to downstream component (Wrana et al, (1994) Nature 370:341-347) then.In a word, the member of TGF beta superfamily has the cysteine residues of one group of 9 high conservative, they with ripe monomer in and disulfide bond between monomer form relevantly, be Dimerized signal-proteins (Griffith etc. (1996) PNAS 93:878-883; Luo etc. (1995) PNAS 92:11761-11765; Schlunegger etc. (1993) J.Mol.Biol.231:445-58; Daopin etc. (1993) Proteins17:176-92; Murray-Rust etc. (1993) Structure 15:153-9; Archer etc. (1993) Biochemistry 32:1164-71; Daopin etc. (1992) Science257:369-373; Schlunegger etc. (1992) Nature 358:430-434; Hinck etc. (1996) Biochemistry 35:8517-34; Mittl etc. (1996) Protein Sci.5:1261-71).

Transforming growth factor family is the very protein of extended familys, comprises TGF β subfamily, bone morphogenetic protein matter (BMP) subfamily, nandrolone phenylpropionate subfamily and other.The TGF β subfamily member of illustrative comprises TGF-β-1 ,-2 ,-3 ,-4 and-5.The BMP subfamily member of illustrative comprises BMP 1 (OP-1, BMP-7) and BMP-9.(Ren etc. (2000) Neuropharmacology 39:860-865; Lopez-Coviella etc. (2000) Science 289:313-316; Withers etc. (2000) Eur.J.Neurosci.12:106-116).

VEGF (VEGF) family is the excretory protein of a class, works as strong mitogen in embryo and body angiogenesis.The vegf protein matter that comprises VEGF itself is in the cell surface receptor combination of kinase domain receptor family (KDR) and fms-sample tyrosine kinase (Flt receptor).Vegf protein matter forms the homodimer that has the cystine knot structure.Platelet-derived somatomedin (PDGF) and the only limited sequence similarity (19%) of VEGF, but substantial structural similarity is arranged.PDGF and relevant family member also be cystine knot (knot) protein and in a similar manner with its receptors bind.(Lobsiger etc. (2000) Glia 30:290-300; Sun etc. (1995) Annu.Rev.Biophys.Biomolec.Struct.24:269-291; Muller etc. (1997) Structure 5:1325-1338; Jiang etc. (2000) EMBO J.19:3192-3203; Muller etc. (1997) PNAS 94:7192-7197).

Interleukin is excretory polypeptide factor, the signal conduction between the mediation immunocyte.Known many interleukin have effect to brain, particularly IL-1 α and β, IL-6 and IL-11.((1999) J.Neuroimmunol.100:124-139 such as Van Wagoner; Ling etc. (1998) Exp.Neurol.149:411-23; Mehler etc. (1993) Nature 362:62-5).What is interesting is that IL-6 and IL-11 partly work by receptor protein gp130, and gp130 is the receptor of ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF) and oncostatin M.Therefore, all these factors may all have similar effect in regulating neuronal function and growing.(Benigni etc. (1995) Mol.Med.1:568-75; Benigni etc. (1996) Blood 87:1851-4; Murphy etc. (1997) Prog.Neurobiol.52:355-78).

Table 1: polypeptide growth factor

Family	The illustrative subfamily	Illustrative member
			FGF		bFGF、aFGF、FGF-3、FGF-4、FGF- 5、FGF-6、FGF-7、FGF-8、FGF-9
			Neurotrophin		NGF、BDNF、NT3、NT4/5、NT-6
			IGF		IGF-1、IGF-2
			EGF		EGF, TGF α, HB-EGF, amphiregulin, β tunicin, cowpox somatomedin and neu
			TGF-β	TGF-β	TGF-β-1 ,-2 ,-3 ,-4 and-5
				BMP	OP-1、BMP-9
				Nandrolone phenylpropionate	Inhibin β A, inhibin β B and inhibin β C
			VEGF		VEGF

			PDGF		PDGF
			LIF		LIF
			CNTF		CNTF
			Interleukin		IL-1α、IL-1β、IL-6、IL-11
			Oncostatin.		Oncostatin M

In addition, the nomenclature in polypeptide factor field is very complicated, mainly is because many factors are to be separated independently by on the same group research worker not, and in history, names according to detecting used types of organization in the purification of factor process.Basic FGF has many different names and multiple family member is arranged in scientific literature.These comprise the leukemia somatomedin, macrophage growth factor, the deutero-angiogenesis factor 2 of embryo kidney, prgf, the astroglia growth factor-2, endothelial cell growth factor (ECGF), the chondrosarcoma somatomedin, the deutero-somatomedin 1 of cartilage, the deutero-somatomedin 1 of eye, heparin-bounding somatomedin 11 types, the myogenicity somatomedin, the factor of people's Placenta Hominis purification, the deutero-somatomedin in uterus, the deutero-somatomedin of embryonal carcinoma, people's hypophysis cerebri somatomedin, the adipocyte somatomedin, the deutero-somatomedin of the prostate osteoblast factor and breast tumor.Therefore, any factor of arbitrary aforementioned name referring all within the scope of the present invention.In addition, carried out many effort and named to the factor to use the title that can both accept, the listed any factor of this paper all is considered to be within the purview, no matter and its whether with different names for known to other people.

The present invention also uses the bioactive analogue of aforementioned somatomedin, and they have one or more biological functions of the described factor.Example is bFGF, and it can be polymorphic, and observed molecular weight is 17.8,22.5,23.1 and 24.2kDa; All these forms all has biologic activity, and can be used for the present invention.Can identify the bioactive analogue of the aforementioned factor.Described analog when keeping at least a activity of natural polypeptides through design, thinks that then it is a function equivalent.Bioactive analogue can also comprise and not be polypeptide but still the active molecule of mimic peptide somatomedin.Bioactive analogue also can have favourable characteristic, for example, and the effectiveness of raising or the stability that more meets the requirements (for example, indirect external pot-life and to the resistance of proteolytic degradation in the body).For example, described analog can or cause described factor structure to destroy or the more stable or less stable of other processing of inactivation to proteolytic degradation.Therefore short-half-life can produce ofer short duration biological action, can be in particular organization or more closely control protein level on every side.The long half-life can be improved the effectiveness of the described factor.

In specific embodiment, biologically active polypeptide of the present invention comprises the polypeptide that at least 30% identical aminoacid sequence is arranged with the bFGF sequence of one of SEQ.ID.No.1-3.In preferred embodiments, described biologically active polypeptide contains and draws together the polypeptide that at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identical aminoacid sequence is arranged with the bFGF sequence of one of SEQ.ID.No.1-3.

The method of producing described bioactive analogue is well known in the art.Usually, change the variant that can produce polypeptide factor by in the nucleotide sequence of the described factor of coding, importing.The nucleic acid that can express described change then can detect the various characteristics of described polypeptide then with mutagenic polypeptide.Can one by one in nucleotide sequence, change to import specific required change.Perhaps preparation partly produces variant library, sifting property then at random.

The method that produces potential source biomolecule active analogue thereof library has many.In an illustrative embodiment, arrange the aminoacid sequence of bFGF homologue or other associated protein populations, preferably improve the probability of maximum homology.The population of described variant can comprise, for example from for example bFGF homologue of Mus and chicken of one or more species, perhaps from same species but because of the different bFGF homologue of sudden change.The aminoacid that screening occurs in each position of the sequence of arranging is to produce one group of sequence of degeneracy.In preferred embodiments, by the different libraries that produce the bFGF variant at the nucleic acid level combinatorial mutagenesis, the different libraries of different then gene library coding bFGF variants.For example, synthetic oligonucleotide mixture enzymatic can be connected in the gene order, perhaps be expressed as one group of bigger fusion rotein (for example phage display) that contains the bFGF sequence set so that the potentiality bFGF sequence of degeneracy group can be expressed as single polypeptide.

Available automatic dna synthesizer chemosynthesis degeneracy gene order is connected to synthetic gene in the suitable expression vector then.The purpose of degeneracy group gene is all sequences that the required bioactive analogue of coding is provided in a mixture.The synthetic method of degenerate oligonucleotide be well known in the art (for example referring to, Narang, SA (1983) Tetrahedron 39:3; Itakura etc. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos.Macromolecules, ed.AG Walton, Amsterdam:Elsevier pp273-289; Itakura etc. (1984) Annu.Rev.Biochem.53:323; Itakura etc. (1984) Science 198:1056; Ike etc. (1983) Nucleic Acid Res.11:477.Described technology has been used for other proteinic direct evolution (for example referring to, Scott etc. (1990) Science 249:386-390; Roberts etc. (1992) PNAS 89:2429-2433; Devlin etc. (1990) Science 249:404-406; (1990) PNAS87:6378-6382 such as Cwirla and United States Patent (USP) 5,223,409,5,198,346 and 5,096,815).

Outside combinations thereof mutation, also has additive method.For example, by (Ruf etc. (1994) Biochemistry 33:1565-1572 such as alanine scanning mutagenesis; Wang etc. (1994) J.Biol.Chem.269:3095-3099; Balint etc. (1993) Gene 137:109-118; Grodberg etc. (1993) Eur.J.Biochem.218:597-601; Nagashima etc. (1993) J.Biol.Chem.268:2888-2892; Lowman etc. (1991) Biochemistry 30:10832-10838; (1989) Science244:1081-1085 such as and Cunningham), by joint scanning mutagenesis (Gustin etc. (1993) Virology193:653-660; Brown etc. (1992) Mol.Cell Biol.12:2644-2652; McKnight etc. (1982) Science 232:316); By saturation mutagenesis (Meyers etc. (1986) Science 232:613); By PCR mutation (Leung etc. (1989) MethodCell Mol Biol 1:11-19); Perhaps by random mutagenesis (Miller etc. (1992) AShort Course in Bacterial Genetics, CSHL Press, Cold SpringHarbor, NY; (1994) Strategies in Mol Biol 7:32-34 such as and Greener) can produce the bFGF analog.

Also available said method produces other polypeptide factors except that bFGF.

Produced one or more bioactie agents, can in all sorts of ways and identify variant with desirable characteristics.Produce in cell in the mode similar and reply or suppress described ability of replying by assessing described variant peptides, can determine in a kind of peptide ammino acid sequence it is one or a plurality of change produces bioactive analogue at an easy rate to wild type peptide.In addition, can also determine the ability of described polypeptide in conjunction with its receptor.For example, combine with receptor FGFR1 and FGFR2 under the bFGF normal condition.This combination also can stimulate in conjunction with obtaining by heparin.Can detect these characteristics to determine the activity of bFGF variant.

The various technology that are used to the cDNA library of screening the gene outcome of combinatorial library and being used to screen the gene outcome with particular characteristics are known in the art.The most widely used technology that is used for screening big gene library generally includes is cloned into reproducible expression vector with gene library, vector library with gained transforms suitable cell, detect therein then under the condition of carrier that required activity helps relatively easily separating the gene that its product of coding need detect, express described combination gene.Hereinafter each illustrative detection method can be used for a large amount of variant sequences that needed efficient analysis screening is produced by the combinatorial mutagenesis technology.

In a kind of possible screening detects, gene library is expressed as the lip-deep fusion rotein of virion.For example, under the situation of filobactivirus system, the exogenous peptide sequence can be expressed on the surface of infectiousness phage.These phagies can be used for affinity substrate with high concentration, to screen a large amount of phagies simultaneously.If from affinity substrate, reclaim specific phage with low-yield, can pass through suitable host, for example another in the escherichia coli taken turns and infected the described phage of amplification.One group of escherichia coli filobactivirus M13, fd. and fl much at one is the most frequently used in phage display library, and available phage gIII or gVIII coat protein produce the fusion rotein that do not destroy final virion packing (the open WO 90/02909 of PCT such as Ladner; Garrard etc., the open WO 92/09690 of PCT; Marks etc. (1992) J.Biol.Chem.267:16007-16010; Griffiths etc. (1993) EMBO J 12:725-734; Clackson etc. (1991) Nature 352:624-628; With (1992) PNAS 89:4457-4461 such as Barbas).

In another embodiment, (for example natural the same) that combinatorial library is designed to that the extracellular exists with it or, randomly, can be excretory (for example the polypeptide in all libraries includes a signal sequence).Can with the transfection of described library in eukaryotic cell, then described eukaryotic cell be cultivated with the cell of the functional receptor of expressing required bioactive fragment.For example, people can identify the biological activity variant of bFGF with the cell of expressed bFGF receptor.Bioactive analogue by the emiocytosis of expressing described combinatorial library will be diffused into adjacent receptor positive cell, induce phenotypic alternation then.Can detect phenotypic alternation with for example antibody at generation or destructive epi-position in factor processing answering.

Can screen in these analog the other biological activity of each subsequently.For example, with respect to described proteinic wild type form, can detect from the isolating receptor of combinatorial library-in conjunction with the influence of analog on cell proliferation.Perhaps people can screen the external of described analog or body internal stability.Available animal model is assessed the activity of described analog.For example can in the rat apoplexy model, assess analog and improve the ability of function of nervous system to confirm that analog has suitable biological activity.

Many dissimilar sudden changes can produce bioactive analogue.For example, the conservative change in the expection aminoacid sequence can obtain keeping one or more bioactive analog.Also can reasonably expect to replace leucine, replace aspartic acid, replace threonine or the similar replacement (promptly conservative replacement) carried out with structurally associated aminoacid does not have the biological activity of gained molecule and seriously influences with serine with glutamic acid with isoleucine or valine.Conservative replacement is the replacement that occurs in the class of amino acid of its side chain.The aminoacid of genetic coding can be divided into four classes: (1) acidity=aspartic acid, glutamic acid; (2) alkalescence=lysine, arginine, histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan and (4) uncharged polar=glycine, agedoite, glutamine, cysteine, serine, threonine, tyrosine.Sometimes phenylalanine, tryptophan and tyrosine are classified as aromatic amino acid together.Use similar mode, whole aminoacid can be divided into (1) acidity=aspartic acid, glutamic acid; (2) alkalescence=lysine, arginine, histidine; (3) aliphatic=glutamic acid, alanine, valine, leucine, isoleucine, serine, threonine also can be divided into aliphatic-hydroxyl separately with serine and threonine; (4) aromatic=phenylalanine, tyrosine, tryptophan; (5) amide=agedoite, glutamine; (6) sulfur-bearing=cysteine and methionine.(referring to for example Biochemistry, 2nd ed., Ed.by L.Stryer, WH Freeman and Co.:1981).

In other embodiments, the bioactie agent of available chemical modification.Polypeptide can be carried out chemical modification with by with other chemical parts, for example glycosyl, lipoid, phosphoric acid, acetyl group etc. form conjugate covalency or accumulative and produce derivant.Covalence derivative can prepare by chemical part is linked to each other with functional group on described polypeptide amino acid side chain or N-terminal or the C-terminal.For example, can produce comprise except that with the natural relevant sequence of described protein, in conjunction with extracellular matrix components and can improve partly the bioactie agent of described analog in cell surface location.For example, can be with sequence derived from Fibronectin " repetition of III type ", for example tetrapeptide array R-G-DS ((1985) EMBO 4:1755-9 such as Pierschbacher etc. (1984) Nature 309:30-3 and Kornblihtt) is added in the polypeptide factor to support adhesion (Ruoslahti etc. (1987) the Science 238:491-497 of chimeric molecule by carrying out with cell in conjunction with the ECM component; Pierschbacher etc. (1987) J.Biol.Chem.262:17294-8; Hynes (1987) Cell 48:549-54 and Hynes (1992) Cell 69:11-25).

Perhaps, being used for polypeptide growth factor of the present invention can be made up of the active fragment of the factor.The technology of use-case such as above-mentioned description is easy to determine any given segmental activity.The fragment of bFGF for example when using according to method of the present invention, is compared in function test with by using the performance that total length bFGF polypeptide produced, and shows that performance increases, and is bFGF " active fragment " therefore.Described active fragment is described in for example Baird and Gage (1997) Proc.Natl.Acad.Sci.U.S.A., 94 (13): among the 7047-52.Those skilled in the art have the ability to determine polypeptide growth factor, and whether its size no matter has kept the functional activity of total length wild type peptide somatomedin.

Be used for polypeptide factor of the present invention preferably from its source material purification basically, material can be cell culture, tissue sample, biological fluid etc.Basically material at least 60% weight (dry weight) of the finger purification of purification is required polypeptide, for example the bFGF polypeptide.Preferred described polypeptide forms at least 75%, and more preferably at least 90%, most preferably at least 99% heavily is required polypeptide.Purity can be by suitable standard method, and for example column chromatography, polyacrylamide gel electrophoresis or HPLC analyze and record.Then can with pure basically polypeptide with other required components for example carrier or cell fit over and obtain polypeptide and be lower than 60% compositions, as long as when using to the patient, the concentration of described polypeptide is the concentration that is enough to produce effectiveness.

Be used for polypeptide factor of the present invention and can be natural, synthetic or by the recombinant molecule of forming with the second portion of the heterozygosis of a part that for example contains bFGF or chimeric polyeptides and another kind of polypeptide.Can with these factors from biological sample purification, chemosynthesis, perhaps by the standard technique recombinant production (referring to Current Protocolsin Molecular Biology such as for example Ausubel, New York, John Wiley and Sons, 1993; Cloning Vectors:A Laboratory Manual such as Pouwels, 1985, Suppl.1987).

Although with regard to being used in combination with cell of the present invention, polypeptide growth factor is at present best, the other treatment form that is considered to the nerve stimulation mode also can with cell coupling of the present invention.For example, stride cranium magnetic and stimulate to raise monoamine in the brain, therefore be expected at cell and can have useful effect when co-administered.

The one group of non-polypeptide Dexedrine that can be used as Dexedrine is neurotransmitter agonist or antagonist.Example is for example Prozac of antidepressant, amphetamine, and methylphenidate and tricyclic antidepressants be Elavil for example.

Other useful molecules are tretinoins that differentiation factor for example can trigger cell be divided into functional neurosurgery unit.

Another kind of molecule is so-called guide molecule, and they are class proteins, normally finds in extracellular matrix, and its function is meant that guided cell or cell prominent (aixs cylinder) arrive the suitable function of performance desired position.Example is semaphorins, netrins, neuropilins and ephrins.

Except that above-mentioned Dexedrine, they all have clear and definite effect to brain, the selective bioactive compound that is not considered to Dexedrine also can with the cell coupling.These other chemical compounds normally have known effect, find again the influential chemical compound of CNS cell recently other parts of health.

One group of selective chemical compound comprises the immunosuppression molecule that is used to suppress the alloplast rejection at present.The preferred described molecule of one class is an immunophilin, for example cyclosporin, FK506 and Sa Li polyamines.These molecules have the rejection that suppresses transplanted cells and neuroprotective function dual function are provided.Another kind of alternative stimulant is a Tetracyclines, usually with its antibiosis known to being, but required neuroprotective is arranged also.

4.4 cell

Many dissimilar cells or its mixture can be administered to individuality.Although do not wish to be limited by theory, the cell that requires to be used can influence brain in many ways.Cell itself can be established in brain and form function with neuron and be connected.In addition or alternatively be that cell can produce stimulates endogenous neurocyte to form the new prominent and factor that is connected.At last, the chemical compound inactivation that inhibition recovers can be removed or remove or make to cell from the CNS damage.With regard to these probabilities, should understand any cell, particularly stem cell that has one of above-mentioned character in fact but the cell of potential even final differentiation may all have favorable influence to brain function.The example of the cell type of known final differentiation with useful removing ability is activated lymphocyte and macrophage.

In specific embodiment, cell of the present invention preferably can produce the stem cell of brain cell in vivo.Particularly preferred cell is a pluripotent stem cell.Damaging cells can be used for clinical in growth in vitro.In preferred embodiments, can be used for the stem cell that stem cell type of the present invention comprises neural stem cell, hematopoietic stem cell, embryonic stem cell, lopsided cancerous cell line and other types.

Term used herein: " stem cell " refers to can be infinitely or the self renewal that prolongs, thereby can produce the cell of more than one filial generations of more breaking up.Preferred stem cell can and still keep cells and characteristic of stem through at least 10 cell divisions (under suitable condition).Particularly preferred stem cell can through at least 25,50 or 100 take turns the division and can not lose cells and characteristic of stem.With regard to cell, not only refer to intermediary daughter cell with term " generation ", also refer to finally can follow all cells of progenitor division to that class cell." generation " also refers to the contingent change that does not have the cell type of cell division incident.The cell of some differentiation also has the ability that produces than partial gigantism potentiality cell.Described ability can be natural under specific environment, perhaps can be manually inductive after handling with the different factors.Under both situations, with regard to the object of the invention, can think that described cell is a kind of stem cell.Described stem cell can be called " inductive stem cell " or " stem cell of differentiation "." finished stem cell " refers in any way the stem cell from its n cell environmental disorder.This comprises centrifugal, disassociation, disperses or other processing.Stem cell contained in undressed tissue sample is not considered to " finished stem cell ".

Stem cell normally with the rare cell type of other mixing with cells of more breaking up.In order to reach purpose of the present invention, the available cell suspending liquid that only contains the small part stem cell.Described method is specially adapted to use derived from stem cell enrichment tissue, for example bone marrow.In preferred embodiments, stem cell enriched so that they are at least 50% purely, this means that when using at least 50% cell is a stem cell to individuality.In particularly preferred embodiments, stem cell is at least 60%, 70%, 80% or 90% pure.

4.4.1 be used for the conventional method that stem cell is cultivated and breeds

Can be with various technical points from stem cell of the present invention.Usually, separate described stem cell from tissue sample (for example blood, bone marrow, fetus or adult brain tissue etc.), required stem cell only accounts on a small quantity in described tissue sample.In preferred embodiments, tissue sample is assigned in the cell suspending liquid, and it is stem cell enriched randomly in all sorts of ways.The method for optimizing of separate groups tissue samples is the method that the least possible cell death is arranged.For example, can pass through mechanical means, for example use the pipet mechanical shearing can be from tissue sample separate stem cells.In other cases, available enzyme digestion separates stem cell and surrounding tissue.Can with the liquid tissue sample for example blood separate by centrifugal classification, if suitably can suspend specific components then again.By also separable dissimilar cell of density gradient centrifugal or for example Ficol1 and extracellular material.According to stem cell population continuous growth and special cells labelling, for example use affine isolation technics or fluorescence-activation cell divide art (FACS) but the described stem cell population of enrichment.

There are a large amount of culture medium to can be used for cultivating zooblast at present.Some complexity wherein, some is simple.Although the expection stem cell can grow, preferably explant can be remained on simple culture media usually, for example in the Dulbecco ' s minimal medium (DMEM), so that can control the activation of specific cells population in the tissue sample more accurately in the culture medium of complexity.Culture can be remained in any suitable culture container, 12 or 24 hole microplates for example can remain on then and are used under the typical condition of culture of the cell of same animals separation, and for example 37 ℃, 5%CO ₂Can be with culture vibration to improve ventilation, for example with the velocity fluctuation of 12rpm.

Usually, detecting and classify according to the feature of identifying required cell can be stem cell enriched.For example, monoclonal antibody is specially adapted to identify the labelling (surface membrane protein for example receptor) relevant with specific cells pedigree and/or differential period.The method of separating individual progenitor comprises that magnetic separates, and utilizes the magnetic bead of antibody sandwich, affinity chromatography and with adhering to solid matrix, for example " moving lens graphics (panning) " or other routine techniquess of the antibody of flat board.Provide the technology of clean cut separation to comprise fluorescence amplifying cell separator, this technology can have different complexities, for example many Color Channels, low angle and blunt light scattering sense channel, impedance channel etc.

Can with antibody and labelling for example magnetic bead combine, so that directly separate, biotin can be with removing with bonded avidin of holder or Succ-PEG-DSPE, fluorescent dye can use with fluorescence amplifying cell separator, perhaps other material combinations are so that more easily separate the cell of particular type.Any technology that can too damage cell viability all can not used.

Except that using antibody, also can use and bonded other protein of required cell surface.For example, if the EGF receptor is expressed on required cell-specific ground, then the EGF of serviceable indicia detects those cells in the mode identical with using above-mentioned antibody.Therefore the specific dyestuff specific cell population that also can dye can be used as the part of the method that obtains required cell.Stem cell also has different forms usually.Stem cell has the Cytoplasm of macronucleus and relatively small amount usually.

Above-mentioned screening technique can be used in combination so that further enrichment to be provided with the selective growth condition.For example, can provide natural and recombined engineering cell the layer of feeding as instantaneous cultivation.Described cell also can produce the extracellular matrix that can be used as the screening technique substrate.

Also cell mixture can be contacted with the reagent that causes one or more cell population propagation.Can for example use, mitogen, for example a kind of material of mitosis and the cell transformation of particular type stem cell of inducing causes the amplification of this population.In this way, specificity factor is not made the cell of replying and tend to not divide, and the cell division of replying and become major part in the cell population.

After the enrichment, confirm that importantly the cell that obtains has suitable feature.According to characterizing cell of the present invention to somatomedin, concrete gene expression, antigenic mark, dyeing and/or the grown form on the described cell surface.It is also important that the type of determining the producible cell of specific stem cell population.

Can induce described differentiation of stem cells to become dissimilar cells by changing environmental condition.For example, can whether can instruct adult cell to grow altogether and being divided into so that understand described embryonal tissue individual progenitor and embryonal tissue's reorganization accordingly.Stem cell can be implanted a kind of in the used multiple regenerating model in this area, for example neural stem cell will be transplanted in the rat brain that damages and be grown and differentiation (Gage etc. (1995) Proc.Natl.Acad.Sci.USA, 92:11879-11883; Flax etc. (1998) Nature Biotechnology 16:1033-1039).But with one section foreign DNA transfection genetic marker stem cell.This labelling makes and stem cell filial generation and host cell can be separated.Perhaps, but the differentiation factor of progenitor and one or more growths or inducing cell differentiation can be contacted.With with identify the identical conventional method of stem cell, for example cell surface marker, dyeing etc. can be identified the cell type of differentiation.

Under specific situation, can measure cell proliferation.Described method generally includes the DNA composite character of measuring cellular replication most.It is synthetic that this area has many methods can measure DNA, can be with any according to the present invention.In embodiments of the invention, the radioactive label that is used to detect ( ³The H-thymidine) or the nucleotide analog of labelling (BrdU) determined that by immunofluorescence DNA is synthetic.

In culture medium, also can provide somatomedin optionally to expand the specific cells population or to impel the production of noble cells.

Can be by the positive and negative screening with cell divide.For example, with one or more biotinylated antibody, be the specific positive or negative screening of finishing for the lip-deep factor of target cell.In biotinylated antibody transfered cell culture.After specific temperature retention time, rinse any biotinylated antibody that does not form complex with target cell.Then fixed avidin substrate is added in the cell suspending liquid.Fixed avidin substrate combines with biotinylated antibody/antigen complex.Then that this suspension is centrifugal with division avidin substrate.Perhaps, avidin can be combined with magnetic bead so that the cell with antibodies can be separated through magnetic with unconjugated cell.If described screening is male, will be resuspended in the Nutrient medium that is used for continuous growth with the cell of antibodies.If screening is negative, remove bonded cell, then resuspended remaining unconjugated cell is with further growth.

Obviously, available many other technologies are carried out the positive and negative screening, as long as can provide required affine by described screening element.

Hematopoietic stem cell

The mammal hemocyte provides the cell type of very various scope.Three main pedigrees of hemocyte comprise the lymphocyte pedigree, for example B cell and T cell, myelocyte sample pedigree, mononuclear cell for example, granulocyte and megalokaryocyte, and cell born of the same parents class pedigree, for example erythrocyte.Hematopoietic stem cell (HSCs) is except that generation is equal to the multipotency daughter cell, can also produce the cell of at least two kinds of pedigrees in the above-mentioned pedigree.In preferred embodiments, HSCs can produce three kinds of main hemocyte pedigrees.Except that producing hemocyte, HSCs can also be divided into the cell of many other types, comprise brain cell (Eglitis and Mezey (1997) Proc.Natl.Acad.Sci.USA, 94:4080-4085).

Can from different tissues, separate HSCs.Medullary cell is the good source of HSCs.Can be from derived from bone marrow, for example crista iliaca, tibia, femur, spinal column or other bone cavity.Other sources of human hematopoietic stem cell comprise embryo's blastular, fetal livers and adult's spleen and blood, comprise into human peripheral.

Identify HSCs by cell type and various cytological marker that HSCs produces.HSCs usually extrudes particular dye, for example Hoechst 33324 and rhodamine B 123 (Bhatia etc. (1998) Nature Med.4:1038).Other cells in available described dyeing CHARACTERISTICS IDENTIFICATION HSCs and the blood circulation.Available antibody with the specific cells labeled reactant is identified and purification HSCs.For example, think that mAb AC133 combines (Miraglia etc. (1997) Blood 90:5013) with the HSCs specificity.The Thy-1 molecule is the protein of the high conservative that exists in rat, mice and people's brain and the hemopoietic system.In rat, mice and people HSCs, identify the Thy-1 molecule and can use it for evaluation HSCs (United States Patent (USP) 5,914,108).Many HSCs are CD34+ and/or CD38+ (United States Patent (USP) 5,840,580).The HSCs population has some variation in the cell surface marker of being everlasting, therefore can finish positive identification on the basis that has at least two kinds of above-mentioned cytological markers.

Shortage according to specific markers can be separated HSCs with the cell type that other more break up.CD3, CD7, CD8, CD10, CD14, CD15, CD19, CD20 and CD33 normally HSCs are unexistent.The shortage of above-mentioned several labellings has increased the credibility that HSCs identifies.By above-mentioned, morphology also helps to distinguish HSC.

Should be appreciated that by many characteristic sets the producible cell type of HSCs of the existence of for example morphology, specific markers, the shortage of other labellings and supposition can be identified HSCs.

All can cultivate HSCs in many ways to produce the stem cell of differentiation.For example, can be with cell culture in culture medium that determine, enrichment for example in the Dulbecco ' s culture medium (IMDM) of Iscove ' s modification, this culture medium is made up of salt, aminoacid, vitamin, antibiotic and hyclone usually.The culture that replenishes with hydrocortisone tends to produce myeloid cell, and the culture of shortage cortisone tends to produce bone-marrow-derived lymphocyte.In order to show that HSCs can grow, and can use various conventional methods in the cell of cell born of the same parents class pedigree.For example, cultivation can stimulate the erythrocytic formation of class on the methylcellulose culture medium.(United States Patent (USP) 5,840,580 and 5,914,108; Metcalf (1977) In:Recent Results in Cancer Research61.Springer-Verlag Berlin, pp.1-227).

Neural stem cell

Neural stem cell is derived from the cell of the tissue of adult or developmental nervous system, can be divided into one of following at least basic neural pedigree: neuron, oligodendroglia and astroglia.In addition, the design stem cell also can produce the new NSCs with similar potentiality.In preferred embodiments, neural stem cell is polyenergic and produces most of in the basic neural pedigree or all.

By detecting the pedigree specific protein, and can distinguish each basic neural pedigree by morphology.By detecting, for example microtubule-associated protein 2 (MAP2), tau, specific 'beta '-tubulin (for example TuJ1, 'beta '-tubulin III type), specific neural thread protein NTP (for example neural thread protein NTP L or M), neuronspecific enolase or NeuN can discern neuron.By detecting galactocerebrosidase (GalC), CNPase, myelin basic protein or 04 albumen, can discern oligodendroglia.Can discern the star neurogliocyte by detecting glial fibrillary acidic protein (GFAP).Can discern specific NSCs itself by Vimentin or nestin.Usually utilize the antibody of identification desired protein to finish detection (Villa etc. (2000) Exp.Neurology 161:67-84) by the standard immunoassay staining technique.The antibody of above-mentioned each labelling can buy from following one or more companies: Chemicon, Sigma-Aldrich, Boehringer-Mannheim, Santa Cruz Biotechnology, Dakopatts AB (Sweden).Also the expression of gene of available code pedigree specific protein is distinguished the cell of different pedigrees.Also can comprise in situ hybridization, fluorescence in situ hybridization, quantitatively rtPCR, the gene expression of Northern trace mensuration by the various technology of knowing.

The optimization technique that is used for separating and breeds NSCs is described in the United States Patent (USP) 5,958,767 of following document: Snyder etc.; The United States Patent (USP) 5,270,191 of McKay etc.; Johe, the United States Patent (USP) 5,753,506 of K.; Carpenter, the United States Patent (USP) 5,968,829 of M., the United States Patent (USP) 5,750,376 of Weiss etc.All documents all are incorporated herein by reference.

Usually, there being one or more somatomedin, for example under the condition of bFGF, EGF, TGF-α, LIF or aFGF neural stem cell is remained on state propagation, undifferentiated.The preferred factor is bFGF or EGF.Take out the cell that the described factor can be divided into different pedigrees.The pedigree that forms depends on environment.For example, import the specific environment that the specific NSCs of brain found according to each cell itself and can form all dissimilar brain cells.In cultivation, many factors can influence development pathway.For example, CNTF can induce and be divided into astrocyte, and PDGF can induce neuronic formation, and thyroxin (T3) can be induced the formation of oligodendroglia.

In preferred embodiments, press United States Patent (USP) 5,958,767 description can obtain neural stem cell.At this concise and to the point example of describing this method as the concrete grammar that is used to prepare NSCs.Should be understood that has many such methods at present, and the details that can improve this method is to obtain similar result.In brief, the neurocyte suspension for preparing primary separation from the akrencephalon of 15 all pregnant fetuses.This suspension is layered on the additional not bag that Dulbecco ' s Modified Eagle Medium (DMEM) adds F12 culture medium (1: 1) that contains of N2 culture medium (Gibco) is organized in the plate, to wherein adding bFGF and heparin or EGF.When the cell aggregation thing grows into diameter greater than 10 cells, separate described cell aggregation thing.Finish separation with trypsin, the NSC cell suspending liquid is resuspended in the growth medium.Isolating stem cell can be layered on the slide of the poly-L-Lysine bag quilt in the DMEM+ hyclone to promote differentiation.Can stimulate the astrocyte differentiation by cultivating altogether with the primary separation culture of newborn CD-1 mouse brain.But transfectional cell makes cell need not add somatomedin at in-vitro multiplication so that express the fissional gene of promotion.The method that produces transfectional cell is well known in the art.In preferred embodiments, duplicate-incompetent retroviral vector transfectional cell, express the mitogenesis gene then from viral LTR district with amphophilic.Preferably, promote the fissional gene Dexedrine of not encoding.Preferred gene to be expressed is vmyc, SV-40T antigen, ras oncogene, polyoma large T antigen, neu oncogene or its combination.Preferably at vivoexpression or activate described propagation-promotion gene and protein, but it is expressed or non-activity in vivo hardly.As if the vmyc gene self regulation in this way.The inducible promoter that needs the external factor that provides to express with stimulated gene perhaps is provided.

Other stem cell

Specific embryo's tumor contains the pluripotent cell of many types.In specific embodiments, use the cell line of establishing from these tumors a part as the method for treatment CNS damage.Useful cell line derived from embryo's tumor has been described.For example NT2/Tera cell line can be divided into all main neural pedigrees (United States Patent (USP) 5,175,103).

By all separable described cell of describing among above-mentioned conventional method and United States Patent (USP) 5,175,103 and Andrews (1984) Dev.Biol.103:285-293 of any method.In a word, people's deformity cancerous cell line (Ntera 2/C1.DI or NT2 cell) can be grown on tretinoin and be formed intensive multilamellar culture.The culture that these are intensive is bed board again.The NT2-N cell of small and dense collection does not have confidential relation with following confluent monolayer cells.Can be easy to be removed, enrichment, produce and to have the little of some flat contamination of cells and the light cell culture of basically identical (round phase).By with mitotic inhibitor, for example cytarabin combines further enrichment of N T2-N cell of cultivation.Required cell of taking turns has resistance to this processing, and flat cell is not bred.The NT2-N cell enrichment thing that tends to the neurodevelopment approach is dyeed with anti--NF-L antibody (neural thread protein NTP of small-molecular weight), and undifferentiated NT2 cell (flat cell) the Cam5.2 dyeing of reacting with keratin 8 and 18.

Non-cancer embryonal tissue also is the source of stem cell.The body early embryo cell is all-round, can produce all adult's organisms.As a result, can cultivate described cell to obtain all-round or the height pluripotent stem cell.Available embryonic stem cell is used for the treatment of the part of the method for CNS damage as the present invention.According to condition of culture, these cells can finally produce the cell type of more typing and the cell type of final specific differentiation.According to (1998) Science 282:1145-1147 such as Thomson; Evans etc. (1981) Nature 292:154; Martin, G. (1981) Proc.Natl.Acad.Sci.USA 78:7634 is described to obtain and cultivates embryonic stem cell.

4.5 use

In all sorts of ways and can finish using of cell and other treatment agent, described method does not need each component identical.Usually, when therapeutic agent is chemical compound,, comprise in intravenous, mouth or the brain that (for example in Intraventricular, the sheath or in the brain pond, or directly be passed in the brain) can use described molecule by any known route of administration.Dosage changes (seeing Table 2) with medication.The dosage of determining in the rat is generally used for man-hour and amplifies in proportion.Used ratio depends on medication.If stimulant is whole body administration (for example, per os or intravenous), then determine ratio according to body weight, wherein rat body weight be 300 grams and usually body weight for humans be 70kg.If chemical compound is delivered in the cerebrospinal fluid (for example intracisternal, ICV), presses the brain surface area and amplify.Usually the brain surface area of rat is 1cm ², and the surface area of human brain is 1000-10 usually, 000cm ², depend on that various pleats in all gyrus whether all are calculated in.If described chemical compound is to be passed in the cerebral tissue, amplify in the ratio of brain quality.Usually the brain of rat is 2g, and human brain is 2kg usually.Therefore, be effectively if in rat, use 0.5 μ g in brain pond, then in people patient an intracisternal injection to should be 0.5mg be effective.Certainly, accurate dose can be adjusted according to weight in patients and other standards.Expect that the effective dose of all three kinds of general route of administration is the total amount that is administered in spinal fluid or the cerebral tissue and reaches 0.001-1000mg.In preferred embodiments, described dosage can be from 0.01-100mg, 0.1-10mg or 0.5-5mg.

Table 2: the magnification ratio of the dosage of cell and stimulant

Individual	Medication
					(ratio in body weight is amplified) of general	To spinal fluid (ratio in the brain surface area is amplified)	To cerebral tissue (ratio in brain weight is amplified)
Rat	300g	1cm 2	2g
The people	70kg	1000-10,000cm 2	2kg

With the single dose administered compound or with a series of less dosed administrations.For example, administration is by for example in the back 6 hours shot of damage in the brain pond, for example 24 and 48 hours the double injection in damage back is formed, perhaps in the therapeutic scheme that after the ischemia incident, was carried out at least 6 hours if desired, carry out the series injection to the patient, for example 0.1mg/ injection, perhaps twice (for example every 3-4 days) inject 1mg weekly.How all therapeutic scheme is sustainable.

In specific embodiment, preferably cell directly for example is administered in apoplexy chamber, the spinal fluid in Intraventricular, sheath or in the brain pond.Cell can be carried by pharmaceutically acceptable fluid medium, also can contain bioactive molecule.As selection, cell (mixing separately or with stimulant) can be administered to the apoplexy chamber or be administered to (for example administration in the sheath or the brain pond) in the brain that is full of spinal fluid.Also can be in damage location brain zone on every side with injection cell.But the general dosed cells supposes that specific stem cell can move to the suitable location in the brain.If in the apoplexy chamber, in the ventricles of the brain or the cerebral tissue, on the entity frame that then patient's the head standard of being fixed on will be become, the position of dosed cells is by standard CT or MRI scanning location with injection cell.Bore a small bore at head, injection cell is arrived the desired position with syringe.Amplify the cell of using in proportion by the medication of describing in detail in the table 2.Usually, total cell concentration of using is 10 ⁶-10 ¹², preferred 10 ⁷-10 ¹¹, the best is 10 ⁸-10 ¹⁰Usually to inject 2-7 days at least interval dosed cells repeatedly.

Cell and therapeutic agent use preferred after damage a few hours or a couple of days whenever carrying out in several weeks even several months to the apoplexy.In preferred embodiments, after taking place, damage finished administration at least in 6,10,12 or 24 hours.The exact dose that expection can be adjusted cell and Dexedrine by the doctor according to patient's particular demands and feature.Usually anticipated optimal set dosage to being enough to effectively but be low to moderate the dosage of the undue inflammatory reaction that is enough to avoid hard to bear, can be productive on the contrary (counter-productive) for high.By determining the level of inflammatory reaction, people can determine whether Tai Gao and can not obtain optimum efficiency of given dose rate.The method that this paper proposes is preferred, because they can accurately control and adjust dosage level, and because can carefully control cell and the applied zone of stimulant.In preferred embodiments, Dexedrine is not to be produced by transgenic contained in one or more dosed cells.

The chemical compound that other can be meeted the requirements is used with described cell and Dexedrine.For example, respectively immunosuppressant and antibiotic are used to suppress transplant rejection and infection.In addition, as discussed above, the chemical compound of these types also has other useful effects.

Individual giving of describing in detail of this paper, particularly the individual human conventional method of using cell of the present invention and bioactie agent comprises cell and/or Dexedrine injection or implants the target site of described individuality.The cell of the present invention and the factor are inserted into by in the transfer device that helps importing in injection or the implantation individuality.Described transfer device comprises pipe, and conduit for example is used for cell and liquid infusion in the body of receptor.In preferred embodiments, described pipe also has pin, and for example syringe can import cell of the present invention individual by it in the desired position.Cell of the present invention and the factor can be inserted into described transfer device for example in the syringe with multi-form.For example, in the time of in being contained in described transfer device, cell or the factor can being suspended in the solution or implanting in the supported matrix.As described herein, term " solution " comprises pharmaceutically suitable carrier or the diluent that cell wherein of the present invention can maintain vigour.Pharmaceutically suitable carrier and diluent comprise saline, water-containing buffering liquid, solvent and/or disperse medium.The use of described carrier and diluent is well known in the art.Described solution is sterilized water and liquid preferably.Preferably, described solution is stablely also can be such that it will prevent for example pollution of antibacterial and fungus of microorganism by using for example parabens, methaform, phenol, ascorbic acid, thimerosal etc. under preparation and storage requirement.By in pharmaceutically suitable carrier or diluent, mixing progenitor by described herein, also can mix above-mentioned other compositions on demand, filtration sterilization can prepare solution of the present invention then.

Randomly, cell can be placed on the supported matrix and use.The supported matrix that wherein can mix or implant cell comprises the receptor compatibility and is degraded into substrate to the harmless product of receptor.Natural/or the synthetic biodegradable substrate example that is described substrate.Natural degradable substrate comprises that plasma clot is for example derived from mammal and collagen stroma.Synthetic biodegradable substrate comprises synthetic polymer for example polyanhydrides, poe and polylactic acid.Other synthetic polymer examples and the method for cell being mixed or implants these substrate are known in the art.For example referring to United States Patent (USP) 4,298,002 and United States Patent (USP) 5,308,701.These substrate are supported in the body and protection for cell provides.

Cell of the present invention and Dexedrine can be used with pharmaceutical composition.Suitable compositions can comprise all compositionss that are generally used for whole body or local application medicine.Pharmaceutically suitable carrier should be inert basically, so that can not have an effect with active component or the interference cell viability.Suitable inert carrier comprises water, alcohol, Polyethylene Glycol, propylene glycol etc.

In order to prepare pharmaceutical composition of the present invention, as active component and pharmaceutically suitable carrier combination, according to the form of the required preparation of administration, described carrier can be taked various forms with the Dexedrine of effective dose and cell.These pharmaceutical compositions meet the requirements with single dose form, are particularly suitable for applied dermally or parenteral injection.For example under the situation of suspension, syrup, elixir and solution, can use the drug media of any routine, for example water, glycol, oils, alcohols etc. at oral liquid; Perhaps under the situation of powder, granule, capsule and tablet, use solid carrier for example starch, saccharide, Kaolin, lubricant, binding agent, disintegrating agent etc.For parenteral composition, described carrier will comprise sterilized water usually, be most of at least, although also contain other compositions, for example to help solubility and cell survival.Other compositions comprise antioxidant, viscosity stabiliser, chelating agen, buffer agent, antiseptic.If desired, other compositions can mix described compositions, for example antiinflammatory, antibacterial, antifungal, disinfectant, vitamin, antibiotic.

The example of antioxidant comprises butylated hydroxy-methylbenzene, butylated hydroxyanisol, propyl gallate, citric acid and ethoxy quinoline; The example of chelating agen comprises disodiumedetate and ethane hydroxy bisphosphate; The example of buffer agent comprises citric acid, sodium citrate, boric acid, Borax and sodium hydrogen phosphate; The example of antiseptic is methyl parahydroxybenzoate, ethylparaben, dehydroactic acid, salicylic acid and benzoic acid.Can prepare Injectable solution, for example wherein carrier comprises the mixture of saline, glucose solution or saline and glucose solution.Also injectable suspensions can be prepared, wherein appropriate liquid carrier, suspending agent etc. can be used.The preparation that also comprises solid form is converted into liquid absorption member before use.In being applicable to the compositions of percutaneous dosing, carrier also can randomly comprise the reagent that improves penetrance and/or suitable wetting agent, randomly can use with a spot of any natural suitable additive combination, described additive does not have significant illeffects for skin.

Particularly advantageous is compositions of the present invention to be mixed with unit dosage form use and the dosage homogeneous being easy to.Used unit dosage form refers to the suitable unit that physically separates as single dose in this description and claims, and what each unit contained the amount of pre-determining can produce the active component of required therapeutical effect and required pharmaceutical carrier as calculated.The example of described dosage unit form is capsule, Injectable solution or suspension, one capacity, tablespoonful etc., and separated many doses.

The particular composition that is used for the inventive method is that wherein Dexedrine is mixed with those of the compositions that contains liposome.Liposome is by amphiphatic molecule, for example polarity lipoid, for example artificial capsule of phosphatidyl choline, ethanolamines and serine class, sphingomyelins, cuorin, plasmalogen, phosphatidic acid and cerebiosides formation.When suitable amphiphatic molecule can expand, when forming usually the liquid crystal (being also referred to as coarse liposome) of the multiple structure of forming by many bilayers that separate by hydrous material each other, formed liposome in water or aqueous solution.The known liposome of being made up of single double-deck tunicle hydrous material of another kind of type refers to thin stratiform capsule.If comprise water-soluble material at aqueous phase in the liposome expansion process, they just are absorbed in the water-bearing layer between the class lipid bilayer.

Water-soluble active ingredient is coated in the water-containing space between molecular layer.The for example organic analogies of the lipid solubility active component of Dexedrine mainly insert in the lipoid layer, although polar head group may be stretched out water-containing space from the lipoid layer.Many methods all can be wrapped by these chemical compounds.The most frequently used method comprises by evaporation from organic solvent at flask walls direct casting monophosphatide thin film.When this film is dispersed in the suitable water-bearing media, form multilamellar liposome.Behind suitable sonication, coarse liposome forms the capsule of less similar sealing.

Disperse casting film can mix water-soluble active ingredient usually by aqueous solution with described chemical compound.Remove non-encapsulated chemical compound by centrifugal, chromatograph, dialysis or other proper methods known in the art then.Usually with the active component of lipid solubility before encapsulated membranes, mix by it is dissolved in the organic solvent with phospholipid.If described material the dissolubility of lipoid in mutually amount that be no more than other or that exist be no more than its can with the bonded amount of lipoid, then the liposome by method for preparing contains major part bonded material in the class lipid bilayer usually, does not need liposome and non-encapsulated material are separated.

The conventional especially method that is used to prepare the liposome formulation form of Dexedrine is at EP-A-253, the method for describing in 619 (being incorporated herein by reference).In the method, by lipid constituent is dissolved in the organic media, under pressure, the organic solution of lipid constituent is expelled in the aqueous components and simultaneously organic and aqueous components mixed with high speed homogenizer or mixed method, so spontaneous formation liposome, and preparation contains single lipid bilayer body of the active component of sealing.

Can will contain the single lipid bilayer body and the mixing with cells of the Dexedrine of sealing, directly use then, perhaps it be used at the suitable pharmaceutically suitable carrier that is used for site-specific delivery of drugs.For example xanthan gum, hydroxy propyl cellulose, HYDROXY PROPYL METHYLCELLULOSE and its mixture can improve the viscosity of liposome by adding one or more suitable thickening agents.Electrolyte, buffer system and other compositions, for example antiseptic be formed or can be contained to aqueous components can separately by water.Spendable suitable electrolyte comprises slaine such as alkali metal salt and alkali salt.Preferred slaine is calcium chloride, sodium chloride and potassium chloride.Electrolytical concentration can be 0-260mM, preferred 5mM-160mM.Aqueous components is placed in the suitable container, and described container can adapt in injection organic component process because of influencing the homogenate that big turbulent flow is carried out.The homogenate of two kinds of components can be finished in described container, perhaps aqueous components and organic component is expelled to respectively in the outer mixing arrangement of described container.Under a kind of situation in back, form liposome with blended method, transfer to another container that is used for collecting purpose then.

Organic component is by suitable non-toxicity, acceptable solvent for example ethanol, glycerol, propylene glycol and Polyethylene Glycol, and the suitable phospholipid that is dissolved in this solvent is formed.Spendable suitable phospholipid comprises for example lecithin, phosphatidylcholine, Phosphatidylserine, PHOSPHATIDYL ETHANOLAMINE, phosphatidylinositols, LYSO-PHOSPHATIDYLCHOLINE LYSOPC and phosphatidyl glycerol.Also can use other lipophilic additives so that the feature of modified liposome optionally.The example of described other additives comprises stearylamine, phosphatidic acid, tocopherol, cholesterol and lanoline extract.

In addition, other compositions that prevent phospholipid oxidation can be added in the organic component.The example of described other compositions comprises tocopherol, butylated hydroxyanisol, Yoshinox BHT, ascorbic palmitate and oleic acid acid ascorbyl ester.Also can add antiseptic for example benzoic acid, methyl parahydroxybenzoate and propyl p-hydroxybenzoate.

Can provide introduction method by rechargeable or biodegradable device.With regard to medicine, comprise that the control of protein-based bio-pharmaceutical transmits, developed various slow release polymers devices in recent years and tested in vivo.Can use various biocompatible polymer (comprising hydrogel), comprise that biodegradable and non-degradable polymer is to be formed for the implant at particular target site slow releasing bioactivity factor.

The basic feature of the particular of implant is the linear therapeutic agent that discharges, and can reach described purpose by operation polymer composition and form.By selecting monomer composition or polymerization technique, the may command water yield, porosity and so permeability characteristics.According to disease to be treated and each reaction, but the method that selected shape, size, polymer and determining implanted on the basis of individuality.The preparation of described implant is known in the art.Referring to for example ConciseEncylopedia of Medical ﹠amp; Dental Materials, ed.by David Williams (MIT Press:Cambridge, MA, 1990); United States Patent (USP) 4,883,666 with Sabel etc.

In another embodiment of implant, cell is encapsulated in the doughnut or similar substance that can make implantation.Described fiber can spin in advance, with the back loading cell source (United States Patent (USP) 4,892,538 of Aebischer etc.; The United States Patent (USP) 5,106,627 of Aebischer etc.; Hoffman etc. (1990) Expt.Neurobiol.110:39-44; Jaeger etc. (1990) Prog.Brain Res.82:41-46; With (1991) J.Biomech.Eng.113:178-183 such as Aebischer), perhaps stretch out (the United States Patent (USP) 4,391,909 of Lim altogether with the polymer that forms the polymerization tunicle at cell peripheral; The United States Patent (USP) 4,353,888 of Sefton; Sugamori etc. (1989) Trans.Am.Artif.Intern.Organs 35:791-799; Sefon etc. (1987) Biotehnol.Bioeng.29:1135-1143; With (1991) Biomaterials 12:50-55 such as Aebischer).Then can be with described cell of sealing and Dexedrine combination.

Expection, for convenience, what meet the requirements is that Dexedrine and cell are packaged together in the test kit.Test kit can comprise the dosage-size-specific bottle or the equal portions of cell and/or Dexedrine.Test kit also can contain device used when using the administering drug combinations component.This device has been described above.In specific embodiment, wherein cell is obtained, cultivates from the patient, and then be administered to described patient.Described test kit can contain and is useful on the device that obtains cell sample from the patient, and stem cell will be cultivated from this cell sample.

In particular aspects, unless stated otherwise, practitioner of the present invention can use cytobiology, cell culture, molecular biology, genetically modified organism, microbiology, recombinant DNA and immunologic routine techniques, and these are all in the technology of this area.Described technical description is in document.Referring to for example Molecular Cloning A Laboratory Manual, 2nd Ed., ed.bySambrook, Fritsch and Maniatis (Cold Spring Harbor LaboratoryPress:1989); DNA Cloning, volume I and II (D.N.Glover ed., 1985); Oligonucleotide Synthesis (M.J.Gait ed., 1984); The United States Patent (USP) 4,683,195 of Mullis etc.; Nucleic Acid Hybridization (B.D.Hames﹠amp; S.J.Higgins eds.1984); Transcription And Translation (B.D.Hames ﹠amp; S.J.Higgins eds.1984); Culture Of Animal Cells (R.I.Freshney, Alan R.Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B.Perbal, A Practical Guide ToMolecular Cloning (1984); The treatise, and Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors ForMammalian Cells (J.H.Miller and M.P.Calos eds., 1987, ColdSpring Harbor Laboratory); Methods In Enzymology, Vols.154 and155 (Wu et al.eds.), Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D.M.Weirand C.C.Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

Now the present invention has been done general description, will be more readily understood the present invention with reference to the following example, described embodiment only is for particular aspects of the present invention and embodiment are described, rather than for the present invention is construed as limiting.

5. embodiment

Embodiment 1: neural stem cell in the brain pond (NSC) and somatomedin improve apoplexy and recover

In the present embodiment, recover in the model in the apoplexy of rat, with tire mice neural stem cell (NSC) with or in the brain pond, do not use with basic fibroblast growth factor (bFGF).1 week was handled the male Sprague-Dawley rat of 300-350 gram before operation.Rat is performed the operation in apoplexy and accepts antibiotic, cefazolin sodium (40mg/kg, lumbar injection) the previous day.Performed the operation the same day in apoplexy, be used in 2% halothane anesthesia animal in nitric oxide/oxygen mixture (2: 1).Press described (Kawamata et al. (1999) Exp.Neurol.158,89-96 in the past; Tamura etc. (1981) J.Cereb.Blood Flow Metab.1 53-60) coagulates the middle part cerebral arteries by contiguous electricity and finishes focus cerebral infarction (apoplexy).Specifically, will from the nervi olfactory bundle be near-end to hypencephalon venous blood vessel graft, need not remove cheekbone bow or cross section nerve.This technology in back of the body outside cortex and following striatum, produce health with reproducible infraction, perhaps regional cell death.Inject a cefazolin sodium (40mg/kg, lumbar injection) immediately again after operation, for animal.Animal is waken up from anesthesia.

Apoplexy was performed the operation back 24 hours, accepted injection in the animal brain pond: (1) carrier, (2) NSC (10 ⁶Individual cell), (3) bFGF (0.5 μ g), or (4) NSC+bFGF.Under halothane anesthesia, finish the intracisternal injection of totally 50 μ l volumes to Da Chi by percutaneous injection.Repeat same process after 2 days so that animal was received treatment in 1 and 3 day after apoplexy.In process of the test, use 10mg/kg (lumbar injection) cyclosporin, a kind of immunosuppressant is so that Therapy lasted.

Cause the sensorimotor function imbalance of opposition side hind leg and forelimb by the cerebral infarction of described method generation.The sensorimotor function of multiple neurological test with assessment opposition side limb finished in after apoplexy the second month.These tests comprise and detect that animal replys that vision, sense of touch, proprioception and whiskers stimulate and forelimb and hind leg that limbs are placed on the ability on the desktop are placed test.In addition, carry out the health rotation test, it was laterally preferential when went up on the table top when animal keeps being suspended on by its tail to measure.At last, carry out spontaneous limb service test, probe into the tendency of narrow glass cylinder spontaneous each forelimb of use when inner takeofing to measure animal.Place forelimb and hind leg test and spontaneous limb service test have reflected cortex and striatal function.It mainly is the tolerance of striatum function that health revolves the shape test.

The results are shown among Fig. 1 of these tests.What figure A and B provided is the placement activity of affected forelimb and hind leg (offside of apoplexy side in the brain).Figure C shows the health rotation test, and figure D shows spontaneous limb service test.The data indication that obtains when in all cases, normal behavior is used in that day (1 day) before the operation.In each case, animal all demonstrates the significant abnormal behavior that day after operation.Then, a slow spontaneous incomplete recovery is arranged.Fig. 1 shows that all three kinds of processing are that NSC, bFGF and its combination have all strengthened recovery compared with the control significantly in limb placement test.A similar tendency is arranged in spontaneous limb service test.Observing compared with the control in the health rotation test, each does not have difference between handling.In addition, though these right and wrong are significant, compare in as seen tendency with the bFGF group towards excellent ground enhancement function in conjunction with group with independent NSC.

After apoplexy one month, animal is killed, take out brain, section and also use H﹠amp; E dyeing.(Kawamata et al. (1996) J.Cereb.Blood Flow Metab.16,542-547 as described; Kawamata et al. (1997) Proc.Nat.Acad.Sci.94 8179-8184), determines Infarction volume by image analysis.With regard to Infarction volume, do not see tangible difference between each group, though a tendency towards slightly little Infarction volume is arranged in accepting the group of NSC.The stem cell of transplanting contains the lacZ reporter gene and expresses beta galactosidase.Do the X-gal histochemistry to check the position of transplanting these cells of back.Really, cell has been moved to the position around the focus apoplexy in the right hemisphere from their fixed positions among Da Chi.

In a word, this experiment has shown in apoplexy and has begun to give in the pond the estheticokinetic recovery that NSC and/or bFGF can strengthen the offside limb significantly one day after.This improvement is limited to the test that reflects function of cortex to a great extent.With regard to Infarction volume, do not see tangible difference between each group, illustrate that NSC and bFGF are by other mechanism but not prevent that cell death from producing the promotion restitution.These mechanism can be included in the not part of infringement of brain and set up new connection.In addition, in this first test, it seems that the combination of NSC and bFGF be better than their individual processing separately a little.

Embodiment 2: directly give to give in NSC and the pond bFGF in the brain and recover to strengthen in the rat apoplexy model

In second experiment, NSC is injected directly in the brain to focus apoplexy tissue on every side.BFGF such as before carry out injecting in the pond.In this experiment, only give NSC or bFGF one day after in apoplexy.Under these conditions, we clearly observe the superiority of comparing NSC+bFGF with individual processing separately.

In this experiment, handle animal the last week in operation.In addition, be that pawl stretches out in the test and trains them preceding 10 days of operation an other test.As preceding, they accept cefazolin sodium (40mg/kg, lumbar injection) before operation.Performed the operation the same day in apoplexy, and be close to the arteriocerebral electricity in middle part and coagulate effect, (Kawamata et al. (1996) J.Cereb.Blood Flow Metab.16,542-547 as previously mentioned; Kawamata et al. (1997) Proc.Nat.Acad.Sci.94,8179-8184; Kawamata et al. (1999) Exp.Neurol.158,89-96).Animal is injected a cefazolin sodium (40mg/kg, lumbar injection) again after operation.

In apoplexy one day after, animals received: (1) injection carrier is to the infarction surrounding tissue and inject carrier in Da Chi, (2) injection NSC (10 ⁶Individual cell) arrive infarction surrounding tissue and injection carrier in Da Chi, (3) injection carrier is to the infarction surrounding tissue and inject bFGF (0.5 μ g) in Da Chi, or (4) inject NSC (10 ⁶Individual cell) arrives the infarction surrounding tissue in conjunction with injecting bFGF (0.5 μ g) in Da Chi.

These injections are respectively carried out with 25 μ l volumes under 2% halothane anesthesia.NSC is injected in the striatum tissue at the edge of focus infarction.BFGF is by percutaneous injection (injection in the pond), (Kawamata et al. (1996) J.Cereb.Blood FlowMetab.16,542-547 as previously mentioned in Da Chi; Kawamata et al. (1997) Proc.Nat.Acad.Sci.94,8179-8184; Kawamata et al. (1999) Exp.Neurol.158,89-96).Rat is also accepted cyclosporin at whole experimental session, a kind of immunosuppressant (every day 10mg/kg, intraperitoneal injection).

As preceding, many behavior tests have carried out for after apoplexy the second month.These tests comprise forelimb and hind leg placement test, health rotation test and spontaneous limb service test, as described in example 1 above.In addition, having carried out another test is that pawl stretches out test.Before apoplexy operation in this test animal training, when experiment finishes, test once then.This test is to detect animal to extend through the ability that the cage grid remove to grab food food grain with (offside) fore paw that injures.Usually, animal has about 100% accuracy when finishing this task.After the apoplexy, it has dropped to about 10%.

The results are shown in Fig. 2 and 3 of these behavior tests.Once more, all three processed group are that NSC, bFGF and its combination are all demonstrating superiority compared with the control aspect the recovery in forelimb and hind leg placement test.Once more, in conjunction with group, there is one towards the tendency of preferably recovering.In the health rotation test, NSC handles and does not demonstrate advantage compared with the control separately, but bFGF and combination group have demonstrated.In spontaneous limb service test, have only in conjunction with group to have demonstrated towards the tendency of improving the result.At last, stretch out in the test, compare, demonstrated superiority in conjunction with group with individual processing at pawl.The Histological evaluation of these brains remains unsettled.

In a word, in this experiment, NSC is injected directly in the tissue at focus apoplexy edge.Give bFGF in the pond.When giving separately, these are handled in some tests and have all improved behavior outcome.For each test, it seems and all be better than individual processing in conjunction with handling.This point is stretched out in the test obvious especially in spontaneous limb use and pawl.Such idea has been supported in this experiment, and promptly stem cell and somatomedin are better than each individual processing in conjunction with processing in strengthening the apoplexy recovery.Above-mentioned two embodiment only carry out with the NSC and the somatomedin of a dosage.Further research is being carried out to determine this interactional dose response feature.

Equivalent

Those skilled in the art will recognize that or only use normal experiment can determine the equivalent of many and specific embodiments of the present invention described herein.These equivalents are included in the scope of following claims.

Claims (48)

1. treatment suffers from the method for the individuality of CNS damage, and described method comprises to described patient to be used:

-cell; With

-Dexedrine

The wherein co-administered influence that improves the CNS damage of cell and Dexedrine.

2. the process of claim 1 wherein that described cell can produce neuron, oligodendroglia, astroglia and/or microgliacyte.

3. the process of claim 1 wherein that described cell is a stem cell.

4. the process of claim 1 wherein that described cell is a neural stem cell.

5. the process of claim 1 wherein that described cell is a hematopoietic stem cell.

6. the process of claim 1 wherein described cellular expression vmyc gene, and wherein said gene makes described stem cells hyperplasia at vivoexpression, and wherein after cell implants, described gene is expressed hardly so that the proliferation in vivo rate of cell reduces or stop.

7. the process of claim 1 wherein described cell-derived cell from described individuality.

8. the process of claim 1 wherein that described Dexedrine comprises biologically active polypeptide.

9. the method for claim 8, wherein said biologically active polypeptide comprises polypeptide growth factor.

10. the method for claim 9, wherein said polypeptide growth factor is selected from: fibroblast growth family member, neurotrophin family member, insulin-like growth factor family, ciliary nerve nutrition growth factor family member; EGF family member, TGF 'beta ' family member, leukaemia inhibitory factor (LIF); Oncostatin M, interleukin-6, interleukin 11; The member of platelet-derived growth factor family and VEGF family member.

11. the method for claim 9, wherein said polypeptide growth factor are the polypeptide that is selected from bFGF, aFGF, NGF, BDNF, NT-3, OP-1, FGF-3, FGF-4, FGF-5 and EGF.

12. the method for claim 9, wherein said polypeptide growth factor are the members of FGF family.

13. the method for claim 9, wherein said polypeptide growth factor are with the bFGF polypeptide of one of SEQ.ID.Nos.1-3 at least 30% identical polypeptide to be arranged.

14. the method for claim 12, wherein said polypeptide is identical with the bFGF polypeptide of one of SEQ.ID.Nos.1-3.

15. the process of claim 1 wherein that described Dexedrine is selected from: neurotransmitter, neurotransmitter agonist, neurotransmitter antagonists, differentiation factor, guide molecule and stride cranium magnetic and stimulate.

16. the method for claim 8, wherein said biologically active polypeptide are not to be produced by transgenic contained in one or more cells of using jointly.

17. treatment is suffered from the method for the individuality of brain injury because of apoplexy, described method comprises to described patient to be used:

-stem cell; With

-Dexedrine

The wherein co-administered influence that improves brain injury of cell and Dexedrine.

18. the method for claim 17, wherein said therapeutic alliance are beginnings at least 6 hours after being diagnosed as apoplexy.

19. the method for claim 17, wherein said cell can produce neuron, oligodendroglia and/or astroglia.

20. the method for claim 17, wherein said cell is a neural stem cell.

21. the method for claim 17, wherein said cell is a hematopoietic stem cell.

22. the method for claim 17 is wherein said cell-derived since the described individual cell that obtains.

23. the method for claim 17, wherein said biologically active polypeptide comprises polypeptide growth factor.

24. the method for claim 23, wherein said polypeptide growth factor is selected from: fibroblast growth family member, neurotrophin family member, insulin-like growth factor family, ciliary nerve nutrition growth factor family member; EGF family member, TGF 'beta ' family member, leukaemia inhibitory factor (LIF); Oncostatin M, interleukin-6, interleukin 11; The member of platelet-derived growth factor family and VEGF family member.

25. the method for claim 23, wherein said polypeptide growth factor are the polypeptide that is selected from bFGF, aFGF, NGF, BDNF, NT-3, OP-1, FGF-3, FGF-4, FGF-5 and EGF.

26. the method for claim 23, wherein said polypeptide growth factor are the members of FGF family.

27. the method for claim 23, wherein said polypeptide growth factor are with the bFGF polypeptide of one of SEQ.ID.Nos.1-3 at least 30% identical polypeptide to be arranged.

28. the method for claim 23, wherein said polypeptide is identical with the bFGF polypeptide of one of SEQ.ID.Nos.1-3.

29. the method for claim 17, wherein said Dexedrine is selected from: neurotransmitter agonist, neurotransmitter antagonists, differentiation factor, guide molecule and stride cranium magnetic and stimulate.

30. the method for claim 17, wherein said biologically active polypeptide are not to be produced by transgenic contained in one or more cells of using jointly.

31. the process of claim 1 wherein the administration in intravenous, brain, in Intraventricular or the brain pond of described Dexedrine.

32. the process of claim 1 wherein that described cell uses in intravenous, brain, in Intraventricular or the brain pond.

33. the process of claim 1 wherein that described cell uses in brain, described Dexedrine is used in the brain pond.

34. the process of claim 1 wherein that described cell and described Dexedrine all use in the brain pond.

35. the process of claim 1 wherein that described CNS damage results from apoplexy, wound, hypoxgia, Alzheimer, Huntington, amyotrophic lateral sclerosis, multiple sclerosis or parkinson.

36. be used for the treatment of the test kit of brain injury, contain:

-stem cell; With

-Dexedrine.

37. the test kit of claim 36 also contains the device that is useful on the device of using described stem cell and is used to use described Dexedrine.

38. the test kit of claim 36, wherein said Dexedrine comprise with the SEQ.ID.Nos.1-3 polypeptide at least 30% identical polypeptide is arranged.

39. the test kit of claim 36, wherein said Dexedrine comprises the polypeptide of SEQ.ID.Nos.1-3.

40. the test kit of claim 36, wherein said stem cell contains the neural stem cell of expressing the vmyc gene, and wherein said gene breeds described stem cell rapidly at vivoexpression, and implants that the described gene in back is expressed hardly so that the proliferation in vivo rate of cell reduces or stop at cell.

41. be used for the treatment of the test kit of brain injury, contain:

--be used for containing the device of stem cell sample from the individuality acquisition; With

--Dexedrine.

42. the test kit of claim 41, wherein said Dexedrine comprise with the SEQ.ID.Nos.1-3 polypeptide at least 30% identical polypeptide is arranged.

43. the test kit of claim 41, wherein said Dexedrine comprises the polypeptide of SEQ.ID.Nos.1-3.

44. contain the pharmaceutical composition of Dexedrine, stem cell and one or more pharmaceutically suitable carrier.

45. the pharmaceutical composition of claim 44, wherein said Dexedrine is selected from: fibroblast growth family member, neurotrophin family member, insulin-like growth factor family, ciliary nerve nutrition growth factor family member; EGF family member, TGF 'beta ' family member, leukaemia inhibitory factor (LIF); Oncostatin M, interleukin-6, interleukin 11; The member of platelet-derived growth factor family and VEGF family member.

46. comprising, the pharmaceutical composition of claim 44, wherein said Dexedrine be selected from following polypeptide: bFGF, aFGF, NGF, BDNF, NT-3, OP-1, FGF-3, FGF-4, FGF-5 and EGF.

47. the pharmaceutical composition of claim 44, wherein said Dexedrine comprises the member's who is FGF family polypeptide.

48. the pharmaceutical composition of claim 23, wherein said polypeptide comprise with the polypeptide of one of SEQ.ID.Nos.1-3 at least 30% identical polypeptide is arranged.

Images