CN1278736C - Novel dopaminergic neuron protective factor, and its application - Google Patents

Abstract

The present invention discloses a new method for treating central nervous system degenerative diseases, particularly a Parkinson disease, by utilizing CYSC, a method for screening and preparing a compound for treating central nervous system degenerative diseases by utilizing CYSC, or a new application of a medicinal composition, and simultaneously discloses a medicinal composition for treating nerve degeneration, particularly a Parkinson disease, and promoting the regeneration of the central nerves.

Description

The new dopaminergic neuron protection factor and an application thereof

Technical field

The present invention relates to biological engineering and medical domain, the concrete new dopaminergic neuron that the present invention relates to is protected the factor---Sai Sidating C (cystatin C) and application thereof.

Background technology

Parkinson disease (Parkinson ' s disease, be a kind of comparatively common central nervous system degenerative disease of current society PD), this disease is sent out in person in middle and old age well, and in the U.S., all groups' average attack rate is about 1 ‰, and the sickness rate more than 60 years old then is 1%.In China, this sick sickness rate is similar to western countries because of factor sickness rate such as the aging of populations, and the trend that rises is gradually arranged.The paleocinetic ability of parkinson disease patient is lost gradually, occur comprising tremble, various clinical symptom such as tetanic, slow movement, pathological characters is substantia nigra zona compacta dopaminergic (Dopaminergic, the Lewy corpusculum of sour characteristic appears having a liking in DA) neuron degeneration at the black substance neuron of remnants.The neuronic death of DA causes serious exhaustion (OlanowCW, Tatton, WG, Etiology and pathogenesis of Parkinson disease.1999, the Annu.Rev.Neurosci.22:123-44 of striatum dopamine; Blandini F, Nappi G, Tassorelli C, et al.Functional changesof the basal ganglia circuitry in Parkinson ' s disease.2000, Prog.Neurobiol., 62:63-88).

Up to now, though the research of its genesis mechanism of neuroscience bound pair has obtained remarkable break-throughs, but still lacks complete understanding, aspect treatment, except that the certain methods of alleviating patient symptom, lack basic especially and effective means both at home and abroad.At present, it has been recognized that the process that Parkinsonian nervus retrogression changes can delay.There is data to show, behind about 60-70% black substance DA neuronal death, Parkinsonian clinical symptoms just can occur, show black substance---the huge plasticity of striatum system and the compensatory capacity on the function, therefore, if can effectively slow down the process of parkinson disease degeneration variation and/or strengthen the function that remains dopaminergic neuron, just might significantly improve clinical symptoms, thereby improve patient's quality of life.From this point, find the factor that effectively to protect midbrain dopaminergic neuron a kind of Therapeutic Method that big application prospect is arranged of can yet be regarded as.

At present, it has been found that multiple and protection dopaminergic neuron relevant neurotrophic factor, as the neurotrophic factor (GDNF) of glia cell line-derived and other factor of family thereof or the like, they all demonstrate characteristic (the Airaksinen MS that promotes and protect dopaminergic neuron in the checking of various experiments, Saarma M, The GDNF family:signaling, biological functions and therapeutic value, 2002 Nature Review Neuroscience 3; 383-394).But their this effect just part is effective.It is generally acknowledged, reach higher levels of cytoprotective, need uniting use and could realizing of a plurality of factors.On the other hand, along with raising to parkinson disease pathogeny understanding, comprise that mitochondrial injury, oxidative stress (ROS), glutamate toxicity, genetic factor and apoptosis etc. are considered to Parkinsonian very confidential relation (Olanow CW be arranged, Tatton WG, Etiology and pathogenesis of Parkinson ' sdisease.1999, Annu.Rev.Neurosci.22:123-44), the multiformity and the complexity that show the parkinson disease mechanism of causing a disease.Because Parkinsonian generation may relate to above-mentioned a plurality of factor independently separately simultaneously, therefore, block each link that disease takes place simultaneously and could effectively control its development, thereby reach best therapeutic effect.Therefore, press for the new molecule that can effectively protect dopamine neuron of research at present, and kind should be The more the better, to adapt to the needs of clinical practice as far as possible.

Existing studies show that, PD rat model model that 6-hydroxy dopamine (6-OHDA) is caused and parkinson disease patient's striatal extract all have effect (the Zhou J of the veutro midbrain dopaminergic neuron survival that promotes In vitro culture, Shen Y, Tang Z, Xu L.et al.Striatal extracts promote the survival and phenotypic expressionof rat fetal dopaminergic neurons in vitro.2000, Neurosci.Lett.292:5-8).In the striatum of this rat model; a plurality of molecules in glial cell line-derived neurotrophic factor (GDNF) family; as GDNF; neurturin; persephin; the mRNA expression of artemin significantly rises; the death of prompting substantia nigra dopaminergic neuron can cause some factors in the striatum of denervation the mRNA level significantly improve (Zhou J; Yu; Y; Tang; Z; Shen Y and Xu L.Differential expression of mRNAs of GDNF family in the striatumfollowing 6-OHDA-induced lesion.2000, Neuroreport, 11:3289-3293); and the part in these factors oneself confirmed relevant with neuroprotective by other people; because they may be a group a part in the neuroprotective molecule is arranged, therefore, study this group factor in great detail and help to set up the novelty Therapeutic Method relevant with parkinson disease with exploitation.

In view of this, we by the method that suppresses subtractive hybridization obtain a series of in rat Parkinson disease model striatum Cloning of Differentially-expressed Genes.Sai Sidating C (cystatin C is called for short CYSC) is one of them.This gene is X05607 in the accession number of GenBank.

CYSC is a member in the Sai Sidating superfamily II class family, has the effect of inhibition cysteine proteinase (cysteineprotease).The II type Sai Sidating superfamily in people source comprises Sai Sidating C, D, and S, SN, and the E/M and the F that find recently, they are No. 20 chromosomal Sai Sidating polygenic locuses in seat all.People source CYSC maturation protein contains 120 aminoacid, and originally synthetic precursor protein contains 26 amino acid whose signal peptides.It is cysteine proteinase papain, cathepsin B, H, the inhibitor of L and S etc. (Mason RW, Johnson DA, Barrett AJ, Chapman HA.Elastinolytic activity of human cathepsin L, 1986, Biochem is J.233:925-7; BaricoSWH, Zhou YW, Fuerst RS, Barrett AJ, Shah SV.The role of aspartic and cysteineproteinases in albumin degradation by rat kidney cortical lysosomes.1987, ArchBiochem Biophys.256:687-91; Turk D, Podobnik M, Kuhelj R, Dolinar M, Turk V.Crystalstructures of human procathepsin B at 3.2 and 3.3 Angstroms resolution reveal aninteraction motif between a papain-like cysteine protease and its propeptide.1996.FEBS Lett.384:211-4).

Nearest result also shows, cofactor (Taupin P when CYSC is basic fibroblast growth factor promotion cell proliferation of nerve cord, Ray J, Fischer WH, Suhr ST, Hakansson K, Grubb A, Gage FH.FGF-2-responsiveneural stem cell proliferation requires CCg, a novel autocrine/paracrine cofactor.2000, Neuron.28:385-97), and this effect of CYSC need the glycosylation of CYSC to realize.

CYSC is distributed widely in various tissues, comprise in the body fluid, as urine, blood and cerebrospinal fluid (Barrett AJ, Davies ME, Grubb A, The place of human gamma-trace (cystatin C) amongst the cysteine proteinaseinhibitors.1984, Biochem Biophys Res Commun.120:631-6; Moller CA, Lofberg H, GrubhAO, Olsson SO, Davies ME, Barrett AJ.Distribution of cystatin C (gamma-trace), aninhibitor of lysosomal cysteine proteinases, in the anterior lobe of simian and humanpituitary glands.1985, Neuroendocrinology 41:400-4).There is report to show that CYSC may participate in some process of brain injury and cerebral disorders.As in of short duration cerebral ischemic model, in pyramidal cell in the Hippocampus and the active star spongiocyte specific up-regulated expression pattern (Palm DE is arranged, Knuckey NW, Primiano MJ, Spangenberger AG, Johanson CE.Cystatin C, a protease inhibitor, in degenerating rat hippocampalneurons following transient forebrain ischemia.1995, Brain Res.691:1-8), and response to oxidative stress also can make level rising (the Nishio C of the neuron expression CYSC of In vitro culture, Yoshida K, Nishiyama K, Hatanaka H, Yamada M.Involvement of cystatin C in oxidativestress-induced apoptosis of cultured rat CNS neurons.2000, Brain Res.873:252-62), prompting CYSC has participated in causing apoptotic adjusting by oxidative stress.CYSC may participate in the process of neural plasticity (Ying GX behind the central nervous system injury in the process of going entorhinal cortex domination Hippocampus, Huang C, Jiang ZH, Liu X, JingNH, Zhou CF.Up-regulation of cystatin C expression in the murine hippocampusfollowing perforant path transections.2002 Neuroscience 112:289-98).Also have data to show recently, in alzheimer disease patient's cerebrospinal fluid, the level of CYSC is than matched group height (Levy E, Sastre M, KumarA, Gallo G, Piccardo P, Ghetti B, Tagliavini F, Codeposition of cystatin C withamyloid-beta protein in the brain of Alzheimer ' s disease patients.2001, JNeuropathol Exp Neurol.60:94-104).Above-mentioned data prompting CYSC and central nervous system have confidential relation, and it may participate in the degeneration process of cerebral neuron.But definite function and the meaning of CYSC under these pathological conditions is not clear, and it has any related with parkinson disease or dopaminergic neuron also not have the data prompting.

Summary of the invention

One object of the present invention just provides a kind of new CYSC that utilizes and treats the especially Parkinsonian method of central nervous system degenerative disease.

Another object of the present invention provides a kind of new purposes of CYSC.

A further object of the present invention provides a kind of treatment nerve degeneration and promotes the nervus centralis especially Parkinsonian pharmaceutical composition of dirt again.

A first aspect of the present invention; a kind of method for the treatment of central nervous system degenerative disease is provided, and described method comprises step: the dopaminergic neuron protection factor---the Sai Sidating C (cystatin C) that uses safe and effective amount to the patient of the described treatment of needs.In a preference, described central nervous system degenerative disease is parkinson disease.Preferable, described Sai Sidating C is locally applied to affected area.

A second aspect of the present invention provides the purposes of a kind of Sai Sidating C, and described Sai Sidating C is used to prepare the pharmaceutical composition for the treatment of central nervous system degenerative disease.Preferably, described central nervous system degenerative disease is parkinson disease.

In a preference, described pharmaceutical composition is an injection.

A third aspect of the present invention provides a kind of pharmaceutical composition, and described pharmaceutical composition contains Sai Sidating C and the pharmaceutically acceptable excipient or the carrier of safe and effective amount.

Preferable, the dosage of described pharmaceutical composition is 0.01 microgram/kg body weight-Yue 100 micrograms/kg body weight.

A fourth aspect of the present invention provides a kind of Sai Sidating C the purposes of (cystatin C), and described Sai Sidating C is used to screen the chemical compound of treatment central nervous system degenerative disease.

A fifth aspect of the present invention provides a kind of health product, and described health product contain Sai Sidating C.

As used herein, term " Sai Sidating C albumen " " cystatin C " and " CYSC " are used interchangeably.

Particularly, Sai Sidating C albumen of the present invention can be by introducing corresponding coding sequence host cell (directly introducing or contain by introducing the carrier of Sai Sidating C coded sequence), and under appropriate condition, cultivate transformed host cells to express Sai Sidating C albumen, separation and purification are attended the competition then, and this reaches court of a feudal ruler C albumen.

The transformant that obtains can be cultivated with conventional method, expresses Sai Sidating C albumen.According to used host cell, used culture medium can be selected from various conventional culture medium in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promoter of selection with suitable method (as temperature transition or chemical induction), cell is cultivated a period of time again.

The extracellular can be expressed or be secreted into to recombiant protein in the above methods in cell or on cell membrane.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, high performance liquid chroma-tography (HPLC) and other various liquid chromatography (LC) technology and these methods.

Particularly, Sai Sidating C albumen of the present invention is of use in many ways.These purposes include, but is not limited to: treatment nerve degeneration and promotion nervus centralis are regenerated and are used to screen antibody, polypeptide or other part that promotes Sai Sidating C protein function.The peptide molecule that can suppress or stimulate people Sai Sidating C protein function that can be used for seeking therapeutic value with the reorganization Sai Sidating C protein screening peptide library of expressing.

Utilize albumen of the present invention,, can filter out with Sai Sidating C albumen interactional material takes place, as inhibitor, agonist or antagonist etc. by various conventional screening techniques.When screening, Sai Sidating C albumen can be added in the bioanalysis mensuration, determine the influence of this chemical compound by the interaction between mensuration compounds affect Sai Sidating C albumen and its substrate.In addition, also test compounds can be applied to laboratory animal with Sai Sidating C albumen, the variation of axoneure survival number can show compared with the control, and this chemical compound has promotion or suppresses the proteic effect of Sai Sidating C.

In addition, the expression vector of also cDNA of Sai Sidating C gene can being packed into, the transfection mammalian cell strain, preparation high expressed Sai Sidating C proteic cell strain: and be target site with the Sai Sidating C albumen in this cell strain, screening has Sai Sidating C albumen and activates or inhibiting medicine.And in the proteic cell strain culture fluid of described expression Sai Sidating C, add test compounds, detect the variation of Sai Sidating C expressing quantity.Promote the chemical compound of Sai Sidating C protein expression to promote the regenerated chemical compound of nervus centralis exactly.

Albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, the inert and pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although pH value can be with being changed to some extent by preparation Substance Properties and disease to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intravenous, subcutaneous, oral or topical.Normal Sai Sidating C albumen can be directly used in disease treatment, for example, and the treatment central nervous system degenerative disease.

The present invention also provides a kind of pharmaceutical composition, and it contains Sai Sidating C albumen, its code nucleic acid and/or the antisensenucleic acids of safe and effective amount, and pharmaceutically acceptable carrier or excipient.This class carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by conventional method with normal saline or the aqueous solution that contains glucose and other adjuvant.Pharmaceutical composition such as tablet and capsule can be prepared by conventional method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of active component is the treatment effective dose, for example every day about 0.01 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, albumen of the present invention also can use with the other treatment agent.

When making pharmaceutical composition, be that the Sai Sidating C albumen of safe and effective amount or its antagonist, agonist are applied to mammal, wherein this safe and effective amount is usually at least about 0.01 microgram/kg body weight, and in most of the cases be no more than about 10 mg/kg body weight, preferably this dosage is about 0.01 microgram/kg body weight-Yue 100 micrograms/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

Recombined human Sai Sidating C gene can be packaged in the liposome in addition, and then is transferred in the cell.Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.

Sai Sidating C albumen of the present invention and antagonist thereof not only can be used for treatment, also can be used for promoting neuranagenesis.Therefore, the present invention also provides a kind of health product, and it contains mammiferous Sai Sidating C albumen or its coded sequence, or its antagonist (as antisensenucleic acids).Health product of the present invention, method that can be by the field of health care products routine, by with mammiferous Sai Sidating C albumen or its coded sequence, or its antagonist (as antisensenucleic acids) mixes with suitable diluent, food etc. and prepares.Preferred health product are tablet, granule, oral agents form.

The inventor has confirmed the dependency of CYSC and dopaminergic neuron and the specific rise pattern in the PD rat model model thereof by the kinds of experiments means.By the cell in vitro cultivation with in the body zoopery, confirmed that CYSC has tangible promotion survival and reduces the effect of neurotoxin to its murder by poisoning dopaminergic neuron, points out this molecule having potential application prospect aspect the Parkinsonian treatment.

As previously mentioned, CYSC has the characteristic of cystatin, but definite function and the meaning of CYSC under the central nervous system disease state is not clear, and it has any related with parkinson disease or dopaminergic neuron also not have the documents and materials prompting so far.Inventor's result of study shows, the mRNA of CYSC intrastriatal expression in different times PD rat model model brain significantly raise (Fig. 1, Fig. 2).The immunohistochemical staining result shows that the high expressed of CYSC is distributed in striatal tail shell nuclear and pallidum (Fig. 3, table 1), and derives from the various kinds of cell composition, i.e. neuron, star spongiocyte and microglia (Fig. 4).At black substance, its up-regulated equally also clearly.CYSC has the effect of significant its survival of promotion to embryo's veutro midbrain dopaminergic neuron of In vitro culture, compares with the cellular control unit survival, reaches more than 2 times.Known neurotoxin N-methyl-4-benzene pyridine (N-methyl-4-phenylpyridinium, MPP ⁺) dopamine neuron is had the toxic action of high special, the dopaminergic neuron Mpp of In vitro culture ⁺After the processing, cell survivaling number is about 50% of a matched group, but CYSC (20ng/ml) and MPP ⁺(as 20 μ M) add in the dopamine neuron of cultivating simultaneously then can significantly resist MPP ⁺This toxic action, illustrate that CYSC has the protective effect (Fig. 5) to dopamine neuron.For verifying that further can CYSC resist another kind of neurotoxin 6-hydroxy dopamine at dopamine neuron, we are injected into black substance with CYSC (every animal 5 micrograms or 15 micrograms) in advance, and then give 6-hydroxy dopamine at same position, animals survived is after 4 weeks, the residual dopaminergic neuron of matched group black substance is about 6.9% (with conforming to of reporting in the document) of normal control, and in two treated animals that give 5 micrograms or 15 microgram CYSC respectively the residual dopaminergic neuron of black substance respectively to 27% and 28%, 4 times (Fig. 6, tables 2) that are about matched group.The The above results prompting; because it is simultaneously underproduce that damaged brain self increases the response lag of synthetic CYSC; thereby; be not enough to due to the antineurotoxin to the toxic action of dopamine neuron; the exogenous CYSC that gives then helps to protect dopaminergic neuron, shows the potential value of this molecule as clinical treatment.It this act as research treatment nerve degeneration and promotes the nervus centralis novel process for regenerating that brand-new data is provided.

Description of drawings

Fig. 1. show the variation of Sai Sidating C mRNA level in the PD rat model striatum with Northern blot blotting.After 2 weeks of damage and 5 weeks, the Sai Sidating C mRNA level (arrow indication) of damage side has been compared obviously with offside and has been increased.The latter half of figure shows total rna level of each sample, to show each swimming lane applied sample amount basically identical.

Fig. 2. the in situ hybridization Faxian be shown in the PD rat model model tail shell nuclear (A, B) and pallidum (C, D) variation of CYSC mRNA.

(B, D) (A C) increases the cell of expression CYSC mRNA (blue point-like thing, each point is represented a cell) the damage side, shows that CYSC may be relevant with brain injury than the damage offside.

Fig. 3. show that with immunohistochemical method CYSC expression in the PD rat model striatum has rising.(F) (A, C E) increase the cell number (brown point-like thing) of expression CYSC the damage side than the damage offside for B, D.

Fig. 4. the immunofluorescence double labelling shows the source of CYSC.

A-C:CYSC and glial fibrillary acidic protein (glial fibrilary acid protein, GFAP, the mark of star spongiocyte) coexist in part cell (arrow indication), show that star spongiocyte is one of source of CYSC.

D-F:CYSC and RIP albumen (mark of oligodendrocyte) do not coexist in any cell, show that oligodendrocyte is not the source of CYSC.

G-I:CYSC and Integrin α M (mark of microglia) coexist in part cell (arrow indication), show that microglia also is one of source of CYSC.

Fig. 5. Sai Sidating C has facilitation to the survival of dopaminergic neurons of In vitro culture.

To take from 14 days veutro midbrain (containing dopaminergic neuron) of embryo cultivated 3 days in suitable culture medium, when the time comes these cultured cells are fixed with paraformaldehyde, use tyrosine hydroxylase (TH) antibody to carry out immunohistochemical staining then, carry out cell counting subsequently.

A: blank.

Cell culture after B:CYSC handles is compared with A, and the dopaminergic neuron of visible survival (the dark person of color) number increases.

Cell culture after C:Leupeptin (as positive control) handles is compared with A, and the dopaminergic neuron of same visible survival (the dark person of color) number increases.

The dosage effect figure of D:CYSC.

The dosage effect figure of E:Leupeptin.

F: at 0-20 μ M MPP ⁺Exist down, CYSC can partly block its toxic action, saves dopaminergic neuron.

Fig. 6. under the body situation, Sai Sidating C intracerebral injection is to the protective effect of dopaminergic neuron.

A, C, E, G: the dopaminergic neuron that at first in rat striatum, passes through the reverse labelling black substance of injection fluorogold, the cell that has been labeled can be excited at 330nm wavelength place and present green, thereby it is used to the dopaminergic neuron of labelling black substance survival.

B, D, F, H:, observed the survival of dopaminergic neurons situation by the immunohistochemical dyeing of tyrosine hydroxylase (TH).Under the exciting of 530nm light wave, being taken on a red color by the dopaminergic neuron of tyrosine hydroxylase antibody labeling, (F H), has the neuron of intact cell form all to count interior for B, D.The cell number numeration of damage offside is 100%, and the residual neuron of matched group damage side is 6.9%, and the result of this and bibliographical information conforms to, and illustrates that our Preparation of model is successful.The animal of 5 microgram dosing groups and 15 microgram dosing groups, the cell of damage side survival is respectively 27% and 289%, and (F H), has shown the protective effect of CYSC to impaired dopaminergic neuron from another angle.

The specific embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Experiment material:

TOTALLY RNA separating kit (Ambion, Austin, TX, USA), and in vitro transcription test kit (Roche, USA), sheep blood serum (Invitrogen company, USA), anti-RIP antibody (doctor Xu Xiaoming of U.S. University of Louisville is so kind as to give), anti-Integrin α M antibody (Chemicon, CA, USA), and people source Sai Sidating C (Calbiochem, USA), fluorogold (Flurogold, Flurochrome, CA, USA), DMEM/F12 (GIBCO, Invitrogen, USA) following reagent all from U.S. Sigma company (St.Louis, MO):

Biotinylated goat anti-rabbit igg, biotinylated sheep anti-mouse igg, the sheep anti-mouse igg of fluorescein (FITC) labelling, the goat anti-rabbit igg of Texas's red marker, the Avidin of Cy3 labelling, and the Avidin of DTAT labelling (Jackson Immuno Research, USA), anti-tyrosine hydroxylase (TH) monoclonal antibody, anticol cell plastid original fiber acid protein (GFAP) antibody.

Used rat comes from Chinese Academy of Sciences's Shanghai Experimental Animal Center (Songjiang Jiu Ting).

Embodiment 1 PD rat model Preparation of model

With body weight is that the female Sprague-Dawley rat that 180-220 restrains is fixed on the animals stereo position finder after anesthesia.Utilize microsyringe with 4 microlitre 6-hydroxy dopamine (Sigma, USA) (2.5 micrograms/microlitre is dissolved in 0.2 mg/ml ascorbic acid (Sigma, USA)) be injected into rat right side medial forebrain bundle (with respect to the elements of a fix of bregma: AP,-4.4mm, ML ,+1.2mm, DV, + 7.8mm), injection speed is 0.4 mul/min, finish injection back let the acupuncture needle remain at a certain point and again syringe was slowly withdrawed from 10 minutes.Operation back one all lumbar injection apomorphines (Sigma, USA) (0.05 milligram/kg body weight), the rotation of test rat, the above person of rotation per minute 6 circles is qualified PD rat model model.

Embodiment 2 Northern blot traces

Utilize TOTALLY RNA separating kit (TX USA) extracts total RNA in Adult Rat Brain, utilize capillarity that total RNA is transferred to nylon membrane behind electrophoresis for Ambion, Austin, then with ³²The CYSC probe of phosphorus labelling, exposed 3 days with the X-ray sheet down at-80 ℃ after rinsing jointly 42 ℃ of down hybridization 16 hours, promptly obtained Fig. 1 after the development.Can be clear that the variation of Sai Sidating C mRNA level in the PD rat model striatum in the figure.After 2 weeks of damage and 5 weeks, the Sai Sidating C mRNA level (arrow indication) of damage side has been compared obviously with offside and has been increased.The latter half of figure shows total rna level of each sample, to show each swimming lane applied sample amount basically identical.

Embodiment 3 in situ hybridization analyses

At first according to gene (accession number X16957.1) the sequential design primer of the CYSC of login among the European molecular biology data base (EMBL) and utilize PCR to obtain the long cDNA segment of one 690 bases, utilize in vitro transcription test kit (Roche, USA) the cRNA probe of synthetic corresponding digoxigenin labeled.With the brain of PD rat model model with 4% paraformaldehyde fixing after, section, hybridize down at 50 ℃ with the cRNA probe of CYSC then and spend the night, hybridization solution contains 50% Methanamide, 10% dextran, 10mM Tris-hydrochloride buffer (pH 7.5), 600 mM sodium chlorides, 1 x Denhardt ' s solution and, 400 mcg/ml milt DNA.After hybridization finishes,, hatch, can manifest the positive cell (Fig. 2) that is labeled after adding suitable substrate with anti digoxin antibody through rinsing.With this method can be presented at delicately the PD rat model model tail shell nuclear (A, B) and pallidum (C, D) variation of CYSC mRNA.As seen, (B, D) (A C) increases the cell of expression CYSC mRNA (blue point-like thing, each point is represented a cell) the damage side, shows that CYSC may be relevant with brain injury than the damage offside in Fig. 2.

Embodiment 4 immunohistochemical stainings

The brain sheet is fixed through conventional method, with after the phosphate buffer rinsing that contains 0.01% triton x-100 with 4% sheep blood serum (Invitrogen company, USA) hatch 30 minutes, hatched 48 hours with the first antibody that contains 1% sheep blood serum down at 4 ℃, rinsing, in biotinylated second antibody, hatched 1.5 hours then, hatched 1 hour with the horseradish peroxidase that is combined with avidin again, add substrate DAB chromogenic reaction at last.Carry out cell counting with the optics microscopically through amplifying 200 times.Result visible (Fig. 3), damage side (B, D, F) (A, C E) increase the cell number (brown point-like thing) of expression CYSC than the damage offside, many at damage side black substance and striatal CYSC immunoreation positive cells than the offside number, and color depth, illustrate that the expression of the impaired CYSC of causing of substantia nigra dopaminergic neuron rises, show that further CYSC may be relevant with brain injury.The results are shown in Table 1 for detailed cell counting.

The double-colored labelling dyeing of embodiment 5 immunofluorescences

The brain sheet is fixed through conventional method, with hatching 30 minutes with 4% sheep blood serum after the phosphate buffer rinsing that contains 0.01% triton x-100, hatched 48 hours with the first antibody that contains 1% sheep blood serum down at 4 ℃, rinsing, (Sigma USA) is hatched 1.5 hours, rinsing with brain sheet and the bonded second antibody of biotin, hatched rinsing 1 hour with the fluorescein that is combined with avidin again.4% paraformaldehyde is fixed 20 minutes then, rinsing, hatched 30 minutes with 4% sheep blood serum, then with the antibody incubation of various types of cells labelled molecule, rinsing, hatch rinsing with corresponding fluorescein-labeled second antibody (as the goat anti-rabbit igg of fluorescein-labeled sheep anti-mouse igg, Texas's red marker, the Avidin of the Avidin of Cy3 labelling or DTAT labelling) then.With fluorescence mountant mounting, fluorescence microscope is observed down, takes a picture.The result shows, striatum CYSC immunoreation positive cell from form except that neuron is arranged, star spongiocyte and microglia are also expressed CYSC.Appear at (Fig. 4) in dopaminergic neuron and the microglia at black substance CYSC.

First antibody albumen English name	The corresponding second antibody of using	The purposes of first antibody
			Cystatin C(Caibiochem， USA)	Anti-rabbit igg (Sigma, USA)	The cell of recognition expression cystatin C
Anticol cell plastid original fiber acid protein (GFAP) antibody: Glial fibrillary acid protein	Anti-mice IgG (Sigma, USA)	The identification star spongiocyte

(Sigma，USA)
			Anti-tyrosine hydroxylase (TH) monoclonal antibody: Tyrosine hydroxylase (Sigma, USA)	Anti-mice IgG (Sigma, USA)	The identification dopaminergic neuron
Integrin αM(Chemicon， CA，USA)	Anti-mice IgG (Sigma, USA)	The identification microglia
			RIP (doctor Xu Xiaoming of U.S. University of Louisville is so kind as to give)	Anti-mice IgG (Sigma, USA)	The identification oligodendrocyte

Former being commissioned to train of embodiment 6 fetal rat veutro midbrain cells supported the observation that reaches the CYSC effect

Get conceived 14 days the pregnant Mus of Sprague-Dawley (day of finding cloudy bolt is conceived the 0th day) the embryo place the D-Hanks buffer of refrigerative in advance no calcium, magnesium ion, separation veutro midbrain under anatomic microscope.Organize 2 times with the washing of D-Hanks buffer earlier, add trypsin 25 mcg/ml again) hatched 5-10 minute under the room temperature, with 10% hyclone cessation reaction, DMEM/F12 (1: 1) (GIBCO, Invitrogen, USA) the washing tissue is 2 times, softly dispels piece of tissue, leave standstill and got supernatant cell suspension in 5 minutes, trypan blue dyeing back living cell counting number.With every square centimeter 1 * 10 ⁵The density kind of individual cell goes into to spread in advance 96 well culture plates of poly-D-lysine, and culture medium is the DMEM/F12 that contains 10% hyclone.Be replaced by the DMEM/F12 that contains 1%N2 after 4 hours, add CYSC simultaneously.The cell culture fluid volume is 100 microlitres, changes 24 hours fixed cells behind the liquid.As seen from Figure 5, compare with matched group, CYSC can reach 2.2 times to the effect of the promotion survival of dopaminergic neuron.

For observing CYSC to N-methyl-4-benzene pyridine (MPP ⁺) (Sigma, USA) whether the veutro midbrain cell of the In vitro culture of Chu Liing has the survival of promotion effect, and former being commissioned to train of 14 days rat veutros of embryo midbrain cell supported as above, is replaced by after 4 hours to contain 1%N ₂DMEM/F12, add MPP+ immediately or CYSC adds MPP+, paraformaldehyde is fixed after 36 hours, carries out immunohistochemical staining with the TH monoclonal antibody.The result shows, at variable concentrations MPP ⁺Under the situation about existing, 20 nanograms/milliliter CYSC have the toxic effect of tangible antagonism MPP+ (Fig. 5 F) to the midbrain dopaminergic neuron of cultivating.

The intracerebral injection of embodiment 7 CYSC is to the observation in the protective effect of body animal substantia nigra dopaminergic neuron

The Sprague-Dawley female rats, body weight 180-220 gram is fixed in after the anesthesia on the position finder, and as preceding, nasal height is made as 0.By following coordinate in the fluorogold of striatum two side injections 0.2 microlitre with 2% normal saline preparation: the elements of a fix with respect to bregma are: AP ,+0.5mm; ML, ± 3.4mm; DV ,-5.0mm.After one week, injection CYSC is divided into 5 microgram groups, 15 microgram group and matched groups.Matched group is injected 0.1% bovine serum albumin (being dissolved in 0.1 mole of phosphoric acid salt buffer).Each is organized the injection volume and is 3.4 microlitres. and coordinate is: AP ,-5.4mm; ML ,-2.2mm; DV ,-8.5mm.Nasal height for-the 3.3mm. injection speed is 0.4 mul/min, the slowly withdraw of the needle that let the acupuncture needle remain at a certain point after 5 minutes.After 6 hours, every rat contains 6-hydroxy dopamine 2 micrograms/3.4 microlitres of the normal saline configuration of 0.02% ascorbic acid, the same PD model of performing the operation in same coordinate points injection.After 4 weeks sacrifice of animal, perfusion and preparation cerebral tissue are cut into slices.

Two kinds of methods of identification dopamine neuron on the brain sheet: the one, by injecting fluorogold at striatum, the retrograde dopamine neuron that is transported to black substance in one week back, by the exciting of ultraviolet light (330nm wavelength) can under fluorescence microscope, see by the cell of fluorogold labelling (Fig. 6 A, C, E).The 2nd,, dopamine neuron itself has abundant tyrosine hydroxylase, by immunohistochemical staining can show (Fig. 6 B, D, F).These two kinds of methods are separate, can weigh the effect behind the CYSC intracerebral injection more objectively.

By fluorogold labelling result as seen, the damage offside is complete in the distribution of black substance by the cell of fluorogold labelling, and cellular morphology is complete, and quantity is 100%.Be apoptotic state (Fig. 6 C) by the cell of fluorogold labelling majority in the matched group, the cell of complete form is about 6.9% of offside, and this result with bibliographical information 6.1% conforms to.5 microgram administration groups and 15 microgram administration groups damage side cell number are approaching, are about 28% and 27.1% of matched group, and the protective effect of CYSC pair cell is described.On the other hand, by the immunohistochemical staining of tyrosine hydroxylase (TH), we have investigated the survival condition of TH immunoreactive positive neurons.Under the exciting of 530nm light wave, taken on a red color by the dopaminergic neuron of TH labelling, have only the complete neuron of cellular morphology just to list numeration in.The TH positive cell number of damage offside is made as 100%; and residual 6.9% (Fig. 6 D) of TH immunoreactive positive neurons for contrasting of matched group damage side; cell in 5 microgram administration groups and the survival of 15 microgram administration groups damage side is respectively 27% and 28% (Fig. 6 F; 6H; table 2), shown the protective effect of CYSC to impaired dopaminergic neuron.The results are shown in Table 2 for cell counting in detail.

Embodiment 8 preparation of drug combination

CYSC albumen is made injection, during use subcutaneous injection is carried out in the affected part, directly protect the axoneure of affected area, the central nervous system degenerative disease that makes patient particularly parkinson disease improves, thereby reaches the purpose of treatment.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Claims (5)

1. the purposes of a people Sai Sidating C is characterized in that, is used to prepare the pharmaceutical composition for the treatment of central nervous system degenerative disease.

2. the purposes of the described people Sai Sidating of claim 1 C is characterized in that, described central nervous system degenerative disease is parkinson disease.

3. the purposes of claim 1 or 2 described people Sai Sidating C is characterized in that described pharmaceutical composition is an injection.

4. a pharmaceutical composition is characterized in that, it contains people Sai Sidating C and the pharmaceutically acceptable excipient or the carrier of safe and effective amount.

5. the purposes of a people Sai Sidating C is characterized in that, is used to screen the chemical compound of treatment central nervous system degenerative disease.

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