CN1678336A - Treatment of disease by inducing cell apoptosis - Google Patents

Abstract

The present invention relates generally to the treatment and prevention of diseases characterized by excess cell proliferation and/or activation. In particular, the present invention provides compositions and methods to suppress the activation and/or proliferation of various cells. In some preferred embodiments, the present invention provides compositions and methods to suppress the activation and/or proliferation of mesenchymally derived cells (including, but not limited to hepatic stellate cells), as well as cells with abnormal growth characteristics. In some particularly preferred embodiments, the present invention provides compositions and methods to inhibit or eliminate fibrosis. In alternative preferred embodiments, the present invention provides compositions and methods to induce fibrosis. In still further embodiments, the present invention provides methods and compositions to treat and/or prevent cancer.

Description

With apoptosis-inducing method treatment disease

Technical field

The present invention relates to excessive cell proliferation and activation is the treatment of diseases and the prevention of feature.Distinctive feature of the present invention is to provide some preparations and method, is used to suppress the activation and/or the propagation of the cell of multiple variety classes.These preparations provided by the invention and method preferentially are used for suppressing an activation of the derivative cell of matter and/or propagation (including but are not limited to the liver spider cell), also are used to suppress those cells with misgrowth feature (as tumor cell).The present invention also provides some preparations and method, special blocking-up or the elimination fibre modification of preferentially being used in addition.In addition, the present invention also provides preparation and method to induce fibre modification.The present invention also further provides method and formulation for treatment for cancer and prevention.

Background technology

The abnormality proliferation of cell is a kind of sign of multiple different pathological process.Under given conditions, out of control and some fibre modification disease association (as hepatic fibrosis) of cell growth is also with other multiple diseases and comprise that the abnormal cell proliferative disease reason condition of cancer is relevant.

With the chronic hepatitis is example, and this is a kind of inflammatory hepatopathy of not getting better June and changeing that reaches at least.Chronic hepatitis c is a type of chronic hepatitis.What remain further verification is that chronic hepatitis c may develop into liver cirrhosis and massive hepatic necrosis.Although chronic hepatitis c is often relevant with the type i collagen deposition, thereby causes hepatic fibrosis, its fiber formation mechanism is not found out as yet.Really, at present still there is not definite Therapeutic Method [referring to document as Maher and McGuire, J.Clin.Invest.86:1641-48 (1990) for excessively producing disease that relevant liver fiber generation causes with type i collagen; Chojkier " Pathogenesisof hepatic fibrosis " .In Extracellular Matrix, Marcel Dekker Inc., New York, NY, pp541-57 (1993)].However, the S-6 kinases of still having found out at present multiple ribosomal protein is for comprising that the existence such as the various kinds of cell of liver spider cell is vital, thereby and the liver spider cell has caused the generation of liver cirrhosis just by its excessive generation fibrous tissue.Although but, still need advanced preparation and method at present, so that be used for suppressing effectively to comprise the paracytic activation and/or the hypertrophy of cancer cell having dropped into a large amount of funds and research effort aspect the fibrosis lesion (changing) as fibroid.

Summary of the invention

Main purpose of the present invention is for being the treatment of diseases of feature and preparation and the method that prevention provides a kind of new advanced person with excessive cell proliferation and activation, so that be used for suppressing effectively to comprise the paracytic activation and the hypertrophy of cancer cell.

The invention provides a kind of method of promoting apoptosis process, this method possess following some: a) precondition is: i) at least one cell (intend or a cell colony); Ii) a kind of polypeptide that comprises one or more tetrapeptide arrays of while, these tetrapeptide arrays then are selected from (1) KAVD (lysine-alanine-valine-aspartic acid, as follows) and (2) KKPA (lysine-Lys-Pro-alanine, as follows); B) handle the apoptosis that cell promotes this cell thus with these polypeptide formulations.Also can adopt similar aminoacid in this method utilization.For example: the alanine in KAVD (this sequence is available from typical mice and human C/EBP β sequence) tetrapeptide and KKPA (this tetrapeptide array is available from the typicality rat C/EBP β sequence) tetrapeptide seemingly can be by other any tyrosine that removes, and the aminoacid outside threonine and the silk ribbon propylhomoserin is replaced.According to this kind skill, then the alanine in KAVD or the KKPA tetrapeptide is like can preferentially using arginine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tryptophan, perhaps valine is replaced.Under the situation more preferably, the alanine among above-mentioned KAVD and the KKPA should be replaced by any aminoacid except that cheese, Soviet Union, silk ribbon, Guang, bright, paddy and aspartic acid.Though the present invention is not limited at a certain particular cell types, specifically, the cell that the present invention is directed to is chosen to be such one group of cell, comprising: hepatocyte, pneumonocyte, nephrocyte, Skin Cell, muscle cell, heart cell, glial cell, eyes cell, vascular cell, neurocyte, epithelial cell, endotheliocyte, and Interstitial cell.In above-mentioned cell, what preferentially relate to is cancer cell, then preferentially relates to the metastatic carcinoma cell in cancer cell.In addition, above-mentioned cell involved in the present invention is meant the isolated cells of a certain main body or in somatic cell.

Under some situation, one or more aminoacid will be modified or by the aminoacid of certain synthetic in the aforementioned polypeptides sequence, or other molecules similar on function replace (for example, in order to strengthen modification or the replacement that its stability is in vivo done, etc..).The micromolecule analog also is provided sometimes, and its function and above-mentioned tetrapeptide are same or similar.In some occasion, these molecules are identified with efficient screening technique.But in the other occasion, these micromolecule then produce (for example, carry out chemical based such as similar electric charge, configuration because of replacement) by rational design on one or more aminoacid.

In addition, the present invention also provides a kind of reduction apoptotic method simultaneously, and this method is made up of following several respects: a) precondition is: i) at least a cell; Ii) a kind of polypeptide formulations that contains one or more tetrapeptide arrays, these tetrapeptide molecules are selected from (1) KEVD (relying-the third-figured silk fabrics-aspartic acid) and (2) K-phosphoT-VD (relying-phosphoric acid Soviet Union-figured silk fabrics-aspartic acid), (3) KKPD (Lai-Lai-dried meat-aspartic acid); And b) handling cell with these polypeptide formulations makes the apoptotic process of these processed cells be minimized thus.

The present invention further provides and reduced the method that cancer takes place in certain tissue, its composition is: a) precondition is: a kind of tissue i) is arranged; A kind of polypeptide formulations is ii) arranged, and said preparation contains one or more tetrapeptide molecules, and these tetrapeptide molecules have following sequence (1) KAVD (relying-the third-figured silk fabrics-aspartic acid) and (2) KKPA (Lai-Lai-dried meat-alanine); And b) with these polypeptide formulations go to handle at tissue, the cancer cell in this tissue is reduced.Though the present invention is not limited to a particular studies object, specifically, related here tissue is to come from certain mammal.The mammal main body that preferentially relates to is human.Under certain situation, under a cloud in the tissue of the main body that the present invention relates to have or confirmed cancer cell.Specifically what relate to here organizes, and is selected from following a series of tissue, comprising hepatic tissue, and cerebral tissue, lung tissue, nephridial tissue, breast tissue, prostata tissue, cervical tissue, rectal tissue, pancreatic tissue, gastric tissue, ovary tissue, esophageal tissue, oral cavity tissue, tongue tissue, the nose tissue, skin histology, muscular tissue, gums, heart tissue, bronchial tissue, cartilaginous tissue, osseous tissue, testis tissue, internal film tissue, uterine cancer cell, bladder body, myeloid tissue, lymphoid tissue, spleen tissue, thymic tissue, parathyroid tissue, nervous tissue, gallbladder tissue, ocular tissue, and synovial tissue.

The present invention also provides a kind of method that reduces the tissue fibers degeneration simultaneously, and its composition is: a) supposition: i) at a kind of tissue; A kind of polypeptide formulations ii) is provided, and said preparation contains one or more tetrapeptide molecules, and these tetrapeptide molecules are selected from (1) KAVD; (2) KKPA; And b) with this polypeptide formulations handle at tissue, thereby the fibre modification in the processed tissue is minimized.Here specifically related organizing be meant a kind of mammalian tissues, preferentially refers to a kind of tissue.More specifically, the main body that relates to here is meant and exists or under a cloudly have a fibrotic tissue.What further need specify is again, fibre modification involved in the present invention is optionally to relate to following a series of fibre modification, for example: hepatic fibrosis, pnemnofibrosis (for example emphysema), kidney fibre modification (for example glomerulonephritis) and eyeball fibre modification.When relating to the hepatic fibrosis degeneration, what more preferably relate to is meant the hepatic fibrosis relevant with following concrete condition: for example repulsion of liver transplantation, hepatitis C infection, hepatitis B infection, alcoholism, liver poisoning, hereditary hemochromatosis, leaf quinoline disease, and liver cirrhosis.Another kind of situation is, this fibre modification also with another kind of disease association, this class disease includes cranial glia hypertrophy, Alzheimer, hepatopathy, brain injury, neural wound, neural degeneration, myocardial infarction, arteriosclerosis, fibrosis dermatosis, fibrosis pneumonopathy.

The present invention also provides apoptotic method in a kind of reduction tissue, this method comprise following some: a) supposition have: i) a kind of tissue; Ii) with a kind of polypeptide formulations, said preparation contains one or more tetrapeptide molecules, and these tetrapeptide molecules are selected from (1) (1) KEVD, (2) KphosphoTVD, (3) KKPD and (4) KKPphosphos; B) go to handle related tissue with this polypeptide formulations, thereby reduce the apoptosis of this tissue.Here, what preferentially relate to is a kind of mammalian tissues, and preferentially relates to tissue in mammalian tissues.When relating to these and organize, existed in its tissue or suspected to have apoptosis process.And when further relating to apoptosis, then be meant and wound burn, damage, the apoptosis that environmental toxin or ischemia are relevant.In addition, apoptosis also with a certain tissue in a kind of disease association, this class tissue comprises lung, kidney (as glomerulonephritis), and ocular tissue's (as retinitis pigmentosa), another kind of situation is, apoptosis is also relevant with a kind of hepatopathy, and this hepatopathy comprises one group of disease, comprising: the repulsion of liver transplantation, hepatitis C infection, hepatitis B infection, alcoholism, liver poisoning, hereditary hemochromatosis, leaf quinoline disease.This apoptosis is also organized disease association with another, and this group disease comprises: brain colloid inflammation, Alzheimer, hepatopathy, brain injury, neural wound (as spinal trauma), neuratrophia, myocardial infarction, arteriosclerosis, fibre modification hepatopathy and fibre modification pneumonopathy and parkinson disease.

The present invention also provides a kind of polypeptide formulations, its composition is one or more tetrapeptide molecules, these tetrapeptide molecules are selected from the tetrapeptide molecule of following amino acid sequences: (1) KEVD (this tetrapeptide molecule is available from typical mice and human C/EBP β sequence), (2) KphosphoTVD (available from typical mice and human C/EBP β sequence), (3) KKPD (available from typicality rat C/EBP β sequence), and (4) KKPphosphos (available from typicality rat C/EBP β sequence), here the sequence of being said at least with preceding cysteinyl aspartic acid specific protease Procaspase1 and Procaspase8 in a kind of the combination, this combination then reduces the activation of Procaspase.And the polypeptide that relates to here in most cases contains 20 aminoacid.

The present invention further provides a kind of polypeptide formulations, contain sequential coding 3 (SEQ IDNO:3) in this constituent, this becomes the prominent alanine (SEQ ID NO:3) that becomes corresponding to 217 last threonine (wild type is SEQ ID NO:2) in the murine C/EBP β series.Polypeptide provided by the invention also contains SEQ ID NO:4, corresponding to 217 sudden changes from threonine (wild type is SEQ ID NO:2) to glutamic acid in the sequence.Polypeptide provided by the invention also contains SEQ IDNO:14, this rat CCAAT enhancer binding protein β (C/EBP β) corresponding to modified contains from sequence 105 silk ribbon propylhomoserin (wild type is numbered SEQ ID NO:13) sudden change and the alanine (SEQ ID NO:14) that comes.Polypeptide of the present invention also contains sequential coding 15, and promptly aspartic acid is the silk ribbon propylhomoserin on 105 from rat C/EBP β sequence (wild type is numbered SEQ ID NO:13).Polypeptide provided by the invention also contains following one or more sequences, comprising SEQ IDNO:7,10,20 and 24, represented 266 threonine (wild type from human C/EBP β series respectively, be numbered SEQ ID NO:6,9,19,23) suddenly change and next alanine (SEQ ID NO:7,10,20,24).In addition, polypeptide of the present invention also contains one or more aminoacid sequences, and SEQ ID NO:8 is wherein arranged, SEQ ID NO:11, SEQ IDNO:21, SEQ ID NO:25, represented the threonine (wild type on 266 from human C/EBP β respectively, be numbered SEQ ID NO:6,9,19,23) suddenly change and next glutamic acid (SEQ ID NO:8,11,21,25).

Method and formulation provided by the invention also is applicable to activation and/or the hypertrophy that suppresses cell.These method and formulations can be preferably applied in and suppress Astrocytic activation of liver and/or hypertrophy.The present invention especially preferentially provides a kind of method, can adopt the C/EBP β sequence that contains 217 site mutations (becoming alanine (Ala) from threonine (Thr)) whereby.In addition, the activation process that causes these two kinds of enzymes is subjected to press down thereby endogenous phosphatase and cysteinyl aspartic acid specific protease Caspase1 and Caspase8 link.But, thereby when having experienced, peptide chain suddenlys change after alanine occurring on its 217 site, and this peptide chain just can be competed with the wild type peptide chain, thereby impels the Caspase activation, and causes apoptosis.

The invention provides C/EBP β peptide class preparation through the modification change from different plant species.Under some situation, murine C/EBP β peptide class preferential generation on its 217 suddenlyd change.At this time, the threonine in last 217 sites of Muridae wild type C/EBP β (SEQ ID NO:2) is replaced by alanine (SEQ ID NO:3) sometimes, but also can be replaced as glutamic acid (SEQ ID NO:4) sometimes.The sudden change that takes place on the rat C/EBP β peptide chain then is common in No. 105 silk ribbon propylhomoserins (SEQ ID NO:10) on the amino acid sites and is replaced as alanine (SEQ ID NO:11) sometimes, then is replaced as aspartic acid (SEQ ID NO:12) sometimes.And the sudden change Chang Youxian on the human C/EBP β peptide chain occurs on its No. 266 amino acid sites.When on the human wild type C/EBP β peptide chain undergo mutation in 266 sites, (numbering is respectively SEQ IDNO:6 to threonine on this site, SEQ ID NO:9, SEQ ID NO:19, with SEQ ID NO:23) sometimes by alanine (SEQ ID NO:7, SEQID NO:10, SEQ ID NO:20 and SEQ ID NO:24) displacement, but also may be replaced as glutamic acid (SEQ IDNO:8,11,21,25).

The present invention also notices the sudden change on the C/EBP β peptide chain that occurs in other species.To unique requirement of the C/EBP β peptide chain of these modifieds is that the modification of these peptide chains changes and wants to induce or to prevent that apoptosis from occurring in those interested cells, comprises in cancerous cell and the tissue.And it is worth noting that especially apoptotic bringing out often causes reducing (promptly dwindling), eliminate and/or prevent the fibre modification of cells of interest or tissue.Further it should be noted that this apoptotic normal minimizing and/or the elimination that can cause cancerous cell of bringing out, and/or reduce or prevent cancer cell metastasis.What another merited attention then is that the C/EBP β peptide chain after the change also can be used for bringing out fibre modification (for example promoting wound healing).

In some specific occasions, the invention provides CCAAT/ enhancer binding protein, and this albumen can be induced the generation apoptosis through transforming.These CCAAT/ enhancer binding proteins preferentially are selected from one group through transformed human CCAAT/ enhancer binding protein, and through transformed mice CCAAT/ enhancer binding protein.And more common situation is, has often selected next group aminoacid coding in these protein-bonded specific amino acids sequences, comprising SEQ ID NO:3,4,7,8,11,20,21,24 and 25.

The present invention also provides the method for cell death inducing, promptly will have at least a CCAAT/ enhancer binding protein through modification by a plurality of steps and applies at least a cell.Here, the cell that comparatively preferentially relates to is a kind of Interstitial cell.The cell that further relates to then is to be selected from such one group of cell, comprises liver, lung, kidney, skin, muscle, heart, neuroglia, the cell of organs and tissues such as eye and blood vessel.Some special preferential range of choice of the present invention is by the enforcement of said method, can prevent fibre modification.And another kind of preoption is, the method for above-mentioned cell death inducing has been used to the apoptosis of inducing tumor cell.

The method of cell death inducing provided by the invention is under certain conditions, and aforementioned CCAAT/ enhancer binding protein is used for handling a certain experiment main body, and the endogenous phosphorylation peptide chain molecule of this main body can suppress a kind of caspase of main body at least thus.In some preferred range of choice, the enforcement of this method can cause the apoptosis of selected cell in the relative subject.Some preferential especially range of choice of the present invention is that the enforcement of said method will cause the apoptosis of the tumor cell in the relative subject.

The present invention further provides the method for inducing cell, the composition of this method is: adopt at least by certain part of the CCAAT/ enhancer binding protein of modified, and it is applied at least a cell, the CCAAT/ enhancer binding protein by modified that relates to here then is selected from one group by the mice of modified, rat and human CCAAT/ enhancer binding protein.In application scenario more preferably, the mutational site on the mice CCAAT/ enhancer binding protein sequence of modified mistake is the 217th amino acids, and this 217 site mutation aminoacid then is selected from one group of aminoacid being made up of alanine and glutamic acid.Another preferential concrete Application Example is that it is encoded to SEQ ID NO:3 and SEQ ID NO:4 some aminoacid sequence that the mice CCAAT/ enhancer binding protein of modified mistake contains.In addition, the amino acid mutation site that the human CCAAT/ enhancer binding protein of modified mistake contains is the 266th in its sequence, and here, the aminoacid on 266 sites is to be selected from one group of aminoacid that alanine and glutamic acid are formed.And some aminoacid sequence that the human CCAAT/ enhancer binding protein of modified mistake contains be encoded to SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:11.Yet the mutational site that the rat CCAAT/ enhancer binding protein of modified mistake contains is at the 105th of its sequence, aminoacid on these 105 is the one group of aminoacid that is selected from alanine and aspartic acid composition, and its sequential coding is SEQ ID NO:14 and SEQ IDNO:15.More preferably the instantiation of Xuan Zeing is, the apoptosis that the present invention relates to is induced generation at least in a kind of cell, and this cell just is selected from a kind of in following one group of cell, this group cell comprises: liver, heart, lung, kidney, eye, nerve, muscle, epithelium, endothelium, the cell of organs and tissues such as a matter and skin.Other instantiations of more preferably selecting are to comprise that any type cancerous cell also can be induced the generation apoptosis.The application example of higher preference is that above-mentioned cell all is present in a certain main body.The example of further preferentially selecting for use is that this main body is meant a kind of mammal.And preferentially especially in mammal specify a certain human body.And related here human body is meant to suffer from the diseases related people of fibre modification, is meant one group of following disease and the fibre modification here is diseases related, comprises hepatopathy, brain injury, myocardial infarction, arteriosclerosis, the eyeball fibre modification, fibre modification dermatoses, and fibre modification pneumonopathy.Further concrete example is, above-mentioned apoptosis inducing method has been used for the apoptosis of inducing tumor cell.

The present invention also provides the fibrotic method of inducing, this method comprises: adopt a kind of by the part of the CCAAT/ enhancer binding protein of modified at least, it is acted at least a tissue, here said CCAAT/ enhancer binding protein by modified is meant a kind of in the CCAAT/ enhancer binding protein of one group of modified, and these are conjugated protein then to stem from mice, rat and the mankind.In some preferential example of selecting is implemented, state in the use before the CCAAT/ enhancer binding protein, have at least a kind of selected tissue to show that its wound healing process suffers damage.And in some application example,, the wound healing process of at least a tissue is improved owing to adopt this method to induce fibre modification.In the preferential especially application example of selecting, its amino acid sequence encode of CCAAT/ enhancer binding protein of above-mentioned modified mistake shows it is SEQ ID NO:4.

The present invention also provides the method that suppresses tumor cell proliferation.In some preferential application example of selecting, method of the present invention has been used for those and has had on one's body the main body or patient of chemotherapy and/or radiotherapy tolerance cell.Under certain conditions, adopt the C/EBP β peptide quasi-molecule of sudden change to produce inducing action, thereby successfully treat and/or prevent tumor growth the apoptotic process of specific tumor cell.It is considered herein that by inducing apoptosis of tumour cell and can reach control tumor growth and prevention or suppress neoplasm metastasis.

Definition

For helping the understanding of the present invention, will be defined as follows following term:

The term " matter " that this paper adopts is to produce connective tissue, and produces muscle, hemocyte and vascular tissue, the germinal layer tissue with origin feature of epithelial tissue and some gland tissue.

The term " gene " that this paper adopts is meant the deoxyribonucleotide sequence, the coding region of containing a structural gene in this sequence, comprise that simultaneously other sequences are positioned at 5 ' and 3 ' end of this gene coding region respectively in abutting connection with ground, and can extend to thousands of base pairs respectively, this gene is corresponding to the length of overall length Messenger RNA thus.The sequence that is arranged in coding region 5 ' end also can correspondingly appear at Messenger RNA, is called as 5 ' one end non-translated sequence.The sequence that is positioned at gene coding region 3 ' end or catchment then is called as 3 ' end non-translated sequence when it appears on the Messenger RNA.The genome structure or the clone of a gene include coded sequence, are called exon, and non-coding sequence then is called " intron " or is called " interleaving the district " or " intervening sequence ".Part fragment in gene order of intron, these fragments are transcribed into heterogeneous nuclear RNA (hnRNA).Intron might contain regulating element such as enhancer.Intron is often by from the nuclear transcript or primary transcript is removed or by " shearing is gone out "; Thereby intron does not appear in Messenger RNA (Mrna) transcript.Except containing intron, may comprise some sequences in the genome structure of a gene, these sequences do not occur in the rna transcription thing yet, and are positioned at 5 ' and 3 ' end of the corresponding gene sequences of these rna transcription things.These sequences of not transcribed are called " side " sequence or zone (these flanking sequences are positioned at 5 ' and 3 ' end of that non-translated sequence on the mRNA transcript).The 5 ' flanking region here might comprise regulates sequence for example promoter and enhancer, and these regulate sequence will produce regulation and control or influence to gene transcription.The 3 ' side areas here then visitor can contain some sequences, and these sequences might cause the termination of transcription, cracking and polyadenylic acidization after transcribing.

Term used herein " coding region " is meant those corresponding amino acid whose nucleotide sequences of encoding when it is used to represent a structural gene, it is on the masterplate nascent polypeptide chain that carries out producing behind the protein translation that the aminoacid here is present in mRNA.In eukaryotic cell, 5 ' end of aforesaid coding region is connected with a kind of nucleotide triplet " ATG ", the initial son of this triplet coding methionine, 3 ' end in the coding region then be connected with three kinds of termination codoies (be TAA, TAG, a kind of in TGA).

Here said " structural gene " is meant that an end is the aminoacid sequence of RNA or a kind of protein coding.On the contrary, " regulator gene " then is another structural gene, and the product (as transcription factor) of these regulator gene codings then is responsible for other expression of gene of regulation and control.

Term used herein " regulating element " is meant a kind of gene element, and some position that this element is expressed nucleotide sequence has regulating and controlling effect.For example, a kind of promoter just can be used as a kind of regulating element and can help transcribing of coding region that a kind of operability of starting connects.The other regulating element is a splicing signal, polyadenylation signal, termination signal, enhancer element or the like.Promoter and enhancer constitute a kind of DNA sequence of weak point to be arranged, and relates to the single-minded interaction [Maniatis ef al, Science 236:1237 (1987)] of intracellular protein generation of transcription with this and those.Promoter is separated from different eukaryotic cells with enhancer element, and this comprises yeast, the gene of insecticide and mammalian cell and viral gene (similar controlling element＜be promoter also all in prokaryotic cell, find).The selection of a kind of specific promoter and enhancer depends on that the cell of which kind of type will be used to express protein of interest matter.Some eukaryotic promoter and enhancer have the wide spectrum host type, and the function performance of other promoteres and enhancer then can only show and be only limited to some cell subsets.[referring to summary: Voss et al., Trend Biochem.sci., 11:287 (1986); And Maniatis, et al., Science 236:1237 (1987)].For example, the early gene enhancer of SV40 (simian virus 40) has very strong activity in multiple mammalian cell types, has been widely used in protein expression [Dijkema.etal., EMBO.4:761 (1985)] simultaneously in mammiferous cell.Other express active example from human elongation factor Ia gene [Uetsuki et al., J.Biol.Chem, 264:5791 (1989) about promoter/enhancer element in the wide spectrum mammalian cell; Kimet al., Gene 91:217 (1990) and Mizushima and Nagata, Nucl.Acids.Res., 18:5322 (1990)] and the terminal repetition unit of length of rous sarcoma virus [Gorman et al.,

Proc.Natl.Acad.Sci.USA79:6777 (1982)] and human cytomegalovirus's the terminal repetition unit of length [Boshartet al., Cell41:521 (1985)].

" promoter element " or " promoter " is used for representing the section of DNA sequence, 5 ' of a certain gene end (being front end) and provide an initiation site to mRNA for genetic transcription in this sequence bit DNA polymer.

" interested gene " and " interested nucleotide sequence " represents any gene or nucleotide respectively, can control and obtain ideal results to this gene or sequence for certain reason can adopt the routine techniques of document record.

" expression vector " represents a kind of dna molecular of reorganization here, this dna molecular contains a kind of ideal coded sequence and suitable nucleotide sequence, and this sequence is that to control the coded sequence that is connected be essential in the intravital expression of a specific host machine for carrying out the people.In prokaryote, the necessary for gene expression nucleotide sequence comprises a kind of promoter, a kind of operon sequence of nonessential selection and a kind of ribosome binding site, and other possible sequences.And in eukaryotic cell, known for the necessary for gene expression nucleotide sequence comprises promoter, enhancer is with termination signal and polyadenylation signal.

" modification " vocabulary shows the change to a kind of any structure of nucleotide sequence.Change on the nucleic acid structure comprises the change of covalent bond and non-covalent bond on the nucleic acid chains.These variations can show as sudden change, mismatch, and chain interruption, and between the nucleotide sequence, and covalency and noncovalent interaction (the nucleotide sequence here comprises unmodified and/or adorned nucleotide sequence) between nucleic acid and other molecules.The covalent effect that reaches between nucleotide sequence and other molecules between the nucleotide sequence can bring variation (as forming a kind of thymine glycol) to the base of a nucleotide, in addition, also can produce the covalency interconnection between the double chain DNA sequence, for example the effect by ultraviolet and suitable handkerchief can cause this situation to take place.In addition, covalent interaction also can occur between nucleotide sequence and other molecules, for example can two nucleotide sequences be covalently bound on the psoralen molecule by action of ultraviolet ray.Noncovalent interaction between nucleotide sequence and the another kind of molecule comprises the noncovalent interaction of the molecule of nucleotide sequence and a kind of non-nucleic acid, and the noncovalent interaction between the molecule of nucleotide sequence and another kind of non-polypeptide.Noncovalent interaction between the molecule of nucleotide sequence and non-nucleic acid and non-polypeptide shows the noncovalent interaction between double chain DNA fragment and ethidium bromide or the psoralen.It is considered herein that those modifications that change functional character also usually can change the form of expression of these functional moleculars.

" allelic series " is used for representing the wild-type sequence of gene in this article." modification of allelic series " then represents the two or more nucleotide sequence of a certain gene, and in this two or more nucleotide sequence, each sequence all exists a kind of decorative features at least with respect to the nucleotide sequence of its wild type gene.

" sudden change " is in this expression disappearance, insertion or replacement." disappearance " speech is defined as by losing the nucleotide sequence that causes behind one or more nucleotide and changes." insertion " or " interpolation " then is illustrated in the nucleotide sequence has increased one or more nucleotide." replacement " represents that then one or more nucleotide of wild-type nucleic acid sequence is replaced as different nucleotide respectively.For example thymus pyrimidine is replaced as cytosine, gland fast cry of certain animals, guanine or uracil.Perhaps a kind of nucleic acid is replaced by the nucleic acid of modified, and for example thymus pyrimidine is replaced by thymine glycol.

" mismatch " vocabulary and show the noncovalent interaction that does not carry out between two nucleic acid chains, thereby these two chains all have been attached on the wrong nucleotide sequence separately according to basepairing rule.Occurred the complementary sequence of part thus, between 5 '-AGT-3 ' and 5 '-AAT-3 ', G-A occurred and mismatched.

" nucleic acid " and " unmodified nucleic acid " a kind of (being guanine, the fast cry of certain animals of gland, cytosine and thymus pyrimidine) in four kinds of DNA (deoxyribonucleic acid) bases of this expression." modification of nucleic acids " then represented with respect to unmodified nucleic acid, the nucleic acid that its structure has changed.And the example of this modification might comprise the displacement covalent modification of base, as alkanisation amino and ring nitrogen, and saturation of double bonds.

" modification sexual cell " is meant in the genome sequence of this cell and contains a kind of modification at least.

" nucleotide sequence-dressing agent " is meant that those can make a kind of nucleotide sequence produce the reagent of at least a modification. [for example:N-ethyl-N-nitrourea (ENU) includes but are not limited to chemicals, methyl Asia nitrourea (MNU), preceding kappa Ding Which acid (PRC), triethylene melamine (TEM), acrylamide Single Body (AA), benzoic acid chlormethine (CHL), alkeran (MLP), Ring phosphamide (CPP), diethyl sulphuric acid (DES), ethyl methane sulfonate (EMS), methylmethane sulphonic acid ester (MMS), 6-sulfenyl purine (6MP); Silk rimocidin-c (MMC); Preceding Long Ba Ding (PRC), N-methyl-N '-nitro-N-Asia nitroguanidine (MNNG), heavy water (³H ₂O), and urethane (UR)], and electromagnetic radiation (for example, x-ray radiation, gamma-radiation, and ultraviolet radiation).

" wild type " speech is meant the gene that natural conditions exist when being used for gene is described.The frequency of occurrences of this gene in a colony is the highest, therefore is confirmed as " normally " or " wild type " structure of gene.In contrast, " modified " or " sudden change " represents that then change (being alteration of form) has taken place for gene or gene outcome on its sequence and/or functional characteristic, and this change is for wild type gene or its gene outcome.Noticed to exist the sudden change that takes place under the natural conditions, the existing fact has confirmed this point.The present invention confirms that in some preferential application example of selecting modified gene outcome can be induced really and be produced apoptosis or fibre modification.Therefore in further preferably specifically using, modified C/EBP β provided by the invention can be suitable for inducing the fibre modification tissue to produce apoptosis, thus, fibrotic negative consequence has been produced prevention, alleviates and the elimination effect.Further example is that modified C/EBP β also can be used for inducing fibre modification, thus, has obtained promotion in fibrotic process (as the wound healing process) since those.

" anomaly of β-enhancer binding protein (C/EBP β) " is used in this article defining and a kind ofly compares with C/EBP β wild-type sequence, has one or more aminoacid the C/EBP β of difference to occur in its aminoacid sequence.The anomaly of C/EBP β also comprises the protein and the polypeptide of " modification property C/EBP β ".Under some actual conditions, perhaps variability polypeptide (i.e. modification property polypeptide) has conservative changes, and promptly in this variation, some substitute aminoacid also has similar structure or chemical characteristic (replacing mutually with isoleucine as leucine).Still less the situation of Fa Shenging is, a kind of variability (i.e. modification property) polypeptide has " non-conservation " and changes (as the exchange between glycine and the tryptophan).Similarly secondary variance comprises amino acid whose disappearance or insertion (promptly adding), or both exist simultaneously.Can which with how many amino acid residues can be replaced, insert or lose and not lose its original biology and immunocompetence by known computer program help decision, as DNAstar software.In some preferential especially occasion, the threonine on 127 sites of wild-type mice C/EBP β can be replaced by a kind of alanine or glutamic acid.In further specifically implementing, the silk ribbon propylhomoserin in the 105th site of rat wild type C/EBP β can be replaced by a kind of alanine or aspartic acid.And further, then available alanine or glutamic acid are replaced the threonine on human wild type C/EBP β the 266th site.

" tumor " and " tumor " are meant compares the tissue with fast growth feature with control tissue.Tumor might be benign, but is not limited thereto, and for example, tumor comprises hemangioma, and neuroglia is processed tumor, abnormal born of the same parents' tumor etc.

Tumor also may be transformed into virulent, as a kind of carcinoma, and sarcoma, refreshing The Jiao Quality is because of Fine born of the same parents' tumor, star Fine born of the same parents tumor, neuroblastoma ， Visual Net film is because of Fine born of the same parents' tumor, or the like.

The Evil tumor " and " tumor cancerates " be meant a kind of tumor that contains a kind of cancerous cell at least. " cancerous cell " is meant the cell that has experienced multistep neoplasia such as early; Middle and late phase development.With the microscope image description the early; Middle and late phase feature of this neoplasm development.Cancerous cell comprises displacement in these three equal ubiquity Yi of period of expansion Chang Ran Se Body Group types; Is inverted; And disappearance; Heterochromosome, monomer and chromosome increase.Cancerous cell comprises that " increasing germ cell " promptly is in the early stage cell of development that cancerates, and " Hair educates undesirable cell " promptly is in the neoplasia development cell in mid-term, and the neoplasia cell then is meant the cell in late period of locating to form in development.The neoplasia cell has typical invasive, and promptly these cells both can be invaded adjacent tissue, also can original position come off, and then along with blood and lymph metastasis arrive other position, and begins to form one or more secondary cancers at new position, and this is called as " transfer " .Therefore, " cancer " represents a kind of tumor that cancerates here, and this tumor that cancerates may be metastatic, and also the possibility right and wrong are metastatic.

The present invention does not look to being only limited to cancer or the cancerous cell at a certain particular type.Can go the malignant tumor for the treatment of and identifying with method provided by the invention, comprise a big carcinoid, pulmonary carcinoma for example, breast carcinoma, carcinoma of prostate, cervical cancer, cancer of pancreas, rectal cancer, ovarian cancer, gastric cancer, the esophageal carcinoma, oral cancer, carcinoma of tongue, the dental bed cancer, skin carcinoma (as melanoma, the stromal cell cancer, Kaposi's sarcoma etc.), muscle cancer, heart cancer, hepatocarcinoma, bronchogenic carcinoma, chrondrocarcinoma, osteocarcinoma, carcinoma of testis, renal carcinoma, endometrial carcinomas, uterus carcinoma, bladder cancer, bone marrow cancer, lymphoma, spleen cancer, thymic carcinoma, thyroid carcinoma, the brain cancer, neurocyte Ai ， Inter rind gall, carcinoma of gallbladder, eye cancer (as the cornea cancer, ommochrome Layer cancer Veins Network film cancer, speckle cancer, Bo Li Body liquid cancer etc.) arthrocarcinoma (for example synovial fluid cancer) Cheng Jiao Quality Fine born of the same parents cancer, lymphatic cancer and leukemia, sarcoma (for example osteosarcoma and Kaposi's sarcoma) is the another kind of type of malignant tumor.

" suspection has " speech is used for clinical or pathological condition at a certain study subject (as cancer, fibre modification etc.) time, being meant that a patient or object of study are diagnosed as has certain clinical or pathological symptom, these patients or main body are all gone out to have positive reaction in the one or many diagnostic test, thereby under a cloud have some specific clinical or pathological symptom, (as is exposed to some Environmental Chemistry factor, way of act and the appearance of these diseases may relate to some reason, genetic predisposition, etc.).

" Right cancer susceptible " is meant that a main body or patient have one or more carcinogenic danger factors, accepted treatment of cancer in the recent period, or once diagnosed out cancer, is in Slow now and separates the phase.

" behind the cancer resection surgery " is that expression is excised back one period with the cancerous tissue of performing the operation patient.

" cancer return " is meant that patient is just experiencing the recurrence of cancer, or because the recurrence of the disease that cancer causes.This recurrence is the recurrence (the cancer Fine born of the same parents of previous Class type before promptly relating to) of cancer of the same type, and inferior Grade or Jin deutero-cancer of one step, relates to the cancerous cell of different Class types.

" biological system Elixirs " be meant Yi Seed Zai Broken and imaging side mask the Try Elixirs of some purposes is arranged, and The Try Elixirs can act on Yu Yi Seed Fine born of the same parents, Qi Guan Huo Machine Body ， comprises but does not limit what Drug thing (as Drug Elixirs) to remove a little cells of Guide Zhi that the Gong Neng Productivity of Qi Guan Huo Machine Body gives birth to Changeization.

" Specifications originally " the general De Han of Yong Zai Li Ju You Wide Righteousness, it can be used to represent that Yi Seed controls Treatment De System Elixirs with what, also can represent this Huo of Standard Pei Raising thing in Mou Seed Come source.Biological Specifications originally might take from Move thing (comprising people Class) also Bao Kuo Group Woven (such as this) of Huo Inspection Specifications, Ye Body, Gu Body, Group Woven with Gas Body.Biological Specifications originally also comprises Xue Productivity product, as Xue Pulp, serum or the like.Specifications that the bright Should of this Hair of Dan uses this but be not limited only to the example here).

" chemotherapeutics " represents to suppress the growth of neoplasia cell pathology microbial cell here, or reduces its survival rate, or suppresses the preparation of virus amplification (this and only limit to duplicate, assembling or cell infection).The chemical compound of the present invention's definition comprises the purification product of natural prodcuts, also comprises synthetic and semi-synthetic chemical compound.Therefore, chemical compound provided by the invention is not limited in a certain particular production method or a certain particular source.

" chemical compound " represents any chemical entities, medicament, medicine, and can be used for treating or preventing the material of certain disease and body function imbalance.Chemical compound comprises known simultaneously and the chemical compound of potential therapeutical effect is arranged.Whether a kind of chemical compound has therapeutical effect can be selected by method provided by the invention.A kind of known therapeutic compound " effect is proved (for example by zoopery or the experience used in the human body in the past) to a kind of therapeutic compound of expression to treatment of diseases.In other words, a kind of known therapeutic compound is not limited in when the treatment cancer effective.

" test compound " is meant any chemical compound entity, preparation, and medicine, and can be used for treating and prevent material after certain disease and the body function imbalance.Test compound comprises known simultaneously and has the chemical compound of potential therapeutical effect.Whether a kind of test compound has therapeutical effect can be screened and determine via method provided by the invention.A kind of " known therapeutic compound " is that the treatment and the preventive effect of a kind of therapeutic compound of expression has been proved to be effectively (for example by zoopery or the previous experience of using) in human body.In other words, a kind of known therapeutic compound is not limited in and can treats fibre modification with being effective in.

When a kind of chemical compound might be by any ideal approach (as mouth, vein, subcutaneous, muscle etc.) be applied to a kind of mammal, and when this chemical compound or its active metabolite appeared in the mammiferous blood, this chemical compound should be to be fit to be applied to mammiferous form and to exist with a kind of.With the female mammal time-like of a kind of compound administration, might cause this chemical compound to be passed in the individual fetus body of gestation in pregnancy.

A kind of XXX used herein, the preparation that has comprised a kind of specific polymerized nucleoside acid sequence is used to broadly represent any preparation that contains specific polymerized nucleoside acid sequence.This preparation might contain a kind of aqueous solvent.It is human to contain coding, mice and or rat C/EBP β or the segmental polymerized nucleoside of its part

Acid sequence might be used as a kind of preparation of hybridizing pin that includes.In this application scenario, the poly nuclear former times acid that contains C/EBP β one coded sequence will typically be dissolved in a kind of salt (as sodium chloride Nacl) that contains, detergent (as sodium lauryl sulphate) and other chemical compound (as denhardt solution, drying powdered milk, salmon sperm dna etc.).

" pharmacological preparation " used herein is any preparation or the chemical compound that is fit to be used for treating and preventing the disease of a certain study subject of expression.This paper thinks that this preparation comprises the medicine of can effectively alleviate or eliminate a disease sign and symptom.The present invention is only limited at any specific dosage or application strategies.It is considered herein that this " pharmaceutical formulations " can adopt various using method, comprise, but be not limited to these drug treating schemes, in use promptly, a kind of pharmaceutical formulations that merges use will be by each different pharmaceutical carrier (as the independently property of medicine, pill, medicinal liquid etc. separately).

" dose therapeutically effective " is meant that therapeutic agent can suppress tumor cell growth, or reduces required dosage of its when survival term used herein.

It is used herein that " inferior therapeutic dose " is meant employed a kind of drug concentration or dosage when not expecting to destroy cell fully.

This paper uses " anti-hypertrophy " speech to represent that a kind of therapeutic agent can produce the effect that suppresses or stop to paracytic growth, and necessity is killed it (though it also may kill it).

This paper represents those can cause cell death in the process of curing the disease therapeutic agent with " cell toxicant " medicine or reagent.In some preferential application, cell toxicity medicament is effective especially to rapid splitted cell.

" IC50 " speech is used for expression at this paper and reaches 50% Cytotoxic concentration, i.e. half cell toxicant concentration.Cytotoxicity can adopt the bibliographical information method to add mensuration.List of references is: Alleg etal, and cancer Res, 48:589-601,1988, or Scudiero efal, Cancer Res, 48:4827,1988, or by known suitable method in any document.In a certain concrete application, Cytotoxic mensuration is based on to be observed the mitochondrion acid activity and is reduced to the used drug level of a half.

The animal of any disease development of any possibility represented in this article in " main body " speech, will need (for certain reason) to use method provided by the invention when the employing routine techniques is determined the development of these diseases.In some preferential concrete application, this main body can be any live animal that can accept certain pharmaceutical preparation treatment processing.Under the preferential situation, this main body is a kind of mammal, occasion more preferably, mammal includes, but are not limited to human and inhuman animal such as ape and monkey Class, Rodents Cotton sheep Class, cattle Class ， anti-Chu Class, rabbit Class, pig, goat Class, horse Class Quan Tooth Move thing, the non-human animal that cat family Move Wu ， Birds Class isopreference is selected is some rodent (as mice and a rat).Therefore, chemical compound provided by the invention might be adopted by human health professional and veterinary work personnel.

" hepatitis C " one bragged and is used for representing hepatitis C virus in this article, and this is a kind of single strand RNA virus, and it has resiniferous encapsulation, and is considered to one of member of Flavivirus family.This has comprised various forms of hepatitis C, and acute hepatitis C and various forms of chronic hepatitis c (as chronic active hepatitis and Man Stubborn solidity hepatitis) are wherein arranged.

" hepatitis C symptom " makes a general reference clinical manifestation at this paper, laboratory and image result, and show liver form and tissue pool on one's body with hepatitis C feature in main body.Clinical manifestation may include, but are not limited to, stomachache, jaundice, the big and ascites of Gan Pi Swollen.Experiment and image result can include, but not limited to serum changes plain rising of chloric acid rising ， Bile Red and the rising of gal Mary globulin levels, and at computer picture, it is big that the liver that shows in the magnetic resonance image (MRI) regulating liver-QI ultrasonic image is wanted.The liver form of hepatitis C and histology's shelves are marked and can be comprised, but are not limited to, and find to have the evidence of deposition of fibrous tissue in the hepatic tissue biopsy.

" fibre modification or fibrosis " speech is used for herein representing that the fibrous tissue that a kind of compensatory or reactive process causes forms, this situation betides after wound, inflammation and/or infecting, normal structure is substituted, form the process of scar tissue from face, in some cases, fibroid one speech represents that also normal smooth muscle is replaced by the fibrosis connective tissue.The fibre modification process can occur in any tissue, and its effect can be related to part or extensive region more.Local fibre modification may contain and infraction, abscess, a Gas Guan Expansion Zhang, and/or other pathological symptoms.

" apoptosis " vocabulary shows cell death.Be meant the programmed cell death that is different from the necrocytosis process especially.In this apoptotic process, cell can experience a series of variations, and finally causing cell degradation is the apoptosis body, and then this apoptosis body carries out classical engulfing by other cells.There is bibliographical information to point out, reduces level of apoptosis and can cause cell survival, must (though its possibility is arranged) not promote hyperplasia.The available known literature method of apoptotic mensuration carries out.For example, identify apoptotic process by the amount of measuring the V-type annexin that combines with the green propylhomoserin of the phosphoric acid that is present in the cell serous coat.The increase of V-type annexin amount is an early stage index (the Rudel And Bokoch of apoptosis, the front is addressed) (seeing the C group among Fig. 1), this can divide Election (FACS) technology to observe green by microscope viviperception (see Fig. 1 C group) or Firefly light Pay competent cell and be full of dark brownish green albumen, the growth and decline relation between this transfection index and the annexin V.In addition, mention, also can carry out nucleus and dye the identification of cell apoptosis by what Chester (Hoechsf 33342) dyeing as this paper.

Phrase " fibrosis symptom index " is to be used for representing to be present in the fibre modification in arbitrary tissue and morphology that has and/or histological indices.In preferential especially example, " hepatic fibrosis " is meant form and/or the histological indices that relates to hepatic fibrosis.These indexs include but not limited to, the fibrosis tissue deposition evidence of finding in the liver biopsy, form with the activatory seriality fiber of finding that raises by benzene two acyl dialdehyde-adducts (MDA-adducts), ， is Ji (the enhancing expression of RNA in star albumen of c-myb and X1 (I) type collagen protein of Birds pulpefaction Fine born of the same parents leucovirus oncogene in the spider cell activation.

" treatment preparation " represents a kind of like this preparation in this article, be that its contained chemical compound can exist with a kind of form of accepting from the materia medica viewpoint, simultaneously can prevent and/or reduce fibre modification again, this fibre modification includes but are not limited to hepatic fibrosis, the dosage form feature of therapeutic preparation will arrive and depend on multiple factor, and this comprises the mode of administration of therapeutic agent.Might contain dilution Liu in this therapeutic agent, adjuvant and shape agent, and other compositions.

" non-intestinal carries out ground " is used for expression during to treatment main body medication in this article, medicine use be by except that gastrointestinal tract or lung by way of carrying out.Non-enterally administer commonly used is drawn together but in the Bu Xian Yu Static arteries and veins by way of Bao, intramuscular and subcutaneous tissue.

" therapeutic dose " and " effective dose ", reaching similar terms is to be used for a kind of like this dosage of expression, when promptly using this dosage, a kind of chemical compound or formulation can prevent successfully that fibrotic symptom from taking place, and/or alleviate its symptom.A kind of effective dose for the treatment of preparation may be by multiple factor decision, and these factors comprise that age, immune state, the race and sex of main body is other, with the Fibrotic order of severity and other factors relevant with biological variability.

" a kind of genome that can be used for genetic engineering procedure among the non-human animal " is that the present invention is specially-made by non-human animal's system genitale genome is advanced experimental implementation, these genetic engineering non-human animal also permits by diverse ways and produces, contracted affreightment with a kind of " transgenic " even the method for technology nucleic acid (normally DNA) by the artificial interference addressed as this paper imports a kind of non-human animal embryo's target cell and/or the somatic cell that whole Yu advances the non-human animal and/or the chromosome of system genitale cell with it.Contain a kind of genetically modified non-human animal and be called " transgenic animal ".Transgenic animal are exactly the animal that those its gene organizations are used the transgenic technology change.

" transgenic " is that expression has been inserted a kind of organism with a kind of exogenous gene.This is to obtain after this external source gene is imported a kind of new germ cell or body early embryo." exogenous gene " expression any one we operate by experiment be imported into a certain animal genomic nucleic acid (for example gene order) this comprise that also those find that in animal the gene under the plucked instrument natural conditions compares, exist the site that the gene order of variation has taken place.

" the consolidation type C/EBP β albumen that non-human mammal is expressed " represents that with being used for a kind of express at non-human mammal comparing with wild type produced the C/EBP β albumen that makes a variation herein.The mammal of so-called " forfeiture produces functional C/EBP β ability " is meant that its C/EBP β generation is in inspection and does not go out mammal on the level (being the background level that the generation of C/EBP β surpasses used detection method).Functional C/EBP β albumen is a kind of like this C/EBP β, and its functional character is identical with wild type C/EBP β, and molecular weight is also identical.The functional activity fragment of human C/EBP β can show the identical functions with its total length C/EBP β in some way in external and/or body." activity on the function " also comprises and the relevant normal activity of C/EBP β albumen.

" change " " remodeling " and speech such as " modifications " are used for describing a kind of biochemistry; biology or clinical picture are (as the apoptosis cell survival; cell proliferation; canceration; fibre modification; the change of preceding cysteinyl-aspartic acid specific kinase activity and activation and similar phenomenon) time, be the qualitative change (promptly increase or reduce) of this phenomenon of expression level of living in.Typical apoptosis, survival, propagation, canceration, fibre modification, the mensuration of zymogen activity and activation levels can be by known literature method and method provided herein.

" increase "; the vocabulary that equates on " rising " and " inducing " and the Chinese language thereof; when being used for describing our biochemistry; biology or clinical picture are (as describing apoptosis, survival, propagation; canceration; fibre modification, the increase of preceding cysteinyl-aspartic acid specific kinase activity and activation and similar phenomena) time, be to represent that the variation of matter has taken place the previous level of this this phenomenon.And be more prone to represent a kind ofly thought that by any approved statistical analysis technique rising on the quantity levels of statistical significance is arranged.In preferential application scenario; a kind of enhancing phenomenon has appearred in the sample (as apoptosis; survival, propagation, canceration; fibre modification; before cysteinyl-aspartic acid specific kinase activity and/or its activatory enhancing) be that the same phenomenon that increases the relative comparison sample on the quantity of these phenomenons of purport is come, exceed 50% at least, or 10%; preferably can exceed 50% or 75%, even exceed 90% at least.

" reduction ", " prevention " " minimizing " " inhibition " and at phraseological synonym when being used for representing a kind of biochemistry, biology or clinical picture are (as apoptosis, survival, propagation, canceration, fibrosis, zymogen activity, the minimizing of proenzyme activation and similar phenomena) time, be meant qualitative (being subjective) minimizing or reduction of a kind of phenomenon previous level, tend to simultaneously to represent on the quantity of this phenomenon previous level (promptly objectively) minimizing or reduction again, the minimizing on this amount has the statistical significance that any statistical analysis technique is thought.Describe in a certain sample in some specific application scenario, a kind of phenomenon that has reduced (as apoptosis, survival, propagation, canceration, fibre modification, the reduction of aforementioned zymogen activity and/or its activation levels) promptly be meant compared to other samples, preferably compared to check sample, the quantitative variation that these phenomenons are taken place on previous level, at least reduced by 5% or 10%, even reduced or reduced 50% to 70%, up to having surpassed 90%.The unessential a certain phenomenon of the phenomenon that this level reduces lacks (promptly eliminate or get rid of) fully, though there is this possible.This gallbladder does not also require and is not limited to relate to eliminating fully of phenomenon yet.

" reduce " and " permissions " is that this phenomenon of expression level of living in might rise or living change when coming cheirognomy to the influencing of arbitrary phenomenon." do not raise " on the contrary and represent that then this phenomenon level of living in may reduce or not change.

For example this paper said " minimizing " is meant that fibre modification symptom or other symptoms alleviate to some extent on degree.The reduction of common this degree will show by objective indicator.For example, the liver biopsy sample before and after the medication is compared, can be pointed out the reduction of hepatic fibrosis degree.In addition, alleviating also of symptom can be represented by subjective index, sends out alleviating of stomachache.

" peptide ", " peptide sequence " " aminoacid sequence ", " polypeptide " and " peptide sequence " exchange use in this article, represent to pass through Tai Key or the covalently bound product of Qi Si Key by at least two aminoacid or its homologue with this.The preferential peptide matters of selecting of this paper contains 4 to 3000 aminoacid, further excellently makes 4 to 3000 aminoacid, however further preferential 4 to 500 aminoacid, next 4-100 aminoacid more preferably, 4-50,4-25,4-20,4-10 is until the peptide class of 4 amino acid lengths.The peptide class comprises propylhomoserin, polyamino acid or its homologue.What the peptide class also comprised normal theory contains peptide class about 2 or 20 usually.The peptide class also is commonly used to expression and contains about 20 to 50 amino acid whose peptides in the left and right sides.The peptide class also comprises from about about 50 to 3000 amino acid whose peptide.The aminoacid of formation peptide is the also right Fang aminoacid of left Fang aminoacid.A kind of peptide, polypeptide or protein may be synthetic, reorganization, or abiogenous.A kind of synthetic peptide generates (for example not being synthetic in vivo) in the external use manual method.

" " be used for here representing that cell changes physiology and the progressivity aspect the morphology that is showed at the experience cell division, cell cycle is common to be thought and can be divided into " interval ", " in earlier stage " " mid-term " " late period " and " latter stage " cell proliferation.In addition, also cell cycle called after " M (division stage), " S " (synthesis stage) ", " GO " " GI (gap phase 1) " and " GZ (gap phase 2) ".In addition, this cell cycle also comprises the transition period between above-mentioned each phase.The level of cell proliferation can be determined by the known references method.For example, cell can with Australia's Brdurd (Brdu), this Australia Brdurd can mix the DNA of somatoblast, can detect this process of mixing by the SABC method then.As described herein, proliferation index can calculate (referring to Fig. 3) by the percentage rate of the positive vesicle of Brdu-in the epithelial cell sum in the sample.In addition, the epithelial propagation level of mammals is determined tissue dyeing by using at the antibody of proliferating cell nuclear antigen.

When " be attached to,,,, on ", " with,,, link ", " with,,, interact ", when being used for describing a certain polypeptide of the present invention or other polypeptide (the green propylhomoserin kinases proenzyme 1 and 8 special), be certain interaction of being undertaken by non-covalent Http between expression two peptide species as methionine.The method of this paper explanation can be used to measure the combination between a peptide species and the above-mentioned proenzyme, and shown in Fig. 3 .C group, above-mentioned proenzyme 1 increases when C/EBP β mice is handled with carbon tetrachloride (CCl4) with being associated in mutually of C/EBP β and C/EBPB-PTHR217 to some extent with 8.

" specificity in conjunction with ", " bonded specificity " and phraseological equal vocabulary thereof are expression polypeptide priorities of the present invention when polypeptide that is used for describing this paper and first peptide species (as above-mentioned proenzyme 1 and 8).The effect of mutually combining of above-mentioned first peptide species, this is with for the polypeptide above-mentioned first peptide species mutually combines with respect to polypeptide of the present invention.The specificity combination is relative, it does not also require absolute specificity combination, in other words, " specificity in conjunction with " also do not require polypeptide of the present invention, and be different under the condition that mutually combines between second peptide species of first peptide species and also will mutually combine with first peptide species.On the contrary, come when higher (exceed 10% at least, preferably exceed 20% or further exceed 50% at least) when the level of mutually combining of polypeptide of the present invention and above-mentioned first peptide species so that exceed 75%, until exceeding 90%.Polypeptide of the present invention is to depend on the unique texture that is present on polypeptide of the present invention and/or above-mentioned first peptide species with this combination of " specificity combines " prompting of last first peptide species; In other words, above-mentioned first peptide species can be discerned and be attached on the unique texture of peptide more than the present invention, rather than in common polypeptide or the basic acid.For example, suppose that polypeptide of the present invention is special to " A " structure that is present on above-mentioned first peptide species, when the another kind peptide sequence that contains this A type structure (or unlabelled freely A type structure) needs the reaction that " A " type structure participates in adding so, this peptide species will reduce how too binding capacities of its " A " type structure and the present invention.

When " activatory " speech is used for above-mentioned proenzyme, be meant the enhancing of this proenzyme self lytic activity.The mensuration of above-mentioned zymogen activity can be undertaken by method of the present invention.For example, in Fig. 3 D group, weighing with a small steelyard above-mentioned enzyme source 1 in the cell pyrolysis liquid and 8 activity increases to some extent, the use by oneself C/EBP β of CCL4 processing of this spider cell lysate ^-/-With C/EBP β-ALA ²¹⁷Mice, rather than from C/EBP β ^-/-Mice

" phosphoric acid love body region " and " phosphate acceptors territory " and " phosphoric acid like body aminoacid " are used for representing a seed amino acid (as the green nitrilo acetic acid of threonine etc.), and this seed amino acid is arranged in a kind of sequence can be by phosphorylation under a kind of enzymatic activity effect of phosphokinase.

When a seed amino acid that " can not by phosphorylation " is used for describing in a kind of peptide sequence, be meant that this seed amino acid can not be produced phosphorylation by the enzymatic activity catalysis of phosphokinase.、

" cell ", " at least a cell " and " cell colony " are the plural cells of expression.

In this description and the patent right of enclosing require, " a " of singulative " an " and " the " comprise most reference substances, unless this paper has other clear and definite explanations.

" Or " speech is when being used for expressing " A " or B herein " time, promptly represent a kind of preparation with A and B, disease during product etc., is that expression is a kind of, or another kind of, or two kinds of whiles.

About brief description of the present invention:

The present invention generally relates to those are had the treatment of diseases and the prevention of excessive cell proliferation and/or activation feature.It is to be noted that especially preparation provided by the invention can suppress multiple different cell and/or hypertrophy with this with method.In some preferential application scenario, the derivative cell of matter (including but not limited to the liver spider cell) and have the activation and/or the hypertrophy of misgrowth feature cell between preparation provided by the invention and method are used for suppressing.Some especially preferentially at the application scenario, preparation provided by the invention and method are used for suppressing or eliminating fibre modification.Adding the occasion of some prior applicabilities, this preparation and method are used to induce fibre modification.Further concrete application scenario is, preparation of the present invention and method can be used for treating and prophylaxis of cancer.

Aforementioned CCAAT/ enhancer binding protein β (being called for short C/EBP β) (Descombes efal, GenesDev., 4:1541-1551 (1990); With Akira ef al, EMBO J., 9:1897-1906 (1990) can transmit in the rectum cancer cell by activatory hyperplasia effect (the Chinery ef al. that is caused by anxious anti-Should of oxidation Should and antinuclear antibodies Protein S-6 kinases (RSK) of d-transforming growth factor (TGFX), Nat.med., 3:1233-1241 (1997)), in addition, in the primary cell of mice, also have and sympathize with (Buck ef al. mutually, mol.cell., 4:1087-1092 (1999)). but C/EBP β (LAP, NF-1L6) and the connection mechanism between the hyperplasia not clear.Yet, be not essential for using the present invention to the understanding of this mechanism.

The activation of ERK/MAPK signal pipeline promotes hyperplasia by some mechanism, comprises promoting nucleic acid to synthesize gene expression, the synthetic and cell growth (Whitmarsh and Davis, J, MoL., 74:589-607 (2000)) of albumen.These of MAPK cell growth on, majority is by P90RSK mediation (Nebrda and Gavin, Science) RSK: nuclear powder nucleoprotein S-6 kinases.

C/EBP β: CCAAT/ enhancer binding protein β 286:1309-1310 (1999)).RSK has phosphorylation and deactivation (Bonni ef al., Science 286:1358-1362 (1999)) to deadly Kai Move (BAD) of short apoptosis protein (Pro-apoptotic Protein) Bcl-XL/Bcl-2 Xiang Off; Simultaneously Ke Tong Guo Ring adenosine phosphate Should answers the phosphorylation of Yuan spare Knot hop protein (CREB) and activation and resist-the up regulation effect of having transcribed (Bonn:ef al. sees above-mentioned) of apoptosis gene B lymphocytic lymphoma-2 (Bcl-2); Thereby also can induce chromatinic reconstruction or reinvent and promote gene expression (Sassone-corsi ef al., Science 285:886:891 (1999)) by the phosphorylation of histone H 3.

In the time spent of doing of replying epithelium growth factor, the continuous amplification that above-mentioned Fine Bao Wai Tone Festival Namida enzyme/short Fine born of the same parents divide activated protein kinase (ERK/MAPK) contains to have become regulating action to the whole process of purine nucleic acid, and this need carry out (Graves ef al. by activatory carbamyl phosphate synthetase II, Nafure403:328-332 (2000)). other adjusts mechanism (the Whitmarsh and Davis of cells survival and cell cycle, J, Mol.med., 403:255-256 (1996)) also comprise: the phosphorylation of a＞by transcription factor makes the activation of genetic transcription process, (Whitmarsh andDavis, J.Mol.med., 74:589:607 (1996)); Bhaff and Ferrell, Science 286:1362-1365 (1999)), the phosphorylation of histone H 3 and high speed swimming family 14 (HMG-14) also can activate transcription (Sassenl-corsief al, aforementioned: Thomson ef al., EMBO J., 17:4779-4793 (1999)); B＞promote protein translation (Pyronnef ef al., EMBOJ., 18:270-279 (1999)) by the super phosphorylation that begins factor 4E (elF-4E); C＞by increasing the expression of cyclin D1, promote dna replication dna (Lavoie efal., J.BIOL.Chem., 271:20608-20616 (1996)) .RSK is after it is by phosphorylation ERK/MARK activation, will the ERK/MAPK signal in the process of regulating cell survival and cell cycle, play requisite effect (Bonni ef al., aforementioned; BHAFF AND Ferrell, aforementioned; Gross ef al, Science 286:1365-1367 (1999)) and Sassone-corsi af al., aforementioned).Therefore, forming process capsule of the present invention is once to becoming interested via the C/EBP β pair cell survival effect of RSK mediation.

In production process of the present invention, adopted former generation spider cell of mouse liver, this be because these cells to excessive production (Friedman, ef al., Proc.Nafl, the Acad.Sci.USA 82:8681-8685 (1985) of fibrous tissue; AndAnkoma-Sey, and Friedman, in Sfrain and Diehl (eds), Liver Growth andRepair, Chapman ﹠amp; Hall, London, Pages 512-537 (1998)) be a committed step that develops into behind the liver injury in the liver cirrhosis process.(Chojkier, in Strain and Diehl (eds) Liver Growth and Repair, aforementioned, af pages 430-450). in addition, as normal liver cell, these cells can keep resting state (Leeefal., J, clin, Invesf., 96:2461-2468 (1995); And Ankoma-sey, and Friedman is aforementioned), but when with these cell culture on EHS (Mafregel) substrate the time, they again can be the inducing of the anxious anti-Should of oxidation Should down, activated rapidly down and hypertrophy (friend's L liver function by the effect of type i collagen or TGFa., aforementioned; And Friedrhan ef al., J, Brd.Chem.264:10756-10762).They can adjust with being both C/EBP β cell growth effect (Chinery efal., aforementioned; And Back efal., (1999), aforementioned).Similarly situation is, Astrocytic activation also needs the stimulation of anoxia pressure, the latter sees following two kinds of situations: the one, cause experimental hepar damnification (Houglum ef al. with CCL4, J.clin.Invesf, 96:2269-2267 (1995) is by ethanol, hereditary hemophilia, Porphyria and viral hepatitis cause human hepatopathy (Chojkidr, aforementioned).But before the present invention produces, do not understand when depending on the activatory living necessities of stellate cells C/EBP β (as mice C/EBP β by RSK; (m) phosphorylation of 217 threonine C/EBP β).The same as detailed in this article, liver toxicity CCL4 has induced the activation of RSK, star proliferation of cells in the phosphorylation of Thr217 and the normal mouse on the C/EBP β, but can cause C/EBP β-/-and C/EBP β-Ala217 (a kind of feminine gender can not by the dominance consolidation of phosphorylation) transgenic mice in the apoptosis of this cell.In other words, under the effect of CCL4, induce and produce anoxia pressure in the liver, to cause apoptosis thereupon, but this do not occur in C/EBP β+/+mice, be detected in C/EBP β-/-or C/EBP β-Ala217 (a kind of can not by the consolidation of phosphorylation) transgenic mouse.And then find, C/EBP β-PThr217 and with its phosphorylated analogs C/EBP β-Glu217, rather than C/EBP β-Ala217 can associate mutually with Procaspases1 and 8 in vivo and in vitro, thereby suppress its activity.The phosphorylation that the present invention forms Thr217 on the Notes of Key Data C/EBP β that obtains in the threonine process has produced a kind of functional XEXDCaspase substrate/mortifier box (Kphospho-T ²¹⁷VD). and it can to the similar (KE of C/EBP β-Glu217 ²¹⁷VD).The observed result unanimity is therewith, C/EBP β-/-can under the effect of the logical living KE217VD tetrapeptide of cell or C/EBP β-Blu217, avoid apoptosis with C/EBP β-Ala217 spider cell, will have further this this paper and describe in detail.

There is a kind of new C/EBP β mechanism in the results suggest of gained in the production process of the present invention, in order to the influence of explanation RSK pair cell existence downstream process with to the effect (Thornberry andLazebnik, Science 281:1312-1316 (1998)) that prevents of Procaspases1 and 8 activation processs.These Caspases can activate downstream effect and Procaspases (Earnshaw ef al., Ann.Rev, BIOCHEM.68:383-424 (1999)).Physiological coherent signal approach in spider cell, as CCL4 for mice with type i collagen (Friedman efal., J.Biol.chem.., m264:10756-10762 (1989) in cultivating; With Rudolph ef al., Science 287:1253-1258 (2000)).The phosphorylation that will cause Thr217 on the activation of RSK and the endogenous C/EBP β.C/EBP β-PThr ²¹⁷, but be not the C/EBP β of non-phosphorylating, can associate in Procaspases1 and 8 (as be subjected to epidemic disease fluorescent and coimmunoprecipitation measure and external directly with the recombiant protein association) thereby block apoptotic continuous amplification process, and allow cell be survived.Thereby the preliminary proof of present data will promote it to combine with Procaspases by ERK/MAPK/RSK by way of the phosphorylation of the transcription factor that causes, thereby prevent its activation.Although Thr on the C/EBP β ²¹⁷Phosphorylation may cause a kind of functional XEXD caspase substrate/mortifier case (Thornberry etd.) J.Biol.Chem, 272:17907-17911 (1997); And Blanchard et al., J.Mol.Biol., 302:9-16 (2000) have explained the anti-apoptosis effect of C/EBP β, but illustrating still of its precise mechanism need be carried out structural analysis.But the understanding to it is not that application the present invention institute is essential.

C/EBPB-PThr ²¹⁷Need under being induced the condition of generation, the expression of C/EBP β carry out the inhibition of Procaspases1 and 8 activation processs, for example normal Astrocytic activation or handle mice by liver toxicity CCL4 after produce, perhaps in cell culture, induce generation by type i collagen substrate.In addition, when spider cell during from C/EBP β mice, natural death of cerebral cells can be induced generation.

The above-mentioned discovery of these experiment promptings is not the result of the illogicality of C/EBP β overexpression.The expression of C/EBP β-Ala217 also can (CCl4 be to mice to being exposed to growth stimulation in the transgenic mouse, collagen is to cultured cell) under spider cell induce that to produce natural death of cerebral cells opposite, phosphorylation mutant similar in appearance to C/EBP β-Glu217 can make cell avoid by MEKKI, the negative dominant mutation of IKB α or RSK or with natural death of cerebral cells process (Chenet al., the Nature 374:386-388 (1995b) of albuminous body inhibitor Loctacgstin processing cell induction; Nakajima et al., Cell 86:465-474 (1996), Leeet al., 88:213-222 (1997)) expression of .C/EBP β-Glu217 in spider cell also can prevent the inductive apoptosis of FAS-, this apoptotic process is that activatory Procaspase8 mediates (Ashkenazi and Dixit, Science281:1305-1208 (1998)), but can not prevent the inductive apoptosis of removing serum (Joza et al., Nature 410:549-554 (2001)) of Procaspase9 activation process mediation.These results disclose C/EBP β-PThr217 pair cell apoptosis selectively to be influenced.The activation of C/EBP β and and the aggressiveness zone is unessential to the combination of procaspases1 and 8 or inhibition for it.In addition, carrying out in the preliminary experiment with spider cell, adopted reporter gene (the Descombes et al. of a kind of C/EBP of containing β in conjunction with the territory, aforementioned, with Houglum et al.,, 94:808-814 (1994)), but aspect the activation of not finding to transcribe what difference is arranged between the two J.Clm.Invest. at wild type C/EBP β and its Ala219 phosphorylation mutant.

C/EBP β-Glu ²¹⁷(KE ²¹⁷VD) similar to C/EBP β-PThr217, thereby the view of having affirmed current popular is that mortifier/substrate tetrapeptide of Caspase needs certain structure.This tetrapeptide needs a kind of asparagicacid residue (D) to be positioned at its P1 site (Thornberry and Lazebnik, see before and state), it is aminoacid D219 (Cao et al., Gene that this aspartic acid also appears at C/EBP β the 219th site, Develop., 5:1538-1552 (1991)).With scanning substrate library, a kind of site, found that it is I/L/V (be different bright/rely/valine) (P4 site) EXD that optimal sequence is write in the combination of Caspase8, although I, V, W, T, P and D are (different, figured silk fabrics, color, Soviet Union, dried meat and aspartic acid) also can be accepted by the P4 site, this is based on the downstream cracking site (Thornberry et al. sees before and states) of apoptosis continuous process.These find recently by to studying of the crystal image of Caspase8 to Indeed Recognize (Blanchard et al., Supra).Though the Caspases of III family (comprising caspase 8) more is inclined to little hydrophobic residue and is positioned at the P4 site, yet the research of thermodynamics and crystal image aspect still shows to have only this special II of DEVD family mortifier, it interacts at the enzyme in this site, and its inhibitory action-pile-Su-aspartic acid (IETD) bright identical (Blanchard et al., aforementioned) almost with the mortifier Iso of specificity III family.K216 on the C/EBP β similar to the hydrophobicity of D (Boyle, et al., Meth.Enzymel., 201:110-149 (1991)).In addition, people such as Blanchard think that the S3 of Caspase8 is that interactional site takes place for P3 (not being P4) with tetrapeptide, it also is its specific important determiner, therefore initial classification for Caspases needs to revise, for Caspase8, to repartition (people such as Blanchard is seen before and stated) especially.As the present invention is observed, not only C/EBP β-9Thr217 has the selectively acting feature, and diphtheria toxin, diphtherotoxin A fragment (CrmA) also can optionally suppress Procaspases1 and 8, rather than Procaspases3,6 and 7 (Zhou et al., J.Biol.Chem., 272:7797-7800 (1997)) but its reported literature thinks that CrmA also can suppress Caspases 4,5,9 and 10 (Garcia-Calvo et al., J.Biol.Chem., 273:32608-32613 (1998)).In addition, bar Like virus (baculovirus) P35 is to Procaspases1, and 3,6,8 and 10 is a kind of potential but relative more weak mortifiers of selectivity (Anelrade et al., Immun., 8:451-460 (1998)).The further experiment of the present invention shown K-Plospho-T217VD among the C/EBP β (thereby or the KE217VD preface can combine with ProcaspSES1 and 8 effectively the blocking-up latter activity.

The present invention obtains the strong backing of following experiment, comprises β ~ cell with C/EBP, negative dominant mutation of Ala217 and the positive dominant mutation cell of Glu217, and the experiment that C/EBP β-object of study such as Ala217 transgenic mice are done.C/EBP β-Ala217 sudden change with show as a kind of negative dominant mutation after RSK combines, this show in the cell culture after Ccl4 and type i collagen are handled respectively with the mammal astrocytosis after.The AC-KA217VD-CHO peptide of synthetic promotes the spider cell accent to die, similar in appearance to the dominance cell mutation effect of C/EBP β-Ala217 and C/EBP β 216-256-Ala217.These effects come from the inhibition of RSK and/or the enhancement of Procaspases1 and 8 activation processs, impel himself cracking simultaneously again.But the understanding to this mechanism is unessential by using the present invention.

Although γ C/EBP β has a kind of abiogenous exchange that takes place in 217 sites between Ala and the Thr, the exchange (Buck et al., (1999) are seen before and stated) between a kind of Ser and the Ala also can take place in its 105 site in it.The Ser105 that also once observed endogenous γ C/EBP β is by phosphorylation (being the rat phosphate acceptors congener of RSK).The expression of the positive dominant mutant of γ C/EBP β-Asp105 can effectively be avoided the negative dominant mutant of RSK or handle inductive natural death of cerebral cells with the albuminous body mortifier.The activation of PKC α or MAPK signal pipeline has caused phosphorylation (Trautwein et al., the Nature 364:544-547 (1993) of Ser105 on the γ C/EBP β; And Buck et al., (1999) are seen above-mentioned).In addition, separate the spider cell that obtains from γ C/EBP β-Asp105 transgenic mice tolerance lactocystin pair cell is transferred inducing of dying.As the description to C/EBP β, the γ C/EBP β that contains P-ser 105 or its phosphorylated analogs all can combine with the rat spider cell.These data are pointed out, although C/EBP β or γ C/EBP β can on different amino acid residues phosphorylation take place, two types phosphorylating protein all can effectively be avoided the natural death of cerebral cells process by the Caspase8 mediation.

On the contrary, can not can be induced little broomcorn millet and rat spider cell to take place to transfer and die by the γ C/EBP β of phosphorylation-Asp105 mutant behavior performance as if a kind of negative dominance (contrast) in C/EBP β-Ala217 mutant.The analog KKPD105 of γ C/EBP β-Pser105 sequence contains indispensable D in its P1 site, with that the P of high-priority is arranged on the P2 site is the same, substrate/mortifier XX-dried meat-aspartic acid (XXPD) as Cytotoxic cell proteinase-1 (granzyme B), with regard to (Harris et al. as the substrate library of adopting synthetic determines, J.Biol.Chem., 273:27364-27373 (1998)).Starting material Caspases, Caspase8 and granzyme B, shared IETD tetrapeptide array as the Procaspase3 of substrate/mortifier (Thornberry, et al 1997, aforementioned; Harris et al., aforementioned), point out that thus Caspases8 also may discern relevant XXPDA substrate.This is confirmed by experimental data of the present invention.AC-KKPD105-CHO (γ C/EBP β) tetrapeptide fragment can significantly suppress the Astrocytic accent of C/EBP β ~ mice and die.

The data that obtain in the production process of the present invention relate to the disease that the Interstitial cell activation is caused, and Interstitial cell causes a kind of over-drastic tissue repair mechanism (as cranial glia fibroplasia, liver cirrhosis, lung and kidney fibre modification).Result in this narration points out that C/EBP β RSK phosphate acceptors can be suitable for treatment by the tetrapeptide fragment of phosphorylation, because they have inducing cell to transfer the effect of dying to various kinds of cell after its activation.Really also observe, C/EBP β-Ala217 transgenic mice can tolerate chronic administration CCl4 to the hepatic fibrosis inducing action of (comprising liver cirrhosis).In addition, because IL-I is prompted to relate to acute refreshing The Change sexually transmitted disease (STD) haircut and gives birth to process (Rothwell et al., J.Clin.Invest., 100:2648-2652 (1997)), therefore, Caspase1 activation process is thought the disease that Neng Minus Light is relevant with fibre modification and/or natural death of cerebral cells with it to the inhibition of the processing procedure of IL-I precursor.

Description of drawings

The data that this figure of Fig. 1 provides show that the threonine on the 217th is by phosphorylation among the basic C/EBP β of the Astrocytic survival needs of liver.The A group provides a kind of Phosphorylated Peptide collection of illustrative plates, is illustrated respectively in when cultivating the 4th day, and the interior originality C/EBP β of static and activatory elementary spider cell body internal labeling, these spider cells are to hand over the former substrate separating obtained from EHS of mice or I type.It shown in the arrow threonine that contains phosphorylation in the peptide sequence of activating cell on its 217th.This result points out that type i collagen can induce the phosphorylation of the threonine of the 217th of C/EBP β.The B group provides the Phosphorylated Peptide figure of the C/EBP β of external labelling to boil.Activatory P90RSK produces phosphorylation to C/EBP β, but is not the C/EBP β-LA under the conditions in vitro ²¹⁷(shown in the arrow).The result that the C group provides represents the influence that the type i collagen pair cell after the transfection is cultivated.As shown in the figure, type i collagen is implemented transfection reagent comprise that murine sarcoma virus (MSV)-C/FBPB is counter and anticipate (AS) that MSV-C/EBP β You Yi Righteousness (s) becomes MSV-C/EBP B-Ala ²¹⁷Add huge Fine cellular virus green fluorescent protein (CMV-GFP) simultaneously, and examine the kinase whose 90 Ge amino acid peptide Chain fragments (P90RSK) of sugared Body S6 (every kind of reagent 1Mg). began to measure the combination of film United albumen-V-phosphoethanolamine (annexin-v-PE) to serous coat after the transfection in 4 hours, measured value is recently represented (wherein C/EBP β AS and C/EBP β-Ala with the percentage of annexin-V-PE (positive) (redness) in transfectional cell (green) ²¹⁷The variation P value of percentage ratio is less than 0.05 after the transfection).In the D group, the cell transfecting method is identical with the A group, but used vector expression MEKKI, IkBa, or the positive sudden change of the dominance of PQORSK, or with the cell (real post) of the diligent Ke Tuosaisiting Lactocystin of albuminous body mortifier (10pm) processing and with MSV-C/EBP β-Glu ²¹⁷The cell of (each 1Kg) cotransfection (real white post) is seen diagram.The combination of Annexin-V-PE is pressed the method for A group medium velocity and is measured (C/EBP β-Gla wherein ²¹⁷The P value less than 0.05).The result that the E group provides measures, cell transfecting method and C group are together, but transfection reagent is the male P90RSK of a kind of dominance, earlier handle cell or cultivate 6 hours (real post) earlier at serum-free medium with FS7 Xiang Off surface antigen (FAS) (100n/h 6hour), and with MSV-C/EBP β-Gla ²¹⁷The cell of (each 1 μ g) cotransfection is seen diagram.Dead cell is examined to dye with HOECHSF 33342 and is measured (C/EBP β-the Gla behind the RSK consolidation ²¹⁷In group and the FAS processed group, the P value is less than 0.05).

The result that this figure of Fig. 2 provides represents, from C/EBP β-Gla and C/EBP B-Gla ²¹⁷The liver spider cell of transgenic mouse, its apoptosis increases.A group result relates to the β from C/EBP, C/EPP β and C/EBP β-Gla ²¹⁷Separate in the mice and the elementary spider cell of cultivation in a kind of type i collagen substrate.Cultivate and after 6 hours (C/EBP β and C/EBP β-Ala are measured in the combination of annexin-V-PE ²¹⁷Two groups P value is less than 0.05).The expression of B group is carried out the result that HOECHSF 33342 nuclears dye to above-mentioned (A group) cell.In addition, from C/EBP β-Ala ²¹⁷The cell of mice is handled with following reagent respectively: the cell permeability inhibitor (IETD) of Caspase8 (1.05nM) or use MSV-C/EBP β, C/EBP β-Ala ²¹⁷, C/EBP B-Ala ²¹⁰, or RSK (each 1Mg) is referring to diagram (C/EBP β wherein, C/EBP β-Ala ²¹⁷, C/EBP β ²¹⁷+ RSK+C/EBP β-Ala ²¹⁰The P value of group is less than 0.05).The data that C group provides then are, in above-mentioned A and the B group to elementary week cell carry out annexin-V combination and mitochondrion pick off and Hoechst 33342 nuclears and dye to measure prominent example is provided.As shown in the figure, from C/EBP β-Ala ²¹⁷The spider cell of mice has shown enhanced annexin-V-PE binding characteristic, and the measured value of mitochondrion pick off also changes, and has lost nuclear integrity simultaneously.Arrow has indicated apoptotic cell.

The result that this figure of Fig. 3 provides shows C/EBP β-Thr ²¹⁷Be associated with the confused propylhomoserin kinases 1 and 8 of methionine specificity.The A group is the result be used for representing, by adopting the fibrillary acidic protein (GFAF) of Laser Scanning Confocal Microscope and neuroglia, can show β-Ala from C/EBP ²¹⁷Face be not C/EBP β+/ the 4R mice after CCL4 handles 12 hours, the activation of Caspase3 in its liver spider cell.GFA β copolymerization point and activatory Caspase3 are by shown in the arrow.Asterisk indication C/EBP β+/+mice in the little tube chamber of shape.After being removed, GFAP antibody just can observe background dyeing (redness).Mineral oil can not cause variation.Pass the United Nations General Assembly of B group data representation PGORSK and C/EBP β is increasing.These results show, by to C/EBP β ^{+ /+}, C/EBP β-Ala ²¹⁷, and C/EBP ^{+ /+}(matched group) albumen is not understood the immunoprecipitation of liquid (500Kg), and RSK is done further immunity confirmation experiment, the description A group of sample.Handle C/EBP β with CCL ^{+ /+}With C/EBP β-Ala ²¹⁷Mice can cause hurriedly RSK relatedness rising of isolating star from these mices.Handle C/EBP β with CCL4 ^{+ /+}With C/EBP β-Ala ²¹⁷Mice can make C/EBP β-PThr ²¹⁷Increase with the relatedness of RSK and the relatedness of activatory RSK-phosphorus propylhomoserin 380 (PSer380) (in the RSK immunoprecipitation experiment).C group result represents, handles C/EBP β+/ 4 mices with CCL4, can increase aforementioned kinases gram 1 and 8 and C/EBP β and C/EBP β-PThr ²¹⁷Relatedness (showing in the immunoprecipitation of C/EBP β) mutually the immunoprecipitation of exchange also confirmed these relatednesss.D group result then represents as the spider cell C/EBP β that CCL4 handles that hangs oneself ^-/-With C/EBP β-Ala ²¹⁷Mice, rather than C/EBP β ^{+ /+}During mice, its above-mentioned kinase activity is enhanced.Caspase is active to be measured by 5 * 10 6 chromogenic substrates that cell discharges on each little time point.Measured value is expressed as half of meansigma methods scholar Caspase1 and the active tee pipe range of variation of Capase8, and experiment mice is handled (basic value of untreated mice is as proofreading and correct) through CCL4.

Data show C/EBP β-PThr that this figure of Fig. 4 provides ²¹⁷Combine and suppress the latter's activation with Pro-Caspases1 and 8.The A group provides illustrating of C/EBP β peptide class.Represented activation domain (dotted line bar) among the figure, DNA is in conjunction with territory (solid line bar), dimer territory (grid bar).Result after the transfection of B group expression spider cell, transfection agents is specified grain connection garden CMV-GFP (each 0.3Mg), is incubated at type i collagen substrate (C/EBP β-216-256-Ala after the transfection ²¹⁷P value＜0.05 with RSK consolidation group).C group result is presented at and detects C/EBP β and PROCASPASE8 in the activatory spider cell, and spider cell is separated from C/EBP β ^{+ /+}Mice has been adopted Laser Scanning Confocal Microscope and specific antibody (antibody is available from Santscro Bio-techontegg) during detection. and matched group is C/EBP β ^-/-Spider cell, this cell are the C/EBP β positive, the Procaspase8 positive, and can be brought out apoptosis, the D group provides the immunoblotting result, uses the spider cell of MSV carrier (0.3Mg) transfection of expressing C/EBP β+LA (blood clotting albumen) peptide chain in the experiment.The peptide chain of expressing carries out immunoprecipitation with anti--HA plastochondria.Only in the cell of expressing wild type C/EBP β, available specific antibody is measured C/EBP β-PThr ²¹⁷. the immunoblotting result shows, Procaspases1 and 8 and C/EBP β-PThr ²¹⁷With C/EBP β-Glu ²¹⁷Association be higher than they and C/EBP β-Ala ²¹⁷Between association, RSK is then relevant with all C/EBP β peptide chains.But in the cell of expressing C/EBP β AS, no matter C/EBP β still be Procaspases1 and 8 all can not be by measuring at the immunoprecipitation of C/EBP β.Carry out immunoprecipitation at Pro-caspases and RSK conversely and confirmed above-mentioned relatedness.

This figure result of Fig. 5 shows C/EBP β-Glu ²¹⁷Directly combine with Procases1 and 8.A group provides a kind of immunoblotting result, in the experiment with the recombinant C/EBP β-GLU of purification ²¹⁷With C/EBP β-216-253-Glu ²¹⁷Merge with the reorganization Procaspases1 (0.6mg) of purification and 8 (0.12mg) and to carry out immunoprecipitation, optical density measures, and Procaspases1 and 8 amount are than C/EBP β-HA-Ala ²¹⁷With C/EBP β 216-253-HA-Ala ²¹⁷Exceed 7-12 doubly.The result that the B group provides represents the C/EBP β-HA-Glu of purification ²¹⁷With C/EBP β 216-219-PThr ²¹⁷With-Glu ²¹⁷Synthetic tetrapeptide can be in the autoactivation of vitro inhibition Pracaspases1 and 8.Carry out under the situation that is determined at C/EBP β peptide (200Mg) existence 2 hours or disappearance (matched group) of enzymatic activity.Adopted the activation measurement of a kind of Procaspases1 (1V) (expression) and 8 (0.2Mg).Measured value is expressed as the half of the two-tube variation group of meansigma methods scholar.C group is the measurement result behind the cell culture, and cell culture is a row on type i collagen substrate the time.And added different agent treated cells respectively 4 hours, the N-acetyl that the purified subsides synthetic of tool cell permeability is wherein arranged, the specified recombinant C of C-aldehyde radical C/EBP β tetrapeptide (10 μ M) or/EBP β-HA peptide, then these peptide classes are mixed the back reuse respectively it handles cell with Chariot reagent (Activemotif), control cells is parallel cultivates, but in processing, do not contain above-mentioned peptide class (all contain the peptide class agent treated of Ala217, and its P value is all less than 0.05)

This figure result of Fig. 6 shows that the phosphorylation of rat spider cell endogenous C/EBP β occurs on the Ber105.A group provides cultivates that the Astrocytic phosphoric acid figure of immobilized and activatory liver approaches after four days, and cultural method as shown in Figure 1.These results show that type i collagen can induce the phosphorylation of Ser105 on the rat C/EBP β.The arrow indication is the peptide class that contains phosphoric acid-green propylhomoserin 105 in the activating cell.The B group provides the phosphoeptide collection of illustrative plates of giving birth to group C/EBP β of external labelling.Activatory P90RSK can produce phosphorylation to the C/EBP β of reorganization at body, but must the organize C/EBP β of Ser105 after by the Alu105 displacement do not had this effect (seeing shown in the arrow).

This figure data of Fig. 7 shows after green propylhomoserin on the 105th of rat C/EBP β is by phosphorylation can prevent the spider cell apoptosis.The A group provides the experimental result after the transfection of mice spider cell, transfection method sees before and states (see figure 2), adopted the P90RSK or the Lactacystin of consolidation) 10mm) (closed rod) handle cell, or, see diagram with CMV-rC/EBP β-Asp105 transfect cell (each 1Mg) (open bar) altogether.The combination of Annexin-V-PE is measured with the described method of Fig. 2.B group is carried and has been encircleed the measurement result of anmexin-V-PE in the spider cell, spider cell from C/EBP β+/+the rC/EBP β-Asp of (closed loop) and Lactacysfin (0-30mm) processing ¹⁰⁵In the transgenic mice, the P value is less than 0.05).The C group provides rC/EBP β-HA to make up the Astrocytic result of special (0.3Mg) transfection rat.Adopt anti--HA antibody to carry out immunoprecipitation to the peptide class of having expressed.After adopting specific antibody, rC/EBP β-pser105 only can measure in the cell of expressing wild type C/EBP β.Show they and C/EBP β-Pser at the immunoblotting of Procaspases1 and 8 ¹⁰⁵With rC/EBP β-ASP ¹⁰⁵Association be higher than C/EBP β-Ala ¹⁰⁵The association of peptide.The result of mutual immunoprecipitation has also confirmed these relations of associating.The D group provides Astrocytic experimental result, spider cell separate from C/EBP β-/-mice, cell adopts the N-acetic acid-C-acetaldehyde C/EBP β tetrapeptide (10MM) and the common processing of Chariof reagent 4 hours of synthetic of the purification of specified tool cell permeability in cultivation.Control cells does not contain peptide class (the P value of two peptide species is all less than 0.05) in cultivation.E group provides the experimental result of AC-KE217VD-CHO and AC-KKPD105-CHO (200mm), and these results point out that these tetrapeptides can suppress the activation voluntarily of external Caspase8.Adopt the change color base matter of addressing in the example in the mensuration of enzymatic activity.The active basic value of Caspase8 is that 4.7U (100%) measured value is expressed as the half of meansigma methods scholar to pipe variation value.

Nucleotide sequence that this figure of Fig. 8 provides (SEQ ID NO:1) and aminoacid sequence (SEQ ID NO:2) are had by wild-type mice C/EBP β.

This figure of Fig. 9 provides the aminoacid sequence of modified mice C/EBP β, can find out thus, threonine appears at 217 sites (SEQ ID NO:2) of wild-type mice C/EBP β, it can be replaced (SEQ ID NO:3) by alanine, the another kind that provides among the figure is modified the C/EBPB sequence and is shown, threonine on wild type C/EBP β the 217th site (SEQ ID NO:2) can be held propylhomoserin (SEQ ID NO:4) and replace.

This figure of Figure 10 provides nucleic acid (SEQ ID NO:5) and the aminoacid sequence (SEQ IDNO:6) of human wild type C/EBP β, and this (enters numbering: M83667) conform to the sequence of Genbank.

The aminoacid sequence that this figure of Figure 11 provides human modification to repair C/EBP β, in this sequence, the threonine (SEQ ID NO:6) on the 266th of the wild type C/EBP β is met and can be held propylhomoserin (SEQ ID NO:7) and replace.

This figure of Figure 12 provides two kinds through changing the aminoacid sequence (SEQ ID NO:9) of the human C/EBP β that modifies, in this sequence, the threonine (SEQ ID NO:9) on the 266th of the modification property C/EBP β is replaced by alanine (SEQ IDNO:10) and also can be replaced by glutamic acid (SEQ ID NO:11).

This figure of Figure 13 provides nucleotide sequence (SEQ ID NO:18) and the aminoacid sequence (SEQ ID NO 19) of human wild type C/EBP β,

This sequence is consistent with the sequence in Genbank enters coding X52560.

This figure of Figure 14 provides the aminoacid sequence of a kind of modified human C/EBP β, at this, threonine (SEQ ID NO::19) on the 266th of the wild type C/EBP β is replaced into alanine (SEQ ID NO:20), and is same, also can be replaced into propylhomoserin (SEQ ID NO:21).

This figure of Figure 15 provides nucleotide sequence (SEQ ID NO:22) and the aminoacid sequence (SEA ID NO:23) of human wild type C/EBP β, and this sequence conforms to the sequence that Genbank enters on the numbering NM005194.

This figure of Figure 16 provides the aminoacid sequence of the C/EBP β of human modification, threonine (SEQ ID NO:23) in this sequence on per 266 of the C/EBP β is replaced into alanine (SEQ ID NO:24), equally, this threonine (SEQ IDNO:23) has been replaced as glutamic acid (SEQ ID NO:25).

This figure of Figure 17 provides nucleotide sequence (SEQ ID NO:12) and the aminoacid sequence (SEA IDNO:13) of rat C/EBP β.

This figure of Figure 18 provides the aminoacid sequence of the modified C/EBP β of rat, in this sequence, the 105th of wild type C/EBP β goes up green propylhomoserin (SEQ ID NO:13) and is replaced as alanine (SEQ ID NO:14), and this green propylhomoserin (SEQ IDNO:13) also can be replaced as aspartic acid (SEQ ID NO:15).

The specific embodiment

About detailed description of the invention process:

The present invention generally relates to have the treatment and the prevention of excessive hyperplasia and/or activation feature.Unique distinction of the present invention has provided and has suppressed multiple variety classes cell activation and/or outgrowth preparation and method.In some preferential range of application of selecting, matter derived cell (including but not limited to the liver spider cell) and activation and/or hypertrophy between preparation provided by the invention and method are used for suppressing with cell of misgrowth feature.In some preferential especially application of selecting, preparation provided by the invention and method are used for suppressing or eliminating fibre modification, and in the opposite preferential occasion of another kind, preparation provided by the invention and method are used for bringing out fibre modification.Further the qualified method of also available preparation provided by the invention is treated and prophylaxis of cancer.

The phosphorylation process of Thr217 is essential by spider cell existence on the A.C/EBP β peptide chain.

For the phosphorylation whether test I Collagen Type VI substrate can induce C/EBP β, the elementary spider cell of mouse liver is used ³²P-orthophosphoric acid labelling 12 hours, and, place EHS Ji Quality glue [EHS (Matrigel)] (akinete) or type i collagen (activatory cell) to cultivate as described in the example 5.The phosphoeptide collection of illustrative plates demonstration type i collagen substrate that C/EBP β obtains behind immunoprecipitation is induced a kind of site-specific phosphorylation process that produced, comprise the Thr217 on the endogenous C/EBP β, a kind of p90RSK phosphoric acid body territory (Buck et al., (1999), aforementioned) and be a kind of still unacknowledged phosphate acceptors position (seeing A and B group among Fig. 1).The phosphorylation of C/EBP β is to be difficult for (the seeing Figure 1A group) that quilt is measured when spider cell is incubated in a kind of EHS substrate in the resting stage of mice.

Secondly, dependency is found out between the function of C/EBP β and the phosphorylation of its Thr217.The elementary spider cell of activatory mouse liver has used wild type (meaningful) or anti-meaning (cmtisense) C/EBP β expression vector to come transfection.Spider cell is expressed anti-meaning of C/EBP β when it, rather than (Buck et al. during pleasant RNA, EMBO J., 13:851-860 (1994)), the C/EBP beta protein that shortage can be measured, and can not hypertrophy, because can not survey expression (Bravo et al., the 326:515-517 (1987) of proliferative cell nuclear antigen; Buck et al., (1994), above-mentioned; With Buck etal., (1999), above-mentioned).But these cells demonstrate combining of phosphoric acid silk ribbon propylhomoserin on the V-type annexin that raise and the serous coat, and this is a kind of apoptotic early stage index (Rudel and Bokoch, above-mentioned) (seeing the C group among Fig. 1).

(be C/EBP β-PThr217) whether essential by cells survival, the MSV expression vector that mice spider cell usefulness is combined with the C/EBP β-Ala217 mutant of no phosphorylation probability has carried out transfection for the phosphorylation of determining the Thr217 on the mice C/EBP β.The evaluation of transfectional cell described in example 2, adopts the CMV-GFP (GFP egfp) of three radio frequency channel fluorescence microscope cotransfections to carry out.Find to express cell (the Buck et al. of C/EBP β-Ala217 mutant, (1999), aforementioned) can not hypertrophy, but begin to enter apoptotic process, this is to adopt stroboscopic microscope method (seeing C group among Fig. 1) and detect the result who determines behind transfection index green fluorescent protein and the V-type-annexin with cell divide analysis (FACS).The nuclear of the affirmation of dead cell by Hoechst 33342 dyes and carries out.The RSK of coexpression can not save cell and avoid the inductive apoptotic process of C/EBP β-Ala217 (Fig. 1 C group).

On the contrary, the activation (Beg and Baltimore, the Science274:782-784 (1996) that have the NFKB of anti-apoptosis activity by blocking-up; With Wang et al, Science 274:748-787 (1996)) or the activity of blocking-up RSK (Buck et al., (1999), aforementioned; Bonni et al., aforementioned; Bhatt and Ferrel, aforementioned; Gron etal., aforementioned; With Sasson-Corsi et al., aforementioned) inductive apoptosis can be exempted (Buck et al., (1999), aforementioned) (seeing Fig. 1 D group) by the effect of phosphorylated analogs C/EBP β-GLu217 mutant

It is phosphorylation by NFKB inhibitive factor (IKB) kinases α and β to the activation of Fine karyon factor K appaB (NFKB) that Fine born of the same parents divide activity kinases/Bao Wai Tone Festival kinases [MEK kinases 1 (MEKK1)], and then cause the phosphorylation of IKB to carry out (Lee et.al., Cell 88:213-222 (1997)), this will experience a fast protein hydrolytic process (Chen et al. by ubiquitin-proteasome pathway, Genes Der., 9:1597 (1995a)).Therefore,, it has been done following processing, comprised with the expression vector that contains the negative dominant mutation of MEKK1 IKB α and RSK and carry out transfection, or handle with Lactacystin (a kind of proteasome inhibitors) for inducing Astrocytic apoptosis.In each case, the part that Astrocytic apoptotic process all is subjected to C/EBP β-GLu217 mutant suppresses (Fig. 1 D group), points out thus, and under the condition of activatory NFKB or RSK disappearance, the phosphorylation process of C/EBP β-PThr217 can be promoted cell survival.In addition, the expression of C/EBP β-GLu217, can prevent RSK mutant or the inductive apoptosis of FS7 Xiang Off surface antigen Pei Body (FAS Ligand), but can not prevent serum remove apoptosis (Fig. 1 E group) expression caused this prevent to act on the Caspase activated channel is had certain selectivity.

B. with Ccl4 handle from C/EBP β+/+and the liver spider cell of C/EBP β-Ala217 transgenic mice can make this cell show the apoptosis of rising.

Be the correlation function characteristic of the phosphorylation of finding out mice C/EBP β and Thr217 thereof, analyzed and separated that used mice has passed through at clear handle (the Buck et al., aforementioned) of the targeting of C/EBP β here from the elementary Astrocytic existence situation of the liver of mice.C/EBP β+cell can produce apoptosis rapidly on type i collagen, but C/EBP β+/+cell do not have this phenomenon, can find out this point (A group among Fig. 2) with what stroboscopic microscope was observed annexin-v in conjunction with feature.Be the effect of research C/EBP β-PThr217 to spider cell existence, set up a kind of can have can express a kind of can not be by the C/EBP of phosphorylation β-genetically modified mice of Ala217 feminine gender dominant mutant.Although C/EBP is β-and the growth of Ala217 transgenic mice is normal, and its liver spider cell always is difficult to survived (Fig. 2) under the activation of type i collagen.Its apoptosis phenotype of spider cell from C/EBP β-Ala217 transgenic mice confirms (Green and Reed by mitochondrion sensor determination method, Science 281:1309-1312 (1998)), this shows that its mitochondrial membrane permeability changes (among Fig. 2 C group), and also can be confirmed (B and C group among Fig. 2) by its karyopycnosis staining method.The Astrocytic apoptotic process of C/EBP β-Ala217 can be blocked or blocks (among Fig. 2 B group) by the expression of C/EBP β-Glu217 by the mortifier (IERD) of a kind of cell permeability Caspase8.

In addition, the present invention has confirmed also whether the activation of stellate cells contains by CCl ₄Stimulate and cause, and CCl ₄Be a kind of hepatotoxin, it can induce the anxious anti-Should of liver oxidation Should with He Fiber Victoria (Houglum et al., aforementioned; And Chojkier, aforementioned).Stellate cells can be induced hypertrophy in the liver of C/EBP β+H mice, this only needs the Ccl4 of independent dose to handle and can take place.On the contrary, in the liver of C/EBP β-1-and C/EBP β-Ala217 transgenic mice, only under the condition that activatory effector Caspases exists, Ccl4 just can induce Astrocytic apoptosis (see figure 3).Because activatory pqoRSK can make the outer phosphorylation of the Thr217 Zai Body on the C/EBP β (Buck et al., (1999), aforementioned), thereby whether RSK combines with C/EBP β in these animals and has obtained affirmation.

C.CCl ₄Processing can induce the normal mouse stellate cells all RSK and C/EBP β-Thr217 activation and induce itself and the combining of Procaspases1 and 8.

Because the p90RSK of reactivity can be external with the Thr217 phosphorylation on the C/EBP β (Figure 1B group) (Buck etal., (1999), aforementioned), so the conclusion that whether can combine with C/EBP β to RSK is analyzed.The result points out that RSK raises to some extent with combining really of C/EBP β in the spider cell, but the C/EBP β that this spider cell is handled from Ccl4+/+mice and C/EBP β-Ala217 transgenic mice (B group among Fig. 3).On the contrary, after RSK was by the specific antibody immunoprecipitation, the level of C/EBP β that can see co-precipitation was also higher, this phenomenon be present in all the time the C/EBP β that Ccl4 handles+/+mice and C/EBP β-Ala217 transgenic mice (B group among Fig. 3).As (the B group among Fig. 3) that specific antibody confirmed used at RSK-Pser380, Ccl4 can strengthen expression and the activity of RSK.C/EBP β+/+mice in, CCl4 also can induce the Thr217 on the endogenous C/EBP β to produce phosphorylation, detects this phosphorylation situation (Fig. 3 B group) with a kind of specific antibody through affinitive layer purification at this phosphorylation epitope of new generation.These discoveries point out that RSK is necessary to the stellate cells existence of the phosphorylation of Thr217 on the C/EBP β.

Because containing its phosphorylation of a kind of KT217VD sequence (being same as human C/EBP), the Thr217 phosphate acceptors of supposition C/EBP β depends on RSK, and again on function by C/EBP β-GLu217 sequence KE217VD (Buck et al, (1999), aforementioned), imitate, whether and the latter has promptly found to be present in mortifier and the substrate box-like body of Caspases similar in appearance to a kind of XEXD box-like body, thereby adopt experiment to come Indeed Recognize C/EBP β-PThr217 relevant with Procaspases.Experimental result shows Procaspases1 and 8 (Fig. 3 C group), be present in C/EBP β after Ccl4 handles+/+(contain C/EBP β-PThr217) but this precipitation does not contain Procaspases3,7 or 9 (not video datas) in the Astrocytic C/EBP β immunoprecipitate of mice.From C/EBP β+/+Procaspases1 of mice and 8 levels handle the back at Ccl4 and rise, and contain C/EBP β (Fig. 3 C group) in its immunoprecipitate.Ccl4 handles in the cell of back C/EBP β-Ala217 transgenic mice, and C/EBP β is in baseline values (C group among Fig. 3) with combining of Procaspases1 and 8, identical therewith is with in contrast C/EBP β of mineral oil vehicle processing+/+mouse cell.In addition caspases1 and 8 enzymatic activity (D group among Fig. 3) and less than the enzymatic activity (A group among Fig. 3) of thing Caspase3 Ccl4 handle C/EBP β+or C/EBP β-Ala217 mice in do not have this reaction.The generation of the phosphorylation of Thr217 and two kinds of functional XEXD box-like body on these results suggest C/EBP β expression of the similar C/EBP β of phosphorylation-GLu217 mutant (or with) needs with combining of Procaspases1 and 8 for C/EBP β.These data are also pointed out the processing and the activation of the possible Procaspases1 of the phosphorylation of C/EBP β and 8, neither KE217VD (Wilson et al., Nature 370:270-275 (1994) because the preferential substrate of Procaspases1 and 8 promptly is not K-Phospko-T217VD; Margolin et al., J.Biol.Chem., 272:7223-7228 (1997); Earnshaw et al., Ann.Rev.Biochem., 68:384-424 (1999)), as if the cracking of C/EBP β neither be by Caspase1 or 8 (coming video data).In addition, C/EBP β-Ala217 cell can avoid apoptosis (Thornberry and Lazebnik, aforementioned) (seeing B group among Fig. 2) by the expression that RSK and the key P1 that contains the XEXD box-like body detest winter propylhomoserin residue mutant.

D. the C/EBP β-PThr217 that lacks activation domain and dimer territory also can fully allow the survival of spider cell on type i collagen.

Found to lack the dimer territory but still contain Thr217 or the segmental expression of the C/EBP β 1-253 of GLu217 can compatible cells survival (Descombes et al., (1990), aforementioned) (Fig. 4 A group), but can not activate the situation of Ala217 existence.All pair cell production does not exert an influence C/EBP β 1-215 disappearance DNA in conjunction with the phosphate acceptors (Fig. 4 A group) of territory and dimer territory and KT217VD.Opposite C/EBP β 216-253 Ala217, rather than C/EBP β 216-253 GLu217, when lacking activation domain and dimer territory in the fragment (Fig. 4 A group), just may induce cellular control unit to produce apoptosis, even also be (B group among Fig. 4) like this under wild type RSK expression, in addition, the expression of C/EBP β 216-253 GLu217 also can make cell avoid the inductive apoptotic process (Nakajima of RSK mutant (RSKN ' C ') of hydrolysis deactivation, et al., Cell 86:465-474 (1996)) (Fig. 4 B group).With previous report (Mao et al., J.Biol.Chem., 273:23621-23624 (1998); And Ritter et al., J.Cell Biol., 79:358-364 (2000)) conform to, Procaspases1 and 8 mainly measures in the cytoplasm of activatory spider cell, and this is in identical position (Fig. 4 C group) with C/EBP β.Immunoprecipitation result to spider cell C/EBP β-HA and C/EBP β 216-253-HA shows, with phosphorylated analogs Glu217 can be come out by co-precipitation with Procaspases1 and 8 efficiently (Fig. 4 D group) use antibody to exchange immunoprecipitation at Procaspases1 and 8, also obtain similar result.Therefore, no matter be activation domain or the dimer territory on the C/EBP β fragment, for Astrocytic existence, it is essential all to represent.

E.C/EBP β-PThr217 peptide chain combines with Procaspases1 and 8 and suppresses its activation: further clarified the influence of the autoactivation of recombinate under the conditions in vitro caspases1 and 8 to the recombinant C/EBP β peptide chain of purification.Consistent with result of experiment in the above-mentioned body, C/EBP β-HA-GLu217 and C/EBP β 216-253-HA-GLu217 peptide chain combine with caspases1 and 8, and with respect to C/EBP β-HA-Ala217, efficient is up to 7-12 times (Fig. 5 A group).And find the C/EBP β-GLu217 of reorganization, tetrapeptide molecule AC-K-phosphoric acid-T217VD-CHO that is divided into and AC-KEE217VD-CHO can directly suppress the to recombinate autoactivation (Fig. 5 B group) of caspases1 and 8.The negative dominant mutant peptide of cell permeability section AC-KA217VD-CHO can produce apoptosis by repeatability ground inducing cell, and the inductive degree of its degree and C/EBP β-Ala217 and C/EBP β 216-253Ala217 (Fig. 5 C group) is (Fig. 5 C group) quite.On the contrary, wild type and Gul217 C/EBP β fragments of peptides cell death inducing (Fig. 5 C group) not.

F. rat C/EBP β-Pser105, i.e. congener on the function of mice C/EBP β-PThr217 can combine and suppress its latter's activation in vivo and in vitro with Procaspases1 and 8:

Although the phosphate acceptors of C/EBP β-PThr217 is high conservative during evolution, rat (r) C/EBP β dual variation still occurred in evolutionary process, promptly lacked the Thr217 phosphate acceptors, but compensatory Ser105RSK phosphate acceptors (Descombes has appearred, et al., aforementioned).C/EBP β-PThr217 or rC/EBP β-Pser105 appear at respectively in mice and the rat cell, and this induces critical (the Buck et al., (1999), aforementioned) that produces the liver cell proliferation for TGF γ.Type i collagen can be induced the phosphorylation in 105 site silk ribbon propylhomoserins on the endogenous C/EBP β or other sites in outgrowth rats'liver spider cell.But the resting stage on being incubated at EHS substrate is in the rat spider cell, E/EBP β-faint (Fig. 6 A group) of phosphorylation.Inductive phosphoeptide of type i collagen and aforesaid Pser105 peptide, a kind of P90RSK phosphate acceptors (trautwein et al., Nature 364:544-547 (1993); Buck et al., (1999), aforementioned) the same, have identical mobility.The result who the polypeptide phosphate of γ C/EBP β is carried out the pancreatin hydrolysis analysis shows, P90RSK can C/EBP β 105 site silk ribbon propylhomoserins and other positions carry out phosphorylation; But how too the phosphorylation of γ C/EBP β-Ala105 mutant does not produce this phosphoric acid (Fig. 6 B group), points out this polypeptide fragment to contain Ser105.As described below, adopt and confirmed that at the antigenic specific antibody of this displacement the phosphoric acid flower on the endogenous γ C/EBP β fragment is present in Ser ¹⁰⁵

The experiment of carrying out is used for verifying whether γ C/EBP β-Pser105 can save cell and avoid apoptosis in addition.Induce spider cell generation apoptosis (Nakajima et al., aforementioned) or handle cell so that cause apoptosis by expressing negative dominance RSK mutant by lactacystin.Found that the expression of phosphorylated analogs γ C/EBP β-Asp105 can make mice (Fig. 7 A group) and rat (data not shown) spider cell avoid apoptosis.The γ C/EBP β-Ala105 mutant of no phosphorylation probability as γ C/EBP β-Ala217, has been induced Astrocytic apoptosis, because this mutant does not possess RSK phosphate acceptors site yet.These results point out, γ C/EBP β-Pser105 (or its phosphorylated analogs γ C/EBP β-Asp105) can block apoptotic pathways, its efficient and C/EBP β-PThr217 (or its phosphorylated analogs C/EBP β-Glu217) identical (Fig. 1).

Because γ C/EBP β-Asp105 mutant is avoiding cell to produce in the behavior of apoptosis phosphorylated analogs identical (Fig. 7 A group) with a kind of γ C/EBP β-Asp105, so has developed the transgenic mice of a kind of γ of expression C/EBP β-Asp105.γ C/EBP β-Asp105 transgenic mice is grown normally to the greatest extent, but its spider cell can tolerate lactacytin pair cell apoptosis induced (Fig. 7 B group).Lactacytin handle can induce C/EBP β+/+the remarkable and fast fast reading increase of Astrocytic apoptosis (Fig. 7 B group).γ C/EBP β-HA and γ C/EBP β 92-142-HA show P-SER105 and phosphorylated analogs Asp105, with respect to the possible Ala105 of no phosphorylation (Fig. 7 C group), can be gone out by immunoprecipitation jointly with the Astrocytic Procaspases1 of rat and 8 more expeditiously (Fig. 7 C group).γ C/EBP β-Asp105 does not combine (not video data) with activatory caspases1 and 8.Identical with the effect of rC/EBP β-Pser105 and γ C/EBP β-Asp105 pair cell existence, to just from C/EBP β+mice, isolating spider cell with containing γ C/EBP β (AC-KE217VD-CHO) or γ C/EBP β CAC-KKPD105-CHO) the cell permeability tetrapeptide molecule in the similar territory of phosphorylation handled, can avoid the apoptosis (Fig. 7 D group) that the type i collagen activation is caused by ten these cells.Confirm in addition, synthetic tetrapeptide AC-KKPD-CHO, identical with AC-KE217VD-CHO, all can suppress reorganization caspases8 in external autoactivation (Fig. 7 E group).

The result who obtains in the forming process of the present invention also indicates, and the present invention provides a kind of new method for cells survival.Yet it is necessary to understand its related mechanism the inventive method when utilization not.In any case, knownly make RSK activation will cause Thr217 on the C/EBP β by phosphorylation by the signal pipeline.And C/EBP β-PThr217 can with promoter Procaspases1 and 8 and then suppress its course of processing, promptly a kind of blocking effect of apoptosis tandem reaction sequence, thus allow the spider cell on the activation basis to be survived.This is believed, and the phosphorylation that confirms a kind of transcription factor first realizes by the ERK/MAPK/RSK approach, and this phosphorylation can prevent its activation by means of the association with Procaspases simultaneously.The present invention can not be limited to any specific mechanism, follow the generation of false XEXD box-shaped thing and the phosphorylation of Thr217 in the C/EBP β sequence of carrying out (Thornberry et al., (1997), aforementioned; With Blanchard et al., aforementioned) provide the most appropriate explanation for the anti-apoptosis phenomenon of the transcription factor of this new proposition.

Although C/EBP β-Thr217 is associated with promoter Procaspases1 and 8 (suppressing its processing and activation process whereby), be not incorporated into effector Procaspases3,7 or 9 (Earnshaw et al., aforementioned; Thornberry andLazebnik, aforementioned; And Askkenazi and Dixit., aforementioned), but can block the continuous process of apoptosis effectively, as C/EBP β+/+the liver spider cell in find that the activatory caspases3 of disappearance is pointed, but this phenomenon of nothing in a kind of transgenic mice that contains the possible C/EBP β of no phosphorylation-Ala217 feminine gender dominant mutant.In addition, phosphorylated analogs C/EBP β-activatory spider cell of GLu217 sudden change physical ability avoids the inductive apoptosis of the following factor, as MEKKI, the expression of the negative dominant mutant of IKB α RSK, or by handle (Chen et al., Nature 374:386-388 (1995b) with proteasome inhibitors lactacystin pair cell; Lee et al (1997), aforementioned; AndNakajima et al., aforementioned).Think in addition, contain the C/EBP β-GLu217 of KE217VD and related polypeptide and will be found and be used to save apoptotic cells, so that prevent under certain conditions and/or handle cell for example nerve or hepatocyte, so that cell injury is prevented from or is alleviated.Existing such view, promptly different patients suffers from the injured nerves wound as those, and the patient of neurocyte (degeneration) and acute liver damage will be benefited because of using these peptide molecules.

For with the association of Procaspases1 and 8 and suppress its course of processing, activation domain on the C/EBP β or dimer territory all are not absolutely necessary.What is more important, and the C/EBP β that is same as the cattle and the mankind of mice Thr217 phosphate acceptors essence (Akira et al., aforementioned; Cao et al., aforementioned; With Yamaoka et al., aforementioned).C/EBP β-PThr217 is also with C/EBP β-C/EBP β-Glu217 (KE217VD) (Buck et al., (1999) are aforementioned) thereby further confirm the current understanding that has topology requirement about caspases inhibition tetrapeptide.These tetrapeptide molecules need the P1 position to have aspartic acid (D) residue, and (it is D219 (Cao et al that this residue also appears on 219 of C/EBP beta amino acids sequence for Thornberry and Lazabnik, (1998); , aforementioned, and Back et al., (1999), aforementioned).

Use a kind of site scanning substrate library method, Thornberry and its colleague (Thornberry et al., J.Biol.Chem., (1997)) although proved that ideal sequence is I/L/V (P4 site) EXD for caspases8, I, V, W, T, P and D also can be accepted by the P4 site, and this is based on the downstream cracking site of the continuous process of apoptosis.These discoveries have adopted the crystallography method to be confirmed (Blanchard et al., aforementioned) recently.

A the third, B Radix Asparagi+N, and C half Guang, the D Radix Asparagi, E paddy, the F phenylpropyl alcohol, G is sweet, the H group, I is different bright, and K relies, and L is bright, M egg, N N, P dried meat, Q glutamy, R essence, S silk ribbon, T Soviet Union, V figured silk fabrics, W color, X firefly purine aminoacid.Z paddy+glutamy

The sorting of FACS fluorescence-activation sexual cell

C/EBP β .CCAAT/ enhancer binding protein β

XEXD

EVD

TVD

MAPK urgees cell division activity cell kinase

The silk ribbon propylhomoserin kinases that the Caspase methionine is special

The silk ribbon propylhomoserin kinases proenzyme that the Procaspase methionine is special

Pro-apoptosis

Anti-apoprosis

RSKHE ribosome S 6 kinases

CrmA diphtheria toxin, diphtherotoxin A fragment

Tandem series connection immunoadsorption precipitation

The RIA ria-determination

XA?Factor

Entero kinane enterokinase

Vaccinia Viruses vaccinia virus

Herpes virus herpes

III group caspases (comprising caspase8) has association tendentiousness to the little hydrophobic residue in P4 site, for example these research in used D (Blanchard et al., aforementioned) its K216 to C/EBP β has similar hydrophobicity (Boyle etal., Meth.Enzymol., 201:110-149) (1991)).In addition, these authors think that also the S3 subprovince of caspase8 can interact with P3 (rather than and P4) position of tetrapeptide, this is for determining that it is specific vital, but also think that the early stage classification to caspase need make an amendment, especially ((Blanchard et al., aforementioned) need add modification to the classification of caspase.Similarly, the selectively acting of C/EBP β-PThr217 also shows at this, with respect to Procaspases3, and 6 and 7, CrmA can optionally suppress Procaspase1 and 8 (suppress constant be respectively 0.01 and 0.95nM) (Zhou et al., Immunity 8:451-460 (1998)).But other researcher suggestions CrmA also can suppress caspase4,5,9 and 10 (Garcia-Calro et al., J.Biol.Chem., 273:32608-32618 (1998)).In addition, for Procaspases1,3,6,8 and 10, Baculovirus P35 also is a kind of powerful mortifier (Ki=0.1nm), though its selectivity relatively poor (Andrade et al., Immunity 8:451-460 (1998)).Further also find, the DEVD-CHO that can effectively suppress Procaspases1 and 8 (Ki=17 and 12nm) (Earnshawet al., aforementioned) also is the powerful mortifier of caspases8, it is renderd a service with classical mortifier IETD suitable (Blanchard et al., aforementioned).

Also provide further experimental data to support the present invention in the forming process of the present invention, and discussed at this.

As pointing out at this, the Kphospho-T217VD among the C/EBP β (or KE217VD) can effectively associate Procaspases1 and 8 and suppress its activation.The activation of stellate cells is by handling animal and induced generation with hepatotoxin Ccl4, or by these are carefully experimentized, want with the positive dominant mutant of Glu217 and comprise that the new experiment that C/EBP β-the Ala217 transgenic mice carries out all supported the present invention forcefully with Ala217 negative showing.

Yet, contain a kind of two sudden changes (Ser105 of the Ala217 of the Thr217-raf of mice and mice Ala105 → rat) among the rat C/EBP β and produced required compensatory Ser105 phosphate acceptors (the Buck etal. of a kind of cell growth thus, (1999), aforementioned).Therefore, further whether in cell survival, played similar effect to mice C/EBP β-PThr217 with testing the C/EBP β-Pser105 that solves rat.The expression of the phosphorylated analogs of rat C/EBP β-Asp105 (Trautwein et al., aforementioned; With Buck et al., (1999), aforementioned) be enough to make activatory delomorphous cells avoid the hydrolysis deactivation the inductive cell the 5th of negative dominant mutant RSK vector expression (Nakajima et al., aforementioned; With Buck et al., (1999), aforementioned), or come to handle the apoptosis that is caused with a kind of proteasome inhibitor.The activation of Protein kinase C α (PKC α) (Trautwein et al., aforementioned) or short cell division activity protein kinase (MAPK) (Nebreda and Gavin, aforementioned; With Whitmarsh and Davis, (2000) are aforementioned) the doubly activation of number approach, all might cause Ser105 on the rC/EBP β phosphorylation (Trautwein et al., aforementioned; With Bucket al., (1999), aforementioned).Have been found that the similar negative dominant mutant of behavior of rat C/EBP β-Ala015 mutant, liver activation spider cell that also can inducing mouse produces apoptosis.

The present invention relates to the such human diseases of like liver cirrhosis, obtain the support that experimentizes at stellate cells.Think that in addition the present invention can treat the disease relevant with process of tissue reparation, promptly owing to relating to the disease (including but not limited to brain, liver and nephrocyte) that other Interstitial cell activation cause.For example, just considering the present invention is used for the treatment of picture hepatic fibrosis (emphysema), brain gtelatinous fibre degeneration (as Alzheimer) and this class disease of kidney fibre modification (glomerulonephritis).But this does not refer to that application of the present invention only limits to this.Further consider, produce a kind of false XEXD box-like body by phosphorylation; It is a kind of very common biological mechanism that is present in the evolutionary process.This point obtains the support of following observed result, promptly comprising about 350, among 000 proteinic data base, find the phosphorylation (Phosph-TVD of the false XEXD box-like body of potentiality to threonine, the analog of EVD) site surpasses 22,000 and the phosphorylation (Evphospho-S is similar to EVD) of silk ribbon propylhomoserin has then been surpassed 27,000 sites.Therefore, if 1% the interior phosphorylation of body only takes place in these protein sequences, that also may produce false XEXD box-like body in about 500 protein.

G. typical polypeptide of the present invention, nucleic acid sequence encoding they and host cell.

This paper report can produce the functional XEXD Caspase of serratd edge inhibition box-like body by phosphorylation, and this perhaps is a kind of general biological mechanism in evolving.Really also find to produce threonine phosphorylation (Phospho-TVD having among about 350,000 proteinic data bases, be similar to EVD) the position be accredited as more than 22,000 and the phosphorylation position of silk ribbon propylhomoserin (EV-Phospho-S, be similar to EVD) then exceed 27,000.If have only in these about 35,000 protein sequences 1% at body by phosphorylation, then might in about 500 protein, produce functional XEXD Caspase inhibition box-like body.But, as noted here, unessential by using the present invention to the understanding of related here mechanism.

Such just as discussed in detail, a concrete entity of the present invention is: a kind of peptide sequence or its fragment, itself can encode C/EBP β or its fusion rotein or its functional similarity thing, also may be encoded by a kind of recombinant DNA molecules and be used to instruct suitable host cells to express produces C/EBP β.Because the inherent degradation characteristics of genetic code, thereby the DNA sequence of identical aminoacid sequence might produce and be used to the clone and express C/EBP β on other encode basic identical or functions.

Technology understand as being put down in writing by document, and perhaps more superior is by processing the nucleotide sequence that non-abiogenous coding produces C/EBP β.For example, by a certain specific protokaryon or the preferential selected protein expression speed of selecting that increases of codon energy of eukaryotic host cell, or being used for producing a kind of recombinant RNA transcript, this transcript has certain ideal characteristics, produces the transcript of sequence as long half time naturally in those.

Nucleotide sequence of the present invention can design by the method for generally knowing in the document and build, so as for multiple different reasons to C/EBP β-coded sequence change, this includes, but are not limited to change the clone of gene outcome, processing and/or express.Polymerase chain reaction (PCR) reorganization and the synthetic oligonucleotide of the amiable mobile and genetic fragment of dna fragmentation all can be used to build nucleotide sequence.For example, the sudden change that the site is instructed forms technology can be used to insert new restriction fragment, changes the glycosylation mode, changes the tendentiousness of codon, produce to shear variant or import sudden change, or the like.

Of the present invention another specifically used and is, nature, modification, or the coding C/EBP β nucleotide sequence of reorganization also may be used to be connected on the heterologous sequence of encoding fusion protein.For example, sieve is made peptide library so that when finding out the mortifier of human C/EBP 'beta ' activity, and a kind of chimera protein of encoding is discerned by obtainable antibody on the market.Certain fusion rotein also can be constructed to the albumen that contains a kind of cracking site, and this site is between this C/EBP β coded sequence and relevant heterologous protein sequence, thus C/EBP β can be cleaved and from the allos component purification come out.

Another concrete application of the present invention is, the sequence of coding C/EBP β also may be by synthetic, and the utilization literature method carries out that the synthetic of sequence (for example, can be referring to Caruthers et al. in whole or in part, Nucl.Acids Res.Symp.Ser., 215-223 (1998); With Horn et al., Nucl.Acids Res.Symp.Ser., 225-232 (1980).In addition, the also available chemical method of protein itself is produced, thus the propylhomoserin sequence of synthetic C/EBP β, or its part.For example, peptide class synthetic can use different solid phase technique (Roberge et al., Science 269:202-204 (1995)) and synthetic automatically be realization, the automatic synthesizer that provides on the market for example is provided.These synthetic peptides also can use the preparation high performance liquid chroma-tography to come in addition large-scale purification, and this can carry out with reference to method suitable in any document.The composition of synthetic polypeptide can be examined (for example using Ai Deman (Edman) edman degradation Edman) by amino acid analysis or sequence analysis.In addition, the aminoacid sequence of C/EBP β or its any part all might be carried out change in the process of directly synthesizing and/or merge the use chemical method, can utilize other protein or its arbitrary part to produce different polypeptide.

Such just as detailed in this article, in order to express the C/EBP β of biologically active, the nucleotide sequence of coding C/EBP β or its functional similarity thing is inserted into suitable expression vector (promptly containing the promising carrier of transcribing and translate the necessary element of coded sequence of this insertion).These methods comprise external DNA recombinant technique, synthetic technology and the reorganization of intravital heritability.

Different expression vector/host systems may be used to obtain and express the sequence of coding C/EBP β.Here include but not limited to the antibacterial of small organism as transforming with recombinant phage, plasmid or cosmid (cosmid) DNA expression vector; Use yeast; Insect cell system (as bar Like virus baculovirus) with the virus expression systems infection; Virus expression systems (as Hua cabbage Hua Leaf virus (cauliflower mosaic virus, Camv); Cigarette grass flower Leaf virus (tobacco mosaic virus, TMV)) plant transformed cell system or with bacterial expression system (as Ti or Pbr322 plasmid) plant transformed cell system, or zooblast system.Suitably also consider under the situation to adopt nuclear system element or regulate sequence.

In addition, when selecting for use a kind of host cell to be, consider it is that expression to insertion sequence has adjustment capability in fact, or have working ability for ideal fused protein.This modification of polypeptide is comprised but do not comprise acetylation, carboxylated, glycosylation, phosphorylation, fatization and acyl group effect.The cracked translation post-treatment of proteinic " preceding prototype " structure also may be used to promote correct insertion, fold and/or bring into play its function.Known different host cells have the processing behavior after special cell mechanism and the distinctive mechanism of tool produce this translation in the document, thereby might be used to guarantee that a kind of foreign protein mass-energy obtains correct modification and processing.

In order to make recombiant protein mass-energy, preferably can obtain a kind of stable expression by the production of extended high rate rate.For example, the cell line of stably express C/EBP β might be transformed by a kind of expression vector.This carrier may contain the reproduction element and/or the endogenous Expression element of viral origin, and a kind of selected gene sign, and this sign is present on the identical or different expression vector.The cell that imports behind the expression vector might be in abundant culture medium continued growth 1-2 days, and then changes continued growth in the selective medium over to.Adopt selection marker purpose make it can tolerate alternative condition, the existence of this sign simultaneously allows the cell of those energy successful expression importing sequences to be grown and recover.Resistance clone strain in the stable transformant may make its propagation with the tissue culture technique that is suitable for a certain cell type.Available any amount of selective system is recovered transformant and is held.These include but not limited to simple varioliform exanthema virus purine kinases.Known other different types of suitable systems in adenine phosphotransferase and the document.In addition, antimetabolic, antibiotic or help the antibiosis of careless agent also can become the basis of selection.Point out gene of interest whether to occur although whether this marker gene is expressed, finally still will whether exist and express and examined it.For example, if relevant C/EBP β coded sequence is inserted in a kind of sequence of marker gene, can be identified the coded sequence that contains C/EBP β in the cell of rebuilding, but can not identify the function of marker gene, in addition, a kind of marker gene also can be with C/EBP β coded sequence by in connecing sad sequence, from here under the control of a single promoter.Thereby replying when inducing with selection, the tandem expression of gene has also been indicated in the expression of marker gene usually.

In addition, contain host cell that nucleic acid coding sequence expresses simultaneously C/EBP β again also the distinct program of available known references identified.These programs comprise but are not limited to DNA-DNA, or DNA-RNA hybridization and protein bioassay method, or immunoassay, and these include film, liquid or are the fundamental measurement technology with the chip, for use in to nucleic acid or proteinic detection and/or qualitative.

The expression that has several different methods to be used to detect and measure human C/EBP β as the literature method that adopts in the present invention, utilizes specific polyclonal antibody or monoclonal antibody at related protein.Example comprises enzyme-linked immunosorbent assay (ELISA), cell sorting (FACS) method of radioimmunoassay (RIA) and fluorescence-activation.

The known multiple labelling of document also can be used to carry out different nucleic acid and determined amino acid with coupling technology.Several different methods is used for producing labelling hybridization or PRC probe so that detect the relevant polymerized nucleoside acid sequence of encoding with C/EBP β, and this comprises few labelling, incises translation, end labelling or the pcr amplification that uses certain labeled nucleotide to carry out.Also can adopt additive method,, its coded sequence or this sequence one one can be cloned in a certain carrier in order to obtain certain mRNA probe of C/EBP β.But the known this carrier of document obtains on the market, thereby available its carry out the vitro synthesized RNA probe, if still need in this process suitable RNA polymerase such as T7, T3 or SP6, and the nucleotide of labelling.These programs can be undertaken by multiple commercial reagent box.Adopted suitable reporter molecules or label comprise active nucleus, enzyme, fluorescein, chemiluminescence element, or colour coupler and substrate, common factor, mortifier, magnetic granule or the like.

Host cell with the nucleotide sequence transfer of coding C/EBP β can be cultivated under suitable condition, and this condition should be adapted at the expression and the recovery of related protein in the cell culture.The protein that reconstitution cell produces may be secreted or be included in the cell, and this depends on used sequence and/or carrier.As being understood by document, the expression vector that contains coding C/EBP β polymerized nucleoside acid sequence also may be designed to contain signal sequence, and this sequence can instruct the part fragment of C/EBP β to see through protokaryon or eukaryotic cell membrane is secreted out.

Also can adopt other recombination structures that the coded sequence of C/EBP β is connected with the nucleotide sequence of a kind of polypeptide domain of coding, improve the purification of soluble protein thus.The zone of this promotion purification includes but not limited to metal chelating peptide, as organize a tryptophan assembly, this assembly can allow the purification on solid metallic, or protein A territory, this territory helps solid-phase immunity globulin purification, have and comprise the zone that relates to when adopting FLAGS extension/protein affinity purification system (Immunex Corp., Seattle, WA product).The catenation sequence of cleavable comprises the XA factor or enterokinase Invitrogen, and the sequence that San Diego is special, these sequences promote between zone and the human C/EBP β sequence at purification, and the employing of these catenation sequences may be used for promoting purification.

Production except recombinant products, some fragment of C/EBP β also can see through direct polypeptide synthesize and produce, this can adopt the solid phase technique of having known in the document carry out (referring to, as, Merrifield J.Am Chem.Soc., 85:2149-2154 (1963)).Also can see through craft or automatization focuses and carries out protein synthesis.Any commercially available synthesizer that is suitable for correlation engineering might successfully carry out proteinic synthetic automatically.In addition, the also available chemical method of different C/EBP β is synthetic respectively, and through chemical method why it is formed relevant full length fragment binding molecule.

H. treatment

Such just as in this discussion, considered that C/EBP β peptide chain of the present invention will be used in some treatment of conditions and prevention, these diseases relate to unusual fibre modification.Really, modified CCAAT/ enhancer binding protein (this paper is called modification " C/EBP β ") finds to can be used for relating to fibrotic different occasion.One of them concrete example is that C/EBP β can and use with other conventional pharmaceutical preparations.Merge and use therapeutic agent to be considered to a kind of synergism can be provided, thereby reduce the effective dose of unitary agent and alleviate its side effect with different mechanism of action.Another concrete application is, when the anti-meaning sequence of the relevant numbering C/EBP β polynucleotide of a kind of vector expression, also this carrier may be used for a certain sick body so that treatment and prevention fibre modification.In addition concrete application of another is to adopt this anti-expectation to reach carrier to promote fibre modification (as the wound healing process).

Many expression vectors precursor of deriving is a retrovirus retrovirus, adenovirus, and herpes and vaccinia virus, or different bacterial plasmids, these deutero-expression vectors are used to nucleotide sequence is sent into target organs, tissue and cell colony.After the method for mentioning in other places except this paper, the sophisticated method of knowing in the document also can be used for building the recombinant vector that can express C/EBP β.

A lot of methods can be used to the carrier transfered cell or organize these methods to obtain, and can be suitable for reaching in the body external equally.For carrying out the treatment of inside and outside mode, carrier may need earlier, import from patient's stem cell and carry out the cloning amplification, and then syngenetic graft is returned in the same patient body.Also can import and be written into, can adopt the method for knowing in the document to carry out for this reason by the method for transfection or plasmalogen injection.

Above-mentioned any method all might be used to the main body of this treatment of arbitrary needs.For example comprise: mammal such as Canis familiaris L., cat, cattle, horse, rabbit, monkey, and the mankind more preferably.Noun " is used " (administering) when it relates to a peptide species, and its implication is meant and imports a kind of nucleotide sequence (or importing a kind of host cell that contains associated nucleic acid sequences) and this sequential coding and express related polypeptide.

Modification C/EBP beta polypeptides of the present invention also can be used for different tissues and cell.Consider that also any suitable route of administration will can be used for the present invention.Therefore, in some concrete application, using of polypeptide of the present invention can be adopted gene therapy method.Selectivity polypeptide transfer system, or any other suitable methods are so that be transported to interested position with polypeptide.In some preferential especially concrete application, these polypeptide are delivered into stellate cells.In other preferential concrete application, the using of these polypeptide, then oral administration or local application in further concrete the application without intestinal.

In some concrete application, preparation of the present invention is only given the administered single dose, under other situations a certain main body is then used multidose.Under preferential applicable cases, the drug delivery route that the present invention selects for use comprises: subcutaneous injection, and oral, intravenous drip, intra-arterial medication, lumbar injection, colon administration, intravaginal medication, local application, the intramuscular administration, the nasal cavity medication, the lung innerlich anwenden is (as sucking, spray into, etc.), administration in the trachea, cross percutaneous drug delivery, epidermis administration, administration under the connective tissue, the ophthalmic medication, medication near the eyes, the medication of eyeball Hou, the outer Layer medication of medication Veins arm film under the retina, marrow innerlich anwenden, intracranial medication, ventricle innerlich anwenden, and sheath inner medicine.In other concrete application, another group equipment has been selected in using of medicine for use, comprises the mechanicalness liquid reservoir, apparatus, implant, and paster.In further concrete application, preparation has been selected a kind of in following one group of form for use, comprises pill, capsule, and liquid, colloid, powder, suppository, suspending agent, unguentum is congealed agent, gas atomization agent and food additive.Polypeptide also can ointment in addition, the mode medication of lotion or gel (when promptly being used to handle skin and mucosa zone).Really, the present invention also is not intended to be only limited to any specific application process, because any suitable method all can be used for the present invention.In some concrete application, will have expression vector in the cell of expectation different tissues and express interested gene (promptly in these tissues, expressing interested polypeptide).

In other concrete application, in case these polypeptide mix into cell, they just can be competed with the wild nature type protein of endogenous C/EBP β.And in further concrete the application, the appearance of the C/EBP β peptide molecule of sudden change has caused Astrocytic apoptosis.And in other concrete application, the activation and the hypertrophy of stellate cells are prevented from.In preferential especially concrete application, this cell activation and outgrowth preventing cause the reduction that fibrous tissue forms in the liver and stop fully.In some instances, said method is used for the treatment of patient's chronic hepatopathy, or treatment suspect the sick body suffer from chronic hepatopathy (for example, include but not limited to hepatitis C, hepatitis B,, alcoholism, toxic or heritability hepatopathy).Further also think, the present invention may be used for preventing with liver transplantation after liver repel relevant liver fibre deformation.In further concrete the application, the appearance of C/EBP beta polypeptides sudden change product also can be used for inducing apoptosis of tumour cell.

The concrete application of another kind of the present invention relates to using of a kind of pharmaceutical preparation, and this is relevant with the received carrier of engraving on a kind of materia medica, to reach above-mentioned any therapeutic effect.This medicament can comprise C/EBP β, the antibody of C/EBP β, its analog, its analeptic, its antagonist, or its inhibitor.Said preparation can be used separately, or share with at least a other preparations, and as using with the chemical compound with Stabilization, this stabilizing agent is used for any sterilization, and the medicament carrier of biocompatibility includes but not limited to normal saline, glucose, and water.Said preparation can be used for patient separately, and perhaps with other reagent, medicine or hormone one are used from patient.As this paper point out, being applied to medicament of the present invention can be by above-mentioned any amount of suitable route administration.

Outside above-mentioned active component, these medicaments can contain the receptible load of suitable materia medica, comprise that excipient and adjuvant help active component is processed in the prepared product whereby, thereby can be used as pharmaceutical preparation, known in the document.

Medicament for oral use can be accepted carrier with known pharmacy in the pharmacopeia and be configured by suitable oral dosage.This carrier can make medicament be configured to tablet, pill, and dragee, capsule, liquid, gel, syrup, serosity, suspension, and analog are so that patient swallows.Active ingredient and solid excipient are share in the pharmaceutical preparation of oral use, also can as required mixture be ground, and the processing granular mixture if desired, adds suitable adjuvant, obtains the core of tablet or dragee thus.Suitable excipient is carbohydrate or protein implant, as saccharide, comprises lactose, sucrose, mannitol, or sorbitol; Corn starch, wheaten starch, rice starch, potato starch, or other plant starch; Cellulose, as methylcellulose, hydrogen-oxygen third methylcellulose, or the plain sodium salt of carbon oxygen methyl; The natural gum class comprises arabic gum and Tragacanth; Protein such as gelatin and collagen.Need as crossing, also can add distintegrant or lytic agent, as cross-coupled polyvinylpyrrolidone, agar alginic acid, or the salt of these chemical compounds are as Sodium Alginate.

The medicament of non-enterally administer may preferentially adopt the physiological compatibility buffer to prepare with saying that solution prepares, as " Hanks ' s " solution, Ringer ' s solution, or physiological buffering saline, the water-soluble injection suspension, as sodium carboxymethyl cellulose, sorbitol, or glucosan.In addition, the suspension of active component also can be prepared into oily injection suspensions.Suitable lipophilic solvent or carrier comprise fatty oil; As Oleum sesami or synthetic fatty acid ether, as the ethyl oleate, or glyceric acid, or liposome.As required, relevant suspension also can contain the reagent that suitable stabilizing agent maybe can improve compound dissolution, so that the preparation highly concentrated solution.

For ease of part and nasal cavity applied medicine, in the prescription employing is suitable for making some special barrier can be by penetrating penetrating agent.These penetrating agents can detect in pharmacopeia.

Medicament of the present invention can be made (, dissolving granulating, sugar-coat making, water mill, emulsifying agent, canned capsule, vanning, or lyophilizing processing as by conventional mixing) of known mode in the document.

The supply form of this medicament also can be used as the salt of multiple acid, and these acid include but not limited to hydrochloric acid, sulfonic acid, and acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, etc.Its corresponding radical form of salt analogy more is soluble in aqueous or other proton solvents.In other application scenarios, can preferentially prepare a kind of freeze dried powder, this powder contains any or whole components of following material: the 1-5mM histidine; The sucrose of 0.1%-0.2% and the mannitol of 2-7%, pH4.5-5.5 is incorporated in the buffer earlier with preceding.

After medicament is ready to, it is put in the proper container, and perform labelling for the processing of specified requirements.For the application of C/EBP β, this labelling seemingly should comprise the dosage of use, frequency and method.

Be suitable for medicament of the present invention and comprise such preparation, promptly its active component is contained in and can reaches in certain specific purpose effective dose.And but the decision reference literature of this effective dose carries out.

For any chemical compound, can the measuring by cell culture and/or at animal model according to a preliminary estimate of the effective dose that is used for the treatment of.Animal model also can be used to determine the concentration range and the route of administration that suit.The data of this respect further decides human effective dose and route of administration.A kind of dose therapeutically effective is meant that active component can mitigation symptoms or required dosage during the state of an illness.Therapeutic efficiency and toxicity are promptly carried out (for example, ED50 is a half treatment effective dose) and LD50 (half lethal dose) by the pharmaceutical procedures of standard in cell culture and zoopery.Dosage rate between treatment and the toxic action is therapeutic index (therapeutic index is expressed as LD50/ED50).

When presenting very high therapeutic index, then can preferentially adopt medicament.Cell culture is measured and the zooscopy result is used for using the preparaton weight range for the mankind.The preparation that contains this dosage preferentially is in the scope of circulation composition, and comprises ED50 and toxicity seldom or avirulence in this scope.DM in this scope depends on used dosage form, patient's sensitivity, and route of administration.

Dosage will be determined by the practitioner accurately, so that make decision according to the correlative factor that need carry out the patient of this treatment.Dosage and using method will be adjusted to the level that sufficient bioactive molecule can be provided, or maintain ideal effect.The factor that need consider comprises the seriousness of the state of an illness, patient's general health, and the age, body weight, sex, diet, time and medication frequency, the composition of medicine, the sensitivity of reaction, and to treatment tolerance/reaction.Long-acting medicament can every 3-4 days, weekly, or whenever biweekly, decide according to the half-life and the clearance rate of special formulation.

Normal dose also can be according to patient and route of administration and change.Can find the guide of given dose and medication in the document.The technology of document has adopted different prescriptions to prepare nucleotide preparation, and these prescriptions will be more than the making prescription of protein or its inhibitor.Equally, at special cell, condition, situations such as position also will need the transfer approach of each species specific polynucleotide or polypeptide.

I. diagnosis

In other concrete application, the energy specificity can be used for the diagnosis of the state of an illness or disease in conjunction with the antibody of C/EBP β, these diseases have the feature that fibre deformation increases or reduces, or detect change of illness state (modification C/EBP β for example of the present invention) in C/EBP β treatment back with this assay method.

Be preparation antibody, different hosts comprise goat, rabbit, rat, mice, human or other animals, will by inoculation tool immunogen characteristic or its fragment or its oligopeptide.According to host's kind, can adopt different adjuvants to come the enhance immunity reaction.These adjuvants include, but are not limited to freund adjuvant, and mineral rubber style such as aluminium hydroxide and surfactant are as molten blood lecithin ， Duo From (pluronic polyols), poly Overcast From, peptide class, emulsifier, key hole Destroy worm hemocyanin and dinitrophenol,DNP.Be used for human adjuvant, especially preferentially adopt bacillus calmette-guerin vaccine (BCG) and excellent Like Gan Jun Body.

Monoclonal antibody at C/EBP β can prepare with any technology, and these technology prepare antibody molecule by successive cell culture, include but not limited to hybridoma technology, human B cell hybridoma technology, and EBV-hybridoma technology.

In addition, also having developed the technology of a kind of preparation " chimeric antibody ", is that mice and human antibody gene are stitched together, and the antibody molecule of gained has suitable antigenic specificity and biological activity, can be used for known art in the document.In addition, also can adopt literature method to produce the single-chain antibody of C/EBP β.The antibody that has relative specific but be made up of specific idiotype simultaneously also can pass through the incompatible generation of random group of the medium-chain mobile realization in immunity albumen library of known method in the document.Also can produce antibody in vivo by induction of lymphocyte colony, or the required antibody of immunoglobulin library screening from recombinating, or known one group of binding reagents with high degree of specificity in the document.

Also can produce and contain the antibody fragment that C/EBP β is had the specific bond position.For example, this fragment includes but not limited to that (ab ') 2 fragments, this fragment obtain after by the pepsin hydrolysis antibody molecule F, and the Fab fragment then can (reduction of disulfide bond produces on ab ') 2 fragments by F.The Fab expression library also can be built to allow to identify fast and easily and have desirable specific Fab fragment in the monoclonal antibody in addition, as reporting in the document.

Different method of immunity can adopt and screen and evaluation has desirable specific antibody.Some operating processes are used for competitive combination or the immunoradiometric assay method is used for known specific polyclone or monoclonal antibody.These immunoassay typically relate to mensuration by the complex that forms between C/EBP β and its specific antibody.

The diagnostic assay of C/EBP β comprises utilization antibody and a kind of label, detects C/EBP β in the cell or tissue with this.Antibody in use can be through modification or not modified, and also adopting covalently or non-covalently in the possibility labelling, the key mode is connected with a kind of reporter molecules.A lot of reporter molecules of prophet may be utilized in the document, wherein have some and address in front.Distinct methods informs in the literature, and a kind of basis of the C/EBP of detection β expression is provided, and these methods comprise ELISA, RIA, IFA and FACS.

Another concrete application of the present invention is that the coding polynucleotide of C/EBP β can be used for diagnostic purpose.Can be utilized polynucleotide and comprise oligonucleotide sequence, anti-RNA and the dna molecular of anticipating, and PNAS.Polynucleotide can be used to detect the gene expression amount with the quantitative assay biopsy, and in these tissues, the expression of C/EBP β may alleviate interrelated with the disease and/or the state of an illness.Diagnostic assay may be used to distinguish the disappearance that C/EBP β expresses, occur and overexpression, and monitor therapy interfere in the adjusting of C/EBP β level.

On the one hand, the PCR probe can be surveyed polynucleotide series by hybridization, comprises nuclear group type sequence, the C/EBP β of coding or the molecule that is closely related, and this hybridization technique also can be used to the nucleotide sequence of identification code C/EBP β.The specificity of probe, according to it whether from the high special zone, strict degree with hybridization, or the degree (maximum of amplification, high, centre, or low) will determine relevant probe whether only can identify abiogenous C/EBP β and etc. the coded sequence at seat, or its correlated series (modification C/EBP β for example of the present invention).

Experiment

Following example is used for showing some preferential concrete application example of selecting of the present invention, is not with limiting range of application of the present invention.

In the experiment below, will use following abbreviation: eq (equivalents, homologue, coordinate); MM (millimole concentration, millimolar); μ M (micromolar concentration, micromolar); N (equivalent concentration, Normal); Mol (mole, moles); Mmol (millimole, millimoles); μ m (micromolar, micromoles); Nmol (nM, nanomoles); G (gram, grams); Mg (milligram, milligrams); μ g (microgram, micrograms); Kg (kilogram, Kilograms); L (rises, Liters); ML (milliliter, milliters); μ L (microlitre, microliters); Cm (centimetre or centimetre, centimeters); Mm (millimeter, millimeters); μ m (micron, micrometer); Nm (millimicron, nanometers); H and hr (hour, hours); Min (divides (clock), minutes); S and sec (Seconds); ℃ (degree, degrees Centigrade); V/V (the volume/volume ratio, Volume/Volume); W/V (by weight/volume; Weight/Volume); μ ci (micromicrocurie, microcuries); FDA (Food and Drug Administration, United States Food and Drug Adiminstration); DMEM (Dulbecco ' sModified Eagle Medium); LDL (low density lipoprotein, LDL, Low-density lipoprotein); DMF (dimethyl methylamine N, N-dimethyl-formamide); α-SMA (α-smooth muscle flesh Move albumen, α-smooth muscleactin); MDA (benzene two acyl dialdehyde, malondi-aldehyde); 4-HNE (the 4-hydroxyl, 4-Hydroxynonenal); PBS (phosphate buffer saline, phosphate buffered saline); FBS (calf serum, Fetal bovineserum); K ₂CO ₃(potassium carbonate, Potassium carbonate); NaHCO ₃(sodium bicarbonate, Sodiumbicarbonate); MgSO ₄(magnesium sulfate, magnesium Sulfate); MgCl ₂(magnesium dichloride, MagnesiumChloride): NaOH (sodium hydroxide, Sodium Hydroxide); FeSO ₄(iron sulfate, ferroussulfate): SD or S.D. (standard deviation, standard deviation); SEM (average standard error, Standarderror of the mean); ATCC (the culture aggregation of U.S. typical organization, American Type CultureColleetion, Manassas, VA); The Accurate Chemical ﹠amp of Jing Indeedization Learn scientific ﹠ technical corporation; ScientificCorp. (westbury, NY) precision subject skill company; Becton Dickinson Bake soup, and Dickenson (HuntValley, MD); Amesham (Arlington Heights, IL) the first nurse of Ah not company; Charles RiverBreeding Labs (Wilmington, DE) Charles River's breeding laboratory; Clonetics (Clonetics Corp, San Diego, CA) clone technology company; DuPont (Dupont CO, Wilmington, DE) E.I.Du Pont Company; Hitachi (Hitachi Scientific Instruments, Meuntain View, CA) Tianjin, island scientific instrument company; Hoechst (Santa Cruz Biotechnology, Santa Cruz, CA) Hoechst AG (DE) Frankfurt/Main 80, Federsl Republic of Germany, Sheng Ta-Cruz biotech company; Sigma (Sigma Chemical Company, St.Louis, MO) sigma chemical company; Molecular Probes (Eugene, OR) molecular probe company; Upstate Biotechnology (Lake Placid, NY) A Busidete biotech company, Vector Laboratories (Burlingame, CA) carrier laboratory; And Alexis (SanDiego, CA) A Lekeshesi.

Except as otherwise noted, otherwise the used cell strain of following experiment all obtain from ATCC.Unless explanation is arranged below in addition, experimental result all be expressed as three multiple pipes meansigma methods (± SEM), the difference of meansigma methods adopts and take seven methods of inspection that Xue Shi precisely tests between the assessment different experiments group, the P value is less than being considered to significant difference at 0.05 o'clock.

In the production process of the present invention the different proteins data were carried out retrieval, comprise Atlas recovery system protein data P1R1, P1R2, P1R3 and PATX (http://speedy.mips.biochem.mpg.de/mips/avaible_databases.html) include 347, protein data more than 000, comprise TVD more than 22,000 and the EVS sequence more than 27,000.Parent's apoptosis mutant comprises mice Ala ²¹⁷, human Ala ²⁶⁶And rat Ala ¹⁰⁵

Embodiment 1 cell culture and transfection

Bull Sprague-Dawley rat and bull C/EBP β ^{+ /+}With C/EBP β ^-/-(Screpantietal, aforementioned; And Bucketal., (1999), aforementioned), C/EBP β-Ala ²¹⁷With C/EBP β---Asp ¹⁰⁵Transgenic mice is used for separating liver spider cell of former generation (Houglumetal., aforementioned).Need be Ni Ke Vertical Hereby one step density gradient method (Single stepdensity Nycodenz gradient, Accurate Chemical ﹠amp by literature method then by being soaked in collagenase/pronase when liver spider cell of former generation is separated; Scientific) carry out purification.(see Leeet al., (1995), aforementioned).Cell is positioned over type i collagen machine matter (Becton Dickinson) or EHS substrate (Matrigel, Collaborative Biomedical product).Add 10% hyclone/10% calf serum during cell culture, use lipofection agent (lipofectamine then, GIBCO) with dna vector or peptide matters (10 μ m) transfectional cell is (referring to document Houglum et al., aforementioned with Chariot reagent (Activemotif); Morris etal.J.Biol.chem., 274:24942-24946 (1999)).The transgenic mice that adopts literature method (see Houglum et al., aforementioned and Lee etal., (1995), aforementioned) to produce can be expressed rat C/EBP β-Asp ¹⁰⁵Sudden change or mice C/EBP β-Ala ²¹⁷Mutant is backcrossed this transgenic mouse and FVB mice again.The available Southern trace of the appearance of neomycin phosphotransferase gene method predicts, and with this transgenic mice is identified.The also available specific antibody at HA or C/EBP β of the transfection albumen of endogenous expression shows (Santa Cruz Biotechnology).

During C/EBP β 1-253 expresses, lost a kind of dimer territory (Desconbes et al., aforementioned; Akira et al., aforementioned) (Fig. 4 A group) contain Thr simultaneously again ²¹⁷Or Glu ²¹⁷, but do not contain Ala ²¹⁷, this expression is block cell growth and survival not.Lack C/EBP β 1-215 and the KI of DNA in conjunction with territory and dimer territory ²¹⁷The existence of VD phosphorylation receptor (Fig. 4 A group) pair cell does not have influence.On the contrary, C/EBP β 216-253Ala ²¹⁷, but be not T ²¹⁷Or Glu ²¹⁷, also disappearance activates and the disome territory, but can impel control cells to express the back produces, apoptosis (Fig. 4 B group) at wild type RSK.In addition, C/EBP β-216-253-Glu ²¹⁷Be saving the inductive apoptosis of expression vector (RSKN ' C ') that cell avoids the saltant RSK of hydrolysis deactivation, the point mutation of this RSK occur in ATP in conjunction with the territory (Nakajima et al., aforementioned; With Bucket al., (1999) are aforementioned) (seeing Fig. 4 B group).Adopt the burnt microtechnique of copolymerization to obtain and consistent result (the Mao et al. of previous report, aforementioned and Ritter et al., aforementioned), be Procaspases1 and 8 in activatory Astrocytic nuclear and endochylema by being inspected, they and the C/EBP β same area (Fig. 4 C group) that coexists.Adopt the specific antibody of HA to come C/EBP β-HA or C/EBP β-216-253-HA in the activation spider cell are carried out immunoprecipitation, show the Thr of phosphorylation ²¹⁷With phosphorylated analogs Glu ²¹⁷, both all can be come out by coimmunoprecipitation with Procaspases1 and 8, and its efficient is higher than the possible Ala of no phosphorylation ²¹⁷(Fig. 4 D group).Therefore.The activation of C/EBP β and dimer territory (Descombes et al., aforementioned; With Akira et al., aforementioned) not required for spider cell existence, do not associate mutually with Procaspases1 and 8 yet.

Embodiment 2 fluorescence microscopy

Fluorescent marker adopts triple channel (triple-channel) fluorescence microscope to observe.Fluorochrome is the plain and texas Red (molecule is visited Needles molecular probes) of Iso Hydrogen thiocyanate Fluorescent light.The quantity of annexin V (+) cell (is seen Rudel and Bokoch by measuring at the body microscopy, aforementioned), be determined in all cells and carry out, or in the cell of those expression GFP indicator protein, carry out, this GFP or with C/EBP β albumen cotransfection, or transfection separately.These measured values are expressed as in the cell of expressing transfection DNA, the percentage that annexin V (+) cell is shared.In each experimental period point, at least 1000 cells are analyzed, wherein comprise 100 cell (Buck andChijkier, EMBO J V., 15 of expressing transfection DNA at least; 1753-1765 (1996); Buck et al., (1994), aforementioned; With Buck et al., (1999), aforementioned.Adopted ApoAlert mitochondrion sensor determination method (Clontech) in some experiments, adopted a kind of dye of positive ion in the method, this dyestuff presents red fluorescence when gathering the mitochondrion of healthy cell, and in the endochylema of apoptotic cell, because its mitochondrial membrane changing function, cause dyestuff still to be monomer structure, thereby present green (Green and Reed, aforementioned).The karyomorphism analysis adopts Hoechst33342 (molecular probes) pair cell to dye and carry out.Laser Scanning Confocal Microscope is used for observing spider cell, and the antibody of Cai Yonging has specificity (Santa Cruz Biotechnology) to C/EBP β and procaspases8 simultaneously.

For example, in one group of experiment, research purpose is to attempt to find out ser ¹⁰⁵Phosphorylation whether can make C/EBP β have the ability save cell to avoid apoptosis.Activatory mouse liver spider cell is incubated in a kind of worker's Collagen Type VI substrate, found that, the expression of the positive dominant mutant of RSK (Nakajima et al is aforementioned) or locate cell with Lactacystin (a kind of proteasome inhibitors) all can be induced the generation apoptosis process.And phosphorylated analogs rC/EBP β Asp ¹⁰⁵The transfection of (Trautweinet al., aforementioned) is simultaneously also changeed CMV-GFP and is instructed thing, and discovery can make stellate cells avoid apoptosis, is used in body fluorescence microscope annexin-V-PE and mixes the result and confirmed this point (Fig. 5 B group).With C/EBP β-Ala ²¹⁷Identical, rC/EBP β-Ala ¹⁰⁵Also can cell death inducing, for example through activated mouse liver spider cell of former generation, because the neither one mutant contains the phosphate acceptors territory (Buck et al., (1999) are aforementioned) of a kind of RSK.These results suggest rC/EBP β-Pser ¹⁰⁵(or rC/EBP β-Asp ¹⁰⁵) can block apoptosis pathway, with C/EBP β-PThr ²¹⁷Effectively same, be (Fig. 1) like this at least under the experiment condition of addressing.

Embodiment 3 animal program and tests

As described herein, want to determine whether available CCl by research ₄, a kind of can induce the urgent and Fibrogenesis of liver oxygen (Houglum et al., aforementioned; And Chojkier, aforementioned) hepatotoxin, promote the activation of stellate cells.As described herein, adopt literature method to produce transgenic mice, make it can express C/EBP β-Asp of rat ¹⁰⁵Or C/EBP β-Ala of mice ²¹⁷(Houglum et al, (1995), aforementioned); Lee et al (1995) is aforementioned), then itself and FVB mice are backcrossed.The appearance of neomycin phosphotransferase gene will be used to identify that transgenic mouse, method are the Seuthern traces.

C/EBP β ^{+ /+}, C/EBP β ^-/-With C/EBP β-Ala ²¹⁷Or C/EBP β-Asp ¹⁰⁵Transgenic mice (25 gram) is accepted the CCl of single dose separately ₄Lumbar injection, CCl ₄Be dissolved in mineral oil (1: 1, Vol/Vol) (50 μ l), or only be mineral oil (25 μ l) (Buck et al., (1999) are aforementioned).After 12 hours, animal is used to carry out immunofluorescence analysis, carries out (Buck et al (1994) is aforementioned) under Laser Scanning Confocal Microscope.Astrocytic evaluation has been adopted at the specific antibody of neuroglia fibres acidic protein and Vimentin (Santa Cruz Biotechnology).Activated Caspase3 also observes (Pharmingen) by its specific antibody.

The stellate cells of identifying by glial fibrillary acidic protein and Vimentin dyeing (Ankoma-Sey and Friedman, aforementioned) can be through CCl ₄C/EBP β after single dose is handled ^{+ /+}Induced hypertrophy in the liver of mice.On the contrary, at C/EBP β ^-/-With C/EBP β-Ala ²¹⁷In the liver of transgenic mice, Astrocytic apoptosis can be induced generation (Fig. 3), and this is that generation owing to effector caspase3 is identified.Because activated P90RSK can cause Thr on the C/EBP β external ²¹⁷Phosphorylation, thereby interesting go to find out in these animals whether RSK can associate mutually with C/EBP β, described in example 4.

Embodiment 4 immunoprecipitations and immunoblotting

C/EBP β, Procaspases1 and 8 and the detection of RSK need adopt at C/EBP β and RSK specific antibody (Santa Cruz Biotechnology product) and at the specific antibody (Pharmingen) of Procaspases1 and 8, the lysate that these antibody is acted on cell and liver carries out immunoblotting [Buck etal., (1994), aforementioned], shown by chemiluminescence (Dupout) method then.These results exempt to draw RSK-5 antiserum (the J.Blenis friendliness provides) and anti--RSK R23820 (Transduction Laboratories) mensuration that antiserum carried out to be confirmed by adopting.The activatory P90RSK of friendship can cause Thr on the C/EBP β under isolated condition ²¹⁷Phosphorylation [Buck etal., (1999), aforementioned], thereby interestingly determine whether RSK associates mutually with the C/EBP β of these live animal.In these experiments, adopt specific antibody that C/EBP β is carried out immunoprecipitation, here, C/EBP β is from liver spider cell of former generation, and these cells are the C/EBP β that handles from CCL4 of fresh separated then ^{+ /+}With C/EBP β-Ala ²¹⁷Mice, and in the immunoprecipitation.Its result points out, from the C/EBP β of CCL4 processing ^{+ /+}Mice, rather than C/EBP β-Ala ²¹⁷Mice, the association between RSK in its spider cell and the C/EBP β raises, as shown in Figure 3.The specific antibody (Santa Cruz Biotechnology) at P90RSK, this antibody and P70S6 kinases no cross reaction are adopted in the evaluation of RSK.These results further adopt other two species specific antibodies to be confirmed, wherein a kind of is anti--RSK-5 antiserum (the J.Blenis. friendliness is gifted), and another kind of resisting-RSK antibody (Transduction Laboratories supply).When RSK albumen was come out by the specific antibody immunoprecipitation, C/EBP β was found and is precipitated out by the appearance instrument simultaneously, and this C/EBP β that is handled at CCL4 by the C/EBP β level of co-precipitation ^{+ /+}All the time the phenomenon that presents rising in the mice.Contrast property preimmune serum, the immunoglobulin G of anti--beta galactosidase or purification (in the immunoprecipitate that IgG-produces, contains RSK or C/EBP β that detection does not go out level, thereby has confirmed the specificity of above-mentioned coimmunoprecipitation phenomenon.These find that strong suggestion RSK is to Thr on the C/EBP β ²¹⁷Phosphorylation the existence of stellate cells is absolutely necessary.

The phosphorylation of embodiment 5 C/EBP β

Separate in mice and the rat liver former generation spider cell at EHS (Matriget; Becto-Dickinson) (Couaborative Researdch) (active cell) cultivated the 4th day on (akinete) or the type i collagen substrate, last 30 minutes training liquid with Somci add 20% hyclone then), then the reference literature method make two-phase type collection of illustrative plates (see Trantwein efal, aforementioned; Buck ef al., (1999), aforementioned).

In order to observe the phosphorylation reaction of Rsk in external generation, the reference literature method has detected SPg through the C/EBP of immune purification β, C/EBP β-AThr ²¹⁷, rC/EBP β and rC/EBP β-Ala ¹⁰⁵To containing AThr ²¹⁷Or Pser ¹⁰⁵The evaluation suggestion of polypeptide phosphate adopt the nigration of generally acknowledging polypeptide to carry out and the employing by special people's antibody confirms, the specific antibody of Cai Yonging is revived at the mankind's C/EBP β/rely-green-Lai-third-Lai-Lai-phosphorylation here ²⁶⁶-figured silk fabrics-Radix Asparagi-Lai-group-green-aspartic acid KSKAKK-PhosphoT ²⁶⁶VDKHSD (SEQ ID N0:16) is with the PThr of mice ²¹⁷Homology) and rat C/EBP β (rely-dried meat-green-Lai-Lai-dried meat-phosphorylation is green ¹⁰⁵-Radix Asparagi-cruel-Gan-cruel-figured silk fabrics-green (SEQ ID NO:17)).

In experiment, whether can induce C/EBP β to produce phosphorylation in order to find out type i collagen substrate, former generation spider cell quilt of mouse liver ³²P-Orthophosphate labelling 12 hours, and be placed on EHS (Matrigel) (akinete) or type i collagen (activating cell) substrate and cultivate.The phosphoeptide collection of illustrative plates of the C/EBP β of immunoprecipitation shows that the type i collagen base can be induced site-specific phosphorylation, and this phosphorylation process betides the THr on the endogenous C/EBP β ²¹⁷, the phosphoric acid variant field of a kind of P90RSK (Buck ef al., (1999), aforementioned).

And, shown in Fig. 1 result, another not certified as yet phosphoric acid variant site.Be incubated in the static spider cell of mice of EHS substrate, the phosphorylation process of C/EBP β is faint to the (see figure 1) of can ignoring.

Although C/EBP β-Thr during evolution ²¹⁷The phosphate acceptors high conservative, this comprise human C/EBP β (Cao efal., aforementioned; Akira ef at., aforementioned. and Yamaoko.ef al is aforementioned).Two consolidations have still taken place in (r) C/EBP β of rat, lacked Thr phosphorylation receptor (Descompes ef al, aforementioned; With Pali ef al, aforementioned).C/EBP β-PTh ²¹⁷Or rC/EBP β-Pser ¹⁰⁵For the inductive cell proliferation of extracellular factor is critical (Bucket al., (1999) are aforementioned).The phosphorylation that whether can induce γ C/EBP β for test I Collagen Type VI substrate, isolated former generation spider cell employing 32P-orthophos-phate labelling is 12 hours in the rat liver, and is incubated on EHS (Matrigel) (akinete) or the type i collagen substrate (activating cell).The phosphoeptide collection of illustrative plates of C/EBP β shows that type i collagen substrate can be to Ser105 on the endogenous γ C/EBP β behind the immunoprecipitation, a kind of p90RSK phosphorylatable domain (Buck et al., (1999) are aforementioned) and the still unidentified phosphate acceptors of another kind site (seeing Fig. 6 A group).The rat resting stage spider cell of cultivation on EH3 substrate, its γ C/EBP β content can be ignored (seeing Fig. 6 A group).

In addition, find out that also the functional dependency between the phosphorylation of C/EBP β and its Thr217 adopts wild type (meaningful) or anti-meaning, C/EBP β expression vector the activatory spider cell of mouse liver has been carried out transfection.Found that, can express C/EBP β antisense rather than have the spider cell of adopted RNA (Buck ef al, (1994)) not see propagation, measuring the proteic expression of proliferating cell nuclear, also see analog result (BRAVO, ef al., aforementioned; Buck ef al., (1994), aforementioned; Buckef al., (1999), aforementioned), but this combining to present and increase of the green propylhomoserin of phosphoric acid on the annexin of proliferative cell (annexin-v) and the brains film not, this increasing is that Fig. 1 C group is seen in apoptotic a kind of early stage indication (Rudel and Bokoch, aforementioned)).For finding out Thr on the mice C/EBP β ²¹⁷Phosphorylation for type i collagen, during inductive stellate cells, adopted the MSV expression vector of the C/EBP β consolidation body that can express no phosphorylation probability.The method that the evaluation of transfectional cell adopts triple channel Fluorescent light microscope to observe the CMV-GFP (green Fluorescent photoprotein matter) of cotransfection is carried out.Express C/EBP β-LAa ²¹⁷The cell of consolidation body (Buck ef al (1999), aforementioned) is not seen propagation, becomes apoptotic cells simultaneously, and this point is determined by intravital microscope (seeing Fig. 1 C group) with at the cell sorting method of transfection indicant GFP and annexin V.In contrast, the similar C/EBP β-GLu of phosphorylation ²¹⁷The consolidation body can make stellate cells avoid being induced to produce apoptosis (Buckef al., (1999), aforementioned), but this occurs in anti-apoptosis factor NFRB (Beg and Baltimore, aforementioned and Wang ef al., aforementioned) or RSK (BUCK ef al., (1999), aforementioned, Bonni ef al., aforementioned, Bhattand Ferrell. aforementioned, Gross ef al. is aforementioned; Sassone-Corsi ef al., (1999), aforesaid pay to live (see Fig. 1 D group) when being blocked.

(the Lee ef al., (1997), aforementioned) that MEK silk floss enzyme 1 (MEKKI) needs to the activation of NFKB that phosphorylation by IKB kinases d and β make then that the phosphorylation of IKB carries out; Nakano ef al., aforementioned), in the middle of this by ubiquitin-proteasome by way of having experienced Proteolytic enzyme process (Chen ef al, (1995a), aforementioned) rapidly.Thereby, MEKKI has been adopted in the Astrocytic transfection of activated former generation, the expression vector of the negative dominance consolidation body of IKB2 and RSK, or handle cell with Lactacystin (a kind of proteasome inhibitor) that can cell death inducing.Under situation separately, because C/EBP β is arranged ²¹⁷Thereby expression avoided inducing of spider cell apoptotic process, prompting, when NFKB that swashs when disappearance or RSK, Thr on the C/EBP β ^MusPhosphorylation be enough to keep the existence of stellate cells.

In order to find out the function correspondence of mice C/EBP β, and Thr ²¹⁷Phosphorylation, also the Astrocytic existence of former generation has been carried out investigating (Screpanti ef al., aforementioned), the spider cell is here separated the mice of being removed by targeting from C/EBP β, found that to have only C/EBP β ^-/-Mice, rather than C/EBP β C/EBP β ^{+ /+}The spider cell of mice is insensitive and beginning apoptosis to the growth-promoting effect of type i collagen, and this confirms (Fig. 2 A and C group) by activation microscope memebrane protein in conjunction with Qing Qu.Be research C/EBP β-PThr ²¹⁷To the effect of spider cell existence, develop and a kind of can the expression.

The transgenic mice of the negative dominant mutant of C/EBP β-Ala217 that no phosphorylation is possible.Though this C/EBP β of university-Ala217 transgenic mice grows normal, isolated spider cell can not under the type i collagen activation condition, survive all the time (seeing that Fig. 2 A and C organize) from these animal liverss.By a kind of mitochondrion sensor determination, can predict the increase of mitochondrial membrane permeability.Confirmed to separate Astrocytic apoptosis phenomenon (seeing Fig. 2 C group) thus from C/EBP β-1-and C/EBP β-Ala217 mice.

Embodiment 6

By the apoptosis of inductive matter of C/EBP β-Ala217 and cancerous cell, in these experiments, C/EBP β-Ala217 is used to two kinds of Interstitial cell systems to test the ability of its cell death inducing.Human Interstitial cell is a 1321-N1 (colloid carcinoma cell; Available from Dr.Joan Brown, branch school, Santiago, University of California), RD (skeletal muscle cancerous cell; ATCC (enters number: CCL-136), (the 98VA3N cell is available from Dr.Jerry Vanden Berg, Veterans Administration medical center for the synthetic fibers cell, the Santiago, California), ES2 (ovarian cancer cell line), C3A (hepatoma cell line) and MCF-breast cancer cell are incubated at the minimum essential medium that is supplemented with calf serum by literature method (to be seen, for example, Buck et al., EMBOJ., B:151-160 (1994)).As adopted cell type in the present invention, the present invention is not intended to only limit to these special cell lines.Really, result similarly obtains (for example, CCF-STTG1 cell (ATCC enters numbering: CRL01718)) (not video data) from the cell that adopts other types.

Carried out cotransfection by document known method pair cell, used carrier can be expressed indicator protein GFP and C/EBP β-Ala217 (seeing Buck et al., Mol.Cell, 4:1087-1092 (1999)).Control cells is only carried out transfection with GFP.After the transfection the 4th hour, phosphoric acid silk ribbon propylhomoserin on cell is used to measure its annexin and be exposed to the endochylema film combine feature, it is a kind of early stage index of cell membrane apoptosis, known to document, adopt intravital microscope and commercially available apoptosis test regent box (research and development system house, R ﹠amp; D (1997)).

The induced expression of C/EBP β-Ala217 mutant that no phosphorylation is possible the apoptosis of these Interstitial cells, as shown in table 2 as follows:

Table 1. by C/EBP β-Ala217 cause between matter and cancer cell-apoptosis

Cell line	Apoptosis (%)
			Contrast	????C/EBPβ-Ala217
	????RD	????9			????46*
????1321?N1			????4	????56*
	????98?VA3N	????6			????80*
????C3A			????5	????34*
	????ES2	????7			????44*
????MCF-F			????4	????49*

P＜0.01

Therefore, above-mentioned data point out that C/EBP β-Ala217 will be found and can be used as a kind of therapeutic agent, are used for the treatment of the different tissues that comprises cancerous cell and the fibre modification of cell type.

Embodiment 7

Activation of cysteinyl aspartic acid prokinase (Procaspases) and cysteinyl aspartic acid kinases (Caspase) and activity.

In these experiments, the reorganization of purification and synthetic C/EBP beta polypeptides are used to detect it to the reorganization Procaspases1 of purification and 8 activatory inhibition ability (Alexis Biochemicals).

The regroup C/EBP beta polypeptides of purification is expressed as literature method and (to be seen Descombes et al., Supra; AndBuck et al.; (1994); aforementioned); with the synthetic N-acetyl group of purification, C-aldehyde radical tetrapeptide (200 μ M) (U.S. peptide class company) is used to measure its class Caspases1 to the reorganization of purification (Catalogue#201-056) and 8 (Catalogue#201-0410C005) (Alexis Biochemicals).The sequence of Caspase8 comprises by the clone advances a kind of Ser217 of the expression vector that contains 21 aminoacid junctional complexs at its N end to the sequence fragment between the Asp439.Therefore, (preceding 220 aminoacid are deleted substantially for its forefoot area.This fragment is cleaved in the Asp385 site, simultaneously this activated Caspase and those Caspase basic identical (Stennicke and Salvesen, Meth EnzymoL, 322:91-100 (1997)) that is identified in apoptotic cell.The active mensuration of Caspase is undertaken by the release of phosphoric acid one nitro alanine (P-Nitroalanine) colorimetric (Colormetric) substrate, mensuration to Caspase1 (Catalogue#260-026) or 8 (Catalogue#260-045) all requires to assign to carry out (Thornberry et al. at the line part of power determination, (1997), aforementioned; Stennicke and Salvesen, aforementioned).Caspases1 and 8 active mensuration are undertaken by using quality test kit (Catalogue#850-2110K and 850-221-K) in the cell, and with reference to producer's explanation (Alexis) (see Thornberry et al., (1997), aforementioned).The mortifier IETD of Caspase8 from California biochemical corp (Calbiochem) (Catalogue#218773).

The phosphate acceptors of supposing the cattle and the mankind's C/EBP β contains a kind of KT217VD sequence (Buck et al., (1999), aforementioned), can on function, be simulated (Bucket al. by the inductive phosphorylation of RSK by the KE217VD in C/EBP β-Glu217 sequence, (1999), aforementioned), whether and the latter is again similar in appearance to a kind of XEXD box-like body of finding in mortifier/substrate of Caspase, thereby investigate C/EBP β-PThr217 by experiment and associate mutually with Procaspases.The result shows, adopts specific antibody to carry out immunoprecipitation, can show C/EBP β, this C/EBP β be present in fresh classification from C/EBP β+/+and the liver spider cell of C/EBP β-Ala217 transgenic mice.Adopted specific antibody and identified starting material Procaspases1 and 8 (seeing Fig. 3 D group) but be not effector Procaspases3,6 and 7 (Thornberry and Lazebnik, aforementioned; Ashkenazi and Dixit, aforementioned; And Earnshaw et al., aforementioned), this is at the specific antibody at C/EBP β, rather than contrast IgG, immunoprecipitate in identify, and this immunoprecipitate hang oneself Ccl4 handle C/EBP β+/+spider cell that is activated behind the mice.

In the cell of C/EBP β-Ala217 mice after Cel4 handles, and the C/EBP β after contrast mineral oil is handled+/+cell of mice in, C/EBP β and Procaspases1 and 8 association still are in baseline values (Fig. 3).But the C/EBP β that handles through Ccl4-/-or C/EBP β-Ala217 rather than C/EBP β+/+mice, the activity of starting material caspases1 in its spider cell and 8 (see figure 3)s and effector caspases3 (see figure 3) all raises.The phosphorylation of Thr217 or its phosphorylated analogs C/EBP β-Glu217 mutant need for the association between C/EBP β and Procaspases1 and 8 in these presentation of results C/EBP β sequence, the processing and the activation of pointing out this association might prevent Procaspases1 and 8 simultaneously, because the priority substrate that KPhospho-T217VD or KE217VD all are not caspases1 to be brought disaster upon (Wilson et al., aforementioned; Margolin et al., aforementioned; With Earnshaw et al., aforementioned).

For illustrating the effect of C/EBP β for Procaspases1 and 8 activation processs, the external evoked autoactivation process that has produced regroup Procaspases1 and 8 (Wilson et al., aforementioned; Margolin et al., aforementioned; With Earnshaw et al., aforementioned).Also clarified simultaneously of the effect of the recombinant C/EBP beta polypeptides of purification to this course of processing, this recombinant polypeptide press the preceding method generation (Descombes et al., aforementioned; With Buck et al., (1994) are aforementioned).Consistent with experimental result in the above-mentioned body, C/EBP β-HA-Glu217 and C/EBP β 216-253-HA-Glu217 polypeptide, but be not C/EBP β HA-Ala217 polypeptide, can associate with Procaspases1 and 8 (seeing Fig. 5 A group).C/EBP β-the Glu217 of reorganization and the polypeptide segment AC-KPhospho-T217VD-CHO of synthetic and AC-KE217-VD-CHO all produce inhibitory action (seeing Fig. 5 B group) to the activation process of Procaspases1 and 8.Find that in addition in activatory liver spider cell, cell bpi peptide Segment A C-KA217VD-CHO pair cell apoptotic process produces inducing action (seeing Fig. 5 C group).

All that mention are delivered document and patent and are all compiled in the list of references in the above description.Also will be considered to be in above-mentioned document and the patent conspicuously to the modification of the different modes of method and system of the present invention and change, and thereby not depart from scope of the present invention and violate spirit of the present invention.Although the present invention combines some preferential especially application examples and narrated, these are understood that the present invention when application claim, should only not limited to the concrete practical case of these characteristics inadequately.In fact, the difference that runs through that narrating mode of the present invention changes all can obviously see above-mentioned document and patent, thereby has a mind to these modifications and change are all included within the scope of the present invention.

Claims (49)

1. one kind is used for increasing apoptotic method, it is characterized in that: this method composed as follows:

A) precondition

I) at least a cell; With

An ii) peptide species, this polypeptide is made of one or more tetrapeptide arrays, and in the mentioned herein tetrapeptide array, having one or more sequences at least is to be selected from one group of tetrapeptide array, and contains KAVD and KKPA in this group tetrapeptide array; And

B) thus aforementioned polypeptides is used the cell that language relates to here is increased the apoptosis of this cell.

2. according to claim 1ly a kind ofly be used for increasing in the apoptotic method, it is characterized in that: wherein mentioned tetrapeptide array is KAVD.

3. according to claim 1ly a kind ofly be used for increasing in the apoptotic method, it is characterized in that: wherein said tetrapeptide array is KKPA.

4. according to claim 1ly a kind ofly be used for increasing in the apoptotic method, it is characterized in that: wherein said cell is to be selected from following one group of cell, comprising: liver cell, the lungs cell, kidney cell, Skin Cell, muscle cell, heart cell, neurogliocyte, ocular cell, vascular cell, neurocyte, epithelial cell, endotheliocyte, and Interstitial cell.

5. according to claim 1ly a kind ofly be used for increasing in the apoptotic method, it is characterized in that: wherein said cell is a cancerous cell.

6. according to claim 5ly a kind ofly be used for increasing in the apoptotic method, it is characterized in that: wherein said cancerous cell is metastatic cancerous cell.

7. according to claim 1ly a kind ofly be used for increasing in the apoptotic method, it is characterized in that: wherein said cell be external be cell under the isolated condition.

8. according to claim 1ly a kind ofly be used for increasing in the apoptotic method, it is characterized in that: wherein said cell is meant the intravital cell of a certain master or in somatic cell.

9. one kind is used for reducing apoptotic method, and it is characterized in that: it is composed as follows:

A) precondition:

I) at least a cell; With

Ii) a kind of polypeptide that has one or more tetrapeptide arrays to constitute, in the said here tetrapeptide array, having one or more tetrapeptide arrays at least is to select from following one group of tetrapeptide array, comprise KEVD in this group tetrapeptide array, K-PhosphoT-VD, KKPD, and KKP-PhosphoS; And

B) thus aforementioned polypeptides is applied to said cell is minimized its apoptosis of said cell.

10. according to claim 9ly a kind ofly be used for reducing in the apoptotic method, it is characterized in that: wherein said tetrapeptide array is KEVD.

11. according to claim 9ly a kind ofly be used for reducing in the apoptotic method, it is characterized in that: wherein said tetrapeptide array is K-PhosphoT-VD.

12. according to claim 9ly a kind ofly be used for reducing in the apoptotic method, it is characterized in that: wherein said tetrapeptide array is KKPD.

13. according to claim 9ly a kind ofly be used for reducing in the apoptotic method, it is characterized in that: wherein said tetrapeptide array is KKP-PhosphoS.

14. one kind is used to reduce the method that certain tissue cancerous cell produces, it is characterized in that: this method is composed as follows:

A) precondition is:

I) a kind of tissue; With

An ii) peptide species, this polypeptide is made of one or more tetrapeptide arrays, and at least a or multiple in the said here tetrapeptide array is to be selected from one group of tetrapeptide array being made up of KAVD and KKPA; And

C) thus aforementioned polypeptides is applied to said tissue is minimized the cancerous cell quantity in this tissue.

15. a kind of being used to according to claim 14 reduced the method that certain tissue cancerous cell produces, it is characterized in that: wherein said tetrapeptide array is KAVD.

16. a kind of being used to according to claim 14 reduced the method that certain tissue cancerous cell produces, it is characterized in that: wherein said tetrapeptide array is KKPA.

17. a kind of being used to according to claim 14 reduced the method that certain tissue cancerous cell produces, it is characterized in that: wherein said tissue is present in a certain mammal main body.

18. a kind of being used to according to claim 17 reduced the method that certain tissue cancerous cell produces, it is characterized in that: wherein said mammal main body is meant the mankind.

19. a kind of being used to according to claim 14 reduced the method that certain tissue cancerous cell produces, it is characterized in that: wherein said main body is the main body that cancer cell occurs under a cloud in its linked groups.

20. a kind of being used to according to claim 14 reduced the method that certain tissue cancerous cell produces, it is characterized in that: wherein said main body is meant the main body that has cancerous cell to occur in its relevant tissue.

21. a method that is used for reducing the fibre modification (or fibrosis) of certain operation, it is characterized in that: it is composed as follows:

A) precondition is:

I) a certain tissue; With

Ii) a certain polypeptide, this polypeptide is made of one or more different tetrapeptide arrays, and in the said here tetrapeptide array, at least a or multiple is to be selected from one group of tetrapeptide array being made up of KAVD and KKPA; With

B) aforementioned polypeptides being applied to the above-mentioned fibre modification that makes thus in this tissue of organizing is minimized.

22. a kind of method that is used for reducing the fibre modification (or fibrosis) of certain operation according to claim 21, it is characterized in that: wherein said tetrapeptide array is meant KAVD.

23. described a kind of being used for reduces the method for the fibre modification (or fibrosis) of certain operation according to claim 21, it is characterized in that: wherein said tetrapeptide array is meant KKPA.

24. a kind of method that is used for reducing the fibre modification (or fibrosis) of certain operation according to claim 21, it is characterized in that: wherein said tissue is present in a certain mammal main body.

25. a kind of method that is used for reducing the fibre modification (or fibrosis) of certain operation according to claim 24, it is characterized in that: wherein said mammal main body is meant the mankind

26. a kind of method that is used for reducing the fibre modification (or fibrosis) of certain operation according to claim 21 is characterized in that: wherein said main body is meant the main body that has fibre modification to produce under a cloud in its intravital certain tissue.

27. a kind of method that is used for reducing the fibre modification (or fibrosis) of certain operation according to claim 26, it is characterized in that: wherein said fibre modification is selected from as next group fibre modification, comprising: the hepatic fibrosis degeneration, the lungs fibre modification, kidney fibre modification, or eyeball fibre modification.

28. a kind of method that is used for reducing the fibre modification (or fibrosis) of certain operation according to claim 27, it is characterized in that: wherein said hepatic fibrosis degeneration is repelled relevant with liver transplantation, also infect with hepatitis C, B-mode infection, liver alcoholism, liver poisoning, the hereditary hemochromatosis element is precipitating disease, and porphyria is relevant with liver cirrhosis.

29. a kind of method that is used for reducing the fibre modification (or fibrosis) of certain operation according to claim 26 is characterized in that: a wherein said fibre modification and a heavy disease association, this disease is selected from following gang disease, comprising the cranial glia fibre modification, Alzheimer, hepatopathy, brain injury, the nerve wound, the nerve fiber degeneration, myocardial infarction, arteriosclerosis, fibrosis dermatosis and fibrosis hepatic disease.

30. one kind is used for reducing apoptotic method in a certain tissue, it is characterized in that: it is constructed as follows:

A) precondition is:

I) a certain tissue; With

Ii) a certain polypeptide; This polypeptide constitute one or more tetrapeptide arrays, and at least a or multiple in this tetrapeptide array be to be selected from by comprising KEVD, KPhosphoTVD, KKPD and KKPphosphoS are at one group of interior tetrapeptide array; With

B) aforementioned polypeptides being applied to the above-mentioned apoptosis that makes thus in this tissue of organizing is minimized.

31. a kind of being used for reducing in a certain tissue in the apoptotic method according to claim 30, it is characterized in that: wherein said tetrapeptide array is KEVD.

32. a kind of being used for reducing in a certain tissue in the apoptotic method according to claim 30, it is characterized in that: wherein said tetrapeptide array is K-PhosphoT-VD.

33. a kind of being used for reducing in a certain tissue in the apoptotic method according to claim 30, it is characterized in that: wherein said tetrapeptide array is KKPD.

34. a kind of being used for reducing in a certain tissue in the apoptotic method according to claim 30, it is characterized in that: wherein said tetrapeptide array is KKP-phosphoS.

35. a kind of being used for reducing in a certain tissue in the apoptotic method according to claim 30, it is characterized in that: wherein said tissue is present in a certain mammal main body.

36. a kind of being used for reducing in a certain tissue in the apoptotic method according to claim 35, it is characterized in that: wherein said mammal main body is meant the mankind.

37. a kind of being used for reducing in a certain tissue in the apoptotic method according to claim 30 is characterized in that: exist in its intravital certain tissue of wherein said main body or under a cloudly have an apoptotic phenomenon.

38., it is characterized in that according to described a kind of being used for reducing in a certain tissue in the apoptotic method of claim 37: wherein its said apoptosis and wound, burn, damage, environmental toxin or ischemia are relevant.

39. according to described a kind of being used for reducing in a certain tissue in the apoptotic method of claim 37, it is characterized in that: wherein said apoptosis and a certain disease association, and this disease is present in a certain tissue, and this tissue is selected from following one group of tissue, comprising lung tissue, nephridial tissue and eyeball tissue.

40. according to described a kind of being used for reducing in a certain tissue in the apoptotic method of claim 37, it is characterized in that: wherein said apoptosis is relevant with a kind of hepatopathy, this hepatopathy is selected from following one group of hepatopathy, repel comprising liver transplantation, hepatitis C infects, and is hepatitis B infected, liver alcoholism, toxic hepatic disease, plain hemachromatosis of hereditary hemochromatosis and porphyria.

41. according to described a kind of being used for reducing in a certain tissue in the apoptotic method of claim 37, it is characterized in that: wherein said apoptosis and a kind of disease association, this disease are selected from following one group of disease, comprising brain disease fibre modification, Alzheimer, hepatopathy.Brain injury, nerve damage, neural degeneration, myocardial infarction, arteriosclerosis, fibrosis dermatosis and fibrosis hepatic disease.

A 42. peptide species, it is characterized in that: contain one or more tetrapeptide arrays in the composition of this polypeptide, in the composition of this tetrapeptide, have at least one or more tetrapeptide arrays to be selected from one group of tetrapeptide array forming by KAVD and KKPA, this sequence at least can with a kind of the combining in Procaspases1 and 8, and this binding energy is activated Procaspases.

43. according to the described peptide species of claim 42, it is characterized in that: wherein said tetrapeptide array is KAVD.

44. according to the described peptide species of claim 42, it is characterized in that: wherein said tetrapeptide array is KKPA.

A 45. peptide species, it is characterized in that: contain one or more tetrapeptide arrays in the composition of this polypeptide, in the composition of this tetrapeptide, have at least one or more tetrapeptide arrays to be selected from by KEVD, KPhosphoTVD, one group of tetrapeptide array that KKPD and KKPphosphoS form, and this sequence at least can with a kind of the combining in Procaspases1 and 8, and this combination can reduce the activation of said Procaspases.

46. according to the described peptide species of claim 45, it is characterized in that: wherein said tetrapeptide array is KEVD.

47. according to the described peptide species of claim 45, it is characterized in that: wherein said tetrapeptide array is K-PhosphoT-VD.

48. according to the described peptide species of claim 45, it is characterized in that: wherein said tetrapeptide array is KKPD.

49. according to the described peptide species of claim 42, it is characterized in that: wherein said tetrapeptide array is KKP-phosphoS.

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