JPH08508014A - Oligodendroglia growth factor and its use - Google Patents

Abstract

  (57) [Summary] The 60 kilodalton protein ("oligodendroglial growth factor", OTF) induces oligodendroglial accumulation in brain white matter cultures. The half-maximal increase in oligodendroglial is obtained with 2 ng / ml oligodendroglial growth factor. The oligodendroglial growth factor has a cell division-inducing effect on oligodendroglial progeny and oligodendroglia, but does not stimulate growth on astrocytes, Schwann cells, or nerve fiber inner sheath fibers. Immunological studies with monoclonal oligodendroglial growth factor antibodies have shown that oligodendroglial growth factor is expressed by nerves in the CNS and dorsal roots.

Description

Detailed Description of the Invention               Oligodendroglia growth factor and its useField of the invention   The present invention is directed to oligogodendroglial growth factors and their benefits. It is about usage.Background of the Invention   Demyelinating diseases such as multiple sclerosis and encephalitis are debilitating diseases for patients. Many sides Extensive cerebral and spinal demyelination destabilizes gait at the end of sclerosis , Incontinence, and paralysis are common. In most cases, it is followed by death. Multifaceted Patients with dysplasia often have relapses and remissions over the years. Encephalitis often It is characterized by a monophasic and acute process leading to death. Demyelinosis is generally the major disease As a physical process, there is a defect in the myelin sheath that covers and insulates Axon. Multiple sclerosis has been the subject of exhaustive, but incomplete research. Still this There is no known effective treatment for these diseases.   Oligodendroglial cells produce myelin overlying central nervous system axons . Oligodendroglia are derived from neuroectodermal stem cells of the CNS embryo matrix. gold Goldman J.E et al., J. Neuorolsci 1986, 6, 52-60; LeVine S.M. ) And Goldman, J Comp Neurol 1988 277,441-455. In the process of Rigodendroglia formation, it is small, survives the process, and has a motor ability. Becomes a rapidly growing intermediate. Raff M.C. et al., Nature 1983,303,390- 396; Small R.K. et al., Nature 1987, 328,155-157; Levine and Go. Nordmann, J Comp Neurol 1988, 277, 441-455: Noble M.D. et al., Natur. e1988,333,560-562; Grinspan J.B. et al., J Neurosci 1990,10,186. 61-18873; Hardy (Hardy R.) and Reynolds R., Development  In the 1991,111,1061-1070 rat, cells in this intermediate stage were treated with gangliosides and And monoclonal antibodies (mAbs) that recognize other cell membrane epitopes Also by astrolog An antigen characteristic of the rear (eg glial fibrillary acidic protein (GFAP)), or Antigens characteristic of Rigodendroglia (eg galactocerebroside (galC), mi Erin basic protein (MBP) and lipid protein (PLP) expression loss Can be identified immunohistochemically. The review is by Goldman (1992). this , Motile oligodendroglial origin cells proliferate, migrate, differentiate, and differentiate The survival of oligodendroglia depends on the basic fibroblast growth factor (basic FGF) (macro). McKinnon R.D. et al., Neuron 1990, 5,603-614; Bogler O. Et al., Proc Natl Acad Sci USA 1990,87,6368-6372), platelet-derived growth factor (PDGF ) (Richardson W.D. et al., Cell 1988, 53, 309-319; Noble et al. Nature 1988,333,560-562; Pringle N. et al., EMBO J 1989,8,1049-10. 56; Grinspan et al., J Neurosci 1990, 10, 18661-18873; Barres B.A. et al. , Cell 1992,70,31-46) and insulin-like growth factor-1 (IGF-1) (McMorris (McMorris F.A.) and Dubois-Dalcq M., J Neurosci Re s 1988, 21, 199-209).   Oligos with a molecular weight of 16-18 kDa released into the medium from various neuroblastoma cell lines Dendroglia growth factor is known. Bottenstein J. E.) et al., J Neurosci Res, 1988, 20, 291-303; Hunter S.F. Tostein, J Neurosci Res 1991, 28: 574-582; Giulin D. et al., J. Neurosci 1991,11,327-336 CNS neurons released from the primary culture into the culture medium Growth-promoting factors for cells of the Godendroglia lineage (Levine, Neuron 1989, 3 , 103-113; Guard (Gard A.L.) and Pfeiffer S.E., Neuron 1 990,5,615-625) have not yet been reported at the molecular level.   Tissue culture studies show that oligodendroglial differentiation is in the absence of neurons Get up in Raff M.C. et al., Nature 1978, 274, 813-816; Barbarez ese E.) and Pfeiffer S.E., Proc Natl Acad Sci USA 1981, 78,1953-1957; Zeller N.K. et al., J Neurosci 1985,5,2955-2962; Sanet. (Saneto R.P.) and De Vellis J., Proc Natl Acad Sci USA 1 985,82,3509-3513; Knapp P.E. et al., Dev Biol 1987,120,356-365; Gulin Span et al., J Neurosci 1990,10,18661-18873; Hardy and Reynolds, Develop. ment 1991,111,10 61-1070 However, the oligodendroglia is in contact with an appropriately thick Axon. Can collect myelin and maintain it densely. Wood (Wood P.M.) And Williams A.K., Dev Brain Res 1984,12,225-241; Kid. (Kidd G.J.) et al., J. Neurosci Res 1990,26,409-418. Adhesive interactions between glial MAG and components of the uncharacterized neuronal plasma membrane Stabilized by action. Johnson P.W. et al., Neuron 1989, 3,377. -385, Owens G.C. et al., J Cell Biol 1990,111,1171-1182; Cuale. Quarles R.H. et al., In: Myelin: Biology and Chemistry (Martson enson RE)), pp413-448 Boca-Raton: CRC Press (1992) Second contact Rigodendroglia-Neuron Interactions on the Oligodendroglia Membrane-bound Surface Inhibition of protein-induced axon elongation. Caroni P .) And Schwab M.E., Dev Biol 1989, 136,287-295: Faucet. Fawcett J.W. et al., J Cell Sci1989,92,93-100   Some observations indicate that between oligodendroglial lineage cells and neurons. Presence of a third contact-dependent proliferative interaction, the induction of oligodendroglia mitosis. Is suggested. Dorsal root ganglion neuron The co-cultured oligodendroglial was co-cultured with the axorema-rich membrane fraction. In the case of oligodendroglials; Chen S.J. and Devries (DeVr) ies G.H.), J Neurochem 1989, 52, 325-327; Vick R.S. and Devry. S, J Neurosci Res 1992, 33, 68-74; Proliferates similar to; Wood and Bang (B unge R.P.), Nature 1986,320,756-758; Wood and Bang, Glia 1991,4,225-2. 32. The nature of the axolemma-related oligodendroglial growth-promoting factor has been demonstrated in one study. Suggest acidic fibroblast growth factor (acidic FGF) or its related proteins Vick and Devries, J Neurosci Res 1992, 33, 68-74, another institute. Research shows a novel 50kDal foreign membrane protein with a half-maximal activity of 200ng / ML Although it has been suggested that, Nordland (Mordlund M.) et al., Glia 1992,5,18 2-192, not established yet. This 50 kDa protein is also found in Schwann cells. It is also a source of proliferation. This is replaced by myelin lost in the process of multiple sclerosis Induction of oligodendroglial mitosis is highly desirable.Summary of the invention   Applicants have identified and isolated a novel oligodendroglial growth factor. This tampa The cytoplasm induces oligodendroglial mitosis, and is associated with pleural sclerosis and encephalitis. Applicants to promote the exchange of myelin that is lost during the development of eel demyelinosis Thus indicated.   In one preferred embodiment of the invention, a 0.5 M salt extract of whole mammalian brain is prepared, containing 0.15 Incubate the ion exchange matrix and extract at pH 7.4 in MNaCl at 5 kDa to 600 kDa. Run the unbound supernatant on a size exclusion column with fraction regions, elute the fractions, and Fractions with Rigodendroglial proliferative activity are collected and the active fraction is dissolved on a denaturing gel. A method for isolating oligodendroglial growth factor is provided.   In one preferred embodiment of the invention, a 0.5 M salt extract of whole mammalian brain is prepared, containing 0.15 Incubate the ion-exchange matrix and extract at pH 7.4 in M NaCl, 5 kDa to 600 kDa Run the unbound supernatant on a size exclusion column with fractional regions of, elute the fractions, Fractions with oligodendroglial proliferative activity were collected and the active fraction was dissolved on a denaturing gel. Then, a protein with a molecular weight of about 60 kDa is recovered from the gel, and the oligodendroglog that contacts Effective amounts of recovered protein and oligodendroglia for inducing Lear mitosis A method of inducing mitosis of oligodendroglia consisting of contacting with Law is provided.   In a preferred further aspect of the invention, a 0.5M salt extract of whole mammalian brain is prepared, Incubate the ion exchange matrix and extract at pH 7.4 in 15M NaCl, 5kDa to 600kD Elute the fractions by running the unbound supernatant on a size exclusion column with a fraction area of a. , Collect the fractions with oligodendroglia growth activity and dissolve the active fractions on a denaturing gel. And recovering a protein with a molecular weight of about 60 kDa from the gel, Diseased with recovered protein in an amount effective to induce mitosis in Godendroglia. A method for treating demyelinating disease in a patient with demyelinating disease is proposed. Provided.   In a further preferred embodiment of the invention, about 400 units / ug of oligodendroglial is added. Substantially pure oligodendrocyte consisting of a protein with a molecular weight of about 60 kD that has a fertile activity Glial growth factor is provided.   Furthermore, in a preferred embodiment of the present invention, the oligodeoxynucleotide in a pharmaceutically acceptable carrier is There is provided a drug comprising an androglian growth factor.   Furthermore, in a preferred embodiment of the present invention, oligodendroglial expansion in recombinant host cells is achieved. Part of the DNA encoding oligodendroglial growth factor sufficient for expression of the replication factor Recombinant host cells transformed with.   In another preferred embodiment of the invention, the recombinant DNA transformed in the host cell is It encodes godendroglial growth factor and encodes it in its host cell. Ligated in a form available to appropriate control sequences that can affect expression of the sequence. Culturing a recombinant host cell having a defined DNA sequence A method for producing dolglia growth factor is provided.   Oriented, operably linked to control sequences compatible with the expression systems described herein. It can be expressed in a suitable expression system containing a DNA sequence encoding godendroglial growth factor. Recombinant vectors are also provided in preferred embodiments of the invention.   Furthermore, in a preferred embodiment of the present invention, oligodendroglial growth factor is specifically Hybridoma cell line expressing a monoclonal antibody that recognizes is provided .   The antibody of the present invention is also effective in inducing oligodendroglial mitosis in an embodiment. Contacting a quantity of oligodendroglial growth factor with oligodendroglia A method of inducing mitosis of oligodendroglia is provided.   In a further preferred embodiment of the present invention, the oligodendroglial fibrillation of the central nervous system An amount of oligodendroglial growth factor effective to induce mitosis is administered to patients with demyelinating disease. There is provided a method of treating demyelinating which comprises administering.   Applicants have been inducing oligodendroglial mitosis Growth factors were identified. Oligodendroglial growth factor Isolated from whole mammalian brain on the basis of proliferative activity in loglia.Brief description of the drawings   Figure 1 is a photograph of the results of treating neonatal rat CWM cultures with a brain membrane NaCl extract. Is. A2B5 'cells (panels A and C) or R-mA in neonatal rat CWM cultures Indirect immunofluorescence microscopy was used to identify b'cells (panels B and D). Panel Le C and And D were stored in the medium containing the brain membrane NaCl extract (20 μg / mL) 3 days before microscopic observation. Shown is the culture that was retained, panels A and B are the same with no extract added. It shows the appearance of the culture. Size bar = 36 μm   Figure 2 is a photograph showing the effect of a brain membrane NaCl extract on DNA synthesis in CWM cultures. is there. CWM cultures were treated with 20 μg / mL brain membrane NaCl extract for 2 days, of which 2 days The eyes were kept warm with BrdU (10 μM). Panel A shows R-mAb bound to its plasma membrane Figure 2 shows two oligodendroglia that are present, and Panel B shows those after fixation. Demonstration of anti-BrdU mAb binding to the nuclei of two oligodendroglials . Similar results were obtained when 01 mAb was used in place of R-mAb in such studies. (Not shown).   Figure 3 shows oligodendroglial and 0-2A cells after addition of the brain membrane NaCl extract. It is a figure which compares the DNA synthesis rate of a cell. Brain membrane NaCl extract (20 μg protein / mL ) On day 0 (4 days after CWM cell isolation) and fresh medium on days 3 and 7. Was added again. 24 hours before observing with dual wavelength indirect immunofluorescence microscopy (oligoden Observation of drogria, panel A) or Brd 6 hours before (observation of 0-2A cells, panel B) U was added to the medium. Oligodendroglia identified by R-mAb binding to the surface Was done. 0-2A cells were identified by A2b5 mAb binding to the surface. The data is 4 Mean of independent experiments, vertical lines represent SEMs. White bar = untreated culture; black Rod = culture treated with NaCl extract   Figure 4 is a copy of a silver dye SDS-PAGE gel. Lane 1 of the silver dye gel is 2 μg of S200 Sep Lane 2 was loaded with oligodendroglial growth factor after acrylate. μg of oligodendroglial growth factor after DEAE was applied. S200 Sepha Proteins from parallel unstained lanes of oligodendroglial growth factor after krill Quality elution of all galC⁺Cell aggregation activity of 60kDal (MW_app) The prominent band territory It was shown to be recovered in the area.   Figure 5: MAG immunofluorescence microscopy of CWM cultures treated with oligodendroglial growth factor. It is an image of microscopic observation. This CWM culture shows the oligodendants after S200 Sephacryl. Treatment with roglia growth factor (100ng / mL) for 10 days, R-mAb (panel A) and anti-MAG Immunoglobulin (panel B) binding was observed by double wavelength indirect immunofluorescence microscopy. Size stick = 33 m Ichometer   Figure 6 MBP of CWM cultures treated with oligodendroglial growth factor or PDGF. It is an image of immunofluorescence microscopy. Panels A and C are human PDGF (2ng / mL) for 10 days Phase-contrast image and MBP indirect immunofluorescence image of a region of inter-processed CWM cultures; About half of oligodendroglia are MBP⁺Is. Panels B and D are S200 Cefac CWM culture treated with oligodendroglial growth factor (100ng / mL) for 10 days after rilling The phase contrast image and the MBP indirect immunofluorescence image of the object region are shown. MBP⁺There are only a few cells Existence Size bar = 26 micrometers   Figure 7: CW treated with oligodendroglial growth factor, PDGF, or basic FGF MBP in M culture It is a radioactivity photograph of Northern blot analysis of mRNA. CWM culture , PDGF (2ng / mL), basic FGF (10ng / mL), or oligo after S200 Sephacryl The cells were treated with dendroglia growth factor (100 ng / mL) for 7 days. Total RNA (basic FGF and And oligodendroglia growth factor-treated cultures at 10 μg / lane, PDGF-treated cultures 5 μg / lane) was examined by Northern blotting using MBP cDNA probe . The upper panel is [³²P] -labeled MBP cDNA probe showing radioactivity pictures There is. The signal intensity of MBP mRNA was higher than that of the PDGF lane by the basic FGF and oligodendrocyte. The glial growth factor lane is much weaker. [³²P] -labeled PLP cDNA probe The signal in the PDGF lane was determined by rehybridization of the blot used. Much stronger than basic FGF and oligodendroglial growth factor lanes Was shown (results not shown). The bottom panel shows the same three lanes The methylene blue-stained 28S band is shown showing bFGF and oligodendrogogues. The amount of RNA loaded in the rear lane is almost the same, and the amount loaded in the PDGF lane is almost the same. It is less than this.   Figure 8 shows two panels showing immunohistological localization of oligodendroglial growth factor. Is an image of.   Panel A shows peroxidation after incubation with oligodendroglial growth factor mAbs Du. Shown is a paraffin section of human cerebellum labeled with a dase; Specific staining was performed.   Panel B shows P6 dorsal root nerves labeled with peroxidase after incubation with Du mAb. Nodal cells are shown; sections viewed by Zeiss differential interference contrast optics.Detailed Description of the Invention   In accordance with some embodiments of the present invention, substantially pure oligodendroglial enrichment is provided. A breeding factor (OTF) is provided.   The term "oligodendroglia growth factor" refers to oligodendroglial growth factor. It refers to a protein that has activity. What is oligodendroglia proliferative activity? Inducing somatic division of dendroglia cells above background levels Means Somatic cell division causes the cell's chromosomes to replicate, segregate, and It means the process of division into two daughter cells containing the same set of chromosomes. This, of course, is well understood by those skilled in the art. Increase oligo dendroglia Breeding activity is the background activity that occurs after the addition of oligodendroglial growth factor. Number of additional oligodendroglia more than 100 mm / mm²Is preferable to be referred to by the expression Good. One unit of activity is oligodendroglial cell 1 in the background 0 / mm²Is defined to be equivalent to the addition of.   Oligodendroglia growth factor activates somatic division of oligodendroglia On the other hand, with oligodendroglia, myelin clogs, such as MPB or PLP Stabilizes the interactions between tightly packed lipid bilayers, thereby allowing these Mostly responsible for promoting the first covering of Axon with Rigodendroglia Production of myelin building proteins is not activated.   The oligodendroglial growth factor is associated with neurons. Oligodendro Glial growth factor is followed by a combination of separation by ion and size Can be isolated from whole brain extracts by SDS polyacrylamide gel digestion It Accumulation of oligodendroglia in rat cerebral white matter cultures was 0.5M  Monitoring the process of purification of oligodendroglial growth factor from NaCl membrane extract Can be used as an analytical method. Then extract the DEAE Sephadex ( Incubate with an ion exchange resin such as pH 7.4) in 0.15M NaCl. Combined The empty supernatant is run over a size fractionation column with a fraction width of 5 kDa to approximately 600 kDa. be able to. Elute fractions and collect fractions with oligodendroglial proliferative activity It The proteins contained in the active fraction can be resolved using a non-denaturing gel.   Biologically active oligodendroglial growth factor is DE in 0.15M NaCl at pH 7.4. No binding to AE Sephadex, 5 from globular protein from S-200 Sephacryl column It is eluted in the fraction corresponding to the molecular weight of 0-70 kDa. By SDS polyacrylamide gel For degradation, preparations that have been run through S-200 Sephacryl are It was found to contain a major band with a mass of approximately 60 kDa. Increase oligo dendroglia The reproductive activity was recovered by elution of the 60 kDa band. For cultured tissue of cerebral white matter R-mAb⁺And Ol⁺Half-maximal accumulation of oligodendroglia is 2 ng of SDS-PA Obtained at a concentration of oligodendroglial growth factor protein / mL after GE. This As a general rule, oligodendroglial growth factor is 4,000 times that of the first NaCl extract. Purified to be substantially pure.   In some embodiments of the present invention, "substantially pure" means 85% or more pure. It means that. Among other embodiments of the invention are those that are 90% or more pure. Be preferred. Most preferred is a purity of 95% or higher.   In some embodiments of the invention, the oligodendroglial growth factor is about 50 kD. It has a molecular weight ranging from a to about 70 kDa. In the most preferred embodiment of the present invention , Oligodendroglial growth factor has a molecular weight of 60 kDa. Oligodend Precursors of logulial growth factor are also involved in the biological activity of oligodendroglial growth factor. Included in this as a viable fragment.   The oligodendroglial growth factor of the present invention, when in suspension or in solution, is When it crystallizes or precipitates depending on the pH of the environment, or in the solid state Depending on the pH of the environment of, and in a pharmaceutically acceptable salt or neutral state I think that the. Of course, the free amino groups of the protein are Acids, or organic acids such as sulfuric acid, or for example acetic acid, glycolic acid, succinic acid, Alternatively, acidic salts can be formed with organic acids such as mandelic acid. Liberation The carboxyl groups of are sodium hydroxide, potassium hydroxide, or calcium hydroxide. Inorganic bases such as um, and piperidine, glucosamine, trimethylamine, Forming salts with bases including organic bases such as choline or caffeine Can be. In addition, the protein is associated with other biological substances such as lipids and carbohydrates. Quality, or acetylation of amino groups, phosphorus of the side chain having a hydroxyl group Can be modified by side chain modifications such as oxidation or oxidation of sulfoxyl groups it can.   Modifications of oligodendroglial growth factor are described in As long as the rear growth activity is retained, it falls within the scope of the definition. Finally, A slight modification of Rigodendroglial growth factor can be substantially equivalent or even enhanced. Have been shown to produce proteins with proliferative activity. These modifications are part It is an artifact of position-directed mutagenesis or an oligodendroglial growth factor It may be an incidental derivative, such as a mutation of a host that produces offspring. Proliferative activity All these modifications are included as long as they are retained.   The oligodendroglial growth factor of the present invention may be naturally occurring or recombinant. Produced.   As described, oligodendroglial growth factor can be prepared directly from the whole mammalian brain. It Oligodendroglia growth factor can be analyzed, for example, by autohydrolyzable amino acid analysis. Amino Acids Using a Vessel (Applied Biosystems model 420, ABS, Foster City, CA) Determination of amino acid sequences by standard techniques known in the art such as analysis and And further characterized by compositional analysis. Sequence analysis, for example, pulsed liquid phase Edman degradation using a sequencer (Applied Biosystems model 477A, ABS) Can be done by law. Cysteine residues were added to the M.E. D., S.S. Harwig et al., J. Clin. Inv., 1985, 76 (4): 1436-1439. Reaction of 4-vinylpyridine, followed by reduction of the sulfhydryl group, as described in Identified by origin.   Mass spectral analysis can be used to determine the molecular weight of peptides such as by fast atom collisions. used.   Polypeptides are routinely used, for example in commercially available peptide synthesizers. In a substantially pure form by standard techniques well known in the art. Can be synthesized with.   The oligodendroglial growth factor can also be prepared recombinantly. For example, A cDNA expression library was used to isolate the DNA sequence encoding oligodendroglial growth factor. Ibrary can be used. cDNA expression libraries are commercially available Alternatively, it can be prepared according to methods known in the art. Oligode The Monoclonal antibody against drogria growth factor screens the library Plaques that can be used to can do. Monochrome that specifically recognizes oligodendroglial growth factor Internal antibodies can be used to produce such monoclonal antibodies. Hybridoma cell line is provided by the present invention. When specifically recognized Antibodies to oligodendroglial growth factor, excluding other proteins present It means to combine.   Alternatively, the DNA library specifically targets the oligodendroglial growth factor gene Screening can be performed using a nucleic acid probe that recognizes. Such a nucleus Acid probe prepared based on the amino acid sequence of oligodendroglial growth factor be able to.   The nucleic acid sequence encoding all or part of the oligodendroglial growth factor is Included in some aspects of the invention.   Based on the determined nucleic acid sequence, oligodendroglial growth factor or a fragment thereof Are incorporated by reference as if fully set forth herein. Many well-known sets, such as those described in U.S. Pat. It is believed that it can be prepared by using any modification of the replacement technique. As used herein, "a portion thereof" refers to a desired biological activity, particularly Any peptide of sufficient size to retain oligodendroglial proliferative activity Alternatively, it means a part of nucleic acid.   You can easily transform cells, construct vectors, Jar RNA extraction, cDNA library preparation, and similar Most of the techniques used to implement are widely practiced in the art, and most practitioners Familiar with standard method documentation describing conditions and treatments. However, flights For convenience, it is provided as a guideline in the following paragraphs.   Prokaryotes are most often represented by various strains of E. coli. However While, for example, Bacilli, such as Bacillus subtilis, a variety of Pseudomonas Other microbial strains such as bacteria, such as seeds or other bacterial strains, can also be used. Wear. In such prokaryotic systems, replication points and species derived from species compatible with the host are used. And plasmid vectors containing control sequences are utilized. For example, such "expression As one of the "systems", Bolivar et al., Gene 2:95 (1977) are derived from E. coli seeds. Derivatives of the plasmid pBR322 were used to transform E. coli. pBR322 is Anne Construction of desired vector containing genes resistant to picillin and tetracycline Provides additional markers that can be retained or destroyed during Commonly used Prokaryotic control sequences optionally have an operator along the ribosome binding site sequence. Containing a promoter for transcription initiation with a beta-lactamase (penicillin And the lactose (lac) promoter system (Chang et al., Noture 19 8: 1056 (1977)), and the tryptophan (trp) promoter system (Go Dell et al., Nucleic Acid Res 8: 4057 (1980)) and P from lambda._Lpromotion And N-gene ribosome binding site (Simatalk et al., Nature 292: 128 ( 1981)) and commonly used promoters.   In addition to bacteria, eukaryotic microbes such as yeast can also be used as hosts. Many Other strains of Saccharomyces cerevisiae, which is a baker's yeast, are commonly available. Laboratory strains are also often used. Vector with 2 micron origin of replication illustrated Meanwhile, J.R. Brooch, Meth Enz. 101: 307 (1983), suitable for expression in yeast Other known plasmid vectors are also known (for example, Steinhkoum et al., Na ture 282: 39 (1979), Cemp et al., Gene 10: 157 (1980), and L. Clark. Et al., Meth Enz 101: 300 (1983). Glycolysis-related regulatory sequences for yeast vectors A promoter for the synthesis of the enzyme is included (Hes et al., J. Adv Enzyme Req 7:14. 9 (1968); Holland et al., Biochemistry 17: 4900 (1978)). Known in the art Promoting 3-phosphoglycerate kinase as an additional promoter T. (Hitzmann et al., J. Biol. Chem 255: 2073 (1980), and Glycerarde. Hydr-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, Phosphoglycerate mutase, pyruvate kinase, triose phosphate rearrangement Other glycolysis-related enzymes such as dihydrogen, phosphoglucose transferase, and glucokinase Includes a prime promoter. Additional that transcription is controlled by the proliferative environment Other promoters with significant advantages are alcohol dehydrogenase 2, isocytochrome C , Acid dephosphorylating enzymes, degrading enzymes involved in nitrogen metabolism, and maltose and gala Koutou It is a promoter region for enzymes (Holland, ibid.) Related to the utilization of sucrose. Also , It is believed desirable to have a terminator sequence at the 3'end of the coding sequence. It Such a terminator is a 3'untranslated sequence following the coding sequence of a yeast-derived gene. Found in the area. Many of the vectors exemplified include eno containing the peno46 plasmid. From the lucase gene (M.J. Holland et al., J. Biol Chem 256: 1385 (1981)). Or LEU2 gene obtained from YEp13 (J. Brooch et al., Gene 8: 121 (1978)) The control array of is included. However, a yeast-compatible promoter, origin of replication , And other control sequences are all suitable.   Genes encoding polypeptides in eukaryotic host cell cultures derived from multicellular organisms Of course, it can be expressed. For example, Tissue Cultures, Academic Press, See Cruise and Patterson (1973). Useful host cell lines include Includes VERO, Hela cells and Chinese hamster uterine (CHO) cells. In vivo expression of a gene encoding a polypeptide in a eukaryotic host also A convenient method for preparing gastrointestinal defensin polypeptides.   The expression vector for such cells is typically, for example, Simian virus 40 (SV40) Commonly used early and early species such as Fires et al., Nature 273: 113 (1978). And late promoter, or polyoma, adenovirus 2, bovine papillomawill Or other viral promoters from viruses such as the avian sarcoma virus -, Etc., containing promoter and control sequences compatible with mammalian cells. mammalian General concepts of transformation of cellular host systems are described, for example, in Axel; US Pat. No. 4,39. It is described by No. 9,216. Now the "enhancer" region is optimally expressed Have been found to be important to promoters; enhancers are generally promoter regions. It is a sequence found in the non-coding DNA region upstream or downstream of the region. Origin of replication Obtained from virus sources if needed. However, in eukaryotes, insertion into the chromosome Entry is a common mechanism of DNA replication. Plant cells are now used as hosts Possible, such as nopaline synthase promoter and polyadenylation sequences , Regulatory sequences compatible with plant cells are also available (A. Depicker et al., J. Mol App. l. Gen 1: 561 (1982)).   Depending on the host cell used, transformation is standard for such cells. Done using techniques. For prokaryotes and other cells that have a substantial cell wall barrier Described by N. Cohen, Proc Natl Acad Sci (USA) 69: 2110 (1972), Treatment with calcium chloride as described, or Molecular Clonin g: A Laboratory Manual (1988) Cold Springs Harbor Press. Methods are available. Agrobacterium tumefaciens (CH. Shaw et al., Gene  23: 315 (1983)) is believed to be useful for certain plant cells. For mammalian cells without such cell walls, Graham and Van der Eve, Virolo gy52: 546 (1978), the calcium phosphate precipitation method can be used. cell In addition, the gene incorporated into the vector is described in WO 90/11734 October 18, 1990. By direct introduction into cells within the vessel wall using the published balloon catheter technique. Thus, it can be transformed in vivo.   Transformation into yeast was performed by P. van Solingen et al., J Bact 130: 946 (1977) and And C. L. Xiao et al., Proc Natl Acad Sci (USA) 76: 3829 (1979). It can be carried out.   cDNA and genomic libraries using colony hybridization Can be screened. Generally microtiter plates Each can be duplicated on nitrocellulose filter paper (S & S BA-85 type), Colonies can be grown for 14-16 hours at 37 ° C on L agar containing 50 μg / ml Amp. it can. The colonies were lysed and the DNA was treated with 500 mM NaOH, 1.5M NaCl for 5 minutes. And wash the filter with two washes of 5x standard citrate (SSC) for 5 minutes. Fix it. Filters are air dried and baked at 80 ° C for 2 hours. Duplicate fill Is 10 ml of DNA hybridization buffer per filter [5 x SSC , PH7.0 5 x Denhardt's solution (polyvinylpyrrolidine with Ficoll and bovine blood Clear albumin; 1 x = 0.02% each), 50 mM, pH 7.0 phosphorus Sodium phosphate buffer, 0.02% SDS, 20 μg / ml poly U, and 50 μg / ml denatured solution Pre-hybridize with Sperm DNA] at 42 ° C for 6-8 hours.   The sample is hybridized with the phosphorylated probe under conditions that depend on the desired intensity. You can Typical optimal strength conditions include probe per filter. Use 1-5 ml of DNA hybridization buffer for 24-36 hours at 42 ° C. Can be Higher temperatures and shorter times are used for more intense strength. In general , Filter with 2 x SSC, 0.2% SDS and 50 mM sodium phosphate, pH 7, at 37 ° C Wash 4 times for 30 minutes each, then 2 times with 2 x SSC and 0.2% SDS and air dry. After drying, take an autoradiogram at -70 ° C for 2 to 3 days.   Construction of a suitable vector containing the desired coding and control sequences is within the skill of the art. Well understood standard ligation and restriction methods are used. Isolated Isolated plasmid, DNA sequence, or synthetic oligonucleotide They are tailored, reconnected.   Site-specific cleavage of DNA is performed under suitable conditions under conditions generally understood in the art. This is done by treating the DNA with a restriction enzyme (or enzyme). Identified by the manufacturer of commercially available restriction enzymes. For example New See England Biolabs, Product Catalog. Generally, 1 μg of plasmid or Or the DNA sequence is cleaved with 1 unit of enzyme in approximately 20 μl of buffer solution . A modified method is acceptable, but with an incubation time of 1 to 2 hours at about 37 ° C It is feasible. After each incubation, protein / phenol / Removed by chloroform extraction followed by ether extraction, Sephadex After passing through the G-5 spin column, the nucleic acid was recovered from the aqueous phase fraction by ethanol precipitation. To do. If desired, use standard techniques for size separation of the cleaved fragments. Electrophoresis on a polyacrylamide gel or an agarose gel can be used. Size A general description of separation is given in Methods in Enzymology (1980) 65: 499-560.   The restriction digested fragment contains four types of deoxynucleotide triphosphates (dNTPs). 50 mM Tris pH 7.6, 50 mM NaCl, 6 mM MgCl 2 at 20 ° C to 25 ° C₂, 6mM DTT, and And 5-10 μM dNTPs for about 15 to 25 minutes, a large fragment of E. coli DNA polymerase I (Klenow The end can be blunted by treatment with c). Klenow Fragmen Fills in the 5'sticky end, but the 3'single-stranded end protruding even in the presence of four dNTPs. Go backwards at the end. If desired, only one or selected, limited by the nature of the sticky ends It is possible to perform selective repair by adding only dNTPs. Treated with Klenow After that, the mixture was mixed with phenol / chloroform and Sephadex G-50 spincap. Extract by ethanol precipitation after ramming. S1 Nuclease When used in some cases, the single-stranded portion is completely hydrolyzed.   Synthetic oligonucleotides are described by Metoikki et al., J Am Chem Soc 103: 3185 (1981). Use the triester method or a commercially available automated oligonucleotide synthesizer Can be prepared. Single-stranded phosphorylation prior to annealing or for labeling Is an excess of polynucleotide kinase, eg 50 mM Tris, pH 7.6, 10 mM MgC l₂, 5mM dithiothreitol, 1-2mM ATP, 1.7pmoles γ32P-ATP (2.9mCi / mmole) , 0.1mM spermidine, 0.1mM EDTA in the presence of 0.1nmole substrate to about 10 units. Can be performed using   Ligation should be performed in a volume of 15-30 μl under standard conditions and temperatures such as: Can be done in volume: 20 mM Tris-Cl pH 7.5, 10 mM MgCl₂, 10mM DTT, 33μg / m l GSA, 10 mM-50 mM NaCl, and 40 μM ATP, 0.01-0.02 (Weiss) unit of T4 DN A ligase, 0 ° C (for "sticky end" ligation), or 1 mM ATP 0.3- 0.6 (Weiss) unit of T4 DNA ligase, 14 ℃ (for ligation of blunt ends) ) Either one. Ligation of "sticky ends" in the molecule is typically 33-100 μg / Performed at a total DNA concentration of 5 ml (5-100 mM total end concentration). Intramolecular blunt end ligation (Usually using more than 10-30 times the molar number of linker), 1 μM total end concentration Done in.   When constructing a vector using a "vector fragment", the vector was removed except for the 5'phosphate group. Vector fragment to prevent bacterial binding to the alkaline phosphatase. It can be treated with a Base. Digestion with BAP at pH 8 in approximately 150 mM Tris⁺ And Mg²⁺In the presence of about 1 unit of BAP per μg of vector at 60 ° C React for about 1 hour. To recover nucleic acid fragments, prepare the sample with phenol / chromatography. Extracted with Loform, ethanol precipitated, and loaded onto Sephadex G-50 spin column. To desalt. Or double digest with an extra restriction enzyme that cuts unnecessary fragments This can prevent binding in the vector.   For a portion of a vector derived from cDNA or genomic DNA that requires sequence modification Can utilize mutagenesis with site-specific primers. This is the desired mutation Except for the defined mismatches shown, they are complementary to the single-stranded phage DNA to be mutated. This is done using certain primer synthetic oligonucleotides. Briefly, synthetic oligonuclears Cleotide used as a primer for the direct synthesis of strands complementary to phage The resulting double-stranded DNA is transformed into a host bacterium capable of retaining phage. form The transformed bacterial culture is derived from a single phage-bearing cell, which is a plaque. So that you can do it.   Theoretically, 50% of the new plaques will retain the phage with the variant as a single strand. 50% of the clones appear to contain phage with the original sequence. The resulting plaque is accurate Phosphate at a temperature that will allow pairing, but will also prevent improper pairing with the original sequence Hybridization can be carried out with synthetic oligonucleotides. Then pick out the plaques that hybridize to the probe, incubate, Collect the DNA.   Correct ligation into the construction of the plasmid is first ensured that the appropriate ligation mixture is used to It can be confirmed by transforming the lord. Correctly transformed hosts are Resistance to picillin, tetracycline, or other antibiotics, or As can be seen, selection using other markers depending on the plasmid construction Separate. The transformant-derived plasmid was then cloned by D. B. Clewell et al., Proc Natl. Acad Sci (USA) 62: 1159 (1969), optionally chloramphenico. Amplification (D.B. Clewell, J. Bacteriol 110: 6670 (1972)). Wear. Isolated DNA is analyzed by restriction and / or F. Sanger et al., Proc  Natl Acad Sci (USA) 74: 5463 (1977), and Messing et al., F. Supp. Nucleic  Acids Res 9: 309 (1981) or the dideoxy method or Kitham et al., Sequencing by the method of Methods in Enzymology 65: 499 (1980). Wear.   According to the present invention, an oligodendroglial is provided in an effective amount. A method of inducing oligodendroglial differentiation is provided by contact with growth factors. It In order to induce somatic division in oligodendroglia, an amount of oligo of about ng is required. Godend roglia growth factor was found to be sufficient.   In another aspect of the invention, an effective amount of oligodendrocytes in a patient with demyelinating disease. Provided is a method of treating a patient with demyelinization, the method comprising administering glial growth factor. It   One of ordinary skill in the art, given the disclosure herein, will find effective dosing regimens for the treatment of demyelinating disease. You will understand well. For example, the medication may be intravascular, orally, rectally, etc. There are various modes of administration. Treatment is continued until the desired therapeutic result is obtained. Can be Those of ordinary skill in the art will appreciate that the dosage regimen, dosage, and interval You will recognize that it depends strongly on your physical condition.   The oligodendroglial growth factor is an effective amount of oligodendroglial growth factor. It may be formulated into a pharmaceutical composition comprising a child and a normally harmless carrier, such carrier The body is known to those skilled in the art. The composition is given in a dosage regimen appropriate to its form. An example Such compositions include solutions, suspensions, emulsions, as well as orally, intravenously. Usually in liquid form, including those that can be administered subcutaneously or intramuscularly. It is in the form of a product.   In addition, the treatment of demyelinosis involves intracellular oligodendroglylation from the genome. Expression of growth factor is induced. For the expression of oligodendroglia growth factor As an example of your choice, a portion of the oligodendroglial growth factor cDNA or genomic sequence. Is transformed with in vitro or in vivo. transformed in vitro Cells are introduced into the mammal in a manner familiar to those of skill in the art, such as performing cell transplantation. it can. Transformation in vivo is dependent on the introduction of the recombinant vector previously described. Is made. WO 90/11734 Released October 18, 1990.   The following examples are illustrative, but not intended to limit the invention.Example Example 1 Cell culture   Six-day-old sprag-Dawley rat corpus callosum and surrounding white matter to cerebral white Quality (CWM) cultures were established and maintained in serum-free medium as described above. Greenspan JB, et al. , J Neurosci 1990, 10, 1866. 1-18873. The cells were expanded for oligodendroglia during the first 4 days of culture. Treated with growth factors. Medium (addition of oligodendroglial growth factor) Or not) was replaced at 3 or 4 day intervals. In some experiments (Shown in text and figures), human platelet-derived PDGF (mostly AB heterod Immer; Bowen-Hope DF, et al. J Biol Chem 1989,264. , 2502-2508; biweekly, 1% platelet-poor human blood 2 ng / mL was added with serum; Grinspan JB, et al. J Neurosci   1990, 10, 18661-18873. In other experiments, human recombinant acidic Or 10 ng / mL alkaline FGF (R & D system) is added at 48 hour intervals. I got it. Schwann cells or dorsal root of sciatic nerve in newborn rats monolayer culture of Wood P. M. And Williams A. K. , De v Brain Res 1984, 12, 225-241; and Crader. B. Q., et al. , Brain Res 1981, 207, 433-444. Was established as described in.Example 2 Indirect fluorescent antibody microscopy   Monoclonal antibody (mAb) against glycolipid [A2B5 (neural cytoplasmic antigen ) Eisenverse, et al. , Proc. Natl. Acad. Sci. U. S. A ． 1979, 76, 4913-4917; anti-GD3 antibody (Goldman JE. , Et al. , J Neurosci 1986, 6, 52-60; 01 anti-galactose. Lebroside (oligodendroglia surface); Zomer J. And Schutner M.A. , Dev Biol 1980.83, 311-327; and R-mAb (Galaxy). R. Citcelbroside); , Et al. , Proc Natl Acad Sci USA 1982, 79, 2709-2713; , Et al. , J Neurosci Res 1989, 24, 548-557, or Mi Rabbit antiserum generated against extracellular region of Erin-related glycoprotein; Pedraza L. , Et al. , J Cell Biol 1990,111,2651-2751. , Diluted 1: 100 and used by Dr. James Salder, NYU Medical Sen. Given by courtesy of Ter.] At 4 ° C (A2B5,01, R-mAB) or At 25 ° C. (anti-MAG), it was added to the unfixed culture solution for 25 minutes. Hanks loose Rinse with appropriate salt solution and attach the appropriate fluorescence, rhodamine or Texas red Sub-antisera (obtained from Nordic, Organon Technica, or Jackson Labs Cells were incubated with acid-alcohol (95% ethanol). 5% acetic acid, v / v) was fixed at -20 ° C for 10 minutes. Anti-MBP mAb; Hicke Lee, et al. J. Histochem. Cytochem., 1983, 31, 11 26-1135; is added after fixing with acetone, and anti-GFAB (gel activated glial fiber is added. Fiber protein) mAb; Lee VM-Y, et al. , J Neurochem 1984 , 42, 25-32; was added after acid-alcohol fixation. Binds to various antibodies The number of cells was 1 mm on the surface of the cover glass.²The field of view that is 63 times as oily as It was decided by examining 21 pieces. Randomly select the field of view and run under fluorescent illumination Before the examination, it was judged that the density was appropriate under phase contrast. Cell content Cleavage index is at least 4 or 24 hours before fixation in acid-alcohol. Incubation with bromodeoxyuridine (BrdU, 10 μM) Estimated by incubating the nutrient solution. Anti BrdU (Amersham) Indirect fluorescent antibody microscopic observations using a strain showing the phenotype of BrdU incorporation into the nucleus. It was used to determine the number of cells. Greenspin J. B. , Et al. , J Neu rosci 1990, 10, 18661-18873.Example 3 Northern blotting   The steady-state amount of mRNA encoding MBP, PLP, or MAG was Zinc blotting revealed oligodendroglial growth factor, PDGF, or Examined in CWM medium maintained with or without Lucari FGF It was Sunbrook J, Frisch EF, Maniatis T Molecular Cl oning. A Laboratory Manual. Second edition cold sprite Ng Harbor Publishing, 1989. To this end, Chomjinsky and sashes , Anal. Biochem., 1987, 162, 156-159. Isolate total RNA from CWM cultures and run on an agarose formaldehyde gel. Separate by electrophoresis and transfer to a Duralon membrane (Stratagene). I was sphered. Prehybridization was performed at 42 ° C for 2 hours with 50% formua. Amide, 5 × SSPE (1 × = 0.15M NaCl, 0.01M phosphate and 1 mM EDTA), 1% SDS, 5 × Denhardt's solution (1 × = 0.02% Equol, 0.02% polyvinylpyrrolidine, 0.02% bovine serum albumin, 100 μg / mL denatured salmon sperm DNA). Rat MBP or LA Full-length PLP cDNA; Defera F. , Et al. , Cell 1985, 43, 7 21-727; Grinspan J., et al. , J Neurocytol Printing (1 992); Camhols J., et al. , J Neurosci res 1992, 31, 231-244; or rat MAG cDNA (J. Salzer, NY Hybridization by the U Medical Center) I went there for 24 hours. Blot once in 2 x SSC at room temperature and in 2 x SSC 65 Wash once at 0 ° C and once at 0.2 ° SSC at 65 ° C to remove Kodak XAR5 The film was exposed at -70 ° C.Example 4 Purification of oligodendroglia growth factor   The whole brain of a Sprague-Dawley rat 3 days after birth was treated with 0.25 M swab. Claus, 0.1 mM MgCl₂, 0.63mM Phenylmethylsulfonyl Solution containing fluoride and 10 mM Tris (pH 7.0) ) ") At 4 ° C. The homogenate is 1 at 2000 xg. It was centrifuged for 0 minutes at 4 ° C. Soluble by resuspension from 130,000 xg precipitate Prepare the extract and incubate it in 0.5M NaCl suspension buffer for 60 minutes at 4 ° C. Cuvated and then centrifuged at 130,000 xg for 60 minutes at 4 ° C. Up Clear with phosphate-buffered 150 mM NaCl, pH 7.0 (PBS) at 4 ° C Dialyzed overnight in Ratner N .; , Et al. , Proc Natl Acad Sci USA 1988, 85, 6992-6996; and then pre-coated with PBS. Overnight with a balanced 1 / 10th volume of DEAE Sephadex (Pharmacia) It was stirred. The suspension was centrifuged. The supernatant was transferred to an S-200 Sephacryl column (Pharma Shear) and the fractions were eluted with PBS. Oligodendroglia growth factor activity The containing fraction was obtained and dialyzed against deionized water at 4 ° C for 16 hours to make it lyophilic. It was saved at. The peptides in these fractions were digested with sodium dodecyl sulfate-polyamine. Separation by chloramide gel electrophoresis (SDS-PAGE); Ramili, U .; K. Nature 1970, 227, 680-685; and visualized with silver stain It was In one experiment, Ponceau staining and densitometry were performed on the tamper in the peptide band. It was used to estimate the mass. Staining with oligodendroglial growth factor Cut out the lane part of SDS-PAGE not perpendicular to the direction of movement , Washed twice with Ham's F-12 medium at 25 ° C for 10 minutes each, then 0.1% (W / v) Shake overnight in Ham's F-12 medium containing bovine serum albumin at 4 ° C. Proteins were extracted from the fragments by and. The remaining SDS is extratigel Removed from the supernatant by passing through a small column (Pierce). Tampa The proteins were eluted from these columns in Ham's F-12 medium and stored at -80 ° C. It was Accumulation of oligodendroglia in rat CWM cultures is described in Example 5. As described above, the oligodendroglial growth factor from 0.5M NaCl membrane extract was purified. Used to monitor the manufacturing process. Increased bioactive oligodendroglia The growth factor binds to DEAE Sephadex with 0.15M NaCl at pH 7.4. Not combined, S in the fraction corresponding to a molecular weight of 50-70 kD (as globular protein) It was eluted from the -200 Sephacryl column. Silver staining after SDS-PAGE Of oligodendroglial growth factor after passage through S., S-200 Sephacryl Has an apparent molecular weight of approximately 60 under reduction, along with several thinner bands. kD It was shown to contain a major protein band of (Fig. 4). Oligodendroglia The growth factor activity was increased by eluting the gel fragment containing the 60 kDal band. Well collected. R-mAb in CWM medium⁺And 01⁺Oligodendrogly The half-maximal accumulation of a is a factor of oligodendroglial proliferation after SDS-PAGE. The offspring protein was obtained at a concentration of 2 ng / mL. Oligodendroglia growth factor No bioactivity was detected in the eluate from other parts of the gel. All refiners Depending on the process, oligodendrogly was added to the first rat brain membrane NaCl extract. (A) The specific activity of the growth factor increased 4,000 times, and the yield was 67%. Table of results Summarized in 1. Example 5 Biological activity of oligodendroglial growth factor   CWM medium maintained in defined medium without the addition of PDGF or FGF Surface 01 in nutrient solution⁺Or R-mAb⁺The maximum increase in oligodendroglia The dose that can be placed at each stage of purification to determine the protein concentration that halves- Measuring oligodendroglial growth factor activity by constructing a response curve . 01 mAb binding to galC, and both galC and sulpholipid The results for R-mAb binding in one direction are: Vansal R, et al. , J Neuro sci Res 1989, 24, 548-557; indistinguishable. Activity 1 unit is mm in oligodendroglial growth factor²10 more per It was defined as the amount of Rigodendroglia produced. Growth factors of oligodendroglia The maximum response to offspring is the untreated background level at each stage of purification. It was about 8 times the bell. Various antibodies were used to enhance the effect of oligodendroglial growth factor activity. It was added to the medium to determine the fruit. Rabbit anti-human PDGF (collaborate Search, 50 μg / mL) along with oligodendroglial growth factor So added. Rabbit anti-basic FGF (R & D, 60 μg / mL) 90 minutes before adding dendroglia growth factor, rabbit acid-fast FGF (R & D, 50 μg / mL) 30 minutes before adding oligodendroglial growth factor. Added Anti-oligodendroglia growth factor mAb, Du (see below) It was used as a dorma supernatant (1: 100, v / v).Example 6 Monoclonal antibody of oligodendroglial growth factor     18 days with oligodendroglial growth factor after passing S200 Sephacryl Immunize mice over time; Broadsky FM, J Cell Biol 1985, 101, 2047-2054; Arai et al., Proc. Natl. Ac ad. Sci. U. S. A., 1990, 87, 2249-2253. Hybridomas were prepared as described above. The clone was isolated, and the supernatant was used for S200 Using oligodendroglial growth factor after passing through Sephacryl as a target antigen, Oh Binding to Rigodendroglia Growth Factor was tested by ELISA; MY et al. , Proceedings of the Natl. Acad. S ci., USA 1988, 85, 1998-2002. 6 ELISA positives Two of the clones (Td and Du) were driven by oligodendroglial growth factor. Induced in the CWM medium⁺Inhibited cell accumulation. Pup with Du Luoxidase anti-peroxidase (PAP) immunocytochemistry, tissue sections and Immune response of oligodendroglial growth factor in dorsal nerve root (DRG) culture Used to position sex. Tissue tissue prepared from human brain with isotonic alcohol It was fixed and cut into 6 μm paraffin sections. P6 rat DRG culture is acid alcohol Fixed in 2% fetal calf serum (v / v) and 0.1 M Tris, pH 7.0. 30% with 0.25% (w / v) cold water fish gelatin Min blocking and then incubating with oligodendroglial growth factor mAb Incubated. Then about the paraffin fragments and DRG cover glass , Peroxidase antiperoxidase immunocytochemical analysis was performed. Carden ( 1987).Example 7 Preliminary characterization of CNS membrane 0.5M NACL extract   The oligodendroglial growth factor activity was measured as described in Example 5. Add NaCl extract to a concentration of 20 μg / mL on days 0 and 3. Added Number of oligodendroglia binds to 01 mAb or R-mAb Determined by counting cells. The number of 0-2A cells in A2B5 mAb is Count the number of cells that bind but not 01 mAb or R-mAb cells Decided. The 01 mAb and R-mAb results were indistinguishable. Anti-G In parallel experiments with FAP, m in both untreated and treated cultures m²It was shown that there were less than one type 2 astrocyte per cell. The result is a standard error Mean of 4 independent experiments including differences. 0 from crude newborn rat CNS membrane Treatment with newer rat CWM cultures by treatment with 0.5M NaCl extract The number of cells generated from godendroglia increased (Fig. 1 and Table 2), and The BrdU nuclear labeling index of such cells was transiently increased (FIGS. 2 and 3).   Number of type 1 astrocytes, microglia, and endothelial cells in rat CWM medium And mitotic index remained unchanged by treatment with 0.5M NaCl extract (Data not shown). Orientation in CWM media mediated by NaCl extract The increase in godendroglia is associated with the increase in basic FGF, acidic FGF, or human PDGF. It was not inhibited by the antiserum that neutralized the work (data not shown).   CWM cultures induced by 0.5M NaCl extract and human PDGF The increase in the number of oligodendroglia in the liquid is additive (Table 3). Na of brain membrane Cl extract (20 μg / mL) and human PDGF (2 ng / mL) on day 0. And added on day 3. Each result is the mean of 4 independent experiments ± standard error. It   The increased activity of 0.5 M NaCl extract against oligodendroglia was It did not pass through the membrane and was not inactivated by trypsin or treatment at 100 ° C for 5 minutes (Data not shown). While doubly labeled with A2B5 and anti-GFAP Immediate immunofluorescence experiments; Grinspan J. et al. B., et al. , J Neurosci., 1 990,10,18661-18873; is less than one type 2 astrocyte / Mm²But Rough M. C. , Et al. , Nature 1983, 303, 390 -396; both untreated and CWM broth treated with 0.5M NaCl extract Were present in the (no data shown).Example 9 Descendants of 0-2A oligodendroglia and oligos by oligodendrogliaDetection of DNA synthesis stimulated by dendroglia growth factor   S200 Sephacryl or SDS-P in sufficient quantity to elicit maximum response Treatment of the CWM broth with oligodendroglial growth factor after AGE, B As a result of observing one day after pulsing with rdU for one day, A2B5⁺Oligodendrogly A descendants and R-mAb⁺BrdU label in both oligodendroglia Le index increased four to five times.   The oligodendroglial growth factor is linked to oligodendroglial during the BrdU pulse. By increasing the number of rapidly proliferating 0-2A cells that differentiate into Rather than increasing the BrdU label index of Godend Roglia; Fresinaud C. , Et al. , J Cell Physiol 1992, 150, 34-44, Mushi At least R-mAb⁺Cells of the oligodendroglial lineage that have reached the differentiation stage Devised two experiments to prove that it exerts a direct division effect on . The first experiment reduced the duration of BrdU pulse from 24 hours to 4 hours, and Therefore, BrdU-labeled 0-2A cells mature into oligodendroglia Limited time available for R-mAb labeled with BrdU⁺Number of cells Was reduced by a factor of 5 in this experiment, but the R-mAb⁺BrdU label index of cells is , Treated with oligodendroglial growth factor compared to control culture Has increased 4 times. The second experiment was PDGF to increase 0-2A cell population. (2 ng / mL) for 3 days was used; B. , Et al ． J Neurosci. 1990, 10, 18661-18873; and then Cultured for 5 days without PDGF, during which period 0-2A cells were oligodendroglial Matured. At this time, these culture solutions were 0-2A cells / mm²Is less than 1 won. These cultures are rich in oligodendroglia and low in 0-2A cells , 200 ng oligodendroglial growth factor after passing through S200 Sephacryl R-mAb⁺Stimulation of BrdU label index of cells 5 times (starting 1 day after adding oligodendroglial growth factor Pulsed for 1 day, n = 3). The results of these two experiments are Glial growth factor is R-mAb⁺Direct cell division inducing effect on oligodendroglia It was suggested that the results would be exerted.   Oligodendroglial growth factor against Schwann cells or inner nerve fiber Sciatic nerve culture has already been described to determine if it is still a mitogen. Clader B. Q. , Et al. , Brain Res 1981, 2 07,433-444; and in medium with or without serum. Rough Pass through 0.5 M NaCl extract (50 μg / mL) or S200 Sephacryl. And added oligodendroglial growth factor (1 μg / mL) to the medium, Two days later it was pulsed with BrdU for 1 day. Crude 0.5M N in serum-free medium The aCl extract increased the BrdU nuclear labeling index of Schwann cells 4-fold (n = 5). I let you. However, the cause of oligodendroglial proliferation after passing through S200 Sephacryl Pups were labeled with BrdU in serum-free medium or in serum-containing medium. Did not increase the proportion of Schwann cells or inner sheath fibers (data not shown). Not).Example 10 The oligodendroglia increased by the oligodendroglial growth factor is MAG Expressing myelin substrate protein (MBP) or proteolipid (PLP) is not synthesized or expressed   CWM broth treated with oligodendroglial growth factor for 3 days or longer R-mAb in⁺Approximately one-third of oligodendroglia has anti-MA on its own cell membrane. G immunoglobulin was coupled (Fig. 5). Northern blot analysis revealed that oligo That the MAG gene is expressed in cultures treated with Androglia growth factor Confirmed (data not shown).   R-mAb in CWM medium treated with human PDGF 2 ng / mL for 10 days⁺ 46% (n = 3) of cells are MBP⁺It was, but 10 days oligodendrogly R-mAb in CWM medium treated with growth factor⁺Only 4% (n = 3) of cells Is MBP⁺(Fig. 6), and this number did not increase after a further 10 days of observation . Steady-state MBP in CWM medium treated with oligodendroglial growth factor And PLP mRNA levels in parallel PDGF or basic FGF The results were compared with those in the culture solution treated with 1. by using Northern blot analysis. PDGF The number of oligodendroglia in the culture solution treated with MBP and PL, as expected from the fact that they were not actually found in Expression of P mRNA was strong in PDGF-treated cultures but not FGF-treated. Was hardly detected in the culture (Fig. 7). Proliferation of oligodendroglia In the culture medium treated with the factor, the galactolipid characteristic of oligodendroglia was formed. Abundant cells expressing but very low MBP and PLP mRNA levels It's (Fig. 7). This is in CWM broth treated with oligodendroglial growth factor. Of low levels of MBP expression in oligodendroglia of Mechanism is involved at least in part.   Oligodendroglial growth factor oligodend in culture medium treated with PDGF To determine whether to suppress the expression of myelin structural proteins by roglia, CWM medium is stimulated with oligodendroglial growth factor and PDGF for 10 days Was processed as follows. Cultures treated with PDGF only by indirect immunofluorescence microscopy R-mAb in these cultures as well as nutrient solutions⁺About half of the cells (49%, n = 3 ) Is MBP⁺It was also.   From previous observations, the synthesis of myelin lipids and myelin structural proteins has been shown to It was suggested that it is deeply involved in the differentiation of ndroglia. Thus, basic In cultures maintained in media containing FGF, oligodendroglial lines Some cells have myelin galactosphingolipids (galC and sulfatide) And the mRNA encoding both MBP and PLP cannot be expressed, while salt In the medium maintained without the addition of basic FGF, the expression of galC and M The mRNAs encoding BP and PLP appeared, and the immune response of MBP and PLP Responses can be done in the next few days. Knapup P. E. FIG. , Et al. , De v Biol 1987, 120, 356-365; Guard A. L. And puff FER S. E. FIG. , Development 1989, 119-132; Ralini, et al. , Development 1992, 116, 193-200; Greenspan J. , Et al. , J Neurocytol. Printing (1992). Shi However, contrary to these suggestions, cultures treated with oligodendroglia growth factor In solution, the coupling of myelin lipid and myelin structural protein synthesis was not found. Seems not to exist; R-mAb⁺And 01⁺Cells accumulate but express MAG Some do not express MBP and PLP. If you think carefully, a new oligode Oligodendroglial growth factor induces expression of PLP and MBP Not guiding means stabilizing the interaction between lipid bilayers in small myelin Proteins that are heavily involved in; A. And brow lock A. E. FIG. , Myelin: Biology and Chemistry (Martenson RE, editor), pp3- 80. Boklerton: CRC Printing (1992); on these oligodendroglia. It may be to promote the first envelopment of Axon by.Example 11 Studies with monoclonal antibodies against oligodendroglial growth factor haveRigodendroglia Growth Factor Shows Neuronal Expression   2 out of 6 ELISA-positive oligodendroglial growth factor mAbs (" Du "and" Te ") are the oligodendroglial growth factors in CWM medium. By A2B5⁺And R-mAb⁺Inhibited cell proliferation. For each of these two And oligodendroglial growth factor 200n after passage through S200 Sephacryl Half the inhibition of maximal bioactivity of g / mL was obtained by diluting 1: 500 (v / v) high. Obtained from bridoma supernatant (data not shown). Du and Te are PDGF Or expansion of oligodendroglial lineage cells induced by basic FGF treatment Does not inhibit addition (data not shown).   In immunoperoxidase experiments, Du was found to be a pellica in paraffin sections of human brain. Axons of Luya and Pelikarya cells, and dorsal roots of newborn rats (dorsal roo t ganglia) was shown to bind to many nerves in acid alcohol fixation medium. (FIG. 8). Fibroblast or Schwann cells in rat DRG culture, or rat No Du binding was detected to any cell type in C. Data not shown).

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI C12N 15/09 9281-4B C12N 5/00 B C12P 21/02 9455-4C A61K 37/02 AAB 21/08 9162 −4B C12N 15/00 C // (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:91) (C12P 21/08 C12R 1:91) (81) Designated country EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AT, AU, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, ES, FI, GB, HU, JP, KP, KR, Z, LK, LU, LV, MG, MN, MW, NL, NO, NZ, PL, PT, RO, RU, SD, SE, SI, SK, TT, UA, UZ, VN (71) Applicant The Trustees of the University of Pennsylvania Pennsylvania, USA 19104-3147, Philadelphia, Market Street 3700, Sweet 300, Center for Technology Transfer (72) Inventor Crader, Barbara Kew, New Jersey 08054, Mount Laurel, Fox Run Drive 2 (72) Inventor Pleasure, David United States Pennsylvania 19096, Winnie Wood, Kent Road 258 (72) Inventor Lee, Virginia M-Wye Pennsylvania, United States 19147, Philadel Bed and breakfasts, Fifth stream door, South 776

Claims (1)

[Claims] 1. Preparing a 0.5M salt extract of whole mammalian brain;     The extract was combined with an ion exchange substrate at 0.14 M NaCl at pH 7.4. Incubate;     Bound onto a size exclusion column with a fraction range of 5 kDa to 600 kDa Not add supernatant;     Elute the fractions;     Collecting fractions with oligodendroglial growth factor activity; and     Separate proteins in active fractions on denaturing gels A method for isolating an oligodendroglial growth factor consisting of. 2. An oligodendroglial growth factor isolated according to the method of claim 1. 3. Preparing a 0.5M salt extract of whole mammalian brain;     The extract was washed with ion exchange substrate at 0.15M NaCl at pH 7.4. Incubate;     Bound onto a size exclusion column with a fraction range of 5 kDa to 600 kDa Not add supernatant;     Elute the fractions;     Collecting fractions with oligodendroglial growth factor activity;     Separate the proteins in the active fraction on a denaturing gel;     Recovering a protein having a molecular weight of about 60 kDa from the gel; and     Recovery of an effective amount to induce cell division of adjacent oligodendroglia Exposed protein to oligodendroglia A method for inducing oligodendroglial cell division comprising. 4. Preparing a 0.5M salt extract of whole mammalian brain;     The extract was washed with ion exchange substrate at 0.15M NaCl at pH 7.4. Incubate;     Bound onto a size exclusion column with a fraction range of 5 kDa to 600 kDa Not add supernatant;     Elute the fractions;     Collecting fractions with oligodendroglial growth factor activity;     Separate the proteins in the active fraction on a denaturing gel;     Recovering the molecular weight protein from the gel; and     Effective to induce oligodendroglial cell division in patients with demyelinating disease Administering recovered protein to patients suffering from demyelinosis What is claimed is: 1. A method of treating myelination deficiency in a patient suffering from demyelination. 5. The method according to claim 4, wherein the demyelinating syndrome is multiple sclerosis. 6. It has an oligodendroglial proliferative activity of about 400 units / μg and a molecular weight of Substantially pure oligodendroglia growth containing approximately 60 kD protein factor. 7. A sequence having a sequence encoding the oligodendroglial growth factor according to claim 6. Rigonucleotide. 8. Oligodendroglia growth factor protein and a pharmaceutically acceptable carrier A therapeutic composition comprising a layer. 9. An oligodefect capable of sufficiently expressing an oligodendroglial growth factor by a host cell. Recombinant host cells transformed with a portion of the DNA encoding Androglian Growth Factor. 10. An oligodendlog produced from the recombinant expression vector according to claim 9. Rear growth factor. 11. Production of oligodendroglial growth factor involving culturing recombinant host cells The recombinant DNA transformed into the above host cell is an oligoden It has a DNA sequence that encodes a droglia growth factor, and To the appropriate regulatory control sequences that can drive the expression of the The production method described above, which is a flow. 12. A recombinant vector that can be expressed in an appropriate expression system, Oligodendroglia operably linked to a control sequence compatible with the stem The above recombinant vector comprising a DNA sequence encoding a growth factor. 13. A monoclonal antibody that specifically recognizes oligodendroglial growth factor Expressing hybridoma cell line. 14. A monoclonal produced from the hybridoma cell line of claim 13. antibody. 15. The oligodendroglia is contacted with an effective amount of oligodendroglial growth factor. A method of inducing cell division of oligodendroglia, comprising: 16. About 10 μg / mL to about 2 ng / mL oligodendroglia growth factor The method according to claim 15, wherein the method is contacted with oligodendroglia. 17． An amount effective to induce cell division of central nervous system oligodendroglia Comprising administering oligodendroglial growth factor to a patient suffering from demyelinating disease. How to treat demyelinating disease. 18. 18. The method of claim 17, wherein the demyelinating disease is multiple sclerosis.