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WO2002053709A1 - Marqueur de cellule cartilagineuse, son procede de production et son utilisation - Google Patents

Marqueur de cellule cartilagineuse, son procede de production et son utilisation Download PDF

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Publication number
WO2002053709A1
WO2002053709A1 PCT/EP2001/015307 EP0115307W WO02053709A1 WO 2002053709 A1 WO2002053709 A1 WO 2002053709A1 EP 0115307 W EP0115307 W EP 0115307W WO 02053709 A1 WO02053709 A1 WO 02053709A1
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Prior art keywords
protein
nucleic acid
acid molecule
antibody
chip
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German (de)
English (en)
Inventor
Wiltrud Richter
Eric Steck
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Cytonet GmbH and Co KG
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Cytonet GmbH and Co KG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]

Definitions

  • the present invention relates to a marker gene for cartilage cells, methods for producing the same and uses of this marker gene.
  • chondrocytes kept in culture which develop from the human mesenchymal stem cells, do not clearly and quickly and efficiently separate from the progenitor cells, ie the mesenchymal stem cells, and from them distinguish mainly forming osteoblasts.
  • the technical problem on which the present invention is based is therefore to provide means and methods by means of which a simple, quick, clear and reliable distinction can be made between chondrocytes or chondrocyte-like cell types which develop from mesenchymal stem cells, in particular cultivated chondrocytes, from mesenchymal stem cells and other cell types developing from the mesenchymal stem cells, such as in particular osteoblasts, is possible.
  • the invention solves this technical problem by providing chondrocyte-specific markers, in particular nucleic acid molecules, proteins and / or antibodies specifically expressed in the chondrocytes, and methods using the aforementioned nucleic acid molecules, proteins or antibodies which specifically chondrocytes, in particular cultured chondrocytes , of mesenchymal stem cells and others derived from them Differentiate mesenchymal stem-developing cells.
  • the present invention solves the technical problem on which it is based, in particular by providing a, preferably completely purified and isolated, nucleic acid molecule which encodes an extracellular matrix protein selected from the group consisting of
  • nucleic acid molecule with one of the nucleotide sequence shown in ' SEQ ID Nos. 1 to 3, 22, 23 and 26 or a fragment thereof;
  • nucleic acid molecule with a nucleotide sequence which encodes a protein or peptide with a sequence shown in SEQ ID No. 4, 5, 24, 25 or 27 or a fragment thereof;
  • nucleic acid molecule which is complementary to a nucleic acid molecule according to a) or b) or a fragment thereof;
  • nucleic acid molecule which can be obtained by substitution, addition, inversion and / or deletion of one or more bases of a nucleic acid molecule according to a) to c);
  • the nucleic acid molecules according to the invention encode the protein CEP (chondrocyte-expressed protein) -68, which is also referred to as CRTAC1 (cartilage acidic protein-1).
  • CEP chondrocyte-expressed protein
  • CRTAC1 cargo acidic protein-1
  • the gene encoding CEP-68 or CRTAC1 consists of 14 exons and encodes an acidic 68 kDa protein of 653 amino acids. No similar genes or proteins are known from other species.
  • the protein includes a 19 amino acid signal peptide at the N-terminus and an EGF-like calcium binding motif near the C-terminus. The presence of the signal peptide indicates that it is a secreted protein.
  • the EGF-like domain of CEP-68 has sequence homology to the EGF domain of the extra-cellular matrix protein fibulin-1.
  • CEP68 (CRTAC1) is therefore also an extracellular matrix protein.
  • CEP-68 The expression of the gene coding for CEP-68 was initially only detected in cultivated chondrocytes and articular cartilages. Later, a transcript of the gene was also shown in lung tissue using Northern blot analysis, while a transcript approximately 500 bp smaller was found in the brain. Investigations showed that the pre-mRNA of the gene coding for CEP-68 (CRTAC1) is subject to an alternative splicing, as was also shown for other genes of higher eukaryotes.
  • the spliced mRNA form CRTAC1-A which is found in cultured chondrocytes, articular cartilage and in the lung, comprises exons 1 to 13 and 14A, while the splice variant CRTAC1-B detected in the brain comprises exons 1 to 13 and the alternative E-xon 14B includes.
  • CEP-68 CEP-68
  • nucleic acid molecules according to the invention with the nucleotide sequences shown in SEQ ID Nos. 1, 2 and 26 and the nucleic acid molecules which encode a protein with the amino acid sequence shown in SEQ ID No. 4 or SEQ ID No. 27, and Fragments thereof and the proteins encoded by the nucleic acid molecules according to the invention have proven to be specific markers for the identification of chondrocytes, in particular cultivated chondrocytes, of preferably human organisms.
  • the endogenously present gene corresponding to these nucleic acids according to the invention is specifically expressed in cultured chondrocytes, but not in cultured osteoblasts, mesenchymal stem cells or stromal cells, that is to say transcribed into mRNA or proteins.
  • nucleic acid molecules with the nucleotide sequences shown in SEQ ID Nos. 1, 2 and 26 and the nucleic acid molecules which encode a protein with the amino acid sequence shown in SEQ ID No. 4 or SEQ ID No. 27 therefore allow a distinction to be made between chondrocytes and osteoblasts and mesenchymal stem cells.
  • nucleic acid molecules with the nucleotide sequences shown in SEQ ID No. 22, 23 or 26 and the nucleic acid molecules which encode a protein with the amino acid sequence shown in SEQ ID No. 24 or SEQ ID No. 25 represent brain-specific markers .
  • RNA molecules also encompassed by the invention, those of the nucleic acid molecules with the nucleotide sequences shown in SEQ ID No. 1, 2 and 26 and the nucleic acid molecules that contain a protein with the one in SEQ ID No. 4 or SEQ Coding the amino acid sequence shown ID No. 27, derived, in particular the mRNA splice variant CRTAC1-A, and the proteins encoded thereby represent highly specific markers for the selective detection and detection of chondrocytes, in particular human cultivated chondrocytes.
  • the invention is based on this
  • the underlying technical problem is also the provision of nucleic acids, designed as DNA or RNA molecules, proteins and / or antibodies encoded thereof, with the aid of which specifically chondrocytes, in particular cultivated chondrocytes, which can be recognized by the chondrocytes, can be recognized -Distinguish specific expression of the endogenously present CEP-68 gene s, in particular have a content of endogenously present CEP-68 mRNA molecules or proteins.
  • the detection of cultured chondrocytes provided according to the invention can be carried out together with a detection of the known chondrocyte marker collagen type 2.
  • a nucleic acid molecule which is specifically expressed in chondrocytes is understood to mean a nucleic acid molecule which, during the development of chondrocytes from mesenchymal stem cells and during the development of osteoblasts from chondrocytes, exclusively in chondrocytes, in particular cultured, developed from mesenchymal stem cells , but not in the mesenchymal stem cells themselves and in osteoblasts to form a gene product, in particular an RNA molecule and / or protein.
  • a chondrocyte-specific nucleic acid is also expressed in articular cartilage to form a gene product.
  • the term “chondrocyte-specific” does not exclude that a nucleic acid can also be expressed to a gene product in tissues that are not related to the development of chondrocytes or osteoblasts, for example in lung tissue.
  • chondrocytes are understood to mean cartilage cells or cells similar to cartilage cells which are present in native cartilage tissue or which have been isolated directly therefrom by enzymatic digestion of the extracellular matrix, without further in vitro cultivation.
  • the chondrocytes lie either individually or in isogenic groups (chondrons) and have a high proportion of extracellular matrix.
  • This matrix consists largely of type 2 collagen, which provides mechanical strength, and glycosaminoglycans, which resist deformation due to compressive forces, and thereby determine the physiological properties of the cartilage tissue.
  • cultured chondrocytes are understood on the one hand to be chondrocytes which have developed from mesenchymal stem cells by in vitro cultivation, the cultivation preferably taking place over a period of 7, 10, 14, 21 or 28 days ,
  • this term also means chondrocytes which have been isolated from cartilage tissue by enzymatic digestion of the extracellular matrix and cultured in vitro.
  • the nucleic acid molecule can be a DNA or RNA molecule, for example an mRNA molecule, in linear or circular form.
  • the nucleic acid molecule comes from a mammal, in particular a human organism.
  • the invention also includes modified nucleic acid molecules which, for example, by substitution, addition, inversion and / or deletion of one or more bases of the nucleic acid according to the invention
  • Molecule in particular within the coding sequence of the nucleic acid molecule, are available, that is also nucleic acid molecules that can be referred to as mutants, derivatives or functional equivalents, ie structurally different, but functionally equivalent derivatives of a nucleic acid molecule according to the invention.
  • Such manipulations of the sequences are carried out, for example, in order to specifically change the amino acid sequence encoded by a nucleic acid.
  • Nucleic acids encoding modified chondrocyte-specific proteins can be used to obtain recombinant proteins with a modified property profile.
  • nucleic acid molecules according to the invention can be provided to modify the nucleic acid molecules according to the invention in such a way that the gene products can be obtained more easily, for example by fusion to secretion signal peptides, which ensure that the gene product is secreted into the extracellular space.
  • Targeted sequence changes can also serve the purpose of to provide suitable restriction interface regions within the nucleic acid sequence or to remove unnecessary nucleic acid sequences or restriction interfaces.
  • the nucleic acid molecules according to the invention are inserted into plasmids and subjected to mutagenesis or a sequence change by recombination using standard methods of microbiology or genetic engineering.
  • the invention relates to the aforementioned nucleic acid molecules, these being labeled.
  • Customary labels such as isotope labels, fluorescent labels, enzyme labels or other types of labels can be used as labels.
  • Nucleic acid molecules labeled in this way enable the detection of the tissue-specific expression of the marker gene according to the invention.
  • the invention also relates to fragments of the nucleic acid molecules according to the invention with a length sufficient for a detectable and chondrocyte-specific hybridization with, for example, endogenously present in cells to be checked and corresponding nucleic acid molecules according to the invention, in particular RNA transcripts, corresponding to the nucleic acid molecules according to the invention.
  • the length of such fragments can be at least 20, preferably at least 30, 40 or 50, in particular at least 100 nucleotides.
  • the invention therefore also relates to fragments of the invention designed as hybridization primers Nucleic acid molecules, in particular in the specified length, which enable the specific detection of nucleic acid molecules according to the invention, in particular RNA transcripts such as the mRNA splice form CRTAC1-A, in cultured chondrocytes.
  • RNA transcripts such as the mRNA splice form CRTAC1-A
  • such fragments have at least the same sequence specificity as the entire chondrocyte-specific CEP-68 gene.
  • the degree of specificity required that is, homology at the nucleic acid level, can be determined by conventional methods and algorithms for determining sequence identities and homologies, for example the BLAST algorithm.
  • nucleic acid molecules or their fragments which hybridize with one of the nucleic acid molecules described above according to a) to d).
  • nucleic acid molecule which hybridizes with a nucleic acid molecule used in connection with the present invention encompasses a nucleic acid molecule or a nucleic acid which preferably hybridizes under moderately stringent conditions with a nucleic acid molecule or a nucleic acid according to a) to d).
  • hybridization with a radioactive gene probe in a hybridization solution 0.5 M sodium phosphate buffer (pH 7.2); 7% SDS; 100 ⁇ g / ml sheared herring sperm DNA; for the composition of the individual components see Sambrook et al., loc. cit.
  • a hybridization solution 0.5 M sodium phosphate buffer (pH 7.2); 7% SDS; 100 ⁇ g / ml sheared herring sperm DNA; for the composition of the individual components see Sambrook et al., loc. cit.
  • unspecifically bound probe is washed twice for 30 min each. in 40 mM Nat- sodium phosphate buffer (pH 7.2), 1% SDS removed at 50 - 55 ° C.
  • stringent conditions are selected according to which the aforementioned hybridization and washing conditions are set, with the exception that the hybridization and washing temperature is 60 to 62 ° C.
  • highly stringent conditions are selected which correspond to the abovementioned stringent conditions, although temperatures of 68 ° C. are selected for the hybridization and the washing step.
  • these nucleic acid molecules have at least 60%, preferably at least 70%, 80%, 85%, 90%, 95%, 97%, 98% and particularly preferably at least 99% homology (identity) to one another at the nucleic acid level.
  • the present invention also encompasses nucleic acid molecules which encode a polypeptide or protein specifically expressed in human chondrocytes, in particular cultivated chondrocytes, the amino acid sequence of which is at least 40%, preferably at least 60%, particularly preferably at least 70%, 80%, 90% , 95% in particular 99% homology to a polypeptide or protein which is encoded by a nucleic acid with one of the sequences shown in SEQ ID No. 1, 2 or 26.
  • the expression “at least 40%, preferably at least 60%, particularly preferably at least 70%, 80%, 90%, 95%, in particular 99% homology” refers to a sequence match at the amino acid sequence level using known methods, for example computer-aided sequence comparisons (Basic local alignment search tool, BLAST, SF Altschul et al., J. Mol. Bio. 215 (1990), 403-410) can be determined.
  • homology denotes the degree of relationship between two or more polypeptide molecules, which is determined by the agreement between the sequences, where agreement can mean both an identical agreement and a conservative amino acid exchange.
  • the percentage of "homology” results from the percentage of regions in agreement in two or more sequences, taking into account gaps or other sequence peculiarities.
  • amino acid exchange means the exchange of an amino acid residue for another amino acid residue, this exchange not leading to a change in polarity or charge at the position of the replaced amino acid, e.g. B. the exchange of a non-polar amino acid residue for another non-polar amino acid residue.
  • the homology between mutually related polypeptide molecules can be determined using known methods. As a rule, special computer programs with algorithms that take account of the special requirements are used.
  • Computer programs for determining homology include, e.g. B. the .GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research, 12 (12) (1984), 387; Genetics Computer Group University of Wisconsin, Madison (WI)); BLASTP, BLASTN and FASTA (Altschul, S., et al., J. Molec Bio 215 (1990), 403-410).
  • the BLASTX program can be obtained from the National Center for Biotechnology Information (NCBI) and other sources (Altschul S., et al., BLAST Handbuch, NCB NLM NIH Bethesda MD 20894; Altschul, S., et al., J. Mol 215: 403-410 (1990)).
  • NCBI National Center for Biotechnology Information
  • the well-known Smith Waterman algorithm can also be used to determine homology.
  • Preferred standard parameters for the amino acid sequence comparison include, for example: Algorithm: Needleman and Wunsch, J. Mol. Biol 48 (1970), 443-453; Comparison matrix: BLOSUM 62 from Henikoff and Henikoff, Proc. Natl. Acad. Be. USA, 89: 10915-10919 (1992); Gap Penalty: 12 Gap Length Penalty: 4 Threshold of Similarity: 0
  • the GAP program is also suitable for using the parameters described above.
  • other algorithms including those described in the program manual, Wisconsin package, version 9 (September 1997) can be used.
  • the choice of programs depends both on the comparison to be carried out and on whether the comparison is carried out between pairs of sequences, GAP or Best Fit being preferred, or between a sequence and an extensive sequence database, with FASTA or BLAST being preferred ,
  • the present invention also relates to a, preferably isolated and partially or completely purified, protein which can be obtained by expression of a nucleic acid molecule according to the invention or a fragment thereof, for example in a host cell or in an in vitro expression system.
  • the protein preferably has the same development and cell or tissue specificity, in particular the same chondrocyte-specific expression pattern as the chondrocyte-specifically expressed CEP-68 protein, in particular the protein which is also from a nucleic acid molecule one of the sequences shown in SEQ ID No. 1, 2 or 26 is encoded.
  • detection methods known per se can be carried out, such as RT-PCR experiments, in situ hybridizations, Western blot experiments or the like.
  • the present invention also includes isolated and fully or partially purified monoclo- nal or polyclonal antibodies or fragments thereof, which react specifically with a protein according to the invention or part thereof and with such an affinity, in particular recognize and bind that, for example, detection and / or purification of this protein is possible.
  • the antibodies or their fragments preferably do not react with any other antigen.
  • Polyclonal antibodies can be produced by immunizing animals with isolated and completely purified CEP-68 or fragments thereof, and antisera with polyclonal antibodies are thus obtained in a manner known per se. Monoclonal antibodies are produced by methods known per se.
  • the invention also encompasses vectors that contain the present nucleic acid molecules or their fragments.
  • the vector can be designed, for example, as a plasmid, cosmid, bacteriophage, virus or liposome.
  • the invention further relates to a construct which contains a nucleic acid according to the invention and / or a fragment thereof, preferably under the control of at least one expression regulatory element.
  • a “construct”, which can also be referred to here as a vector means the combination of a nucleic acid according to the invention or a fragment thereof with at least one nucleic acid additional element, for example a regulatory element, adapter, linker, spacer, selection marker, Replication sequences or the like.
  • regulatory elements are constitutive or inducible promoters for expression in a bacterial, yeast, insect, mammalian or plant cell, such as the E.
  • Antibiotic resistance genes such as the neomycin phosphorus transferase gene, can be used as selection markers, for example.
  • nucleic acid according to the invention or the fragment can be provided with a transcription termination signal.
  • a transcription termination signal Such elements have already been described (see, for example, Gielen et al., EMBO J., 8 (1984), 23-29).
  • nucleic acid additional elements mentioned can be both native (homologous) and foreign (heterologous) to the host organism.
  • sequences mentioned of the nucleic acid additive elements, for example the transcription start and termination regions can be of synthetic or natural origin or contain a mixture of synthetic and natural components.
  • the nucleic acid or fragment can be used in the construct, in particular a plasmid, both in anti Orientation of meaning as well as orientation of meaning to the regulatory element (s) are available.
  • the invention also provides host cells which contain a nucleic acid according to the invention, in particular a nucleic acid with a sequence shown in SEQ ID No. 1, 2 or 26, or a fragment thereof or a construct comprising a nucleic acid according to the invention or a fragment thereof.
  • the host cell according to the invention can be, for example, a bacterium or a yeast, insect, plant or mammalian cell, in particular a human cell.
  • the invention also relates to the use of the nucleic acid molecules, vectors, host cells, proteins and / or antibodies according to the invention for the identification and / or for the detection of the differentiation stage of cell types which develop from mesenchymal stem cells.
  • animal or human chondrocytes in particular are identified and / or detected, in particular in a sample, for example in a mixture of cells to be examined, a tissue, tissue model or transplant, a cell culture or the like Chondrocyte-specific expression of the CEP-68 gene is detected.
  • such a combination of nucleic acid molecules, vectors, host cells, proteins and / or antibodies is used to identify and / or to detect the differentiation stage of cell types that develop from mesenchymal stem cells. allow discrimination between the mRNA splice form CRTAC1-A detected in chondrocytes and the mRNA splice form CRTAC1-B detected in the brain or discriminate between the CEP-68 protein forms coded by both splice forms. The detection of the splice form CRTAC1-A or the protein encoded by it indicates the presence of chondrocytes.
  • nucleic acids according to the invention are used, for example, for a nucleic acid molecule with the nucleotide sequence shown in SEQ ID No. 1, 2 or 26 or suitable fragments thereof in combination with at least one nucleic acid molecule with one of the ones in SEQ ID No. 22 or 23 shown nucleotide sequences or suitable fragments thereof.
  • proteins according to the invention it is therefore provided that a protein with the amino acid sequence shown in SEQ ID No. 4 or SEQ ID No. 27 or a suitable fragment thereof in combination with a protein with the one in SEQ ID No. 24 or SEQ ID No. 25 amino acid sequence shown or a suitable fragment thereof.
  • Another embodiment of the invention relates to the use of the nucleic acid molecules, vectors, host cells, proteins and / or antibodies according to the invention for the isolation of chondrocytes from a cell culture, a tissue, an organ or a cell mixture, a sample such as a cell culture or a cell mixture for example brought into contact with an antibody according to the invention and cells bound to the antibodies isolated from the cell mixture and then separated from the antibodies.
  • nucleic acid molecules, vectors, host cells, proteins and / or antibodies are used for the isolation of chondrocytes which are specifically directed to the detection of the mRNA splice form CRTAC1-A found in chondrocytes or the protein encoded thereby.
  • nucleic acids according to the invention it is therefore provided, for example, to use a nucleic acid molecule with the sequence shown in SEQ ID No. 1, 2 or 26 or a suitable fragment thereof for the isolation of chondrocytes.
  • proteins according to the invention it is therefore intended to use a protein with the amino acid sequence shown in SEQ ID No. 4 or 27 or a suitable fragment thereof for the isolation of chondrocytes.
  • the invention provides for the isolation of chondrocytes to use an antibody which is directed against a protein having the amino acid sequence shown in SEQ ID No. 4 or 27, or a suitable fragment thereof.
  • Yet another embodiment of the invention relates to the use of the nucleic acid molecules, vectors, host cells, proteins and / or antibodies according to the invention for the production of an agent for the identification and / or detection of the differentiation stage of cell types which develop from mesenchymal stem cells, the agent in particular for the identification and / or is intended for the detection of animal or human, in particular cultivated, chondrocytes.
  • This means for identifying and / or for detecting the differentiation stage of cell types developing from mesenchymal stem cells is preferably a biochip, for example a nucleotide chip , a protein chip or an antibody chip.
  • the agent according to the invention can also be a diagnostic composition which comprises, for example, the nucleic acid molecules, vectors, host cells, proteins and / or antibodies according to the invention provided with suitable markers in a suitable buffer system.
  • the agent according to the invention can also be a diagnostic kit, which comprises the nucleic acid molecules, vectors, host cells, proteins, antibodies, nucleotide chips, protein chips and / or antibody chips according to the invention.
  • the invention also relates to the use of the nucleic acid molecules, vectors, host cells, proteins and / or antibodies according to the invention for producing an agent for isolating chondrocytes from a cell culture, a tissue, an organ or a cell mixture. It is preferably this means for isolating chondrocytes around a biochip, for example a nucleotide chip, a protein chip or an antibody chip.
  • the agent according to the invention can also be a kit which contains the nucleic acids, vectors, proteins, antibodies and / or host cells according to the invention, but also the nucleotide chips, protein chips and / or antibody chips according to the invention together with suitable buffer systems and suitable marking systems.
  • the present invention also relates to a diagnostic composition
  • a diagnostic composition comprising at least one biochip according to the invention, in particular at least one nucleotide chip according to the invention, a protein chip according to the invention and / or an antibody chip according to the invention in combination with suitable buffer systems and suitable marking systems.
  • a “biochip” is understood to mean a device which comprises a large number of biological substances, for example nucleotide sequences, proteins or antibodies, in immobilized or fixed form and with the aid of which a small amount of a by means of hybridization and / or binding processes Ligands that can bind to the biological substance under suitable conditions can be detected in a small sample.
  • the present invention relates to a nucleotide chip comprising a solid support and at least one nucleic acid molecule according to the invention fixed to the support and / or at least one vector according to the invention fixed to the support.
  • a “nucleotide chip” is understood to mean a device which contains a large number of different nucleic acids or nucleotide sequences such as DNA or RNA in immobilized form and with the aid of which a small amount of a complementary nucleic acid in a small sample, for example one, by means of nucleic acid hybridization Cell mixture or cell extract, or a small amount of a protein binding to nucleic acids can be detected by means of DNA / protein binding studies.
  • the invention relates to a aforementioned nucleotide chip, which in particular contains nucleic acid molecules with protein-coding nucleotide sequences, for example the nucleic acid molecules with SEQ ID No. 2, 3, 22, 23 or 26, which have the same properties as SEQ ID No. 4, 5, 24 , 25 and 27 respectively encode amino acid sequences shown, or parts thereof or vectors containing these nucleic acid molecules.
  • nucleotide chip which in particular contains nucleic acid molecules with protein-coding nucleotide sequences, for example the nucleic acid molecules with SEQ ID No. 2, 3, 22, 23 or 26, which have the same properties as SEQ ID No. 4, 5, 24 , 25 and 27 respectively encode amino acid sequences shown, or parts thereof or vectors containing these nucleic acid molecules.
  • the presence or absence of chondrocytes in a mixture of cells, a tissue, tissue model or graft, a cell culture or the like to be examined can be specifically detected.
  • a nucleotide sequence according to the invention can be hybridized with labeled mRNA samples, which have been isolated from the aforementioned sources or amplified by means of the PCR method, under suitable conditions.
  • the hybridization of the isolated or the amplified mRNA with the nucleic acid molecules with SEQ ID No. 1, 2 and / or 26 and / or suitable fragments thereof allows the presence of chondrocytes to be detected in a cell mixture.
  • the hybridization of mRNA with the nucleic acid molecules with SEQ ID No. 22 or 23 or suitable fragments thereof shows that the sample contains no chondrocytes, but brain cells.
  • the invention relates to the aforementioned nucleotide chip which contains nucleic acids which represent regulatory elements of the gene coding for CEP-68, that is to say elements which in particular enable the transcription of the protein-coding regions which are functionally linked to these regulatory elements , for example promoters, transcription termination signals, silencers, enhancers etc.
  • the regulatory elements of the CEP-68 gene according to the invention prove to be particularly advantageous insofar as they contain the regulatory sequences necessary for the induction of the chondrocyte-specifically expressed CEP-68 protein comprise and can accordingly be used to identify those proteins which can induce or inhibit the tissue-specific, in particular chondrocyte-specific, transcription of CEP-68 by binding to these regulatory elements.
  • nucleotide chips containing such regulatory elements can be used to identify substances of any kind that can inhibit or promote the interaction between the regulatory elements and the proteins that bind to them.
  • a particularly preferred embodiment comprises the nucleotide chip according to the invention, wherein on the support, in addition to the nucleic acid molecules according to the invention described above, at least one further nucleic acid molecule selected from the group consisting of:
  • nucleic acid molecule with a nucleotide sequence which is naturally necessary for regulating the expression of the second chondrocyte-specific protein, or a fragment thereof;
  • nucleic acid molecule with a nucleotide sequence which encodes a protein expressed during osteogenic differentiation, or a fragment thereof;
  • nucleic acid molecule with a nucleotide sequence which is natural for regulating the expression of the protein expressed during osteogenic differentiation is required, or a fragment thereof;
  • nucleic acid molecule which comprises a vector and at least one nucleic acid molecule according to a) to d),
  • Nucleotide chips which, in addition to the nucleic acid molecules according to the invention, contain nucleic acid molecules which code for a second chondrocyte-specific protein can, according to the invention, be used to characterize and identify different chondrogenic differentiation stages of cells. It is provided according to the invention that the starting cell populations to be characterized, which are induced, for example, chondrogenically, can come from different human or animal tissues or from different cells, for example progenitor cells from adipose tissue and bone marrow.
  • the nucleotide chip according to the invention comprises gene sequences which code for proteins which are expressed during osteogenic differentiation but not during the chondrocyte stage, it is possible according to the invention to differentiate the differentiation stage of cells, in particular cell transition stages Chondrocytes and osteoblasts to be recorded more precisely.
  • the Col2al gene encodes the ⁇ chain of type II collagen, an early marker for chondrocytes. As with CEP-68, the Col2al gene has two mRNA species due to an alternative splicing. The IIA mRNA form contains exon 2. In contrast, the IIB mRNA form lacks exon 2. If the nucleotide chip according to the invention is a chip containing RNA, the invention provides that the chip both mRNA splice Variants of the collagen contains 2-mRNA or parts thereof.
  • the Col9a2 gene encodes the ⁇ -2 chain of collagen type IX, which attaches to the collagen fibrils.
  • the aggregan gene encodes the extracellular matrix molecule aggrecan.
  • the nucleic acid molecule encoding the protein expressed during osteogenic differentiation is, in particular, the nucleotide sequence of the osteopontin gene, the osteonectin gene or the osteocalcin gene.
  • Osteopontin is a multi-phosphorylated glycoprotein that was first detected in bone tissue. Osteopontin is also detected in other tissues, organs, for example in the kidney, but also in body fluids, for example in milk and urine. Osteonectin is a single-chain polypeptide, the role of which in bone formation is still unclear. Osteonectin is expressed in high amounts in osteoblasts, while its frequency in the bone matrix can vary widely. The osteocalcin gene is expressed in a bone-specific manner.
  • the invention relates to the aforementioned nucleotide chip, which has DNA, RNA or PNA sequences as the nucleotide sequence.
  • PNA peptide nucleic acid or polyamide nucleic acid
  • PNA sequences are molecules that are not negatively charged and act in the same way as DNA (Nielsen et al., 1991, Science, 254, 1497-1500; Nielsen et al ., 1997, Biochemistry, 36, 5072-5077; Weiler et al., 1997, Nuc. Acids Res., 25, 2792-2799).
  • PNA sequences comprise a polyamide backbone of N- (2-aminoethyl) glycine units and have no glucose units and no phosphate groups.
  • the nucleic acid molecules according to the invention which can be fixed on a support, can be isolated from natural sources, for example from human and / or animal chondrocytes.
  • the nucleic acid molecules can be isolated and amplified by means of the PCR method, double-stranded molecules being obtained.
  • the nucleic acid molecules according to the invention can also be synthesized in vitro by known methods, single-stranded oligonucleotides or peptide oligonucleotides being obtained.
  • suitable primers desired regions of the nucleic acids according to the invention, that is to say both individual regions and the entire reading frame of the gene coding for CEP-68, can be amplified and isolated.
  • nucleic acid molecules according to the invention Using common molecular biological techniques, it is possible to insert various types of mutations into the nucleic acid molecules according to the invention. This allows, for example, sequence variants to be recorded that occur in different cell isolates or different species. Such mutations covered by the invention can be insertions, deletions, duplications, inversions, additions, exchanges or the like, even of unusual nucleotides. In this way, however, modified oligonucleotides can also be produced with functional groups which enable the oligonucleotide to be covalently bound to the carrier material for the production of the nucleotide chip according to the invention.
  • oligonucleotides with amino modifications or biotin groups can be produced which can covalently bind to chemically reactive groups (epoxies) or streptavidin groups contained on the surface of the carrier material.
  • nucleic acids are provided with nucleoside derivatives containing photolabile protective groups.
  • nucleic acid molecules which are generated by fusing the nucleic acids according to the invention with genes or components of genes from other sources can also be used for the nucleotide chip.
  • nucleic acid molecules with shortened nucleotide sequences can also be used.
  • the shortened nucleotide sequences have a length of at least 15 base pairs, preferably of at least 20 base pairs.
  • the nucleotide chips according to the invention comprise nucleic acid molecules fixed or immobilized on a solid support.
  • the term “solid support” means an insoluble matrix.
  • the solid support consists of a hydrophobic or weakly hydrophilic material, such as transparent glass, silicon dioxide, metal oxides, polymers and copolymers of dextrans or amides, for example acrylamide derivatives , Cellulose, nitrocellulose, nylon, or polymeric materials such as polyethylene terephthalate, cellulose acetate, polystyrene or polymethyl methacrylate or a polycarbonate of bisphenol A.
  • the support material is preferably coated with a surface-activating agent such as poly-L before fixing the nucleotide sequences -Lysine, polyethyleneimine or polyalkylamine pretreated to improve the fixation of the nucleic acids to the carrier material
  • a surface-activating agent such as poly-L before fixing the nucleotide sequences -Lysine, polyethyleneimine or polyalkylamine pretreated to improve the fixation of the nucleic acids to the carrier material
  • glass used as the carrier is provided with a silane coupling agent which has an amino group, an aldehyde group or an epoxy group has been pre-treated, for which he
  • the nucleotide chip according to the invention can also be used for the commercially available, already coated carrier types such as poly-L-lysine (Sigma Diagnostics), super-aldehydes (Telechem), super-amines (Telechem), silanes prep (Sigma), CMT GAPS (Corning ), Type I (Clontech
  • Suitable supports are those which are used for photolithographically produced nucleotide chips, for example those described in Lipshutz et al. (Lipshutz, Fodor, Gingeras and Lockhart, 1999, Nat. Genet., 21, 20-24).
  • poly-prep slides for example poly-prep slides (Sigma Diagnostics), or aminosilanes, such as silane-prep slides (Sigma), CMT GAPS Slides (Corning) and Super Amine (Telechem), or membranes such as CAST Slides or FAST Slides (Schleicher & Schüll) are used.
  • Epoxy-modified surfaces such as ArrayLink Biochip (GeneScan Europe) or epoxysilane slides (Quantifoil) are particularly preferred for immobilizing amino-modified 0-ligonucleotides.
  • the nucleic acid molecules can be bound and fixed to the carrier substrate by chemical or photochemical reactions or by electrostatic interactions.
  • the immobilization or fixation of the nucleic acids on the support surfaces used is carried out via an electrostatic bond or a covalent bond.
  • the nucleic acids have been prepared synthetically and have a functional group, the nucleic acids can be covalently bound and fixed to suitable functional groups on the surface of the carrier material (Lamture et al., 1994, Nucl. Acids Res., 22, 2121- 2125; Guo et al., 1994, Nucl. Acids Res., 22, 5456-5465).
  • the nucleic acids can also be covalently bound to the surface-activated support via spacers or a crosslinking agent, for example a bifunctional crosslinking agent.
  • a crosslinking agent for example a bifunctional crosslinking agent.
  • the invention provides for the nucleotide sequences to be bound to the support in the case of polylysine, aminosilane and membrane-coated nucleotide chips by means of UV crosslinking and in the case of epoxy-modified chips by means of a chemical reaction.
  • the nucleic acids can of course also be bound to the support via photochemical reactions. In the case of such photolithographically produced nucleotide chips, the photolabile protective groups are specifically cleaved off by means of photolysis following the immobilization.
  • a further preferred embodiment of the present invention therefore relates to methods for producing the nucleotide chips according to the invention, comprising the isolation and / or amplification of at least one nucleic acid molecule which encodes in particular the chondrocyte-specific protein form of CEP-68 and / or regulatory elements of this nucleic acid comprises, or the chemical synthesis of this nucleic acid, the modification of the nucleic acid sequence during or after the synthesis or amplification by the incorporation of functional groups or spacer units, the application of an aqueous solution of the isolated or synthesized nucleic acid molecule to a solid support material and the immobilization of the Nucleic acid molecule on the support by means of chemical or photochemical reaction or electrostatic interaction.
  • Another particularly preferred embodiment of the invention relates to protein chips comprising NEN solid support and at least one protein according to the invention fixed thereon or a fragment thereof.
  • a “protein chip” is understood to mean a device which contains a large number of different proteins or peptides in immobilized form and with the aid of which a small amount of a ligand, for example a protein or an antibody, which / which can covalently or non-covalently bind to at least one protein or peptide fixed on the support, can be detected in a small sample, for example a cell mixture, or a sample liquid.
  • a ligand for example a protein or an antibody, which / which can covalently or non-covalently bind to at least one protein or peptide fixed on the support
  • the protein chips according to the invention contain proteins fixed to a solid support, in particular CEP-68 proteins, which are expressed either in human or animal chondrocytes and lung tissue or in human or animal brain tissue, or parts thereof. Examples of these are proteins with the amino acid sequences shown in SEQ ID No. 4, 24, 25 and 27.
  • the protein chips according to the invention can therefore be used for the identification or detection of antibodies which can recognize the chondrocyte-specific or the brain-specifically expressed CEP-68 protein forms or parts thereof and bind to them.
  • the protein chips according to the invention can also be used, for example, to identify and isolate proteins from chondrocytes which interact in vivo with the proteins contained on the protein chip. After identification and isolation of such interacting proteins, the protein chips according to the invention can also be used to identify substances of any kind which can inhibit or promote the interaction between the proteins according to the invention and proteins interacting therewith.
  • a particularly preferred embodiment of the invention relates to a protein chip, a second chondrocyte being present on the carrier in addition to the CEP-68 protein form specifically expressed in terms of chondrocytes and in particular a protein with the amino acid sequence shown in SEQ ID No. 4. specifically expressed protein and / or a protein expressed during the osteogenic differentiation of chondrocytes and / or fragments of such proteins are fixed.
  • the second chondrocyte-specific protein is particularly preferably collagen 2, in particular the chain thereof, the -2 chain of collagen type IX or the matrix protein aggregan.
  • the protein expressed during osteogenic differentiation is preferably osteocalcin, osteonectin or osteopontin.
  • the protein chip according to the invention can also contain derivatives, functional equivalents or variants of the proteins according to the invention.
  • “Derivatives, functional equivalents and variants” are understood to mean in particular those derivatives of proteins, for example proteins with the amino acid sequences shown in SEQ ID No. 4, 24, 25 or 27, which maintain the basic structure These proteins are obtained by substitution of atoms or molecular groups and their amino acid sequences differ from the amino acid sequences shown in SEQ ID Nos. 4, 24, 25 and 27 in at least one position and which essentially have a high degree of homology at the amino acid level , Derivatives, functional equivalents or variants used according to the invention can also have other pre- and / or post-translational modifications.
  • the protein chip also contains derivatives, functional equivalents or variants of other chondrocyte-specific proteins or of the proteins expressed during osteogenic differentiation.
  • the protein chip according to the invention comprises fragments of the proteins with the amino acid sequences shown in SEQ ID No. 4, 24, 25 or 27.
  • “Fragments” are understood to mean, in particular, those isolated regions of a protein which have fewer amino acids than the native protein, but whose length is sufficient for the isolated fragment to have at least one of the functions which are characteristic of the native protein, such as binding capacity to a second protein, can exert a specific catalytic activity etc.
  • the fragment of a protein comprises a protein region which is an antigen-detergent or an epitope and is therefore particularly suitable for binding an antibody.
  • the proteins immobilized on the protein chip according to the invention can have been isolated and purified from natural sources, for example human or animal chondrocytes, using customary methods known in the art.
  • the proteins or fragments used can also be of synthetic origin.
  • the method of Merrifield (1985, Angew., Chem., 97, 801) can be used to synthetically produce peptides, ie fragments of proteins.
  • the proteins or peptides fixed on the protein chip according to the invention can be produced by means of conventional DNA recombination techniques.
  • the same materials as described above for the nucleotide chips according to the invention can be used as solid supports for the protein chips according to the invention, for example glass, silicon dioxide, other silica materials, polymeric materials such as fluoropolymers or metal oxides. These carrier materials are preferably pretreated before the proteins are immobilized, for example with silane coupling agents.
  • those described by Joos et al. described epoxy-modified supports or membranes (Joos et al., 2000, Electrophoresis, 21, 2641-2650) used.
  • the binding and immobilization of the chondrocyte-specific or brain-specific protein forms of CEP-68 or that during the osteogenic Differentiation expressed proteins or fragments thereof on the support material by chemical or photochemical reaction or electrostatic interaction.
  • the proteins and fragments thereof used according to the invention can be bound and immobilized on the support material, for example, by a large number of functional groups and / or spacers or chemical crosslinking agents, such as bifunctional crosslinking agents, which are usually used.
  • suitable functional groups that enable proteins to bind to silanized surfaces can be found, for example, in Weetall, 1996, Advances in Molecular and Cell Biology, vol. 15A, 161-192, JAI Press Inc.
  • the protein to be immobilized is used as GST fusion protein is present, the protein can be bound to the support via GSH units present on the support surface.
  • a pMAL fusion protein can be immobilized via MBP units on the surface of the support material.
  • the immobilization can take place via Ni 2+ - nitrilotriacetic acid surfaces (Ni-NTA) (Adachi et al., Proc. Nat. Acad. Sei. USA, 97, 7243- 7247).
  • a further preferred embodiment of the present invention therefore relates to processes for the production of protein chips, comprising the isolation of at least one chondrocyte-specifically expressed protein form from CEP-68 and the isolation of the other proteins mentioned above from a suitable source or chemical synthesis or recombinant production of these proteins or fragments thereof, the modification of the proteins or fragments during or after isolation, synthesis or production by the incorporation of functional groups or spacer units, the application of an aqueous solution of the isolated or synthesized proteins to a solid Carrier material and the immobilization of the proteins on the carrier by means of chemical or photochemical reaction or electrostatic interaction.
  • a further preferred embodiment of the invention relates to an antibody chip, comprising a solid support and at least one antibody according to the invention fixed thereon, which is expressed against the chondrocyte-specific CEP-68 protein form or the brain-specific CEP-68 according to the invention -Proteinform o- parts of it is directed.
  • chondrocytes in a mixture of cells, a tissue, tissue model or graft, a cell culture or the like to be examined can be detected.
  • proteins can be extracted from the sources mentioned above and, after labeling, incubated with the antibody chip according to the invention.
  • the antibody chips according to the invention can therefore also be used to identify and isolate chondrocytes.
  • the binding of a protein to an antibody immobilized on the antibody chip which is directed against a protein with the antibody shown in SEQ ID No. 24 or 25, or a fragment thereof, shows that the source from which the protein was isolated , no chondrocytes, but contains brain tissue or brain cells.
  • an “antibody” is understood to mean a polypeptide which is encoded by one or more immunoglobulin genes and which recognizes and can specifically bind specific structures on an antigen, in particular an antigen determinant or an epitope
  • antibody includes not only a complete immunoglobulin, but also a series of fragments that can be obtained by cleavage with various peptidases.
  • antibody also includes modified antibodies, such as oligomeric, reduced, oxidized and labeled antibodies.
  • “Antibody” also includes antibody fragments that have been generated both by modifying intact antibodies and using recombinant DNA techniques.
  • antibody in particular also includes fragments such as Fab, F (ab ') 2 or Fvm that can bind to an antigen determinant. Methods for producing such fragments are described, for example, by Harlow and Lane in “Antibodies: A Laboratory Manual ", 1988, Cold • Spring Harbor Laboratory, New York.
  • the expression “antibody which is specifically directed against a protein” or “antibody which specifically binds to a protein” means that an antibody recognizes an antigen determinant or an epitope of a protein under defined immunoassay conditions and binds to it by means of its paratope can.
  • Antigen determinants usually consist of chemically active groups of molecules, such as amino acids or sugar side chains, on the surface of an antigen, for example a protein, and have a characteristic three-dimensional structure.
  • An antibody immobilized on an antibody chip according to the invention can therefore bind to a protein, peptide, carbohydrate, proteoglycan and / or a lipid complex which is specifically related to the CEP-68 protein according to the invention.
  • An antibody used according to the invention can also be directed against structures which are to be regarded as post-translational modifications of this protein.
  • a preferred embodiment of the invention relates to an antibody chip, wherein in addition to the antibodies which are directed against the protein forms of CEP-68 according to the invention or parts thereof, at least one further antibody which is specific to a second ondrocyte-specific protein is fixed on the support or a protein expressed during osteogenic differentiation.
  • the antibody which is directed against a second chondrocyte-specific protein is preferably an anti- body against collagen 2 or against parts thereof.
  • the antibody, which is directed against a protein expressed during osteogenic differentiation is in particular an antibody against osteopontin, osteonectin or osteocalcin or parts thereof.
  • the antibody chip contains both monoclonal and polyclonal antibodies.
  • the materials mentioned above for protein chips can be used as carrier material for immobilizing the antibodies. These carrier materials are preferably pretreated before immobilization of the antibodies, for example with silane coupling agents.
  • the antibody chips described by Joos et al. described epoxy-modified supports or membranes (Joos et al., 2000, Electrophoresis, 21, 2641-2650) used.
  • the binding of the antibodies, which are directed against the above-mentioned proteins, to the carrier takes place via a chemical or photochemical reaction or via electrostatic interactions.
  • the antibodies used according to the invention can be used, for example, with the aid of a large number of commonly used functional groups and / or spacers or chemical crosslinking agents, such as bifunctional crosslinking agents, are bound and immobilized on the carrier material.
  • a further preferred embodiment of the present invention therefore relates to methods for producing antibody chips, comprising the genetic engineering production, isolation or synthesis of at least one antibody which is expressed against the chondrocyte-specific or brain-specifically expressed protein forms of CEP-68 or against other chondrocyte-specific proteins or a protein expressed during the osteogenic differentiation or parts thereof, or a fragment thereof, the modification of the antibody or fragment during or after isolation, synthesis or production by the incorporation of functional groups or spacer units, the Applying an aqueous solution of the isolated or synthesized antibody to a solid support material and immobilizing the antibody on the support by means of chemical or photochemical reaction or electrostatic interaction.
  • Another particularly preferred embodiment of the invention relates to an antibody chip, comprising a solid support and at least one antibody fixed thereon, which is specifically directed against an antibody according to the invention.
  • the antibody immobilized on the antibody chip specifically recognizes an antibody which is against one of the protein forms of CEP-68 according to the invention or another chondrocyte specific protein such as collagen 2 or against a protein expressed during osteogenic differentiation such as osteocalcin. Osteonectin or osteopontin, and can specifically bind to them.
  • a further preferred embodiment of the invention relates to a diagnostic composition
  • a diagnostic composition comprising at least one nucleic acid molecule according to the invention, a vector according to the invention, a host cell according to the invention, a protein according to the invention, an antibody according to the invention, a nucleotide chip according to the invention, a protein chip according to the invention and / or an antibody according to the invention -Chip.
  • the diagnostic composition comprises a combination of such nucleic acid molecules, vectors, host cells, proteins and / or antibodies which allows the two mRNA splice forms of CEP-68 or the two CEP-68 coded by these mRNA splice forms - Distinguish protein forms and, based on the detection of the presence of the splice form CRTACl-A or the protein encoded by it of CEP-68, detect the presence of chondrocytes. If the nucleic acids according to the invention are used, it is therefore provided, for example, that a nucleic acid molecule with the sequence shown in SEQ ID No.
  • proteins of the invention it is therefore intended to use a protein with the amino acid sequence shown in SEQ ID No. 4 or 27 or a suitable fragment thereof in combination with a protein with the amino acid sequence shown in SEQ ID No. 24 or 25 or a suitable fragment thereof.
  • antibodies the invention provides for an antibody which is directed against a protein having the amino acid sequence shown in SEQ ID No. 4 or 27, or a suitable fragment thereof, in combination with an antibody which is against Protein with the amino acid sequence shown in SEQ ID No. 24 or 25, or a suitable fragment thereof.
  • the invention also relates to a method for producing a CEP-68 protein, in particular the chondrocyte-specific expressed protein form, which emerges from the splice form CRTACl-A, the protein being produced by a nucleic acid molecule according to the invention in a host cell of the present invention , in particular a nucleic acid molecule with SEQ ID No. 1, 2 or 26, or its fragment is cultivated under suitable conditions, and expressed and the protein is obtained.
  • the invention also relates to methods for the identification or detection of animal or human, in particular cultivated, chondrocytes, a cell culture being used in a sample, in particular in a mixture of cells to be investigated, a, for example also artificial, tissue or a tissue model or transplant or ancestry Lich the chondrocyte-specific expression of the CEP-68 gene is detected.
  • the chondrocyte-specific splice form of CRTACl-A or the CEP-68 protein derived therefrom is brought into a sample by contacting the sample, in particular a digested and / or extracted or homogenized sample, with the nucleic acid molecules according to the invention.
  • the antibodies according to the invention therefore provides for the identification and / or detection of chondrocytes to use an antibody which is directed against a protein having the amino acid sequence shown in SEQ ID No. 4 or 27, or a suitable fragment thereof, where it is demonstrated that the CEP-68 protein derived from the chondrocyte-specific splice form CRTACl-A is present.
  • the simultaneous use of an antibody which is directed against a protein having the amino acid sequence shown in SEQ ID No. 24 or 25 or a suitable fragment thereof, the absence of the brain-specific mRNA splice form CRTAC1-B can be derived Protein can be detected.
  • the sample is a cell culture which results from mesenchymal stem cells and contains various cell types such as osteoblasts, chondrocytes, adipocytes and fibroblasts.
  • the sample can also be a cell mixture which has been obtained from enzymatic digestion of extracellular matrix from cartilage tissue.
  • the sample can be disrupted in a manner known per se, for example by means of mechanical, chemical and / or physical action, for example a cheeky press, ultrasound, electromagnetic fields or the like.
  • this relates to the identification and detection of human or animal cultivated chondrocytes with the aid of suitable Neter nucleic acid molecules of the present invention, in particular labeled DNA or RNA molecules, which allow detection of the chondrocyte-specific splice form CRTACl-A, in the hybridization methods known per se, for example Northerblott methods, RT-PCR methods or other PCR methods can be carried out.
  • this relates to the identification and detection of human or animal cultivated chondrocytes with the aid of immunological agents of the present invention.
  • the term “immunological agent” means in particular the use of antibodies or antibody chips for the detection of antigens, in particular of CEP-68 protein.
  • the antibodies or fragments thereof according to the invention can be modified, for example conjugated, associated or covalently or non-covalently bound to other molecules or parts thereof, for example with color labels, radioactive labels, measurable enzymes which trigger reactions, such as phosphatases, peroxidases , Enzyme substrates, fluorescent substances, chemiluminescent substances, cytotoxic agents, spacers, carriers or the like.
  • the labeled, conjugated or unmodified antibodies can be in soluble or immobilized form, for example on carrier matrices or beads.
  • the enzyme labeled anti body are used in a second enzyme amplification system.
  • the use of immunological agents means contacting a sample, in particular a digested and optionally extracted sample, that is to say, for example, a synthetic tissue, a cell mixture or a cell culture with an immunological agent, the immunological agent at least one antibody that specifically recognizes the antigen, ie the CEP-68 protein, or an antibody fragment thereof.
  • the methods according to the invention using immunological agents can preferably be carried out as sandwich ELISA, indirect or competitive ELISA, with HTP (high through put) methods being particularly preferred.
  • the at least two different monoclonal antibodies which are preferably directed against different epitopes, but alternatively also the same epitopes, of CEP-68.
  • the at least two antibodies can, for example, be soluble and / or supported in a homogeneous or heterogeneous system.
  • the immunological agents it is of course also possible to carry out the immunological agents as a system of three different antibodies, one of the antibodies being in the heterogeneous phase and the other two antibodies being soluble are. One of the two soluble antibodies is labeled, while the other is unlabeled. In this embodiment, the soluble antibody is directed against the unlabeled antibody.
  • methods carried out according to the invention using immunological agents require — ie the incubation of a cell suspension to be tested, in particular a cell homogenate or extract with at least two different antibodies which are specific for CEP-68 protein forms.
  • one of the two antibodies is bound to a solid phase, this being done in the usual way.
  • the further antibody is advantageously in soluble form and, in a preferred embodiment, bears a label. If further antibodies are used according to the invention, in a preferred embodiment only one of these bears a label.
  • one embodiment provides that either an antibody that is non-specifically bindable with CEP-68 or particularly preferably an antibody that is specifically bindable with CEP-68 protein forms is bound to a solid phase.
  • This antibody bound to the solid phase is then incubated with the cell extract or cell homogenate and, optionally after a washing step, a second antibody which is specifically bindable with CEP-68, is present in a soluble form and bears a label. If the antibody bound to the solid phase is an antibody which is nonspecifically bindable with CEP-68, bind to the Solid phase not only CEP-68, but also other antigens.
  • the second antibody which binds specifically with CEP-68, only reacts specifically with CEP-68 or the complex of CEP-68 and the first antibody, so that only CEP-68 molecules specifically carry a labeled antibody and so after separation of the solid can be quantified by the liquid phase.
  • a non-labeled CEP-68-specific first antibody to a support, for example, in a sandwich ELISA, to add the test solution or suspension, the test solution or suspension being present CEP-68 is bound by the first antibody.
  • a second labeled antibody against CEP-68 is then added, which specifically reacts with CEP-68 or the complex of CEP-68 and the first antibody.
  • the quantity of CEP-68 can be determined by means of the labeling of the second antibody using a calibration solution.
  • the invention also provides that a microtiter plate is coated with a first antibody, which binds specifically or nonspecifically to CEP-68, that the cell extract or the homogenate is subsequently brought into contact with the coated microtiter plate and, after washing, unbound Components, a labeled, for example biotin-labeled, second antibody against CEP-68 is added to the microtiter plate, the microtiter plate being incubated under conditions that the second antibody can bind to CEP-68.
  • the solid phase is then separated from the liquid phase and either the label is directly detected or, if an enzyme-labeled second antibody is used, a substrate is added and the conversion of the substrate is determined quantitatively.
  • the second antibody labeled with biotin reacts with a conjugate of peroxidase (POD) and streptavidin in a next incubation.
  • the peroxidase is able to oxidize the substrate ABTS (2, 2 '-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid) di-ammonium salt). Oxidized ABTS can then be determined photometrically.
  • the first antibody is bound to a support matrix, for example a fleece, tissue or membrane structure, in such a way that it does not represent the bottom of the depression of an ELISA immunoplate, as is usually the case, but is instead integrated directly into the matrix
  • a support matrix for example a fleece, tissue or membrane structure
  • Preferred matrixes are hollow fiber membranes or microporous flat membranes, which can also be provided with ion exchanger groups in an advantageous embodiment of the invention.
  • the proteins according to the invention can also be used as antigen or immunogen in order to stimulate the formation of antibodies in suitable organisms, for example mammals.
  • the antibodies obtained in this way can be used according to the invention for the detection of endogenously present CEP-68 protein.
  • the invention also relates to methods for isolating chondrocytes, in particular cultivated chondrocytes, from a cell culture or a cell mixture, the cell culture or the cell mixture being brought into contact with an antibody of the present invention and cells bound to the antibodies isolated from the cell mixture and then from the antibodies be separated.
  • This method uses the antigens provided according to the invention not only for identification, but also for the isolation and enrichment of cells, in particular cultivated chondrocytes, which expose these antigens to their surface.
  • sequence listing is part of this description and comprises the sequence listing SEQ ID No. 1 to SEQ ID No. 30.
  • SEQ ID No. 1 shows the complete cDNA sequence of the chondrocyte-specific CEP-68 protein according to the invention with nucleotides 1 to 2507.
  • SEQ ID No. 2 shows a protein coding region comprising 1959 nucleotides derived from SEQ ID No. 1.
  • SEQ ID No. 3 shows a region comprising 57 nucleotides derived from SEQ ID No. 1, which encodes the N-terminal signal peptide.
  • SEQ ID No. 4 shows the 653 amino acid sequence, derived from SEQ ID No. 2, of the chondrocyte-specific expressed CEP-68 protein form.
  • SEQ ID No. 5 shows the 19 amino acid sequence of the N-terminal signal peptide of CEP-68, which is derived from SEQ ID No. 3.
  • SEQ ID No. 6 to SEQ ID No. 21 show primer DNA sequences according to the invention.
  • SEQ ID No. 22 shows the complete cDNA sequence of the brain-specific CEP-68 according to the invention resulting from an alternative splicing. Protein with nucleotides 1 to 1947, whereby exon 14A was replaced by exon 14B compared to the chondrocyte-specific cDNA.
  • SEQ ID No. 23 shows the 152 nucleotides of the alternatively used exon 14B.
  • SEQ ID NO. Figure 24 shows the 38 amino acid sequence derived from SEQ ID No. 23 of the alternatively used exon 14B of the brain-specific cDNA of CEP-68.
  • SEQ ID No. 25 shows the 636 amino acid amino acid sequence derived from SEQ ID No. 22 of the brain-specifically expressed CEP-68 protein form.
  • SEQ ID No. 26 shows the 711 nucleotides of exon 14A of the chondrocyte-specific cDNA of CEP-68.
  • SEQ ID No. 27 shows the 54 amino acid amino acid sequence of exon 14A derived from SEQ ID No. 26.
  • SEQ ID No. 28 to SEQ ID No. 30 show the primers hCRTACl F1, hCRTACl E14A-R and hCRTACl E14B-R used to differentiate the two splice variants of CRTAC1.
  • FIG. 1 shows the Zeil-type-specific expression of CEP-68, determined by means of RT-PCR
  • FIG. 2 shows the expression of CEP-68 in mesenchymal stem cells during their differentiation into chondrocytes, determined by means of RT-PCR
  • FIG. 3a Northern blot analyzes of CEP-68 trans and 3b scripts
  • FIG. 4 autoradiographs of in vitro translated CEP-68
  • Figure 5 shows the tissue type-specific expression of the two splice variants CRTACl-A and
  • Figure 6A shows the intron lengths of the human CEP-68 gene
  • Figure 6B shows the genomic structure of the C-terminus of the human CEP-68 (CRTAC1) gene.
  • Bone and cartilage tissue and mesenchymal stem cells from the bone marrow were obtained after obtaining prior written consent from human patients who underwent hip, knee, or shoulder surgery.
  • the cultured osteoblasts and chondrocytes for the RDA were obtained from a 69-year-old male donor with a broken upper arm.
  • RNA from the brain and lungs of a male adult was obtained from BioChain Institute, Inc. (Hayward, California). The donor was a 28 year old male adult. The investigation was approved by the local ethics committee.
  • Cartilage was digested with collagenase (Röche) and hyaluronidase (Serva) in DMEM (Gibco BRL) overnight. Chondrocytes were cultivated at 37 ° C and 5% CO 2 for up to 6 weeks in a single-layer cell position, i.e. in a monolayer, in DMEM, 10% fetal calf serum (FCS) and 1% penicillin / streptomycin. Osteoblasts were cultured from bone biopsies in a monolayer for up to 9 weeks under the same culture conditions as described above. Mesenchymal stem cells were extracted from marrow isolated by density gradient centrifugation and cultivated for 3 weeks as described above.
  • collagenase Rosöche
  • Serva hyaluronidase
  • expanded mesenchymal stem cells were cultivated in DMEM (high glucose content) for a further 2 to 3 weeks, with the DMEM medium 1% penicillin / streptomycin and 0.1 ⁇ M dexamethasone, 1 mM sodium pyruvate, 0.17 mM ascorbic acid 2-phosphate, 0.35 mM proline, 6.25 ⁇ g / ml bovine insulin, 6.25 ⁇ g / ml transferrin, 6.25 ⁇ g / ml sodium selenite, 1.25 mg / ml Contains BSA and 0.01 ⁇ g / ml TGF-ß 3 (Sigma).
  • the RDA is a PCR-based subtractive hybridization method that is able to examine two different mRNA populations with regard to differential gene expression.
  • cDNA-RDA leads to the elimination of the cDNA fragments derived from mRNA which is present in both cell populations. is present and thereby isolates "the cDNA fragments which represent the difference between the two mRNA populations. In this way, tissue or development-specific genes can be identified.
  • cDNA was synthesized using reverse transcriptase (Superscript II, GibcoBRL) and oligo-d (T) primers. 200 ⁇ g of double-stranded cDNA was digested with Dpnll, phenol extracted and ethanol-precipitated. An adapter molecule (R-Bgl-12/24) was then ligated. The DNA was diluted and amplified by means of PCR (polymerase chain reaction) using R-Bam-24 oligonucleotides as primers (in each case 20 cycles of 1 minute at 95 ° C. and 3 minutes at 72 ° C.). In order to obtain sufficient starting material for the RDA, the PCR products were reamplified in a further five-cycle PCR step.
  • reverse transcriptase Superscript II, GibcoBRL
  • T oligo-d
  • the PCR products obtained were phenol-extracted, ethanol-precipitated and resuspended in a concentration of 0.5 ⁇ g / ⁇ l.
  • the representative test and counter-test samples obtained were digested with Dpnll (New England Biolabs) to remove the R adapters, phenol extracted and ethanol precipitated.
  • the representative test samples were cleaned on a gel and ligated to J-Bgl-12/24 adapters.
  • a final PCR amplification step resulted in the formation of DPI.
  • the adapters were replaced as follows: For DP2, J-Bgl against N-Bgl and for DP3 N-Bgl against J-Bgl.
  • the ratios of the counter test to the test sample were 800: 1 for DP2 and 400000: 1 for DP3.
  • the primer and adapter oligonucleotides for the RDA were:
  • R-Bgl-12 5 '-GATCTGCGGTGA-3', (SEQ ID No. 6)
  • R-Bgl-24 5 '-AGCACTCTCCAGCCTCTCACCGCA-3', (SEQ ID No. 7)
  • J-Bgl-12 5 '-GATCTGTTCATG-3'
  • J-Bgl-24 5 '-ACCGACGTCGACTATCCATGAAC-3'
  • SEQ ID No. 8 J-Bgl-24: 5 '-ACCGACGTCGACTATCCATGAAC-3'
  • N-Bgl-12 5 '-GATCTTCCCTCG-3'
  • SEQ ID No. 10 N-Bgl-24: 5 * -AGGCAACTGTGCTATCCGAGGGAA-3 '(SEQ ID No. 11)
  • the oligonucleotides used for RT-PCR and sequencing were:
  • GAPDH forward 5 '-CCACCCATGGCAAATTCCATGGCA-3'
  • CEP-68 SP6 5 '-AATTAGGGAGACTCGTAAGGC-3'.
  • DP3 fragments were cut out from a 3% agarose gel, amplified by PCR, purified and cloned into the pBluescript KS nector (Stratagene). before being transformed into the E. coli strain SR101. Plasmid DNA was prepared using the Quiagen plasmid miniprep kit (Quiagen) and sequenced using standard methods. BLAST algorithms were used to analyze the DNA and protein sequences for correspondence to the National Center for Biotechnology Information (NCBI) public databases. Known protein structures were identified by SMART (Simple Molecular Architecture Research Tool).
  • RNA from cultured chondrocytes and cultured osteoblasts were size-fractionated per lane on a denaturing gel containing 1% agarose and 6% formaldehyde and transferred to Hybond N + membranes (Amersham Pharmacia Biotech).
  • Gene-specific PCR fragments were labeled by random octamer priming (MBI Fermentas, random domamer priming) using [ 32 P] dCTP as the radiolabeled nucleotide.
  • Hybridization was performed in 0.5 M sodium phosphate buffer, 7% SDS, 1 mM EDTA at 68 ° C overnight. The filters were washed twice in 40 mM sodium phosphate buffer, 1% SDS at 68 ° C for 30 minutes.
  • the first strand cDNA was amplified by PCR using gene specific primers.
  • the CEP-68 specific RT-PCR was performed under PCR conditions suitable for long nucleotide sequences using the Expand Long Template PCR system from Röche. After 20, 25, 30 and 35 PCR cycles, an aliquot on ethidium bromide stained agarose gel was checked. In comparison to the GAPDH-specific band intensity, the amount of the cDNA of the first strand was calculated from individual samples in order to be able to use the same amount of DNA template in the gene-specific semi-quantitative RT-PCR experiments.
  • the 5 '/ 3' RACE Kit from Röche was used to fill in the 5 'and 3' ends of the cDNA in accordance with the manufacturer's protocol.
  • the CEP-68 PCR fragment cloned into the pSP64-polyA (Stratagene) expression vector was constructed using the primers CEP-68 forward 2 and CEP-68 reverse 2.
  • In vitro transcription and in vitro translation was carried out in the presence of [ 35 S] methionine using a TNT Sp ⁇ -coupled reticulocyte lysate system (Promega). 10 ⁇ l of the translation product was loaded onto a 10% SDS-PAGE and analyzed by autoradiography. RT-PCR analysis of RNA from cartilage, brain and lung tissue
  • poly (A +) mRNA produced from cartilage or bone, or of 5 ⁇ g of total RNA isolated from the brain and lungs
  • reverse transcriptase SuperScript II Invitrogen, Düsseldorf, Germany
  • Oligo-d (T) primers performed a reverse transcription.
  • the first strand of cDNA was diluted 1: 5 in 1 x TE and subjected to PCR using primers specific for upstream and downstream gene regions.
  • the PCR comprised 30 to 45 cycles, with a 20-second denaturation at 94 ° C., a 20-second primer annealing at 58 ° C. and a 1-minute extension reaction at 72 ° C.
  • PCR reactions for the human CRTACl gene were carried out under PCR conditions suitable for long nucleotide sequences, the Expand Long Range Template PCR system from Röche Diagnostics (Mannheim, Germany) being carried out over 30 to 45 cycles (10 seconds at 92 ° C, 20 seconds at 58 ° C and 2 minutes at 68 ° C).
  • the primers used were designed with an annealing temperature of 58 ° C and were obtained from Interepta (Ulm, Germany). The following primers were used to distinguish between the two splice variants for the human CRTACl gene:
  • hCRTACl F1 with the sequence 5 '-CTC TGG ACG CTA CTC TATCT-3' (forward primer, in exon 4; SEQ ID No. 28) and the two reverse primers hCRTACl E14A-R with the sequence 5'-TGT GTC CTA AGA TAT GGT CAT T-3 '(in exon 14A; SEQ ID No. 29) and
  • the size of the PCR products was determined using a 1% TBE agarose gel. The bands were cut out of the gel, purified using the gel extraction protocol from Qiagen (Hilden, Germany) and sequenced in the German Cancer Research Center (DKFZ, Heidelberg, Germany). BLAST algorithms were used to search for DNA and protein sequence similarities in public databases. Protein motifs were identified using the “Simple Molecular Architecture Research Tool” program SMART.
  • Chondrocyte-specific gene expression was investigated by means of RDA with cDNA from cultured chondrocytes (test sample) and cultured osteoblasts (counter-test sample), each obtained from the same donor.
  • test sample osteoblasts
  • counter-test sample chondrocytes
  • a clearly different pattern of difference products was obtained. Sequence analysis showed that six of the eight chondrocyte-specific fragments identified were assigned to cartilage glycoprotein 39 (GP-39, YKL-40) and one to the human YKL-39 precursor protein.
  • FIG. 1 shows the results of an RT-PCR isolated from RNA from cultured chondrocytes (cl, c2), cultured osteoblasts (bl, b2), cultured mesenchymal stem cells (sl, s2), primary cartilage tissue (C) and bone tissue (B) was obtained using gene-specific Fischer primer.
  • the expression of CEP-68, GP-39 and YKL 39 was examined.
  • the GAPDH bands indicate the use of equal amounts of the cDNA template.
  • FIG. 1 shows that CEP-68 gene expression can only be observed in cultured chondrocytes, but not in cultured osteoblasts or stromal cells.
  • GP-39 and YKL-39 could be identified in chondrocytes as well as some mesenchymal stem cell samples.
  • GP-39 may differentiate cultured chondrocytes from cultured osteoblasts, GP-39 and YKL-39 result in background expression in mesenchymal.
  • CEP-68 can serve as a stable marker for cultured chondrocytes that is not expressed in mesenchymal stem cells or osteoblasts, even after attempting to induce osteoblast differentiation in cultured osteoblast-like cells.
  • FIG. 2 shows cells from the starting culture (passage 0) after four weeks (lane 1), cells which were cultured in expansion medium (passage 1) after a further two weeks in culture (lane 2) and cells cultured in chondrogenic medium for two weeks (lane 3).
  • CEP-68 was only expressed in the cultures which were kept in chondrogenic medium.
  • the GAPDH bands indicate equal amounts of the cDNA template. 35 PCR cycles were carried out for CEP-68 and Col 2, 25 cycles for GAPDH " .
  • RNA from cultured chondrocytes and osteoblasts was isolated, size fractionated on a denaturing formaldehyde agarose gel and blotted onto a nylon membrane.
  • a 3 kb-length transcript was specifically identified in cultured chondrocytes by hybridization with a CEP-68-specific PCR fragment (FIG. 3a).
  • the ethylenium bromide stained gel shown in FIG. 3a shows that equal amounts of RNA were loaded.
  • the autoradiography shown on the right in FIG. 3a shows that by means of hybridization with a CEP-68-specific sample, a transcript of a length of approximately 3 kb can only be detected in cultivated chondrocytes.
  • the audio radiographs were exposed for 24 hours at -80 ° C.
  • FIG. 3b shows a Northern blot with RNA samples from different human tissues (obtained from Clontech). Outside the skeleton, a CEP-68 transcript of 2.7 to 3 kb in length could be detected in the lungs, while no transcript could be detected in the heart, placenta, liver, skeletal muscle, pancreas and blood (data not shown). A smaller 2.4 kb CEP-68 transcript was found in the brain. Outside the skeleton, two different CEP-68 transcripts could therefore be found in the lungs and brain.
  • the autoradiographs were exposed for five days at -80 ° C. Hybridization with ⁇ -actin, shown in the lower part of FIG. 3, indicates the same amounts of RNA samples.
  • the fragment of CEP-68 initially identified by RDA shows sequence similarity to a human cDNA sequence (FLJ10320, AC: AK001182) with an open reading frame for 408 amino acids with unknown function and to a considerably shorter sequence from the rat (W 307, AC: U78304).
  • sequences available in databases three human EST clones: AC: T77333, R60637, Z43948 and three related human genomic clones: AC: AC015630, AL139239, AL358938
  • the genomic structure of CEP -68 can be determined.
  • the gene apparently has 14 exons within a genomic distance of at least 74 kb.
  • CEP-68 After selection of primers, the complete open reading frame of CEP-68 could be amplified by PCR, cloned and the fragment sequenced. To complete the complete mRNA sequence including the 5 'and 3' untranslated regions (UTRs), 5 'and 3' RACE were carried out.
  • the CEP-68 gene has an open reading frame (SEQ ID No. 2) of 1959 nucleotides which code for 653 amino acids (SEQ ID No. 4). This 653 amino acid protein has a calculated molecular weight of 71 kDa. CEP-68 also has a 19 amino acid signal peptide at the N-terminus (SEQ ID No. 5). The molecular mass without the signal peptide was calculated to be 68 kDa. The exon boundaries lie from 5 'to 3' behind nucleotides 201, 397, 534, 691, 826, 972, 1110, 1192, 1294, 1463, 1607, 1652, 1795 (SEQ ID No. 1).
  • CEP-68 also has an EGF-like Ca 2+ binding domain with conserved cysteine residues from nucleotide positions -1651 to 1794 (SEQ ID No. 1).
  • FIG. 4 shows an autoradiogram of CEP-68 labeled in vitro translated [ 35 S] -Met. Neither by homology searches in protein nor in nucleotide databases could similarity or identity to known proteins or protein motifs be found, apart from the EGF domain mentioned.
  • FIG. 5 shows a gel-electrophoretic separation of the transcripts of CEP-68 (CRTACl), it being shown that the splice variant CRTACl-A found in chondrocytes (lane C) and lungs (lane L) is greater than 1.5 kb during the alternative splice variant CRTAC1-B, which is found in the brain (lane B) is less than 1.5 kb.
  • the CRTACl-B protein encoded by the alternative splice variant is 16 amino acids smaller than the protein CRTACl-A, which is derived from the splice variant found in chondrocytes.
  • the two exons 14A and 14B show no significant similarity either with regard to their nucleotide sequence or with regard to the peptide sequence derived from the nucleotide sequence.
  • the EGF-like domain of CRTACl is only encoded by exon 13 and is preserved in both splice variants.
  • the amino acid sequence derived from the nucleotide sequence of exon 14B has a large number of leucine residues (9 out of 38 residues). A homology to the consensus sequences of leucine-rich domains of known proteins, for example the extracellular matrix proteins versican, decorin and fibromodulin, was not found.
  • Splice variants that affect the C-terminus of a protein and that are caused by the alternative use of the last exon are rare.
  • a well-known example of this is the human prolactin reporter gene, which encodes three different subtypes that stem from an alternative splicing of the last two exons.
  • the RP11-19C6 (AL139239) and RP11-459F3 (AL358938) genomic clones contain the complete genomic sequence of the human CRTACl gene including the two exons 14A and 14B. All introns have the consensus splice donor and consensus splice acceptor sequences. The length of the individual introns is shown in FIG. 6A.
  • the genomic structure at the C-terminal end of CRTACl shows that exon 14B is located 447 bp downstream of exon 13, while exon 14A is located 14529 bp downstream of exon 14B ( Figure 6B).
  • the intron 13A, exon 14B and intron 14B are spliced, while in the splice variant CRTACl-B only intron 13A is removed.
  • the expression profile of the two alternative splice variants of CRTACl was determined using exon 14A and exon 14B-specific RT-PCR experiments.
  • the CRTACl-A specific transcript could only be found in cartilage and lung tissue were demonstrated, confirming that a longer transcript is obtained in these two tissues compared to brain tissue.
  • the CRTAC1-B form was brain-specific and encodes a protein of 70 kDa including the signal peptide.
  • Tissue-specific splice variants are known from several human genes, for example the gene for Cu, Zn superoxide dismutase (SOD1). Five different alternative spliced transcripts were identified for this gene. Different splicing products are also known from the type 2 collagen gene.
  • Type 2 collagen is spliced in a development-specific manner, with exon 2 being contained only in early transcripts from prachondigenic tissues, but not in later stages.
  • the peptide encoded by exon 2 interacts with growth factors of the TGF-ß superfamily and is therefore of functional relevance.

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Abstract

L'invention concerne un gène marqueur spécifique des chondrocytes, la protéine codée par celui-ci, des anticorps réagissant avec ce dernier, des biopuces réalisées sur la base de ce gène marqueur, de cette protéine ou de ces anticorps, ainsi que leur procédé de production et leurs utilisations.
PCT/EP2001/015307 2001-01-05 2001-12-24 Marqueur de cellule cartilagineuse, son procede de production et son utilisation Ceased WO2002053709A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2013202145B2 (en) * 2007-04-26 2015-02-26 Genera Istrazivanja D.O.O. Blood biomarkers for bone fracture and cartilage injury

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Publication number Priority date Publication date Assignee Title
EP0705842A2 (fr) * 1994-10-06 1996-04-10 Hoechst Aktiengesellschaft Régulation des gènes par stimulation du chondrocytes avec 1L-1bêta
WO1998032333A1 (fr) * 1996-12-06 1998-07-30 Osiris Therapeutics, Inc. Perfectionnement concernant la differentiation chondrogene de cellules souches du mesenchyme humain
EP1033403A1 (fr) * 1997-11-27 2000-09-06 Chugai Seiyaku Kabushiki Kaisha Gene provenant de chondrocytes foetaux humain

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
EP0705842A2 (fr) * 1994-10-06 1996-04-10 Hoechst Aktiengesellschaft Régulation des gènes par stimulation du chondrocytes avec 1L-1bêta
WO1998032333A1 (fr) * 1996-12-06 1998-07-30 Osiris Therapeutics, Inc. Perfectionnement concernant la differentiation chondrogene de cellules souches du mesenchyme humain
EP1033403A1 (fr) * 1997-11-27 2000-09-06 Chugai Seiyaku Kabushiki Kaisha Gene provenant de chondrocytes foetaux humain

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Title
BAYARSAIHAN D. ET AL.,: "rapid identification of a novel chondrocyte-specific gene by rna differential display", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATION, vol. 220, 18 March 1996 (1996-03-18), pages 449 - 452, XP002196249 *
DATABASE EMBL embl; 14 September 2000 (2000-09-14), STECK E.: "homo sapiens mrna or chondrocyte expressed protein 68 kDa", XP002196343 *
DATABASE EMBL embl; 20 July 2000 (2000-07-20), BOLTON M.C.: "cloning of aspic, a novel protein secreted by human normal and osteoarthritis cartilage identified by 2D electrophoresis and ms", XP002196342 *
STECK E. ET AL.,: "chondrocyte expressed protein-68 (cep-68), a novel human marker gene for cultured chondrocytes", BIOCHEM. J., vol. 353, 8 January 2001 (2001-01-08), pages 169 - 174, XP002196341 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2013202145B2 (en) * 2007-04-26 2015-02-26 Genera Istrazivanja D.O.O. Blood biomarkers for bone fracture and cartilage injury

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