WO2024074119A1 - 一种重组胶原蛋白及其在软骨修复基质中的用途 - Google Patents

一种重组胶原蛋白及其在软骨修复基质中的用途 Download PDF

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WO2024074119A1
WO2024074119A1 PCT/CN2023/122682 CN2023122682W WO2024074119A1 WO 2024074119 A1 WO2024074119 A1 WO 2024074119A1 CN 2023122682 W CN2023122682 W CN 2023122682W WO 2024074119 A1 WO2024074119 A1 WO 2024074119A1
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xaa
recombinant collagen
yaa
amino acid
recombinant
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PCT/CN2023/122682
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English (en)
French (fr)
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WO2024074119A8 (zh
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林海
徐扬
王境
刘展鸿
杨霞
张兴栋
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山西锦波生物医药股份有限公司
四川大学
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Publication of WO2024074119A1 publication Critical patent/WO2024074119A1/zh
Publication of WO2024074119A8 publication Critical patent/WO2024074119A8/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/26—Mixtures of macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3817—Cartilage-forming cells, e.g. pre-chondrocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/48—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with macromolecular fillers
    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/80—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special chemical form
    • A61L2300/802—Additives, excipients, e.g. cyclodextrins, fatty acids, surfactants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction

Definitions

  • the invention belongs to the field of biomaterials, and in particular relates to a recombinant collagen and an application thereof in a cartilage repair matrix.
  • Articular cartilage injury has become a common orthopedic disease. Trauma, arthritis and sports-related injuries can all cause articular cartilage injury or defect.
  • the repair of articular cartilage mainly depends on the proliferation and differentiation of chondrocytes to produce sufficient extracellular matrix to repair cartilage defects.
  • healthy adult chondrocytes are usually static and highly differentiated cells. Because of their low degree of vascularization, their nutrition mainly comes from synovial fluid and subchondral bone. If traumatic or pathological damage occurs, the repair and regeneration of articular cartilage is very limited. At present, some treatment methods used in clinical practice include microfracture, osteochondral transplantation, chondrocyte transplantation, etc.
  • tissue engineering has made considerable progress in obtaining functional articular cartilage.
  • Some bioengineering treatment strategies including stem cell methods and scaffold technology, have been proposed to provide minimally invasive and more effective ways to repair articular cartilage.
  • scaffold materials play an important role in the construction of tissue engineering cartilage. Their mechanical properties and chemical composition will affect cell adhesion, proliferation and cell phenotype.
  • Collagen has become a biomaterial with extensive research in tissue engineering due to its excellent biocompatibility, bioactivity and biodegradability.
  • most traditional collagen comes from animals.
  • the amino acid sequence of animal collagen is highly similar to that of human collagen.
  • the similarity between mammalian pigs and cattle and humans is 95%, and the similarity between fish and humans is 65%.
  • animal collagen when animal collagen is used in biomaterials or medical devices, these differences may bring obvious risks such as immune rejection, sensitization, and viral infection and transmission.
  • the efficiency of extracting collagen from biological tissues depends largely on its natural abundance, and it is also difficult to avoid the presence of a mixture of different subtypes. Therefore, the deficiencies in batch stability, purity, molecular weight and distribution of animal-derived collagen further limit its many applications in the biomedical field.
  • Recombinant collagen is based on the natural human collagen gene sequence, and codons with specific functional expressions are selected. After appropriate modification and editing, they are transcribed into host cells and the recombinant protein material is obtained by large-scale production using biofermentation technology.
  • the technical problem to be solved by the present invention is to provide a recombinant collagen and its use in a cartilage repair matrix in view of the deficiency of animal-derived collagen.
  • the provided recombinant collagen has relatively obvious integrin binding activity, can promote cell adhesion, proliferation and differentiation, and has the effect of repairing cartilage tissue defects.
  • Integrin binding activity is related to the integrin family, which is a widely distributed transmembrane glycoprotein that is the link between the extracellular matrix and intracellular signal transduction. Integrins are composed of ⁇ subunits and ⁇ subunits connected by non-covalent bonds to form heterodimers. Currently, 18 ⁇ subunits and 8 ⁇ subunits have been discovered, which can be combined into at least 24 integrin dimers.
  • the integrin family of cell adhesion molecules is the main connecting substance between the extracellular matrix and parenchymal cells such as inflammatory cells and fibroblasts. It is closely related to cell adhesion, spreading, migration, functional expression, and the occurrence, maintenance and development of tissue fibrosis. It can regulate the interaction between cells and cells and between cells and extracellular matrix, such as recognition and adhesion.
  • the first object of the present invention is to provide a recombinant collagen, wherein the amino acid sequence of the recombinant collagen comprises N basic repeating units, wherein the basic repeating units contain n1 of the following characteristic amino acid sequences: "G-Xaa 1 -Xaa 2 -GE-Xaa 3 "; the 3' end and the 5' end of the basic repeating unit are connected to form the above characteristic amino acid sequence;
  • N is an integer greater than or equal to 4; and n1 is an integer greater than or equal to 3.
  • the N value is an integer of 4 to 300 or more, and further an integer of 4 to 200 or more.
  • the N value can make the molecular weight of the recombinant collagen reach 1kDa to 250kDa, and further reach 3kDa to 150kDa.
  • the characteristic amino acid sequences are arranged continuously or at intervals in the basic repeating unit.
  • amino acid sequence of the recombinant collagen presents the following characteristics:
  • Xaa 1 is a nonpolar hydrophobic amino acid
  • Xaa 2 is one of serine (S), alanine (A), proline (P), and hydroxyproline (O)
  • Xaa 3 is a basic amino acid
  • Each occurrence of Yaa 1 , Yaa 2 , Yaa 3 , Yaa 4 , ... ... , Yaa n , Yaa n+1 is independently selected from none, one or more different or identical amino acids;
  • n2, n3, n4, ..., n are integers greater than 0, and none of them are 0 at the same time.
  • the non-polar hydrophobic amino acid of the present invention includes one of glycine (G), valine (V), phenylalanine (F), alanine (A), leucine (L), isoleucine (I), methionine (M), proline (P), and hydroxyproline (O); and further includes one of phenylalanine (F), alanine (A), leucine (L), isoleucine (I), methionine (M), proline (P), and hydroxyproline (O).
  • the basic amino acid includes one of lysine, arginine and histidine, and is further lysine or arginine.
  • the recombinant collagen sequence does not contain a protein tag.
  • amino acid sequence of the recombinant collagen as mentioned above is shown as SEQID NO.1.
  • the second object of the present invention is to provide a method for preparing the aforementioned recombinant collagen, which uses gene recombination technology to obtain recombinant collagen through host cell expression, extraction, and purification; the amino acid sequence of the recombinant collagen is obtained by repeatedly connecting multiple basic repeating units;
  • amino acid sequence of the recombinant collagen includes N basic repeating units, and the basic repeating unit contains n1 of the following characteristic amino acid sequence: "G-Xaa 1 -Xaa 2 -GE-Xaa 3 "; the 3' end and the 5' end of the basic repeating unit are connected to form the above characteristic amino acid sequence;
  • characteristic amino acid sequences are arranged continuously or alternately in the basic repeating unit.
  • N is an integer from 4 to 300, and further an integer from 4 to 200, including but not limited to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30. «200.
  • the value of N can make the molecular weight of the recombinant collagen reach 1kDa to 200kDa, and further reach 3kDa to 150kDa.
  • N can be taken from the endpoint values at both ends of the range value defined by the present invention, or from any integer in the range value.
  • the value of N can be 1, 3, 10, 50, 70, 90, 95, 100, 125, 150, etc.
  • the recombinant collagen sequence does not contain a protein tag
  • the amino acid sequence of the recombinant collagen presents the following characteristics: [-GE-Xaa3-Yaa1-(G-Xaa1-Xaa2-GE-Xaa3)n2-Yaa2-(G-Xaa1-Xaa2-GE-Xaa 3 ) n3 -Yaa 3- (G-Xaa 1 -Xaa 2 -GE-Xaa 3 ) n3 -Yaa 4- (G-Xaa 1 -Xaa 2 -GE-Xaa 3 ) n4- ............-Yaa n- (G-Xaa 1 -Xaa 2 -GE-Xaa 3 ) n -Yaa n+1 -G-Xaa 1 -Xa 2- ] N
  • Xaa 1 is a nonpolar hydrophobic amino acid
  • Xaa 2 is one of serine (S), alanine (A), proline (P), and hydroxyproline (O)
  • Xaa 3 is a basic amino acid
  • Each occurrence of Yaa 1 , Yaa 2 , Yaa 3 , Yaa 4 , ... ... , Yaa n , Yaa n+1 is independently selected from none, one or more different or identical amino acids;
  • n2, n3, n4, ..., n are integers greater than 0, and none of them are 0 at the same time.
  • the third object of the present invention is to provide the use of the aforementioned recombinant collagen in the preparation of products for regulating cell adhesion, proliferation and differentiation.
  • the fourth object of the present invention is to provide the use of the aforementioned recombinant collagen in preparing a cartilage repair matrix product.
  • the recombinant collagen of the present invention when used in the aforementioned purposes, it can be used in combination with polymers and/or their derivatives, wherein the polymers include natural polymers and synthetic polymers; further, the natural polymers include but are not limited to hyaluronic acid, chitosan, alginate, cellulose, etc.; further, the synthetic polymers include but are not limited to PLA, PGA, PLCL, PVA.
  • the concentration used is 0.01 to 100 mg/mL, for example, it can be 0.01 mg/mL, 0.05 mg/mL, 2 mg/mL, 4.5 mg/mL, 7 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 50 mg/mL, 80 mg/mL, 100 mg/mL, etc.
  • the concentration used is related to the application site and scenario.
  • the fifth object of the present invention is to provide a cartilage repair matrix, comprising a polymer and/or its derivatives, and the aforementioned recombinant collagen.
  • the polymer includes natural polymers and synthetic polymers; further, the natural polymer is at least selected from any one of hyaluronic acid, chitosan, alginate, and cellulose; further, the synthetic polymer is at least selected from any one of PLA, PGA, PLCL, and PVA.
  • the sixth object of the present invention is to provide a method for preparing the aforementioned cartilage repair matrix, comprising the following steps:
  • step (2) adding the recombinant collagen to the reaction system in step (1) to react, dialyzing and drying the solution obtained after the reaction to obtain a composite matrix;
  • Humanized collagen has a small molecular weight and poor mechanical properties. It is difficult to form a gel after modification and cannot meet the needs of cartilage tissue engineering.
  • hyaluronic acid is firstly appropriately chemically modified with methacrylic anhydride to introduce unsaturated carbon-carbon double bonds, and then the carboxyl groups in the hyaluronic acid are activated by adding EDC/sNHS, and then recombinant humanized collagen is added to make the amino groups on the protein and the activated carboxyl groups on the hyaluronic acid undergo amidation reaction to couple the two.
  • the composite hydrogel finally prepared by photocrosslinking not only has good mechanical properties and biocompatibility, but also the photocrosslinking method makes the operation easier, which can meet the needs of cartilage tissue engineering, and also meet the filling of irregular defects in clinical practice.
  • the EDC described in the present invention refers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and NHS refers to N-hydroxysuccinimide.
  • the polymer derivative in step (1) is hyaluronic acid modified with methacrylic anhydride
  • the molecular weight of the modified hyaluronic acid is 100 to 1500 kDa, preferably 300 to 600 kDa, more preferably 300 kDa;
  • the degree of grafting of the hyaluronic acid modification in step (1) is 20 to 60%, preferably 30 to 50%;
  • step (1) after EDC and sNHS are added, the concentration of sNHS is 0.5-5 mM, and the concentration of EDC is 0.5-5 mM; further, the concentration of sNHS is 1-3 mM, and the concentration of EDC is 2-4 mM.
  • step (1) the pH value of the mixed solution after adding the EDC/sNHS solution is 4 to 5, preferably 4.75;
  • the pH value of the mixed solution after the reaction is 7-8, and the reaction time is 2-5 hours; preferably, the pH value is 7.4, and the reaction time is 3 hours.
  • step (2) the mass ratio of recombinant collagen to polymer derivative is 0.5-30:2-10; further 0.5-20:6; further 1-11:6;
  • reaction time in step (2) is 5 to 15 hours, preferably 8 to 12 hours;
  • step (2) the solution after the reaction is placed in a dialysis bag with a molecular weight cutoff of 8000 to 14000, dialyzed with deionized water for 2 to 4 days, and then freeze-dried to obtain a composite matrix.
  • the photoinitiator used in the present invention is at least one of LAP and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959), and the final concentration is 0.01-0.05% by mass volume, preferably 0.05%.
  • the concentration of the composite matrix after adding the photoinitiator in step (3) is 2 to 30 mg/ml, further 5 to 20 mg/ml, and further 7 to 17 mg/ml.
  • the cartilage repair matrix of the present invention is a product containing the recombinant collagen, which can be used as a component of medical devices including but not limited to implant materials, tissue engineering scaffold materials, soft tissue filling materials, cells or other active substance carrier materials.
  • the cartilage repair matrix of the present invention can be prepared into a cartilage repair hydrogel after adding cells, or can be directly applied to the cartilage defect site without adding cells.
  • the seventh object of the present invention is to provide a method for preparing a cartilage repair hydrogel, wherein the cartilage repair matrix prepared by the aforementioned method is compounded with chondrocytes or bone marrow mesenchymal stem cells, injected into the cartilage damage site, and irradiated with light to form a cartilage repair hydrogel.
  • the wavelength of the light is selected adaptively according to the initiator used.
  • the cartilage repair hydrogel of the present invention further comprises a physiologically acceptable carrier material.
  • the carrier materials here include but are not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.).
  • water-soluble carrier materials such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.
  • poorly soluble carrier materials such as ethyl cellulose, cholesterol stearate, etc.
  • enteric carrier materials such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.
  • composition can also be used in combination with other functional materials as needed, such as components with antibacterial and antimicrobial effects, anti-aging components, anticoagulant components, antioxidant components, growth factors, etc.
  • composition can be prepared according to conventional techniques of pharmaceutics or cosmetics, including mixing the recombinant collagen of the present invention as an active ingredient with a carrier, and preparing the desired dosage form according to conventional techniques.
  • the composition of the present invention can be formulated into a variety of dosage forms as required, including but not limited to oral administration preparations, mucosal administration preparations, injection preparations, inhalation preparations, and external preparations.
  • the products described in the present invention include but are not limited to medicines, foods, health products or cosmetics, and daily necessities.
  • the word “may” includes both performing a certain process and not performing a certain process.
  • the recombinant collagen of the present invention and the above-mentioned composition can be directly administered to patients as drugs, or can be administered to patients after being mixed with suitable carriers or excipients.
  • suitable carriers or excipients include but are not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Among them, water-soluble carrier materials are preferred.
  • the suppository can be a vaginal suppository, a vaginal ring, or an ointment, cream or gel suitable for vaginal application.
  • the dosage form can be a common preparation, a sustained-release preparation, a controlled-release preparation and various microparticle delivery systems.
  • various carriers known in the art can be widely used.
  • carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants, such as dry starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate and citric acid, calcium
  • Tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.
  • coated tablets such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.
  • various carriers known in the art can be widely used.
  • Examples of carriers are, for example, diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, Gelucire, kaolin, talc, etc.; adhesives such as gum arabic, tragacanth gum, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc.
  • various carriers known in the art can be widely used.
  • Examples of carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc.
  • an injection preparation such as a solution, emulsion, lyophilized powder injection and suspension
  • all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc.
  • an appropriate amount of sodium chloride, glucose or glycerol can be added to the injection preparation.
  • conventional cosolvents, buffers, pH adjusters, etc. can also be added.
  • colorants, preservatives, fragrances, flavoring agents, sweeteners or other materials can also be added to the pharmaceutical preparation.
  • the above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracisternal injection or infusion, etc.; cavity administration, such as rectal, vaginal and sublingual; respiratory tract administration, such as nasal cavity; mucosal administration.
  • the above-mentioned administration route is preferably injection, and the preferred injection route is subcutaneous injection.
  • the dosage of the recombinant collagen of the present invention and the above-mentioned composition depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight and individual response of the patient or animal, the specific active ingredient used, the route of administration and the number of administrations, etc.
  • the above-mentioned dosage can be administered in a single dosage form or divided into several, such as two, three or four dosage forms.
  • the specific therapeutically effective dosage level must be determined based on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific active ingredient used; the specific composition used; the patient's age, weight, general health, sex and diet; the administration time, route of administration and excretion rate of the specific active ingredient used; the duration of treatment; drugs used in combination or simultaneously with the specific active ingredient used; and similar factors known in the medical field.
  • the recombinant collagen provided by the present invention has relatively obvious integrin binding activity, has the effect of promoting cell adhesion, proliferation, differentiation, and repairing cartilage tissue defects, and has good application prospects.
  • the present invention covalently cross-links hyaluronic acid and humanized collagen to form a stable composite hydrogel matrix with good mechanical properties and can well maintain the morphology of the composite; it has good biocompatibility, and the photocross-linking method makes the operation easier, which can meet the needs of cartilage tissue engineering and also meet the clinical filling of irregular defects.
  • Figure 1 shows the results of fibroblast cell adhesion and growth
  • Figure 2 shows the OD values of fibroblast cell adhesion
  • Figure 3 shows the results of chondrocyte adhesion and growth
  • Figure 4 shows the OD values of chondrocyte adhesion
  • FIG5 is the results of the adhesion experiment of sequence A and sequence B at different concentrations to human gingival fibroblasts
  • FIG6 is a morphological diagram of chondrocyte/hydrogel complexes after 1, 7, 14, and 21 days of in vitro culture;
  • FIG7 shows the OD values of chondrocyte adhesion after 1, 7, and 14 days of in vitro culture of chondrocyte/hydrogel complexes
  • FIG8 is a CCK-8 graph of chondrocytes after 1, 7, and 14 days of in vitro culture of chondrocyte/hydrogel complexes
  • FIG9 is the staining result of chondrocyte/hydrogel complex
  • FIG10 is the result of immunohistochemical staining of type II collagen of chondrocyte/hydrogel complex
  • FIG. 11 shows the quantitative detection results of GAG in chondrocyte/hydrogel complex.
  • a nucleotide sequence encoding the recombinant collagen is constructed, a vector containing the nucleotide sequence is constructed and screened, a host cell (which may be a prokaryotic cell or a eukaryotic cell) containing the vector is constructed and screened, the host cell is cultured under appropriate conditions to express the protein, and the expressed recombinant collagen is collected and purified.
  • the term "vector” is a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage and animal viruses.
  • the vector may contain a variety of elements for controlling expression, including but not limited to promoter sequences, transcription start sequences, enhancer sequences, selection elements and reporter genes.
  • the vector may also contain a replication initiation site.
  • the vector may contain the nucleic acid of the present invention to facilitate introduction into cells for expression.
  • the vector may contain expression control elements operably linked to the nucleic acid, such as promoters, terminators and/or enhancers.
  • the gene recombination and transcription involved in the present invention refers to synthesizing a nucleotide sequence encoding recombinant collagen, cloning the nucleotide sequence into an expression vector by conventional techniques, and then transforming the expression vector into a host cell to construct and screen an engineered strain.
  • the transformation method includes but is not limited to electroporation, CaCl2 transformation, etc.; the expression vector can be a commonly used vector such as pET26, pET32, pGEX-6p, pPIC9, pPIC9K, etc., and can be a plasmid, phage, virus or other vector.
  • the term "host cell” as used herein refers to a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • the host cell can be a eukaryotic cell or a prokaryotic cell.
  • microbial cells such as Enterobacteriaceae cells (e.g., Escherichia coli BL21, DH5 ⁇ , etc.), eukaryotic cells such as yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae, etc.) or CHO cells.
  • the fermentation culture involved in the present invention refers to inducing fermentation culture in a sterile fermentation tank by combining the screened and protein-high expression-identified engineered strain with a suitable fermentation culture medium under suitable temperature, pressure and pH conditions.
  • the separation and purification of proteins involved in the present invention refers to obtaining bacterial liquid after lysing, homogenizing, separating and other treatments of bacteria, and further obtaining recombinant collagen by conventional protein separation and purification techniques. Separation and purification techniques include but are not limited to filtration, centrifugation, salting out, dialysis, liquid chromatography, ion exchange chromatography, affinity chromatography, etc.
  • gene recombination and transcription are performed by taking Escherichia coli as a host cell, specifically:
  • Gene recombination and transcription The DNA fragments were codon optimized and spliced and recombined using the PCR method.
  • the expression vector was constructed using pET-32a and then transferred into the Escherichia coli expression strain BL21. After culture and screening, genetically engineered Escherichia coli bacteria with high protein expression were obtained.
  • Fermentation culture Select a single colony of genetically engineered Escherichia coli from the LB plate, place it in a 100mL Erlenmeyer flask containing 10mL LB medium, and culture it at 37°C, 220rpm for 12-16h; inoculate the bacterial solution into a fermenter containing LB medium at a ratio of 1:100 for scale-up culture, culture it at 37°C, 220rpm for 3h until OD600 is about 0.6, add 0.5mM IPTG, induce culture at 16°C for 20h, and then collect the bacteria by centrifugation.
  • Protein separation and purification After reselecting the bacteria with Tris buffer, stir at high speed to completely dissolve the bacteria, collect the supernatant by centrifugation and cool to 4°C; the supernatant is filtered using 1 ⁇ m, 0.45 ⁇ m and 0.22 ⁇ m filter membranes in sequence and further purified by affinity chromatography to obtain recombinant collagen.
  • Example 1 The method of Example 1 was used to prepare the following three recombinant collagens containing different amino acid sequences.
  • sequence A was linked 16 times, sequence A: GER GAP GFR GPA GPN GIP GEK GPA GER GAP, and its full sequence is as follows (SEQ ID NO.1):
  • sequence B GEK GSP GAD GPA GAP GTP GPQ GIA GQR GVV GLP GQR GER GFP GLP GPS GEP GKQ GPS GAS, and its full sequence is as follows (SEQ ID NO.2):
  • sequence C HHHHHH GER GAP GFR GPA GPN GIP GEK GPA GER GAP, and its full sequence is as follows (SEQ ID NO.3):
  • Example 1 The cell culture method in Example 1 was used to perform fibroblast adhesion test, chondrocyte adhesion test, and human gingival fibroblast adhesion promotion test on the above three collagens.
  • the results in Figure 2 show that the recombinant collagen experimental group obtained by repeatedly connecting sequence A with more characteristic amino acid sequence combinations can best promote the adhesion of fibroblasts, followed by animal collagen and recombinant collagen obtained by repeatedly connecting sequence B.
  • the adhesion amount of fibroblasts in these three groups is significantly higher than that of the recombinant collagen obtained by repeatedly connecting sequence C and the blank control; the adhesion amount of fibroblasts in the sequence C recombinant collagen group is slightly higher than that of the blank control.
  • the results in Figure 4 show that animal collagen is most conducive to promoting the adhesion of chondrocytes, which is significantly higher than other groups; the sequence A recombinant collagen group with more characteristic amino acid sequence combinations is significantly higher than the sequence B recombinant collagen group, sequence C recombinant collagen group and blank control group; the sequence B recombinant collagen group is significantly higher than the sequence C recombinant collagen group and blank control group; the sequence C recombinant collagen group is slightly higher than the blank control group, but there is no statistical difference.
  • Figure 5 The results of the human gingival fibroblast adhesion promotion experiment show that the sequence A recombinant collagen group with more characteristic amino acid sequence combinations is more conducive to the adhesion of human gingival fibroblasts than sequence B at different concentrations; as the concentration of recombinant collagen increases, the adhesion of human gingival fibroblasts also gradually increases.
  • Example 1 The method of Example 1 is used to prepare recombinant collagen that meets the amino acid sequence characteristics of the present invention, wherein the amino acid sequence core sequence of the recombinant humanized collagen is preferably GERGAPGFRGPAGPNGIPGEKGPAGERGAP (sequence A), which is repeatedly connected 16 times and used to promote cartilage repair.
  • the specific steps are:
  • HA-MA methacrylated hyaluronic acid
  • reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000-14000, dialyzed with deionized water for three days, and then freeze-dried using a freeze dryer to obtain freeze-dried sponge HA-rhCol III;
  • step (8) Take the precursor solution in step (7) and place it in a silicone mold with a specification of ⁇ 6mm ⁇ 2.5mm. Irradiate it under ultraviolet light for 1 minute to obtain hyaluronic acid hydrogel containing recombinant humanized collagen (HA-rhCol III).
  • Example 1 The method of Example 1 is used to prepare recombinant collagen that meets the amino acid sequence characteristics of the present invention, wherein the amino acid sequence core sequence of the recombinant humanized collagen is preferably GERGAPGFRGPAGPNGIPGEKGPAGERGAP (sequence A), which is repeatedly connected 16 times and used to promote cartilage repair.
  • the specific steps are:
  • reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000-14000, dialyzed with deionized water for three days, and then freeze-dried using a freeze dryer to obtain the product HA-rhCol III;
  • step (8) Take the precursor solution in step (7) and place it in a silicone mold with a specification of ⁇ 6mm ⁇ 2.5mm. Irradiate it under ultraviolet light for 1 minute to obtain HA-rhCol III hydrogel.
  • Example 1 The method of Example 1 is used to prepare recombinant collagen that meets the amino acid sequence characteristics of the present invention, wherein the amino acid sequence core sequence of the recombinant humanized collagen is preferably GERGAPGFRGPAGPNGIPGEKGPAGERGAP (sequence A), which is repeatedly connected 16 times (SEQ ID NO. 1), and is used to promote cartilage repair.
  • the specific steps are:
  • reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000-14000, dialyzed with deionized water for three days, and then freeze-dried using a freeze dryer to obtain the product HA-rhCol III;
  • step (8) Take the precursor solution in step (7) and place it in a silicone mold with a specification of ⁇ 6mm ⁇ 2.5mm. Irradiate it under ultraviolet light for 2 minutes to obtain HA-rhCol III hydrogel.
  • Example 1 The method of Example 1 is used to prepare recombinant collagen that meets the amino acid sequence characteristics of the present invention, wherein the amino acid sequence core sequence of the recombinant humanized collagen is preferably GERGAPGFRGPAGPNGIPGEKGPAGERGAP, which is repeated 16 times (SEQ ID NO. 1), and is used to promote cartilage repair.
  • the specific steps are:
  • reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000-14000, dialyzed with deionized water for three days, and then freeze-dried using a freeze dryer to obtain the product HA-rhCol III;
  • step (8) Take the precursor solution in step (7) and place it in a silicone mold with a specification of ⁇ 6mm ⁇ 2.5mm. Irradiate it under ultraviolet light for 1 minute to obtain HA-rhCol III hydrogel.
  • the chondrocytes in the HA-MA hydrogel were cultured for 21 days. Due to the lack of adhesion sites, the chondrocytes aggregated and grew in situ. However, the chondrocytes in the HA-rhCol III hydrogel were partially spread out at 7 days. At 14 and 21 days, the cell spreading growth phenomenon was more obvious and evenly distributed. This shows that rhCol III is conducive to cell adhesion, migration and proliferation, and the HA-rhCol III hydrogel can promote the proliferation of chondrocytes better than the HA-MA hydrogel.

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Abstract

一种重组胶原蛋白及其在软骨修复基质中的用途。所述重组胶原蛋白包含以SEQ ID No.1所示的序列；所述重组胶原蛋白的氨基酸序列中，包括N 个基本重复单元，所述基本重复单元中含有n1 个如下特征氨基酸序列："G-Xaa1-Xaa2-G-E-Xaa3"；基本重复单元的3'端和5'端连接形成上述特征氨基酸序列。所述重组胶原蛋白具有较为明显的整合素结合活性，具有促进细胞粘附、增殖、分化，修复软骨组织缺损等的效果，具有较好的应用前景。

Description

一种重组胶原蛋白及其在软骨修复基质中的用途
本申请要求(1)2022年10月08日提交中国专利局、申请号为2022112245930，(2)2022年11月04日提交中国专利局、申请号为2022113785160的优先权。在先申请的全部内容通过引用结合在本申请中。
技术领域
本发明属于生物材料领域，特别是涉及一种重组胶原蛋白及其在软骨修复基质中的用途。
 背景技术
关节软骨损伤已成为骨科常见疾病，创伤、关节炎和运动相关损伤等均可引起关节软骨损伤或缺损。关节软骨的修复主要靠软骨细胞的增殖分化，产生足够的细胞外基质修复软骨缺损。然而健康成人软骨细胞通常是静止的、具备高度分化的细胞，因其本身血管化程度低，营养主要来源于关节液和软骨下骨，如发生创伤性或病理性损伤，关节软骨的修复再生十分有限。目前，临床上用到的一些治疗方法有微骨折法、骨软骨移植法、软骨细胞移植法等，这些方法能在一定程度上修复软骨缺损，缓解患者的痛苦，但是长期效果并不令人满意，重要的原因就是这些方法重建出来的软骨与正常的透明软骨相比，在组织和结构上有很大的差别，从而导致在组织学和生物力学性能上与人的正常软骨存在着差距。
近年来，组织工程在获得功能性关节软骨方面取得了相当大的进展，一些生物工程治疗策略包括干细胞方法和支架技术已被提出，以提供微创和更有效的方式修复关节软骨。支架材料作为组织工程的三大要素之一，在组织工程软骨的构建中发挥着重要的作用，其力学性能和化学组成将影响细胞的粘附、增殖和细胞表型等。胶原蛋白以其出色的生物相容性、生物活性以及生物降解性，成为组织工程研究较为深入的生物材料。但传统的胶原蛋白大部分来源于动物，动物源胶原蛋白的氨基酸序列与人的胶原蛋白相似度较高，如哺乳动物猪、牛与人的相似度为95％，鱼与人的相似度65％，但将动物源胶原蛋白用于生物材料或医疗器械时，这些差异可能带来明显的免疫排斥、致敏反应以及病毒感染和传播等风险。同时，由于不同亚型的天然胶原在组织、器官中分布不均且以复合形态共存，所以从生物组织提取胶原的效率很大程度取决于其天然丰度，也难以避免的存在不同亚型的混合。因此，动物源胶原蛋白的批次稳定性、纯度、分子量及分布等方面的不足进一步限制了其在生物医学领域的诸多应用。
为了解决上述问题，科研人员把目光聚焦到利用基因工程技术制备重组胶原蛋白的研究。重组胶原蛋白以人天然的胶原蛋白基因序列为基础，选择其中具有特定功能表达的密码子，经适当修饰、编辑后转录至宿主细胞并利用生物发酵技术实现规模化生产得到的重组蛋白材料。
发明内容
本发明所要解决的技术问题在于针对动物源胶原蛋白的不足，提供一种重组胶原蛋白及其在软骨修复基质中的用途，提供的重组胶原蛋白具有较为明显的整合素结合活性，可以促进细胞的粘附、增殖、分化，具有修复软骨组织缺损的效果。
整合素结合活性与整合素家族相关，整合素家族是一类分布广泛的跨膜糖蛋白，是连接细胞外基质与细胞内信号传导的纽带。整合素由α亚单位和β亚单位通过非共价键连接成异二聚体，目前已发现18种α亚基和8种β亚基，可组合成至少24种整合素二聚体。细胞粘附分子整合素家族是细胞外基质与炎症细胞、成纤维细胞等实质细胞之间的主要连接物质，与细胞的粘附、铺展、迁移、功能表达以及组织纤维化的发生、维持和发展密切相关，可以调控细胞和细胞之间以及细胞和细胞外基质之间的识别和粘附等相互作用。
本发明的第一目的在于提供一种重组胶原蛋白，所述重组胶原蛋白的氨基酸序列中，包括N个基本重复单元，所述基本重复单元中含有n1个如下特征氨基酸序列：“G-Xaa1-Xaa2-G-E-Xaa3”；基本重复单元的3’端和5’端连接形成上述特征氨基酸序列；
其中，N为4以上的整数；n1为3以上的整数。
进一步地，所以N值为4～300以上的整数，进一步为4～200以上的整数。
所述N值的取值能够使重组胶原蛋白的分子量达到1kDa～250kDa，进一步达到3kDa～150kDa。
进一步地，特征氨基酸序列连续或间隔排列在基本重复单元中。
进一步地，所述重组胶原蛋白的氨基酸序列呈现如下特征：
[-G-E-Xaa3-Yaa1-(G-Xaa1-Xaa2-G-E-Xaa3)n2-Yaa2-(G-Xaa1-Xaa2-G-E-Xaa3)n3-Yaa3-(G-Xaa1-Xaa2-G-E-Xaa3)n3-Yaa4-(G-Xaa1-Xaa2-G-E-Xaa3)n4-…………-Yaan-(G-Xaa1-Xaa2-G-E-Xaa3)n-Yaan+1-G-Xaa1-Xaa2-]N
其中，所述Xaa1为非极性疏水氨基酸；所述Xaa2为丝氨酸(S)、丙氨酸(A)、脯氨酸(P)、羟脯氨酸(O)中的一种；所述Xaa3为碱性氨基酸；
Yaa1，Yaa2，Yaa3，Yaa4，………，Yaan，Yaan+1每次出现分别独立的选自无、一个或多个不同或相同的氨基酸；
n2、n3、n4、……..、n为0以上的整数，两者不同时为0。
本发明所述非极性疏水氨基酸包括甘氨酸(G)、缬氨酸(V)、苯丙氨酸(F)、丙氨酸(A)、亮氨酸(L)、异亮氨酸(I)、甲硫氨酸(M)、脯氨酸(P)、羟脯氨酸(O)中的一种；进一步包括苯丙氨酸(F)、丙氨酸(A)、亮氨酸(L)、异亮氨酸(I)、甲硫氨酸(M)、脯氨酸(P)、羟脯氨酸(O)中的一种。
所述碱性氨基酸包括赖氨酸、精氨酸、组氨酸中的一种，进一步为赖氨酸或精氨酸。
进一步地，所述重组胶原蛋白序列不含蛋白标签。
在本发明的一实施例中，如前所述重组胶原蛋白的氨基酸序列如SEQID NO.1所示。
本发明的第二目的在于提供前述重组胶原蛋白的制备方法，采用基因重组技术，通过宿主细胞表达、提取、纯化获得重组胶原蛋白；所述重组胶原蛋白的氨基酸序列由多次重复的基本重复单元重复连接获得；
进一步地，所述重组胶原蛋白的氨基酸序列中，包括N个基本重复单元，所述基本重复单元中含有n1个如下特征氨基酸序列：“G-Xaa1-Xaa2-G-E-Xaa3”；基本重复单元的3’端和5’端连接形成上述特征氨基酸序列；
其中，N为4以上的整数；n1为3以上的整数；
更进一步地，特征氨基酸序列连续或间隔排列在基本重复单元中。
所以N值为4～300的整数，进一步为4～200的整数；包括但不限于，4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30.......200。
进一步地，所述N值的取值能够使重组胶原蛋白的分子量达到1kDa～200kDa，进一步为达到3kDa～150kDa。
N值的取值可以取自本发明限定的范围值两端的端点值，也可取自范围值里的任一整数，例如，N值可以为1、3、10、50、70、90、95、100、125、150等。
进一步地，所述重组胶原蛋白序列不含蛋白标签；
所述重组胶原蛋白的氨基酸序列呈现如下特征：[-G-E-Xaa3-Yaa1-(G-Xaa1-Xaa2-G-E-Xaa3)n2-Yaa2-(G-Xaa1-Xaa2-G-E-Xaa3)n3-Yaa3-(G-Xaa1-Xaa2-G-E-Xaa3)n3-Yaa4-(G-Xaa1-Xaa2-G-E-Xaa3)n4-…………-Yaan-(G-Xaa1-Xaa2-G-E-Xaa3)n-Yaan+1-G-Xaa1-Xaa2-]N
其中，所述Xaa1为非极性疏水氨基酸；所述Xaa2为丝氨酸(S)、丙氨酸(A)、脯氨酸(P)、羟脯氨酸(O)中的一种；所述Xaa3为碱性氨基酸；
Yaa1，Yaa2，Yaa3，Yaa4，………，Yaan，Yaan+1每次出现分别独立的选自无、一个或多个不同或相同的氨基酸；
n2、n3、n4、……..、n为0以上的整数，两者不同时为0。
本发明的第三目的在于提供前述重组胶原蛋白在制备用于调控细胞粘附、增殖、分化的产品中的用途。
本发明的第四目的在于提供前述重组胶原蛋白在制备软骨修复基质的产品中的用途。
本发明所述重组胶原蛋白前述用途中应用时，可以与高分子和/或其衍生物配合使用，所述高分子包括天然高分子、合成高分子；进一步地，所述天然高分子包括但不限于透明质酸、壳聚糖、海藻酸、纤维素等；进一步地，所述合成高分子包括但不限于PLA、PGA、PLCL、PVA。
本发明所述重组胶原蛋白在前述用途中应用时，使用浓度为0.01～100mg/mL，例如，可以是0.01mg/mL、0.05mg/mL、2mg/mL、4.5mg/mL、7mg/mL、10mg/mL、15mg/mL、20mg/mL、50mg/mL、80mg/mL、100mg/mL等，使用浓度与应用部位和场景有关。
本发明的第五目的在于提供一种软骨修复基质，包括高分子和/或其衍生物、前述的重组胶原蛋白。
进一步地，所述高分子包括天然高分子、合成高分子；更进一步地，所述天然高分子至少选自透明质酸、壳聚糖、海藻酸、纤维素中的任意一种；更进一步地，所述合成高分子至少选自PLA、PGA、PLCL、PVA中的任意一种。
本发明的第六目的在于提供前述软骨修复基质的制备方法，包括如下步骤：
(1)将透明质酸衍生物溶解在缓冲液中，加入EDC/sNHS溶液反应；
(2)将所述的重组胶原蛋白加入到步骤(1)中的反应体系中反应，对反应后得到的溶液进行透析、干燥，得到复合基质；
(3)将复合基质溶解后，加入光引发剂溶液，即得软骨修复基质；
人源化胶原蛋白由于分子量小，机械性能差，经改性之后也难以成胶，不能满足软骨组织工程的需求。本发明实施例中，首先用甲基丙烯酸酐对透明质酸进行适度的化学改性，引入不饱和碳碳双键，然后通过加入EDC/sNHS活化透明质酸中的羧基，再加入重组人源化胶原蛋白使得蛋白上的氨基与透明质酸上活化的羧基进行酰胺化反应使两者偶联，最终通过光交联制备得到的复合水凝胶不仅具有良好的机械性能以及生物相容性，而且光交联的方式使得操作更加简便，能够满足软骨组织工程的需求，同时也满足了临床上对不规则缺损的填充等。
本发明中所述EDC是指1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，NHS是指N-羟基琥珀酰亚胺。
进一步地，步骤(1)所述的高分子衍生物是采用甲基丙烯酸酐进行改性的透明质酸；
进一步地，改性后的透明质酸分子量为100～1500kDa，优选为300～600kDa，更优选为300kDa；
进一步地，步骤(1)所述的透明质酸改性接枝度为20～60％，优选为30～50％；
进一步地，步骤(1)中，将EDC、sNHS加入后，sNHS浓度为0.5～5mM，EDC浓度为0.5～5mM；更进一步地，sNHS浓度为1～3mM，EDC浓度为2～4mM。
本发明中，步骤(1)中，加入EDC/sNHS溶液后的混合溶液pH值为4～5，优选为4.75；
反应后的混合溶液pH值为7～8，反应时间为2～5h；优选pH值为7.4，反应时间为3h。
本发明中，步骤(2)中，重组胶原蛋白：高分子衍生物的质量比为0.5～30:2～10；进一步为0.5～20:6；更进一步为1～11:6；
进一步地，步骤(2)中的反应时间为5～15h，优选为8～12h；
进一步地，步骤(2)中，将反应后的溶液装入截留分子量为8000～14000的透析袋中，去离子水透析2～4天后，冷冻干燥，得到复合基质。
本发明采用的光引发剂至少为LAP、2-羟基-4'-(2-羟乙氧基)-2-甲基苯丙酮(I2959)中的一种，终浓度为质量体积比0.01-0.05％，优选为0.05％。
本发明中，步骤(3)中加入光引发剂后的复合基质浓度为2～30mg/ml，进一步为5～20mg/ml，更进一步为7～17mg/ml。
本发明所述软骨修复基质为一种包含所述重组胶原的产品，可以作为包括但不限于植入材料、组织工程支架材料、软组织填充材料、细胞或其它活性物质载体材料等医疗器械的组成部分。
本发明软骨修复基质在临床引用时可以加细胞后制备成软骨修复水凝胶，或者不加细胞，直接应用在软骨缺损部位。
本发明的第七目的在于提供一种软骨修复水凝胶的制备方法，将前述方法制备得到的软骨修复基质与软骨细胞或骨髓间充质干细胞复合后，注射至软骨损伤部位，光照后形成软骨修复水凝胶。
所述光照的波长根据所用的引发剂进行适应性选择。
本发明所述软骨修复水凝胶还包括生理学上可接受的载体材料。
这里的载体材料包括但不限于水溶性载体材料(如聚乙二醇、聚乙烯吡咯烷酮、有机酸等)、难溶性载体材料(如乙基纤维素、胆固醇硬脂酸酯等)、肠溶性载体材料(如醋酸纤维素酞酸酯和羧甲乙纤维素等)。
所述组合物还可以根据需要与其他功能性材料配合使用，比如具有抗菌抑菌作用的成分、抗衰老成分、抗凝血成分、抗氧化成分、生长因子等。
所述组合物可以按照制剂学或化妆品常规技术制备，包括将作为活性成分的本发明的重组胶原蛋白与载体混合，按照常规技术制成所需要的剂型。可以根据需要，将本发明的组合物配制成制成多种剂型，包括但不限于口服施用制剂、粘膜施用制剂、注射制剂、吸入制剂和外用制剂等。
本发明所述产品包括但不限于药物、食品、保健品或化妆品、日用品。
本说明书中，使用“可以”表示的含义包括了进行某种处理以及不进行某种处理两方面的含义。
在实际应用中，可以将本发明的重组胶原和上述组合物作为药物直接给予患者、或者与适宜的载体或赋形剂混合后给予患者。这里的载体材料包括但不限于水溶性载体材料(如聚乙二醇、聚乙烯吡咯烷酮、有机酸等)、难溶性载体材料(如乙基纤维素、胆固醇硬脂酸酯等)、肠溶性载体材料(如醋酸纤维素酞酸酯和羧甲乙纤维素等)。其中优选的是水溶性载体材料。使用这些材料可以制成多种剂型，包括但不限于片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、脂质体、透皮剂、口含片、栓剂、冻干粉针剂等。其中，栓剂可为阴道栓剂，也可以是阴道环，也可以是适于阴道应用的药膏、乳霜或凝胶。
所述剂型可以是普通制剂、缓释制剂、控释制剂及各种微粒给药系统。为了将单位给药剂型制成片剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如淀粉、糊精、硫酸钙、乳糖、甘露醇、蔗糖、氯化钠、葡萄糖、尿素、碳酸钙、白陶土、微晶纤维素、硅酸铝等；湿润剂与粘合剂，如水、甘油、聚乙二醇、乙醇、丙醇、淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、紫胶、甲基纤维素、磷酸钾、聚乙烯吡咯烷酮等；崩解剂，例如干燥淀粉、海藻酸盐、琼脂粉、褐藻淀粉、碳酸氢钠与枸橼酸、碳酸钙、聚氧乙烯、山梨糖醇脂肪酸酯、十二烷基磺酸钠、甲基纤维素、乙基纤维素等；崩解抑制剂，例如蔗糖、三硬脂酸甘油酯、可可脂、氢化油等；吸收促进剂，例如季铵盐、十二烷基硫酸钠等；润滑剂，例如滑石粉、二氧化硅、玉米淀粉、硬脂酸盐、硼酸、液体石蜡、聚乙二醇等。还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。为了将单位给药剂型制成丸剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如葡萄糖、乳糖、淀粉、可可脂、氢化植物油、聚乙烯吡咯烷酮、Gelucire、高岭土、滑石粉等；粘合剂如阿拉伯胶、黄蓍胶、明胶、乙醇、蜂蜜、液糖、米糊或面糊等；崩解剂，如琼脂粉、干燥淀粉、海藻酸盐、十二烷基磺酸钠、甲基纤维素、乙基纤维素等。为了将单位给药剂型制成栓剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如聚乙二醇、卵磷脂、可可脂、高级醇、高级醇的酯、明胶、半合成甘油酯等。为了将单位给药剂型制成注射用制剂，如溶液剂、乳剂、冻干粉针剂和混悬剂，可以使用本领域常用的所有稀释剂，例如，水、乙醇、聚乙二醇、1,3-丙二醇、乙氧基化的异硬脂醇、多氧化的异硬脂醇、聚氧乙烯山梨醇脂肪酸酯等。另外，为了制备等渗注射液，可以向注射用制剂中添加适量的氯化钠、葡萄糖或甘油，此外，还可以添加常规的助溶剂、缓冲剂、pH调节剂等。此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂、甜味剂或其它材料。
使用上述剂型可以经注射给药，包括皮下注射、静脉注射、肌肉注射和腹腔注射、脑池内注射或灌输等；腔道给药，如经直肠、阴道和舌下；呼吸道给药，如经鼻腔；粘膜给药。
上述给药途径优选的是注射给药，优选的注射途径是皮下注射。
本发明的重组胶原和上述组合物的给药剂量取决于许多因素，例如所要预防或治疗疾病的性质和严重程度，患者或动物的性别、年龄、体重及个体反应，所用的具体活性成分，给药途径及给药次数等。上述剂量可以单一剂量形式或分成几个，例如二、三或四个剂量形式给药。对于任何具体的患者，具体的治疗有效剂量水平须根据多种因素而定，所述因素包括所治疗的障碍和该障碍的严重程度；所采用的具体活性成分的活性；所采用的具体组合物；患者的年龄、体重、一般健康状况、性别和饮食；所采用的具体活性成分的给药时间、给药途径和排泄率；治疗持续时间；与所采用的具体活性成分组合使用或同时使用的药物；及医疗领域公知的类似因素。
例如，本领域的做法是，活性成分的剂量从低于为得到所需治疗效果而要求的水平开始，逐渐增加剂量，直到得到所需的效果。
 有益效果：
(1)本发明提供的重组胶原蛋白具有较为明显的整合素结合活性，具有促进细胞粘附、增殖、分化，修复软骨组织缺损的效果，具有较好的应用前景。
(2)本发明将透明质酸与人源化胶原蛋白共价交联形成稳定形态复合水凝胶基质具有良好的机械性能，能够很好的维持复合物的形态；具有良好的生物相容性，而且光交联的方式使得操作更加简便，能够满足软骨组织工程的需求，同时也满足了临床上对不规则缺损的填充等。
 附图说明
图1为成纤维细胞细胞粘附生长结果；
图2为成纤维细胞细胞粘附的OD值；
图3为软骨细胞粘附生长结果；
图4为软骨细胞粘附的OD值；
图5为不同浓度序列A和序列B对人牙龈成纤维细胞的粘附实验结果；
图6为软骨细胞/水凝胶复合物在体外培养1、7、14、21天后的形态图；
图7为软骨细胞/水凝胶复合物在体外培养1、7、14天后软骨细胞的粘附的OD值；
图8为软骨细胞/水凝胶复合物在体外培养1、7、14天后软骨细胞的CCK-8图；
图9为软骨细胞/水凝胶复合物的染色结果；
图10为软骨细胞/水凝胶复合物的二型胶原免疫组化染色结果；
图11为软骨细胞/水凝胶复合物的GAG定量检测结果。
 具体实施方式
下面结合附图和具体实施例对本发明进行详细说明。本实施例以本发明技术方案为前提进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。
在整个说明书中，除非另有特别说明，本文使用的术语应理解为如本领域中通常所使用的含义。因此，除非另有定义，本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾，本说明书优先。
实施例1
编码所述重组胶原蛋白的核苷酸序列，构建包含该核苷酸序列的载体，构建并筛选包含该载体的宿主细胞(可以是原核细胞或真核细胞)，在适当条件下培养宿主细胞表达蛋白，收集并纯化表达的重组胶原蛋白。
如本发明所述，术语“载体”，是可将多核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白质获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。载体可以含有多种控制表达的元件，包括但不限于启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体可以包含本发明的核酸以便于导入细胞进行表达。载体可以包含与所述核酸可操作连接的表达控制元件，如启动子、终止子和/或增强子。
本发明中涉及的基因重组及转录是指合成编码重组胶原的核苷酸序列，通过常规技术将核苷酸序列克隆到表达载体中，然后将表达载体转化到宿主细胞中，构建并筛选获得工程菌株。转化方法包括但不限于电穿孔、CaCl2转化等；表达载体可以是pET26、pET32、pGEX-6p、pPIC9、pPIC9K等常用载体，可以是质粒、噬菌体、病毒或其它载体。
本发明所述，术语“宿主细胞”，是已经通过分子生物学技术将核酸分子引入的细胞。这些技术包括转染病毒载体，用质粒载体转化，以及通过电穿孔、脂转染、和粒子枪加速引入裸DNA。宿主细胞可以是真核细胞或原核细胞。例如，肠杆菌科细胞(例如大肠杆菌BL21、DH5α等)等微生物细胞、酵母(例如毕赤酵母、酿酒酵母等)或CHO细胞等真核细胞。
本发明中涉及的发酵培养是指在无菌发酵罐中，将筛选并经过蛋白高表达鉴定的工程菌株与适宜的发酵培养液在适宜的温度、压力和pH条件下进行诱导发酵培养。
本发明中涉及的蛋白的分离与纯化是指菌体经溶菌、均质、分离等处理后获得菌液，进一步通过常规的蛋白分离和纯化技术获得重组胶原蛋白。分离和纯化技术包括但不限于过滤、离心、盐析、透析、液相层析、离子交换层析、亲和层析等。
在本发明实施例中，基因重组及转录以大肠杆菌为宿主细胞为例，具体为：
1、大肠杆菌发酵制备重组胶原蛋白
基因重组与转录：利用PCR的方法将DNA片段进行密码子优化和拼接重组，选择pET-32a构建得到表达载体后转入大肠杆菌表达菌株BL21中，经培养、筛选后获得具有蛋白高表达的大肠杆菌基因工程菌。
发酵培养：从LB平板中挑取优选后的大肠杆菌基因工程菌单菌落，置于装有10mL LB培养基的100mL三角瓶中，于37℃、220rpm培养12-16h；将菌液按照1:100的比例接种到装有LB培养基的发酵罐中放大培养，于37℃、220rpm培养3h至OD600约为0.6时，加入0.5mM IPTG，在16℃诱导培养20h后离心收集菌体。
蛋白的分离与纯化：用Tris缓冲液重选菌体后，高速搅拌使菌体完全溶解，离心收集上清液并降温至4℃；上清液采用1μm、0.45μm和0.22μm的滤膜依次过滤后进一步采用亲和层析纯化得到重组胶原蛋白。
2、细胞培养方法
1)试样制备：重组胶原蛋白用PBS配制成一定浓度溶液(如0.5mg/ml)，动物源胶原蛋白溶液用PBS稀释至相同浓度(如0.5mg/ml)，PBS为空白对照组；
2)铺板：将实验溶液分别加入到96孔板中，每孔加入100μL，每组5个复孔，在4℃条件下静置过夜；
3)封闭：待铺板结束后，移除孔板中的液体后用200μL PBS溶液清洗2次，加入100μL经热灭活的1％BSA-PBS溶液，在37℃、5％CO2的培养箱中孵育1h；
4)细胞接种：待孵育结束后，移除孔板中的液体后用200μL PBS溶液清洗2次，每孔加入100μL密度为5×104-1×106个/ml的细胞悬液，在37℃、5％CO2的培养箱中孵育1h；
5)检测：待孵育结束后，移除孔板中的液体后用200μL PBS溶液清洗2次，加入150μL CCK-8，在37℃、5％CO2的培养箱中孵育1h后，从中吸取100μL检测液加入到新的96孔板中于450nm检测吸光度值(OD)。
试验例1：
采用实施例1的方法，制备下列3种含有不同氨基酸序列的重组胶原蛋白。
具有上述多个特征氨基酸序列组合的重复单元直接重复连接16次获得的重组胶原蛋白，提高细胞的粘附、迁移、功能表达等系列实验。重复连接序列A16次，序列A：GER GAP GFR GPA GPN GIP GEK GPA GER GAP，其全部序列如下(SEQID NO.1)：
另一种具有不同上述特征氨基酸序列的重复单元直接重复4次获得的重组胶原蛋白，对细胞的粘附、增殖作用有所差异。重复连接序列B 4次，序列B：GEK GSP GAD GPA GAP GTP GPQ GIA GQR GVV GLP GQR GER GFP GLP GPS GEP GKQ GPS GAS，其全部序列如下(SEQID NO.2)：
上述重复单元重复连接16次的重组胶原蛋白加入标签后，对细胞的粘附、增殖作用明显降低；重复连接序列C16次，序列C：HHHHHH GER GAP GFR GPA GPN GIP GEK GPA GER GAP，其全部序列如下(SEQID NO.3)：
采用实施例1中细胞培养方法对上述3种胶原蛋白进行成纤维细胞粘附、软骨细胞粘附实验、人牙龈成纤维细胞促粘附实验，
实验结果如图1～3。
图1结果表明，具有较多特征氨基酸序列组合的序列A重复连接得到的重组胶原实验组中有十分明显的成纤维细胞粘附且细胞形态良好；特征氨基酸序列减少后的序列B重复连接得到的重组胶原实验组和动物胶原组具有类似的成纤维细胞粘附与细胞形态；而添加标签后序列C重复连接得到的重组胶原组的成纤维细胞粘附明显减少，空白对照组的成纤维细胞粘附很少。
图2结果表明，具有较多特征氨基酸序列组合的序列A重复连接得到的重组胶原实验组最能促进成纤维细胞的粘附，其次是动物胶原和序列B重复连接得到的重组胶原，该三组中成纤维细胞的粘附量均显著高于序列C重复连接得到的重组胶原和空白对照；序列C重组胶原组中成纤维细胞的粘附量稍高于空白对照。
图3结果表明，动物胶原组中软骨细胞的粘附最明显，几乎铺满了整个视野；其次是序列A重组胶原组，软骨细胞粘附明显且相对较为集中，细胞形态良好；序列B重组胶原组中粘附的软骨细胞较为分散，仅有少部分集中，细胞形态良好；序列C重组胶原组中粘附的软骨细胞分散分布且数量较少；空白对照组中仅有少量的软骨细胞粘附且分散分布。
图4结果表明，动物胶原最有利于促进软骨细胞的粘附，显著高于其它各组；具有较多特征氨基酸序列组合的序列A重组胶原组显著高于序列B重组胶原组、序列C重组胶原组和空白对照组；序列B重组胶原组显著高于序列C重组胶原组和空白对照组；序列C重组胶原组稍高于空白对照组，但无统计学差异。
图5人牙龈成纤维细胞促粘附实验结果表明，具有较多特征氨基酸序列组合的序列A重组胶原组在不同浓度情况下均比序列B更有利于人牙龈成纤维细胞的粘附；随着重组胶原浓度的提高，人牙龈成纤维细胞的粘附也逐渐增加。
实施例2重组胶原蛋白在促进软骨修复中的应用
[根据细则91更正 13.12.2023]
采用实施例1的方法，制备符合本发明氨基酸序列特征的重组胶原蛋白，其中重组人源化胶原蛋白的氨基酸序列核心序列优选为GERGAPGFRGPAGPNGIPGEKGPAGERGAP(序列A)，重复连接16次，将其用于促进软骨修复。具体步骤为：
(1)将分子量为300KDa，改性程度为37％的甲基丙烯酸化透明质酸(HA-MA)，溶解在MES缓冲溶液中，得到1mM的甲基丙烯酸化的透明质酸溶液；
(2)将固体形态的sNHS和固体形态的EDC依次加入到混合溶液中，使得sNHS浓度为2mM，EDC浓度为3mM，并用1.0M NaOH溶液或1.0M HCl溶液将混合溶液的pH维持在4.75；
(3)室温下搅拌反应2h；
(4)用1.0M NaOH溶液调节反应体系pH为7.4；
(5)将重组人源化胶原蛋白加入到反应体系中，使得混合溶液中重组人源化胶原蛋白与甲基丙烯酸化的透明质酸的质量比为1:6，搅拌反应8h；
(6)将反应后的溶液装入截留分子量为8000-14000的透析袋中，去离子水透析三天后，用冷冻干燥机冻干，得到冻干海绵HA-rhCol Ⅲ；
(7)取步骤(6)中的冻干海绵溶解在0.01M PBS中，并加入LAP光引发剂溶液(终浓度为质量体积比0.05％)，使得HA-rhCol Ⅲ溶液终浓度为7mg/ml；
(8)取步骤(7)中前驱液置于规格为Ф6mm×2.5mm的硅胶模具中，在紫外灯光照下光照1min即得含重组人源化胶原的透明质酸水凝胶(HA-rhCol III)。
实施例3重组胶原蛋白在促进软骨修复中的应用
[根据细则91更正 13.12.2023]
采用实施例1的方法，制备符合本发明氨基酸序列特征的重组胶原蛋白，其中重组人源化胶原蛋白的氨基酸序列核心序列优选为GERGAPGFRGPAGPNGIPGEKGPAGERGAP(序列A)，重复连接16次，将其用于促进软骨修复。具体步骤为：
(1)将分子量为100KDa，改性程度为50％的甲基丙烯酸化的透明质酸HA-MA，溶解在MES缓冲溶液中，得到2mM的甲基丙烯酸化的透明质酸溶液；
(2)将固体形态的sNHS和固体形态的EDC依次加入到混合溶液中，使得sNHS浓度为3mM，EDC浓度为5mM，并用0.5M NaOH溶液或0.5M HCl溶液将混合溶液的pH维持在4.75；
(3)室温下搅拌反应2h；
(4)用1.0M NaOH溶液调节反应体系pH为7.4；
(5)将重组人源化胶原蛋白加入到反应体系中，使得混合溶液中重组人源化胶原蛋白与甲基丙烯酸化的透明质酸的质量比为11:6，搅拌反应12h；
(6)将反应后的溶液装入截留分子量为8000-14000的透析袋中，去离子水透析三天后，用冷冻干燥机冻干，得到产物HA-rhCol Ⅲ；
(7)取步骤(6)中的冻干海绵溶解在0.01M PBS中，并加入LAP光引发剂溶液(终浓度为质量体积比0.01％)，使得HA-rhCol Ⅲ溶液终浓度为17mg/ml；
(8)取步骤(7)中前驱液置于规格为Ф6mm×2.5mm的硅胶模具中，在紫外灯光照下光照1min即得HA-rhCol Ⅲ水凝胶。
实施例4重组胶原蛋白在促进软骨修复中的应用
[根据细则91更正 13.12.2023]
采用实施例1的方法，制备符合本发明氨基酸序列特征的重组胶原蛋白，其中重组人源化胶原蛋白的氨基酸序列核心序列优选为GERGAPGFRGPAGPNGIPGEKGPAGERGAP(序列A)，重复连接16次(SEQID NO.1)，将其用于促进软骨修复。具体步骤为：
(1)将分子量为600KDa，改性程度为30％的甲基丙烯酸化的透明质酸HA-MA，溶解在MES缓冲溶液中，得到1mM的甲基丙烯酸化的透明质酸溶液；
(2)将sNHS和EDC依次加入到混合溶液中，使得sNHS浓度为1mM，EDC浓度为3mM，并用1.0M NaOH溶液或1.0M HCl溶液将混合溶液的pH维持在4.75；
(3)室温下搅拌反应3h；
(4)用1.0M NaOH溶液调节反应体系pH为7.4；
(5)将重组人源化胶原蛋白加入到反应体系中，使得混合溶液中重组人源化胶原蛋白与甲基丙烯酸化的透明质酸的质量比为0.5:6，搅拌反应8h；
(6)将反应后的溶液装入截留分子量为8000-14000的透析袋中，去离子水透析三天后，用冷冻干燥机冻干，得到产物HA-rhCol Ⅲ；
(7)取步骤(6)中的冻干海绵溶解在0.01M PBS中，并加入LAP光引发剂溶液(终浓度为质量体积比0.05％)，使得HA-rhCol Ⅲ溶液终浓度为7mg/ml；
(8)取步骤(7)中前驱液置于规格为Ф6mm×2.5mm的硅胶模具中，在紫外灯光照下光照2min即得HA-rhCol Ⅲ水凝胶。
实施例5重组胶原蛋白在促进软骨修复中的应用
[根据细则91更正 13.12.2023]
采用实施例1的方法，制备符合本发明氨基酸序列特征的重组胶原蛋白，其中重组人源化胶原蛋白的氨基酸序列核心序列优选为GERGAPGFRGPAGPNGIPGEKGPAGERGAP，重复连接16次(SEQID NO.1)，将其用于促进软骨修复。具体步骤为：
(1)将分子量为1000KDa，改性程度为40％的甲基丙烯酸化的透明质酸HA-MA，溶解在MES缓冲溶液中，得到0.6mM的甲基丙烯酸化的透明质酸溶液；
(2)将sNHS和EDC依次加入到混合溶液中，使得sNHS浓度为5mM，EDC浓度为5mM，并用0.2M NaOH溶液或0.2M HCl溶液将混合溶液的pH维持在4.75；
(3)室温下搅拌反应3h；
(4)用0.5M NaOH溶液调节反应体系pH为7.4；
(5)将重组人源化胶原蛋白加入到反应体系中，使得混合溶液中重组人源化胶原蛋白与甲基丙烯酸化的透明质酸的质量比为11:6，搅拌反应12h；
(6)将反应后的溶液装入截留分子量为8000-14000的透析袋中，去离子水透析三天后，用冷冻干燥机冻干，得到产物HA-rhCol Ⅲ；
(7)取步骤(6)中的冻干海绵溶解在0.01M PBS中，并加入I2959光引发剂溶液(终浓度为质量体积比0.03％)，使得HA-rhCol Ⅲ溶液终浓度为17mg/ml；
(8)取步骤(7)中前驱液置于规格为Ф6mm×2.5mm的硅胶模具中，在紫外灯光照下光照1min即得HA-rhCol Ⅲ水凝胶。
试验例2：
实施例2制备的重组人源化胶原蛋白修饰的透明质酸水凝胶，在细胞实验中结果如下：
结合图6和图7结果，HA-MA水凝胶中软骨细胞培养至21天，由于缺乏粘附位点，软骨细胞原位聚集成团生长。而HA-rhCol Ⅲ组水凝胶中软骨细胞在培养至7天时，呈现部分铺展状态，在14和21天时，细胞铺展生长现象更加明显，且分布均匀。说明rhCol Ⅲ有利于细胞的粘附，迁移和增殖，且HA-rhColⅢ组水凝胶较HA-MA组水凝胶更能促进软骨细胞的增殖。
图8结果显示，HA-rhCol Ⅲ组水凝胶中细胞核并未出现变形、固缩等现象，且在培养至7和14天时，两组水凝胶中细胞的肌动蛋白呈梭形，说明细胞在凝胶内部得到较好的铺展，而HA-MA组在各时间点，细胞均以细胞团形态原位增殖。该结果表明rhCol Ⅲ为细胞粘附提供了适宜的位点，可以让软骨细胞更快的粘附并为细胞增殖提供条件。
组织学和二型胶原免疫组化染色(图9-10)结果表明，HA-rhCol Ⅲ凝胶中有更明显的多糖和蛋白易染区域，表明rhCol III的加入可以促进软骨细胞的功能表达。GAG定量检测结果(图11)进一步证实，rhCol Ⅲ的加入更有利于促进软骨细胞外基质的分泌。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。

Claims (19)

  1. 一种重组胶原蛋白，其特征在于，所述重组胶原蛋白的氨基酸序列中，包括N个基本重复单元，所述基本重复单元中含有n1个如下特征氨基酸序列：“G-Xaa1-Xaa2-G-E-Xaa3”；基本重复单元的3’端和5’端连接形成上述特征氨基酸序列；
    其中，N为4以上的整数；n1为3以上的整数。
  2. 根据权利要求1所述的重组胶原蛋白，其特征在于，N为4～300的整数。
  3. 根据权利要求1所述的重组胶原蛋白，其特征在于，N为4～200的整数。
  4. 根据权利要求1所述的重组胶原蛋白，其特征在于，特征氨基酸序列连续或间隔排列在基本重复单元中。
  5. 根据权利要求1所述的重组胶原蛋白，其特征在于，所述重组胶原蛋白的氨基酸序列呈现如下特征：
    [-G-E-Xaa3-Yaa1-(G-Xaa1-Xaa2-G-E-Xaa3)n2-Yaa2-(G-Xaa1-Xaa2-G-E-Xaa3)n3-
    Yaa3-(G-Xaa1-Xaa2-G-E-Xaa3)n3-Yaa4-(G-Xaa1-Xaa2-G-E-Xaa3)n4-…………-Yaan-(G-Xaa1-Xaa2-G-E-Xaa3)n-Yaan+1-G-Xaa1-Xaa2-]N
    其中，所述Xaa1为非极性疏水氨基酸；所述Xaa2为丝氨酸(S)、丙氨酸(A)、脯氨酸(P)、羟脯氨酸(O)中的一种；所述Xaa3为碱性氨基酸；
    Yaa1，Yaa2，Yaa3，Yaa4，………，Yaan，Yaan+1每次出现分别独立的选自无、一个或多个不同或相同的氨基酸；
    n2、n3、n4、……..、n为0以上的整数，两者不同时为0。
  6. 如权利要求1所述的重组胶原蛋白，其特征在于，所述重组胶原蛋白序列不含蛋白标签。
  7. 如权利要求1～6任一项所述的重组胶原蛋白，其特征在于，所述重组胶原蛋白的氨基酸序列如SEQID NO.1所示。
  8. 权利要求1～7任一项所述的重组胶原蛋白的制备方法，其特征在于，采用基因重组技术，通过宿主细胞表达、提取、纯化获得重组胶原蛋白；所述重组胶原蛋白的氨基酸序列由多次重复的基本重复单元重复连接获得。
  9. 如权利要求8所述的重组胶原蛋白的制备方法，其特征在于，所述重组胶原蛋白的氨基酸序列中，包括N个基本重复单元，所述基本重复单元中含有n1个如下特征氨基酸序列：“G-Xaa1-Xaa2-G-E-Xaa3”；基本重复单元的3’端和5’端连接形成上述特征氨基酸序列；
    其中，N为4以上的整数；n1为3以上的整数。
  10. 如权利要求9所述的重组胶原蛋白的制备方法，其特征在于，特征氨基酸序列连续或间隔排列在基本重复单元中。
  11. 如权利要求8所述的重组胶原蛋白的制备方法，其特征在于，所述重组胶原蛋白序列不含蛋白标签；
    所述重组胶原蛋白的氨基酸序列呈现如下特征：
    [-G-E-Xaa3-Yaa1-(G-Xaa1-Xaa2-G-E-Xaa3)n2-Yaa2-(G-Xaa1-Xaa2-G-E-Xaa3)n3-
    Yaa3-(G-Xaa1-Xaa2-G-E-Xaa3)n3-Yaa4-(G-Xaa1-Xaa2-G-E-Xaa3)n4-…………-Yaan-(G-Xaa1-Xaa2-G-E-Xaa3)n-Yaan+1-G-Xaa1-Xaa2-]N
    其中，所述Xaa1为非极性疏水氨基酸；所述Xaa2为丝氨酸(S)、丙氨酸(A)、脯氨酸(P)、羟脯氨酸(O)中的一种；所述Xaa3为碱性氨基酸；
    Yaa1，Yaa2，Yaa3，Yaa4，………，Yaan，Yaan+1每次出现分别独立的选自无、一个或多个不同或相同的氨基酸；
    n2、n3、n4、……..、n为0以上的整数，两者不同时为0。
  12. 权利要求1～7任一项所述的重组胶原蛋白在制备用于调控细胞粘附、增殖、分化的产品中的用途。
  13. 权利要求1～7任一项所述的重组胶原蛋白在制备软骨修复基质的产品中的用途。
  14. 一种软骨修复基质，其特征在于，包括高分子和/或其衍生物、权利要求1～7任一项所述的重组胶原蛋白；所述高分子包括天然高分子、合成高分子。
  15. 如权利要求14所述的软骨修复基质，其特征在于，所述天然高分子至少选自透明质酸、壳聚糖、海藻酸、纤维素中的任意一种；所述合成高分子至少选自PLA、PGA、PLCL、PVA中的任意一种。
  16. 权利要求15所述的软骨修复基质的制备方法，其特征在于，包括如下步骤：
    (1)将高分子衍生物溶解在缓冲液中，加入EDC/sNHS溶液反应；
    (2)将权利要求1～7任一项所述的重组胶原蛋白加入到步骤(1)中的反应体系中反应，对反应后得到的溶液进行透析、干燥，得到复合基质；
    (3)将复合基质溶解后，加入光引发剂溶液，即得软骨修复基质。
  17. 如权利要求16所述的软骨修复基质的制备方法，其特征在于，步骤(1)所述的高分子衍生物是采用甲基丙烯酸酐进行改性的透明质酸；改性后的透明质酸分子量为100～1500kDa；所述的透明质酸改性接枝度为20～60％；加入EDC/sNHS溶液后的混合溶液pH值为4～5；将EDC、sNHS加入后，sNHS浓度为0.5～5mM，EDC浓度为0.5～5mM；反应后的混合溶液pH值为7～8，反应时间为2～5h；
    步骤(2)中，重组胶原蛋白：高分子衍生物的质量比为0.5～30:2～10；反应时间为5～15h；将反应后的溶液装入截留分子量为8000～14000的透析袋中，去离子水透析2～4天后，冷冻干燥，得到复合基质；
    步骤(3)中加入光引发剂后的复合基质浓度为2～30mg/ml。
  18. 如权利要求17所述的软骨修复基质的制备方法，其特征在于，步骤(1)中，改性后的透明质酸分子量为300～600kDa；透明质酸改性接枝度为30～50％；
    步骤(2)中，重组胶原蛋白：高分子衍生物的质量比为0.5～20:6，反应时间为8～12h；
    步骤(3)中加入光引发剂后的复合基质浓度为5～20mg/ml。
  19. 一种软骨修复水凝胶的制备方法，其特征在于，将权利要求16～18任一项所述方法制备得到的软骨修复基质与软骨细胞或骨髓间充质干细胞复合后，注射至软骨损伤部位，光照后形成软骨修复水凝胶。
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