Protein composition as well as preparation method and application thereof

Abstract

The present invention discloses a novel protein composition comprising a first protein or polypeptide selected from a specific group. The protein composition has a broad-spectrum and efficient treatment effect on inflammatory diseases. The invention also discloses a preparation method of the protein composition and application of the protein composition in preparation of medical implants, medical devices or drugs.

Description

Technical Field

[0001] The present application belongs to the biological field, and specifically relates to a protein composition and a preparation method thereof. The present application also relates to the application of the protein composition in the preparation of medical implants, medical devices or medicines. Background Art

[0002] Generally speaking, inflammation is a normal defensive reaction of the body to remove pathogens or damaged cells in the body to help repair tissues. During the inflammatory response, various tissue cells and immune cells in the local vascular fluid play a role in rapidly destroying and eliminating foreign invading bacteria and damaged cells, and isolating infected or injured tissues from other parts of the body. However, when inflammation becomes uncontrolled, inflammatory diseases occur. Inflammatory diseases increase and persist inflammation through the secretion of a variety of inflammatory mediators by target cells activated by external stimuli, thereby destroying healthy tissues and threatening human life.

[0003] Immune system disorders often lead to abnormal inflammation. When the immune system mistakenly triggers an inflammatory response in the absence of infection, many inflammatory diseases will occur. Immune-mediated inflammatory diseases have become the leading cause of death from disease worldwide. Although the clinical symptoms of such diseases seem different, they have many similarities in genetic background and pathophysiological pathways. Regardless of the complexity of their etiology, the pathogenesis of the disease mostly involves inflammation and immunity. For example, inflammatory skin disease is a common skin disease mediated by immune cells and keratinocytes. Abnormal reactions of the innate immune system, abnormal activation of T lymphocytes, and various inflammatory cytokines and their target cells (keratinocytes) play an important role in the pathogenesis of inflammatory skin diseases. The immune balance between cell subsets is disturbed, causing abnormal secretion of inflammatory cytokines such as interleukins, interferons, and tumor necrosis factors. These inflammatory cytokines further act on keratinocytes to induce damage to the skin barrier, leading to inflammatory skin diseases.

[0004] Immunosuppression or immunomodulation is currently the main treatment for inflammatory diseases. However, almost all immunosuppressants have obvious and serious side effects. For example, widely used oral immunosuppressants such as cyclosporine A, methotrexate and azathioprine, used for severe inflammatory skin diseases, may lead to toxic blood levels and organ damage, especially in the liver and kidneys. In addition, the rapid development of biomedicine in recent years has provided more and more optional monoclonal antibodies, immunoglobulins and biological agents that specifically target immune molecules. Although the efficacy is certain, its cost is high, systemic adverse reactions cannot be ignored, and the safety of long-term use is unclear. Although some inflammatory diseases involve multi-system systemic diseases, most inflammatory diseases manifest as local diseases, such as allergic rhinitis and rheumatoid arthritis. For such local inflammatory diseases, systemic administration such as intravenous and oral administration is used clinically for treatment, which often brings various drug side effects.

[0005] In addition, with the development of biomaterials and cell therapy, hematopoietic stem cell transplantation, autologous somatic cells or stem cell therapy, collagen, etc. have been applied in clinical practice. It is worth mentioning that the extracellular matrix (ECM) is a fibrous gel mesh structure secreted by cells into the extracellular space. The main components include collagen, fibronectin, elastin, polysaccharides, etc. See review Tara E. Sutherland et al., The extracellular matrix and the immune system: A mutually dependent relationship. Science 379, eabp8964 (2023). DOI: 10.1126 / science.abp8964. ECM has support, protection, and nutrition for cells, and is closely related to physiological activities such as cell metabolism, proliferation, and tissue trauma repair. However, although ECM accounts for at least one-third of the tissue structure, due to its complex and constantly changing structure, its role in immunology has not been fully studied, and there has been no report on the systematic use of ECM for the treatment of immune-mediated inflammatory diseases.

[0006] Therefore, there is an urgent need in this field to develop a new therapy that is more broad-spectrum and highly effective for inflammatory diseases, especially local inflammatory diseases.

[0007] In addition, the advantage of the present invention is that it provides a protein composition, which has a wide range of therapeutic effects not only on inflammatory diseases, but also on other related diseases, including but not limited to mycoplasma infection diseases, skin and mucous membrane surface viral infection diseases, functional lubricating fluid deficiency diseases, hypertrophic scars, and alopecia diseases.

[0008] Another advantage of the present invention is to provide an efficient, large-scale production process for producing protein compositions by mass expansion of fibroblasts. Brief description of the invention

[0009] In one aspect, the present invention provides a protein composition comprising the first protein or polypeptide, wherein the first protein or polypeptide is selected from the group consisting of: Type VI collagen α3 chain, Sushi, von Willebrand factor type A, EGF and valerin domain-containing protein 1, 72kDa collagenase type IV, Interstitial collagenase, and mixtures thereof, The concentration of the first protein or polypeptide in the composition is greater than 1 ug / mL.

[0010] In some embodiments, the protein composition further comprises a second protein or polypeptide different from the first protein or polypeptide, wherein the second protein or polypeptide is selected from the group consisting of: Sulfhydryl oxidase 1, Decorin, Fibronectin, Vimentin, Fibroin-1, Metalloproteinase inhibitor-1, Type I collagen αI chain, Type I collagen alpha II chain, and mixtures thereof.

[0011] In some embodiments, the total protein concentration is detected by BCA method, and the single protein concentration (such as the concentration of the first protein or polypeptide, the concentration of the second protein or polypeptide) is detected by liquid chromatography-mass spectrometry.

[0012] In another aspect, the present invention provides a method for preparing the protein composition, the method comprising: a) obtaining human fibroblasts from human skin tissue by using tissue blocks or digestion method; b) The human fibroblasts were cultured in vitro at 37°C and 3-10% CO 2 Under culture conditions, in vitro expansion was performed in the initial culture medium; c) obtaining a large amount of cell culture supernatant while expanding in vitro and collecting it; and d) filtering the collected cell culture supernatant using a 0.1-0.3 μm microporous filter membrane or filter element to obtain a sterile filtrate, The obtained sterile filtrate is the protein composition.

[0013] In some embodiments, the present invention provides a method for preparing a protein composition comprising the aforementioned first protein or polypeptide and the second protein or polypeptide, the method comprising: a) obtaining human fibroblasts from human skin tissue by using tissue blocks or digestion method; b) The human fibroblasts were cultured in vitro at 37°C and 3-10% CO 2 Under culture conditions, in vitro expansion is performed in an initial culture medium containing fetal bovine serum or a serum-free supplement; c) obtaining and collecting a large amount of cell culture supernatant during in vitro expansion; d) filtering the collected cell culture supernatant using a 0.1-0.3 μm microporous filter membrane or filter element to obtain a sterile filtrate; and e) The sterile filtrate obtained in step d) is ultrafiltered and concentrated through an ultrafiltration membrane to obtain an ultrafiltration concentrate, wherein: When the initial culture medium contains fetal bovine serum, the molecular weight of the ultrafiltration membrane is ≥80KD; when the initial culture medium contains serum-free additives, the molecular weight of the ultrafiltration membrane is ≥1KD. The obtained ultrafiltration concentrate is the protein composition, wherein the concentration of the second protein or polypeptide in the protein composition increases by more than 5 times relative to its concentration in the initial culture medium of the control group.

[0014] In another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of the aforementioned protein composition.

[0015] In another aspect, the present invention provides a medical device comprising an effective amount of the aforementioned protein composition.

[0016] In another aspect, the present invention provides use of the aforementioned protein composition for preparing a medical implant, a medical device or a medicine.

[0017] In another aspect, the present invention provides the aforementioned protein composition for preparing a medical device or medicine for treating an inflammatory disease. In another aspect, the present invention provides the aforementioned protein composition for treating an inflammatory disease. In another aspect, the present invention provides a method for treating an inflammatory disease using the aforementioned protein composition. The inflammatory disease is preferably a local inflammatory disease. In some embodiments, the inflammatory disease is selected from inflammatory skin diseases, autoimmune diseases, sterile inflammation, or a combination thereof.

[0018] In another aspect, the present invention provides the aforementioned protein composition for use in preparing a medical device or medicine for treating mycoplasma infection-related diseases. In another aspect, the present invention provides the aforementioned protein composition for use in treating mycoplasma infection-related diseases. In another aspect, the present invention provides a method for treating mycoplasma infection-related diseases using the aforementioned protein composition.

[0019] In another aspect, the present invention provides the aforementioned protein composition for preparing a medical device or medicine for treating viral infections of the skin and mucous membranes. In another aspect, the present invention provides the aforementioned protein composition for treating viral infections of the skin and mucous membranes. In another aspect, the present invention provides a method for treating viral infections of the skin and mucous membranes using the aforementioned protein composition.

[0020] In another aspect, the present invention provides the aforementioned protein composition for use in preparing a medical device or medicine for treating functional lubricating fluid deficiency diseases. In another aspect, the present invention provides the aforementioned protein composition for use in treating functional lubricating fluid deficiency diseases. In another aspect, the present invention provides a method for treating functional lubricating fluid deficiency diseases using the aforementioned protein composition.

[0021] In another aspect, the present invention provides the aforementioned protein composition for use in preparing a medical device or medicine for treating hypertrophic scars. In another aspect, the present invention provides the aforementioned protein composition for use in treating hypertrophic scars. In another aspect, the present invention provides a method for treating hypertrophic scars using the aforementioned protein composition.

[0022] In another aspect, the present invention provides the aforementioned protein composition for use in preparing a medical device or medicine for treating alopecia. In another aspect, the present invention provides the aforementioned protein composition for use in treating alopecia. In another aspect, the present invention provides a method for treating alopecia using the aforementioned protein composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Attached Figure 1 The following are photos of patients treated for senile arthritis according to an embodiment of the present invention: 1A-before the first treatment, 1B-two months after the treatment.

[0024] Attached Figure 2 2A-before the first treatment, 2B-two months after treatment.

[0025] Attached Figure 3 3A-before the first treatment, 3B-third month of treatment, 3C-fourth month of treatment, 3D-sixth month of treatment, and 3E-eleventh month of treatment.

[0026] Attached Figure 4 4A-before the first treatment, 4B-after two treatments.

[0027] Attached Figure 55A-before the first treatment, 5B-after 40 days of treatment.

[0028] Attached Figure 6 6A-right eye before first treatment, 6B-right eye two months after treatment, 6C-left eye before first treatment, and 6D-left eye two months after treatment.

[0029] Attached Figure 7 7A-before the first treatment, 7B-two months after treatment.

[0030] Attached Figure 8 8A-before the first treatment, 8B-two months after the treatment. DETAILED DESCRIPTION OF THE INVENTION

[0031] The protein composition of the present invention comprises a first protein or polypeptide selected from a specific group, and a certain concentration value must be reached. The applicant was surprised to find that after the protein composition of the present invention is applied to the human body, it has a broad spectrum and efficient therapeutic effect for inflammatory diseases. Not bound by theory, the applicant believes that the therapeutic effect of the protein composition of the present invention for inflammatory diseases and related diseases mainly lies in the role played by human immune cells, body fluids and / or cells in situ. Specifically, due to its specific components and protein concentration, after being applied to the human body, the protein composition of the present invention can promote the migration and chemotaxis of immune cells, thereby facilitating the performance and enhancement of cellular immune function. Generally speaking, whether cells migrate and the migration speed in the extracellular matrix depends on the level of integrin and its corresponding ligand and its interaction. Integrin is a receptor located on the surface of the cell membrane, and the protein composition of the present invention, in particular, the protein or polypeptide contained therein is equivalent to a ligand, and the combination and interaction of the two are important mechanisms for determining cell adhesion and migration. Therefore, after the present invention is applied to the human body, the anchoring and migration of immune cells are increased, which is beneficial to its performance, thereby enhancing cellular immune function. At the same time, the injection of the protein composition of the present invention increases the content of extracellular fluid and accelerates the circulation, which is beneficial to the enhancement of humoral immune function. It is worth mentioning that compared with the systemic administration of traditional immunotherapy, the protein composition of the present invention can be administered locally to induce the local aggregation of immune cells, thereby effectively treating local inflammatory diseases and reducing and avoiding side effects caused by systemic administration.

[0032] In addition, after being applied to the human body, the proteins or polypeptides contained in the present invention are absorbed by in situ cells, i.e., dermal fibroblasts, through collagen receptors (such as matrix metalloproteinase 14 (MT-MMP1) and mannose receptor type C-2 (MRC2)) in the body, and reassembled into collagen and other ECM components, thereby increasing local tissue elasticity, repairing skin damage, and facilitating hair growth and the production of functional lubricating fluid.

[0033] Based on the above mechanism, the applicant has found that the protein composition of the present invention can not only be used to treat inflammatory diseases, but also has a wide range of therapeutic effects on other related diseases, including but not limited to mycoplasma infection diseases, viral infection diseases on the skin and mucous membrane surfaces, functional lubricating fluid deficiency diseases, hypertrophic scars, and alopecia diseases. definition

[0034] The term "protein" as used in this application refers to a macromolecule composed of one or more long chains of amino acids, usually containing 50 or more amino acids. The term "polypeptide" refers to a linear amino acid chain, usually containing 2-50 amino acids. The protein or polypeptide in this application can be in vivo, naturally secreted, or artificially recombinant.

[0035] The term "collagen" as used in this application is the main structural protein in the extracellular matrix of various connective tissues in the human body. Amino acids combine to form a triple helix of long and thin fibers, called the collagen helix. Collagen is the most abundant protein in the human body and is mainly found in connective tissues such as cartilage, bone, tendons, ligaments and skin.

[0036] The term "fibroblast" used in this application is the most common cell in the connective tissue of animals, a cell type that synthesizes extracellular matrix and collagen and plays a key role in wound healing. Fibroblasts typically have a branched cytoplasm surrounding an oval, speckled nucleus with two or more nucleoli. The source of fibroblasts includes connective tissue such as skin.

[0037] The term "medium" used in this application refers to a substance that supplies cell nutrition and promotes cell reproduction and proliferation in cultured cells, and is also a living environment for the growth and reproduction of cultured cells. Culture medium includes basal culture medium and complete culture medium, and complete culture medium is prepared by adding serum of animal origin such as fetal bovine serum or serum-free additives (also known as "blood substitutes") to the basal culture medium. Basal culture medium is usually composed of amino acids, vitamins, carbohydrates, inorganic ions, etc., and additives such as fetal bovine serum or serum-free additives contain proteins required for cell growth and reproduction, and the two are mixed to prepare complete culture medium. The "initial culture medium" referred to in this application is actually the initial complete culture medium, that is, the complete culture medium before adding human fibroblasts. The "control group initial culture medium" referred to in this application refers to the same as the initial culture medium in the preferred preparation method of the present invention, and has undergone the same subsequent steps (including but not limited to culturing, refrigeration, filtration, concentration, bacteria / virus inactivation, etc.) The initial culture medium is used as the control group of the protein composition of the present invention.

[0038] The term "fibroblast conditioned medium" or "human fibroblast conditioned medium" used in this application refers to a large amount of cell culture supernatant produced during the process of in vitro culturing, amplification, and preparation of cell suspension of human fibroblasts, which contains proteins or polypeptides secreted by fibroblasts, that is, the culture supernatant produced by in vitro amplified human fibroblasts.

[0039] As used in this application, the term "medical implant" refers to a device or tissue placed in or on the body. Many implants are prostheses, which are used to replace missing body parts; other types of implants are used to deliver drugs, monitor body functions, or provide support for organs and tissues.

[0040] The term "medical device" as used in this application refers to an article, instrument, apparatus or machine used to prevent, diagnose or treat disease, or to detect, measure, restore, correct or modify body structure or function to achieve certain health purposes. Generally, the purpose of a medical device is not achieved by pharmacological, immunological or metabolic means.

[0041] The term "drug" used in this application refers to a composition or compound used to treat or prevent a disease, relieve symptoms, or help diagnose a disease, and the term "pharmaceutical composition" refers to a composition comprising an effective amount of one or more active pharmaceutical ingredients and a pharmaceutically acceptable excipient, carrier, or diluent. Generally, the therapeutic or preventive purpose of a drug is achieved by pharmacological, immunological, or metabolic means.

[0042] As used herein, the term "treatment" refers to any administration of a therapeutic agent by a therapeutic regimen that achieves the desired effect, i.e., partially or completely alleviates, improves, alleviates, inhibits, delays the onset of, reduces severity, and / or reduces the incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Such treatment may be directed to subjects who do not exhibit the associated disease, disorder, and / or condition and / or to subjects who only exhibit early signs of the disease, disorder, and / or condition, or to subjects who exhibit one or more determined signs of the associated disease, disorder, and / or condition. In some embodiments, treatment may be directed to subjects who have been diagnosed with the associated disease, disorder, and / or condition. In some embodiments, treatment may be directed to subjects known to have one or more susceptibility factors that are statistically associated with an increased risk of the development of the associated disease, disorder, and / or condition.

[0043] As used herein, the term "effective amount", "therapeutically effective amount" or "pharmaceutically effective amount" refers to the amount of a therapeutic agent that confers a therapeutic effect on the patient being treated at a reasonable benefit / risk ratio applicable to any drug treatment. Such a therapeutic effect can be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject gives an indication or feels the effect). In some embodiments, a "therapeutically effective amount" refers to an amount of a therapeutic agent or composition that effectively treats, improves or prevents (e.g., delays onset) a related disease or condition and / or exhibits a detectable therapeutic or preventive effect, such as by improving symptoms associated with the disease, preventing or delaying the onset of the disease, and / or also reducing the severity or frequency of disease symptoms.

[0044] As used herein, the term "mixing" refers to adding ingredients together and achieving homogeneity, and the term "mixture" refers to a homogeneous mixture of ingredients.

[0045] In the present application, when a composition is “substantially free” of a specific ingredient, it means that the composition contains less than a trace amount, or less than 0.1%, or less than 0.01%, or less than 0.001% of the specific ingredient by mass of the composition.

[0046] In this application, unless the order is particularly clear, the terms "a)", "b)" and "c)" used to refer to the steps in the preparation method do not indicate the order. For example, step a) may occur before step b) or after step b), or steps b) and c) may occur simultaneously, or step b) may occur after step c).

[0047] The terms "comprising", "including", "comprising", "including", "comprising", "including", "containing", "including" and "containing" used in this application are intended to be non-limiting, that is, other ingredients and other steps that do not affect the end result may be added. Protein composition

[0048] The protein composition of the present invention comprises a first protein or polypeptide, and the concentration of the first protein or polypeptide in the composition is greater than 1 ug / mL. The protein composition can be in any suitable form such as liquid, gel, etc., but is preferably a liquid.

[0049] As for its preferred preparation method (but not limited to this preparation method), the protein composition of the present invention is essentially a human fibroblast conditioned medium, which contains one or more proteins or polypeptides secreted during in vitro expansion of fibroblasts. As mentioned above, the applicant has found that a specific protein component (i.e., the first protein or polypeptide) and its concentration determine the therapeutic effect of the composed protein composition. Without being bound by theory, the applicant has found that when the solute concentration of the specific component reaches a higher level, the protein composition is more easily diffused through the intercellular space after being applied to the human body, so the clinical treatment effect is better.

[0050] In some embodiments, the total protein concentration of the protein composition of the present invention is greater than 5 mg / mL, preferably greater than 8 mg / mL, preferably greater than 10 mg / mL, preferably greater than 11 mg / mL, preferably greater than 12 mg / mL, preferably greater than 12.5 mg / mL, preferably detected according to the BCA method. When containing 2 or more proteins or polypeptides, the total protein concentration of the protein composition of the present invention refers to the sum of the protein concentrations of the proteins or polypeptides contained therein. The method for detecting protein concentration is shown below.

[0051] The protein composition of the present invention may include 1 or 2 or more proteins or polypeptides. In some embodiments, the protein composition of the present invention includes at least 2 proteins or polypeptides, preferably at least 3 proteins or polypeptides, preferably at least 4 proteins or polypeptides, preferably at least 5 proteins or polypeptides, preferably at least 8 proteins or polypeptides, preferably at least 10 proteins or polypeptides. Preferably, at least 1 of them is collagen, preferably at least 2 collagens, preferably at least 3 collagens, preferably at least 4 collagens, preferably at least 5 collagens, preferably at least 8 collagens, preferably at least 10 collagens. For the sake of clarification, the number of proteins or polypeptides referred to above includes the first protein or polypeptide.

[0052] In other embodiments, the protein composition of the present invention comprises at least 3 proteins or polypeptides and at least 1 of them is collagen, preferably comprises at least 4 proteins or polypeptides and at least 2 of them are collagen, preferably comprises at least 5 proteins or polypeptides and at least 3 of them are collagen. First protein or polypeptide

[0053] The protein composition of the present invention comprises a first protein or polypeptide, wherein the first protein or polypeptide is selected from the group consisting of: Collagen alpha-3 (VI) chain (COL6A3), Sushi, von Willebrand factor type A, EGF and pentraxin domain-containing protein 1 (SVEP1), 72kDa type IV collagenase (MMP-2), Interstitial collagenase (MMP-1), and mixtures thereof.

[0054] In one embodiment, the first protein or polypeptide of the present invention is type VI collagen α3 chain. In another embodiment, the first protein or polypeptide is Sushi, von Willebrand factor type A, containing EGF and pentyl protein domain 1. In another embodiment, the first protein or polypeptide is 72kDa type IV collagenase. In another embodiment, the first protein or polypeptide is interstitial collagenase. Preferably, the first protein or polypeptide of the present invention is a mixture of 2 or more of the aforementioned proteins or polypeptides, such as type VI collagen α3 chain and Sushi, von Willebrand factor type A, containing EGF and pentyl protein domain 1, type VI collagen α3 chain and 72kDa type IV collagenase, 72kDa type IV collagenase and interstitial collagenase. More preferably, the present invention comprises a mixture of the aforementioned 4 proteins or polypeptides: type VI collagen α3 chain, Sushi, von Willebrand factor type A, containing EGF and pentyl protein domain 1, 72kDa type IV collagenase, and interstitial collagenase.

[0055] As far as the preferred preparation method is concerned (but not limited to the preparation method), the first protein or polypeptide of the present invention is a new protein or polypeptide secreted when human fibroblasts are expanded in vitro, and it is not present in the initial culture medium (the specific content will be described in detail in the "Preparation Method" section below). In other words, the first protein or polypeptide constitutes the difference between the human fibroblast conditioned medium obtained according to the preferred preparation method and the initial culture medium, and is also the key to the protein composition of the present invention to achieve the aforementioned therapeutic effect. Without being bound by theory, the applicant believes that after being administered to the human body, the aforementioned specified first protein or polypeptide can be reused by fibroblasts, undergo endocytosis and exocytosis, and then reassemble and secrete active new proteins in the cells to achieve specific therapeutic effects.

[0056] In the present invention, the concentration of the first protein or polypeptide in the protein composition is greater than 1ug / mL, preferably greater than 1.5ug / mL, preferably greater than 2ug / mL, preferably greater than 3ug / mL, preferably greater than 5ug / mL, preferably greater than 8ug / mL, preferably greater than 10ug / mL, preferably greater than 12ug / mL. When two or more first proteins or polypeptides are included, the "concentration" of the first protein or polypeptide referred to in this application refers to the sum of the concentrations of the two or more first proteins or polypeptides in the protein composition. In addition, the applicant has found that the higher the concentration of the first protein or polypeptide in the protein composition, the higher the colloid osmotic pressure, the wider the diffusion range in the body, and the better the therapeutic effect of the protein composition. Therefore, the present invention does not require an upper limit on the concentration of the first protein or polypeptide.

[0057] In some embodiments, the first protein or polypeptide of the present invention is type VI collagen α3 chain, and its concentration in the protein composition is greater than 2ug / mL, preferably greater than 3ug / mL, and preferably greater than 4ug / mL. In other embodiments, the first protein or polypeptide of the present invention is Sushi, von Willebrand factor type A, containing EGF and pentane protein domain 1, and its concentration in the protein composition is greater than 3ug / mL, preferably greater than 4ug / mL, and preferably greater than 5ug / mL. In other embodiments, the first protein or polypeptide of the present invention is 72kDa type IV collagenase, and its concentration in the protein composition is greater than 1.5ug / mL, preferably greater than 2ug / mL, and preferably greater than 2.5ug / mL. In other embodiments, the first protein or polypeptide of the present invention is interstitial collagenase, and its concentration in the protein composition is greater than 1ug / mL, preferably greater than 1.5ug / mL, and preferably greater than 2ug / mL.

[0058] The protein composition of the present invention may include any mixture of the aforementioned first proteins or polypeptides, as long as the sum of their concentrations in the protein composition is greater than 1ug / mL; of course, when two or more of the aforementioned first proteins or polypeptides are included, their concentrations in the protein composition may reach a higher level, preferably greater than 3ug / mL, preferably greater than 5ug / mL, preferably greater than 7ug / mL. In some embodiments, the present invention includes two first proteins or polypeptides: type VI collagen α3 chain and Sushi, von Willebrand factor type A, containing EGF and pentane protein domain 1, and the sum of the concentrations of the two first proteins or polypeptides in the protein composition is greater than 5ug / mL, preferably greater than 6ug / mL, preferably greater than 8ug / mL. In other embodiments, the present invention includes two first proteins or polypeptides: type VI collagen α3 chain and 72kDa type IV collagenase, and the sum of the concentrations of the two first proteins or polypeptides in the protein composition is greater than 3ug / mL, preferably greater than 4ug / mL, preferably greater than 5ug / mL. In other embodiments, the present invention comprises two first proteins or polypeptides: 72kDa type IV collagenase and Sushi, von Willebrand factor type A, containing EGF and pentyl protein domain 1, and the sum of the concentrations of the two first proteins or polypeptides in the protein composition is greater than 4ug / mL, preferably greater than 6ug / mL, preferably greater than 7ug / mL.

[0059] In some preferred embodiments, the present invention comprises a mixture of the aforementioned four proteins or polypeptides: type VI collagen α3 chain, Sushi, von Willebrand factor type A, EGF and pentyl protein domain 1, 72kDa type IV collagenase, and interstitial collagenase, and the sum of the concentrations of the four proteins or polypeptides in the protein composition is greater than 8ug / mL, preferably greater than 10ug / mL, and preferably greater than 12ug / mL. Second protein or polypeptide

[0060] Preferably, in addition to the aforementioned first protein or polypeptide, the protein composition of the present invention further comprises a second protein or polypeptide different from the first protein or polypeptide. The second protein or polypeptide is selected from the group consisting of: Sulfhydryl oxidase 1 (QSOX1), Decorin (DCN), Fibronectin (FN), Vimentin, Fibulin-1 (FBLN1), Metalloproteinase inhibitor 1 (TIMP1), Type I collagen alpha I chain (Collagen alpha-1(I) chain, COL1A1), Type I collagen alpha II chain (Collagen alpha-2(I) chain, COL1A2), and mixtures thereof.

[0061] In one embodiment, the second protein or polypeptide of the present invention is sulfhydryl oxidase 1. In another embodiment, the second protein or polypeptide is decorin. In another embodiment, the second protein or polypeptide is 72kDa type IV collagenase. In another embodiment, the second protein or polypeptide is fibronectin. In another embodiment, the second protein or polypeptide is fibulin-1. In another embodiment, the second protein or polypeptide is type I collagen αI chain. In another embodiment, the second protein or polypeptide is type I collagen αII chain. Preferably, the second protein or polypeptide of the present invention is a mixture of 2 or more of the aforementioned proteins or polypeptides. The mixture can be any combination of the listed proteins or polypeptides, such as sulfhydryl oxidase 1 and decorin, sulfhydryl oxidase 1 and fibronectin, fibulin-1 and type I collagen αII chain, sulfhydryl oxidase 1, decorin and fibronectin, or sulfhydryl oxidase 1, fibronectin and type I collagen αII chain. In some preferred embodiments, the present invention comprises a mixture of 3 of the aforementioned proteins or polypeptides, preferably a mixture of 5 of the aforementioned proteins or polypeptides, and preferably a mixture comprising all 8 of the aforementioned proteins or polypeptides.

[0062] As far as the preferred preparation method is concerned (but not limited to the preparation method), the second protein or polypeptide of the present invention is a protein or polypeptide that grows rapidly when human fibroblasts are expanded in vitro. Although it is also present in the initial culture medium, its content increases by dozens or even hundreds of times when the cells are expanded (the specific content will be described in detail in the "Preparation Method" section below). In other words, based on the aforementioned first protein or polypeptide, the second protein or polypeptide constitutes a further difference between the human fibroblast conditioned culture fluid obtained according to the preferred preparation method and the initial culture medium. Based on this preferred protein composition, it has a better therapeutic effect on inflammatory diseases. Without being bound by theory, the applicant believes that, similar to the role played by the aforementioned first protein or polypeptide in the human body, after the aforementioned second protein or polypeptide enters the body, the denatured protein or polypeptide can also be reused by fibroblasts to re-secrete active new proteins, thereby further enhancing the therapeutic effect.

[0063] In the present invention, the concentration of the second protein or polypeptide in the protein composition is greater than 5ug / mL, preferably greater than 10ug / mL, preferably greater than 20ug / mL, preferably greater than 30ug / mL, preferably greater than 50ug / mL. When two or more second proteins or polypeptides are included, the "concentration" of the second protein or polypeptide referred to in this application refers to the sum of the concentrations of the two or more second proteins or polypeptides in the protein composition. In addition, the applicant has found that the higher the concentration of the second protein or polypeptide in the protein composition, the better the therapeutic effect of the protein composition. Therefore, the present invention does not require an upper limit on the concentration of the second protein or polypeptide.

[0064] In some embodiments, the second protein or polypeptide of the present invention is sulfhydryl oxidase 1, and its concentration in the protein composition is greater than 5ug / mL, preferably greater than 10ug / mL, preferably greater than 15ug / mL. In other embodiments, the second protein or polypeptide of the present invention is decorin, and its concentration in the protein composition is greater than 5ug / mL, preferably greater than 7ug / mL, preferably greater than 10ug / mL. In other embodiments, the second protein or polypeptide of the present invention is fibronectin, and its concentration in the protein composition is greater than 50ug / mL, preferably greater than 100ug / mL, preferably greater than 300ug / mL, preferably greater than 500ug / mL, preferably greater than 700ug / mL, preferably greater than 900ug / mL. In other embodiments, the second protein or polypeptide of the present invention is vimentin, and its concentration in the protein composition is greater than 3ug / mL, preferably greater than 5ug / mL, preferably greater than 7ug / mL. In other embodiments, the second protein or polypeptide of the present invention is fibula protein-1, and its concentration in the protein composition is greater than 5ug / mL, preferably greater than 8ug / mL, preferably greater than 10ug / mL. In other embodiments, the second protein or polypeptide of the present invention is metalloproteinase inhibitor-1, and its concentration in the protein composition is greater than 0.5ug / mL, preferably greater than 1ug / mL, preferably greater than 1.2ug / mL. In other embodiments, the second protein or polypeptide of the present invention is type I collagen αI chain, and its concentration in the protein composition is greater than 7ug / mL, preferably greater than 10ug / mL, preferably greater than 13ug / mL. In other embodiments, the second protein or polypeptide of the present invention is type I collagen αII chain, and its concentration in the protein composition is greater than 10ug / mL, preferably greater than 15ug / mL, preferably greater than 20ug / mL, preferably greater than 25ug / mL.

[0065] The protein composition of the present invention may include any mixture of the aforementioned second proteins or polypeptides. When two or more of the aforementioned second proteins or polypeptides are included, their concentration in the protein composition may reach a higher level, preferably greater than 10ug / mL, preferably greater than 15ug / mL, preferably greater than 20ug / mL, preferably greater than 30ug / mL, preferably greater than 50ug / mL, preferably greater than 100ug / mL. In some embodiments, the present invention includes two second proteins or polypeptides: sulfhydryl oxidase 1 and core protein polysaccharide, and the sum of the concentrations of the two second proteins or polypeptides in the protein composition is greater than 10ug / mL, preferably greater than 13ug / mL, preferably greater than 18ug / mL. In other embodiments, the present invention includes two second proteins or polypeptides: sulfhydryl oxidase 1 and fibronectin, and the sum of the concentrations of the two second proteins or polypeptides in the protein composition is greater than 500ug / mL, preferably greater than 700ug / mL, preferably greater than 900ug / mL. In other embodiments, the present invention comprises two second proteins or polypeptides: fibula protein-1 and type I collagen αI chain, and the sum of the concentrations of the two second proteins or polypeptides in the protein composition is greater than 10ug / mL, preferably greater than 15ug / mL, preferably greater than 20ug / mL. In other embodiments, the present invention comprises two second proteins or polypeptides: fibula protein-1 and type I collagen αII chain, and the sum of the concentrations of the two second proteins or polypeptides in the protein composition is greater than 10ug / mL, preferably greater than 20ug / mL, preferably greater than 30ug / mL.

[0066] In some preferred embodiments, the present invention comprises 3 or more of the second proteins or polypeptides, and the sum of the concentrations of the 3 or 3 second proteins or polypeptides in the composition is greater than 10ug / mL. More preferably, the present invention comprises 5 or more of the second proteins or polypeptides, and the sum of the concentrations of the 5 or 5 second proteins or polypeptides in the composition is greater than 20ug / mL. More preferably, the present invention comprises a mixture of the aforementioned 8 proteins or polypeptides: sulfhydryl oxidase 1, core protein polysaccharide, fibronectin, vimentin, fibulin-1, metalloproteinase inhibitor-1, type I collagen αI chain, and type I collagen αII chain, and the sum of the concentrations of the 8 proteins or polypeptides in the protein composition is greater than 300ug / mL, preferably greater than 500ug / mL, preferably greater than 900ug / mL.

[0067] In a preferred embodiment, the present invention comprises the aforementioned first protein or polypeptide and the second protein or polypeptide, wherein the total protein concentration of the protein composition is greater than 12 mg / mL, the concentration of the first protein or polypeptide in the composition is greater than 3 ug / mL, and the concentration of the second protein or polypeptide in the composition is greater than 10 ug / mL.

[0068] In a preferred embodiment, the present invention comprises the aforementioned first protein or polypeptide and the second protein or polypeptide, wherein the first protein or polypeptide is type VI collagen α3 chain, and its concentration in the composition is greater than 3ug / mL, preferably greater than 4ug / mL, and the second protein is fibronectin and / or type I collagen αII chain, and its concentration in the composition is greater than 20ug / mL, preferably greater than 25ug / mL, and the total protein concentration of the protein composition is greater than 12mg / mL. Other components

[0069] In addition to the aforementioned first protein or polypeptide and the second protein or polypeptide, the protein composition of the present invention may also include other components. In some embodiments, the protein composition further includes a group selected from the following proteins or polypeptides: Pentraxin-related protein PTX3, Adipocyte enhancer-binding protein 1, Type III collagen alpha I chain (Collagen alpha-1(III) chain), Adiponectin, Complement factor H-related protein 1, Cystatin-C, Ectonucleotide pyrophosphatase / phosphodiesterase family member 2, Extracellular matrix protein 1, Fetuin-B, Follistatin-related protein 1, Galectin-1, Glia-derived nexin, Histone H2B type 2-E, Histone H4, Immunoglobulin kappa variable 3D-15, Laminin subunit beta-1, Latent-transforming growth factor beta-binding protein 1, Microfibril-associated glycoprotein 4, Plasminogen activator inhibitor 1, Prelamin-A / C, Procollagen C-endopeptidase enhancer 1, Sex hormone-binding globulin, SH3 domain-binding glutamic acid-rich-like protein 3, Calcium-binding protein S100A6 (Protein S100-A6), Cathepsin B, Serine protease HTRA1, Transforming growth factor-beta-induced protein IG-H3, Apolipoprotein C-III, and mixtures thereof.

[0070] As for its preferred preparation method (but not limited to the preparation method), the protein composition of the present invention is essentially a fibroblast conditioned medium, which may also contain one or more components derived from the initial culture medium. Preferably, the initial culture medium contains fetal bovine serum or serum-free additives. Therefore, the protein composition may also contain one or more components contained in a conventional basal culture medium, fetal bovine serum or serum-free additives. Such components include but are not limited to: growth factors such as natural growth factors or synthetic growth factors, amino acids such as glutamine, leucine, lysine, serine, inorganic salts, glucose, vitamins, and buffer systems such as HEPES and sodium bicarbonate. In some embodiments, the protein composition of the present invention may contain proteins, hormones, lipids, etc. derived from fetal bovine serum, and in other embodiments, there may also be proteins, lipids, etc. derived from serum-free additives. Preparation method

[0071] The protein composition of the present invention can be prepared by any conventional method known in the art, as long as the obtained composition contains the aforementioned specific protein or polypeptide and reaches a specific concentration. For example, one or more recombinant proteins or polypeptides, such as recombinant fibronectin, can be purchased and mixed with deionized water in a specific ratio to obtain the protein composition of the present invention. It will be understood by those skilled in the art that even if certain proteins or polypeptides are difficult to purchase directly, the required proteins or polypeptides can be obtained indirectly by purchasing the corresponding raw materials. For example, type VI collagen α3 chain can be obtained by recombinant type VI collagen, and type I collagen αI chain can be obtained by recombinant type I collagen.

[0072] Preferably, the protein composition of the present invention is a fibroblast conditioned medium, i.e., a culture supernatant produced by human fibroblasts expanded in vitro under specific culture conditions. Compared with the conventional preparation method mentioned above, this method has low production cost and can achieve efficient and large-scale preparation of the protein composition of the present invention.

[0073] In one embodiment, the present invention provides a method for preparing the protein composition, the method comprising: a) obtaining human fibroblasts from human skin tissue by using tissue blocks or digestion method; b) The human fibroblasts were cultured in vitro at 37°C and 3-10% CO 2 Under culture conditions, in vitro expansion was performed in the initial culture medium; c) obtaining a large amount of cell culture supernatant while expanding in vitro and collecting it; and d) filtering the collected cell culture supernatant using a 0.1-0.3 μm microporous filter membrane or filter element to obtain a sterile filtrate, The obtained sterile filtrate is the protein composition.

[0074] The aforementioned step a) is primary cell culture, and primary cell culture refers to the process of obtaining target cells from tissues after dispersing them into cell suspensions, usually by tissue block method or digestion method. In the preparation method of the present invention, the tissue is human skin tissue, and the target cells are human fibroblasts, that is, step a) is primary human fibroblast culture. Preferably, the aforementioned human skin tissue is obtained from routine HIV, HBV, HCV, and syphilis serological tests that are negative, more preferably mycoplasma negative and bacteria negative, by separate aseptic excision of the skin or skin discarded or deliberately excised by eye bag excision, double eyelid surgery, wrinkle removal, foreskin and other surgeries. Preferably, the obtained human skin tissue is subjected to primary fibroblast culture using a conventional tissue block method or an enzyme digestion method to obtain human fibroblasts.

[0075] The aforementioned step b) is cell expansion in vitro, and any conventional method can be used for cell expansion, for example, ordinary cell culture bottles can be used for cell expansion, and roller bottles and hollow fiber tube cell culture systems can also be used for cell expansion in vitro. The primary human fibroblasts obtained in the aforementioned step a) are expanded in vitro in the initial culture medium under the culture conditions of 37° C. and 3-10% CO2 in vitro. Preferably, the culture conditions are 37° C. and 4-8% CO2, and more preferably 37° C. and 5% CO2.

[0076] The initial culture medium provides the basic environment necessary for cell growth, proliferation and functional maintenance, and can be selected from any conventional initial culture medium, and can be commercially available such as purchased from Sigma-Aldrich or Thermo Fisher. In some embodiments, the initial culture medium can be prepared by adding serum, serum-free additives, or mixtures thereof from animal sources to the basal culture medium, and the basal culture medium and its serum or serum-free additives can be purchased in a matching manner. In some preferred embodiments, the basal culture medium can be selected from Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), F-10, F-12, RPMI1640, Glasgow's Minimal Essential Medium (GMEM), α Minimal Essential Medium (αMEM), Iscove's Modified Dulbecco's Medium, or M199. In some preferred embodiments, the serum of animal origin is selected from fetal bovine serum (FBS), rabbit serum, goat serum, horse serum, sheep serum, pig serum, chicken serum, etc., preferably fetal bovine serum, preferably 5-10% fetal bovine serum, preferably 8-10% fetal bovine serum. More preferably, the initial culture medium contains fetal bovine serum or serum-free supplement, preferably fetal bovine serum.

[0077] In the aforementioned step c), a large amount of cell culture supernatant is obtained and collected while the fibroblasts are expanded in vitro. In some embodiments, the fibroblast supernatant is collected when the confluence coverage is ≥90%. Preferably, the collected fibroblast supernatant is stored in a 4°C refrigerator. During storage, the fibroblast supernatant collected from a single culture can be stored separately, or the supernatants collected from multiple cultures can be mixed and stored, or the supernatants from multiple generations of fibroblasts can be mixed and stored.

[0078] In the above step d), the collected cell culture supernatant is filtered using a 0.1-0.3 μm microporous filter membrane or filter element to obtain a sterile filtrate. Preferably, the pore size of the microporous filter membrane is 0.1-0.25 μm, preferably 0.1 μm or 0.22 μm. The main purpose of this microfiltration step is to filter bacteria and fungi. The sterile filtrate obtained by step d) is the protein composition, but in some preferred embodiments, the sterile filtrate can also be subjected to other subsequent processing steps (described in detail below) to obtain a more concentrated or sterile protein composition.

[0079] In some preferred embodiments, the aforementioned preparation method further comprises: e) The sterile filtrate obtained in step d) is ultrafiltered and concentrated through an ultrafiltration membrane to obtain an ultrafiltration concentrate.

[0080] The above step e) is after the above step d), and its main purpose is to filter water for protein concentration. The ultrafiltration membrane used in step e) may have a specific pore size to filter out components larger than a specific molecular weight, and ultrafiltration membranes of different pore sizes may be selected according to the initial culture medium. When the initial culture medium contains fetal bovine serum, the molecular weight of the ultrafiltration membrane is preferably ≥80KD, more preferably ≥90KD, and more preferably ≥100KD. When the initial culture medium contains serum-free additives, the molecular weight of the ultrafiltration membrane is ≥1KD, more preferably ≥3KD, and more preferably ≥5KD.

[0081] In some preferred embodiments, the aforementioned preparation method further comprises: f) performing bacterial / viral inactivation on the obtained sterile filtrate or ultrafiltration concentrate.

[0082] The above step f) can be performed after the above step d), that is, the obtained sterile filtered liquid is subjected to bacterial / viral inactivation, preferably after the above step e), that is, the obtained ultrafiltration concentrate is subjected to bacterial / viral inactivation. Any conventional sterilization method can be selected for sterilization, preferably heating method and / or radiation sterilization method are used for bacterial / viral inactivation, more preferably heating method and radiation sterilization method are used for bacterial / viral inactivation, and even more preferably heating method is used first, and then radiation sterilization method is used. The heating method is preferably a constant temperature water bath, preferably the heating temperature is 60±5°C, the continuous duration is 8-12 hours, and the more preferably heating temperature is 60±0.5°C, and the continuous duration is 10 hours. The radiation sterilization method is preferably 60Co-r rays ≥25KGy.

[0083] Preferably, some or all of the steps of the aforementioned preparation method are carried out in an ultra-clean workbench, preferably a Class 100 ultra-clean workbench.

[0084] As described above, in combination with the aforementioned preparation method, the first protein or polypeptide of the present invention is a new protein or polypeptide secreted during in vitro expansion of human fibroblasts, which is substantially absent in the initial culture medium or the initial culture medium of the control group, or has only a very low content or cannot be detected in the initial culture medium or the initial culture medium of the control group. Preferably, in the preparation method of the present invention, the concentration of the first protein or polypeptide in the initial culture medium does not exceed 0.1 ug / mL, preferably does not exceed 0.01 ug / mL, and preferably does not exceed 0.001 ug / mL.

[0085] On the other hand, in combination with the aforementioned preparation method, the second protein or polypeptide of the present invention is a protein or polypeptide that grows rapidly or even doubles when human fibroblasts are expanded in vitro, although it is also present in the initial culture medium, and its content increases by tens or even hundreds of times when the cells are expanded. Preferably, in the method for preparing a protein composition comprising a first protein or polypeptide and a second protein or polypeptide, the concentration of the second protein or polypeptide in the protein composition increases by more than 5 times, preferably more than 10 times, preferably more than 30 times, preferably more than 50 times, preferably more than 80 times, preferably more than 100 times relative to its concentration in the initial culture medium of the control group. Because of the rapid growth of the second protein or polypeptide during cell expansion, the protein or polypeptide presents a relatively high concentration in the obtained protein composition, preferably a concentration in the protein composition greater than 5ug / mL, preferably greater than 10ug / mL, preferably greater than 20ug / mL, preferably greater than 30ug / mL, preferably greater than 50ug / mL, preferably greater than 50ug / mL. Uses and Indications

[0086] The protein composition of the present invention has a wide range of therapeutic uses for inflammatory diseases and other related diseases. In one aspect, the protein composition of the present invention is used to prepare medical implants, medical devices or medicines, preferably for preparing medical devices or medicines. The protein composition of the present invention can also be used in cosmetics. In some embodiments, the protein composition is the sterile filtered liquid obtained in step d) of the aforementioned preparation method, preferably the ultrafiltration concentrate obtained in the aforementioned step e), and more preferably the sterilized concentrate obtained in the aforementioned step f).

[0087] When used for the aforementioned purposes, the protein composition of the present invention can be directly applied to the patient. In another embodiment, the medical device or medicine or pharmaceutical composition used for the aforementioned purposes comprises an effective amount of the protein composition of the present invention, and a pharmaceutically acceptable excipient, carrier or diluent. The "pharmaceutically acceptable excipient, carrier or diluent" includes but is not limited to any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier, etc., which are permitted by the relevant government management department to be acceptable for use by humans or livestock.

[0088] Protein composition of the present invention can be applied to the patient by multiple approaches, these approaches are such as transdermal, corium, subcutaneous, intranasal, intravenous, intramuscular, intrathecal, regional or local (for example mucous membrane). The most suitable route of administration in any given case will depend on the experimenter, the nature and severity of the disease, and the physical condition of the experimenter etc. Accordingly, according to different modes of administration, medicine of the present invention can be prepared as different dosage forms. For example, in one embodiment, the medical device or the medicine of the protein composition of the present invention comprising effective dose can be prepared as injection, gel etc. Preferably, protein composition of the present invention is injected in local affected part by subcutaneous or corium.

[0089] In one aspect, the protein composition of the present invention is used to prepare a medical device or medicine for treating an inflammatory disease. In another aspect, the protein composition of the present invention is used to treat an inflammatory disease. In another aspect, the present invention provides a method for treating an inflammatory disease using the aforementioned protein composition. Preferably, the inflammatory disease is a local inflammatory disease. In some embodiments, the inflammatory disease is selected from inflammatory skin diseases, autoimmune diseases, sterile inflammation, or a combination thereof.

[0090] The aforementioned inflammatory skin disease can be any inflammatory skin disease, preferably an inflammatory skin disease related to immunity. Preferably, the inflammatory skin disease is selected from dermatitis, acne, sensitive skin, rosacea, chloasma, lichen planus, lichen sclerosus, lichen sclerosus, seborrheic dermatitis, hidradenitis suppurativa, or a combination thereof. Wherein, the dermatitis is selected from atopic dermatitis, eczema, contact dermatitis, seborrheic dermatitis, perioral dermatitis, neurodermatitis, or a combination thereof. More preferably, the inflammatory skin disease is selected from atopic dermatitis, eczema, sensitive skin, rosacea, chloasma, or a combination thereof. Not bound by theory, the applicant believes that the therapeutic effect of the protein composition of the present invention for inflammatory skin diseases mainly lies in its effect on human in situ cells. Specifically, when applied to the human body, such as subcutaneous or dermal injection, the protein or polypeptide contained in the protein composition is absorbed by dermal fibroblasts in vivo and reassembled into collagen, therefore, local tissue elasticity can be increased, and skin damage can be repaired.

[0091] Autoimmune diseases refer to disease states in which the body's immune system responds to its own components and causes damage to its own tissues. The autoimmune diseases to which the present invention is applicable may be any autoimmune diseases. Preferably, the autoimmune diseases are selected from ankylosing spondylitis, allergic rhinitis, rheumatoid arthritis, ulcerative colitis, Crohn's disease, systemic lupus erythematosus, dermatomyositis, multiple sclerosis, Sjögren's syndrome, vasculitis, psoriasis, vitiligo, or a combination thereof. More preferably, the autoimmune diseases are selected from ankylosing spondylitis, allergic rhinitis, rheumatoid arthritis, or a combination thereof. Without being bound by theory, the applicant believes that the therapeutic effect of the protein composition of the present invention on autoimmune diseases mainly lies in its effect on human immune cells and body fluids. When applied to the human body, the protein composition of the present invention, in particular the proteins or polypeptides contained therein, increase the anchoring and wandering of immune cells, and promote the circulation of local extracellular fluid, thereby enhancing humoral and cellular immune functions, and playing a role in alleviating and treating autoimmune diseases.

[0092] In particular, with regard to ankylosing spondylitis, its pathogenesis is mainly derived from genetic factors, chronic inflammation, and the periodic tension generated by people walking upright. Under the combined action of these factors, in order to achieve mechanical balance, the cells of the soft tissue around the damaged spine continuously secrete type I collagen, invade the intervertebral joints to form scar tissue, and try to achieve the purpose of stabilizing the joints, so ankylosing spondylitis continues to spread upward, eventually leading to the formation of bamboo-like changes in the spine. In this regard, the applicant was surprised to find that the implantation of the protein composition of the present invention not only brings about the enhancement of humoral and cellular immune functions, but also the newly produced matrix metalloproteinase (MMP) can degrade collagen, reduce the production of type I collagen, increase degradation, and reduce scars in various segments of the spine, thereby alleviating pain and relieving symptoms.

[0093] Aseptic inflammation refers to an inflammatory reaction caused by various non-biological factors or unknown causes. The aseptic inflammation to which the present invention is applicable may be any aseptic inflammation. Preferably, the aseptic inflammation is selected from migraine, finger tenosynovitis, facial paralysis, scapulohumeral periarthritis, or a combination thereof. Without being bound by theory, the applicant believes that the therapeutic effect of the protein composition of the present invention on aseptic inflammation is similar to the aforementioned therapeutic effect on autoimmune diseases, mainly in its effect on human immune cells and body fluids.

[0094] On the other hand, the present invention provides the aforementioned protein composition for preparing a medical device or medicine for treating mycoplasma infection diseases. On the other hand, the present invention provides the aforementioned protein composition for treating mycoplasma infection diseases. On the other hand, the present invention provides a method for treating mycoplasma infection diseases using the aforementioned protein composition. Preferably, the mycoplasma infection diseases are selected from cervical hypertrophy, uterine prolapse, hemorrhoids, or a combination thereof. Without being bound by theory, the applicant believes that in addition to the local swelling caused by mycoplasma infection, the aforementioned diseases are also caused by the gravity factor of the upright walking position, which causes the original swelling to continue to increase, and the extracellular fluid circulation slows down, thereby further aggravating the symptoms. However, by injecting the protein composition of the present invention, the extracellular fluid circulation of the above-mentioned parts is accelerated, and the body fluid and cellular immunity are enhanced, which is conducive to the removal of mycoplasma infection and the subsidence of swelling.

[0095] On the other hand, the present invention provides the aforementioned protein composition for preparing a medical device or medicine for treating viral infections on the skin and mucous membranes. On the other hand, the present invention provides the aforementioned protein composition for treating viral infections on the skin and mucous membranes. On the other hand, the present invention provides a method for treating viral infections on the skin and mucous membranes using the aforementioned protein composition. Generally speaking, the viral infections on the skin and mucous membranes are caused by human papillomavirus and / or other viral infections of different types. Preferably, the viral infections on the skin and mucous membranes are selected from flat warts, cervical filiform warts, female genital tract HPV infection, or a combination thereof. Without being bound by theory, the applicant believes that the protein composition of the present invention can help or achieve the effect of clearing such viruses by enhancing humoral and cellular immunity.

[0096] On the other hand, the present invention provides the aforementioned protein composition for preparing a medical device or medicine for treating functional lubricating fluid deficiency diseases. On the other hand, the present invention provides the aforementioned protein composition for treating functional lubricating fluid deficiency diseases. On the other hand, the present invention provides a method for treating functional lubricating fluid deficiency diseases using the aforementioned protein composition. The functional lubricating fluid deficiency diseases applicable to the present invention may be any known such diseases, preferably, the functional lubricating fluid deficiency diseases are selected from senile arthritis, dry eye, or a combination thereof. When used to treat senile arthritis, the preferred injection level of the protein composition of the present invention is located in the joint capsule (rather than the joint cavity). Without being bound by theory, in the case of senile arthritis, its pathogenesis lies in the inability of the synovial cells of the joint capsule wall to produce enough synovial fluid to lubricate the articular cartilage. The applicant has found that after the protein composition of the present invention is injected into the joint capsule for four weeks, it will increase the extracellular fluid and accelerate blood circulation, so that the synovial cells can produce enough synovial fluid, thereby achieving the purpose of nourishing the articular cartilage and treating senile arthritis.

[0097] On the other hand, the present invention provides the aforementioned protein composition for preparing a medical device or medicine for treating hypertrophic scars. On the other hand, the present invention provides the aforementioned protein composition for treating hypertrophic scars. On the other hand, the present invention provides a method for treating hypertrophic scars using the aforementioned protein composition. The hypertrophic scars to which the present invention is applicable may be any known such disease. Preferably, the hypertrophic scars are selected from thyroid hyperplasia, breast hyperplasia, prostatic hyperplasia, uterine fibroids, facial hypertrophic scars, or a combination thereof. Without being bound by theory, studies have shown that hypertrophic scars of skin wounds are characterized by abnormal proliferation of type I collagen secreted by fibroblasts. The implantation of the protein composition of the present invention can allow the regeneration of matrix metalloproteinases, reduce the production of type I collagen, and increase degradation, thereby alleviating hypertrophic scars.

[0098] In another aspect, the present invention provides the aforementioned protein composition for preparing a medical device or medicine for treating alopecia. In another aspect, the present invention provides the aforementioned protein composition for treating alopecia. In another aspect, the present invention provides a method for treating alopecia using the aforementioned protein composition. The alopecia to which the present invention is applicable may be any known such disease, or the present invention may be used for the purpose of hair growth in any part of the human body (e.g., eyelashes). Preferably, the alopecia is selected from alopecia areata, diffuse alopecia, cicatricial alopecia, or a combination thereof. Without being bound by theory, the applicant believes that the protein or polypeptide contained in the present invention is reassembled into collagen by in situ cells in vivo, thereby improving the growth environment of hair follicles and allowing resting hair follicles to grow hair again. Protein concentration detection method

[0099] In the present application, the total protein concentration and the individual protein concentration were determined using different detection methods, namely, the BCA method and the liquid chromatography-mass spectrometry method, which are described in detail below. 1. Determination of total protein concentration by BCA method

[0100] Any conventional BCA method can be used to determine the total protein concentration of the protein composition of the present application. The test process is as follows: After the sample is diluted 50 times with PBS diluent, the total protein concentration of the sample is determined by using a BSA protein standard as a standard curve with a standard curve range of 0.05-0.5 mg / mL. The BCA protein concentration determination kit used can be purchased from Solebao. 2. Determination of individual protein concentrations by liquid chromatography-mass spectrometry

[0101] Any conventional liquid chromatography-mass spectrometry detection method can be used to measure the concentration of a single protein or polypeptide (such as a first protein or polypeptide, a second protein or polypeptide) in the protein composition of the present application, and a suitable instrument can be selected. In some embodiments, the following instruments can be used: an electronic balance (Mettler Toledo Instrument (Shanghai) Co., Ltd., model: XPE205), an ELISA reader (Molecular devices, model: Spectramax M5), and a two-in-one high-resolution mass spectrometer (ThermoScientific, model: Exploris 480).

[0102] The test and analysis process is as follows:

[0103] 1) Sample processing

[0104] Use 50 mM NH 4 HCO 3 The sample was diluted with the solution, and the standard protein was added as the internal standard. DL-dithiothreitol and iodoacetamide were used for reduction-alkylation, and the supernatant was taken for liquid chromatography-mass spectrometry analysis after trypsin hydrolysis. Among them, the standard proteins are Protein_AG, Thioredoxin, Klenow, Carbonic, IGF-I_LR3 and Protein_G, DL-dithiothreitol can be purchased from Sigma-Aldrich, and iodoacetamide can be purchased from Sigma-Aldrich.

[0105] 2) Liquid chromatography conditions: Column: BEH C18 2.1mm×150mm,1.7μm; Column temperature: 60°C; Mobile phase: A: 0.1% formic acid-water; B: 0.1% formic acid-acetonitrile; Flow rate: 0.2 mL / min;

[0106] 3) Mass spectrometry conditions: Detection mode: positive ion; Spray voltage: 3.8 kV; Capillary temperature: 320℃; Evaporation temperature: 350℃; Resolution: primary 60,000@m / z 200; secondary 15,000@m / z 200; Precursor ion scan range: m / z 300-1600.

[0107] 4) Qualitative and quantitative analysis of protein components

[0108] The experimental mass spectrometry data were retrieved by Proteome Discoverer 2.5 (Thermo Fisher) software. The database was the human library uniprotkb_taxonomy_id_9606_reviewed_2023_11_27 (a total of 20,429 proteins) downloaded from uniprot on November 27, 2023 plus the sequence of the Thermo standard. The search parameters were set as follows: the enzyme cleavage method was trypsin, with high specificity and a maximum number of missed cleavages of 2; the minimum length of the peptide was set to 6 amino acid residues; the mass error tolerance of the primary parent ion was set to 10 ppm, and the mass error tolerance of the secondary fragment ion was set to 0.02 Da. The variable modifications of the peptide were acetylation of the N-terminus, methionine oxidation, asparagine and glutamine deamidation, and cysteine ​​alkylation was set as a fixed modification.

[0109] According to the total ion flow chart, base peak chart and secondary mass spectrum of the sample's liquid chromatography-mass spectrometry, the protein components can be qualitatively and quantitatively analyzed. If two or more characteristic peptides are detected in the protein / protein peptide chain, and the abundance detected in the sample is High, it can be confirmed that it has been detected. Because the peak area of ​​the peptide mass spectrum is linearly related to the protein concentration, the amount of protein in the sample is calculated based on the mass spectrum peak area of ​​the standard peptide and sample peptide with known concentrations (Formula 1), and then the mass of the protein and its content in the sample are calculated (Formula 2). The average value of the quantitative results of the above six standard proteins is taken as the sample protein content (Formula 3), and the protein concentration in the sample is calculated according to Formula 4. DETAILED DESCRIPTION

[0110] The examples herein are intended to illustrate the invention but are not intended to limit or otherwise define the scope of the invention.

[0111] Examples 1-6 and 8-10 are examples of the protein compositions of the present invention, Example 7 is the preparation method of Examples 1-6, Examples 12-14 are the preparation methods of Examples 8-10, and Comparative Examples 11 and 15 are control groups and their preparation methods. Examples 16-23 are experimental data of the therapeutic effects of Examples 6 and Examples 8-10. Examples 1-6: Protein compositions

[0112] The protein compositions shown in Table 1 consisted of the listed ingredients at the listed concentrations (ug / mL). Table 1 Example 7: Preparation method of the protein composition of Examples 1-6

[0113] The first protein or polypeptide and the second protein or polypeptide listed in Table 1 are purchased from commercial channels and mixed with deionized water in a specific ratio to achieve the protein concentration listed in the table. Among them, type VI collagen α3 chain is derived from recombinant type VI collagen, which can be purchased from MyBioSource.com; Sushi, von Willebrand factor type A, containing EGF and pentyl protein domain 1 is a recombinant protein of human SVEP1 control fragment, which can be purchased from Sigma-Aldrich; 72kDa type IV collagenase can be purchased from Sigma-Aldrich, MDL number MFCD01324322; interstitial collagenase can be purchased from MyBioSource.com; sulfhydryl oxidase 1 recombinant protein can be purchased from MyBioSource.com; decorin can be purchased from Sigma-Aldrich, MDL number MFCD00283027; fibronectin can be purchased from Sigma-Aldrich, MDL number MFCD00131062; vimentin can be purchased from MedChemExpress; fibulin-1 is a recombinant human fibulin 1C protein, which can be purchased from R&D Systems; metalloproteinase inhibitor-1 can be purchased from MedChemExpress; type I collagen αI chain and type I collagen αII chain are derived from recombinant type I collagen and can be purchased from MyBioSource.com. Examples 8-11: Protein compositions

[0114] The protein compositions shown in Table 2 comprise listed ingredients and water according to listed concentrations (ug / mL), and may also comprise other proteins or polypeptides in addition. Wherein, protein compositions 8-10 are protein compositions of the present invention, and composition 11 is a control group. Compared with control group 11, protein composition of the present invention comprises specific proteins or polypeptides, and the content of some proteins or polypeptides in protein composition of the present invention is much higher than its content in the control group. The concentration of listed components is measured by aforementioned liquid chromatography-mass spectrometry detection method.

[0115] According to the aforementioned BCA method, the total protein concentrations of Examples 8, 9, and 10 were 14.1 mg / mL, 12.9 mg / mL, and 13.4 mg / mL, respectively. Table 2 Example 12: Preparation method of the protein composition of Example 8

[0116] The preparation method of the protein composition of Example 8 is as follows:

[0117] 1. Before the patient receives treatment, routine HIV, HBV, HCV, and syphilis serological tests are performed. If the test result is negative, the sample is retained, and if the test result is positive, it is discarded.

[0118] 2. Skin that is individually and aseptically excised, or skin that is discarded or intentionally retained during surgeries such as blepharoplasty, eye bag surgery, wrinkle removal surgery, and circumcision surgery, and is used for in vitro cell culture;

[0119] 3. Primary fibroblasts were cultured by conventional tissue blocks or enzyme digestion method in a 37°C, 5% CO2 incubator. The initial culture medium was DMEM:F12 at a 1:1 ratio, supplemented with 10% fetal bovine serum;

[0120] 4. Use the rotating bottle method to expand cells in vitro;

[0121] 5. When the confluence rate is ≥90%, subculture and collect the supernatant of 6th generation fibroblasts. Store the collected cell culture supernatant in a 4°C refrigerator;

[0122] 6. Filter the stored cell culture supernatant using a 0.22 μm microporous filter membrane to obtain a sterile filtrate;

[0123] 7. The obtained sterile filtered liquid is subjected to protein concentration through an ultrafiltration membrane of ≥80KD to obtain an ultrafiltration concentrate;

[0124] 8. The ultrafiltration concentrate was first heated and then sterilized by radiation to inactivate bacteria / viruses, thereby obtaining the protein composition of Example 7. The heating conditions of the heating method were 60±0.5°C for about 10 hours, and the radiation sterilization method used 60Co-r rays ≥ 25KGy.

[0125] Some or all of the above steps are carried out in a Class 100 clean bench. Example 13: Preparation method of the protein composition of Example 9

[0126] The method for preparing the protein composition in Example 9 is the same as that in Example 12, except that in step 6, the supernatant of 8th generation fibroblasts is collected. Example 14: Preparation method of the protein composition of Example 10

[0127] The method for preparing the protein composition in Example 10 is the same as that in Example 12, except that in step 6, the supernatant of 11th generation fibroblasts is collected. Example 15: Preparation method of control group 11

[0128] The preparation method of the control group 11 is as follows:

[0129] 1. Take the same initial culture medium as in Example 12-14, i.e., a culture medium with a ratio of DMEM:F12 of 1:1 and add 10% fetal bovine serum (but without fibroblasts), place in a 37°C, 5% CO2 incubator for culture, and then refrigerate for the same period of time as in the aforementioned Example 12-14;

[0130] 2. Filter the refrigerated initial culture medium using a 0.22 μm microporous filter membrane to obtain a sterile filtrate;

[0131] 2. The obtained sterile filtered liquid is concentrated by protein through an ultrafiltration membrane of ≥80KD to obtain an ultrafiltration concentrate;

[0132] 3. The ultrafiltration concentrate was first heated and then sterilized by radiation to inactivate bacteria / viruses, to obtain the control protein composition of Example 11. The heating conditions of the heating method were 60±0.5°C for about 10 hours, and the radiation sterilization method used 60Co-r rays ≥ 25KGy. Example 16: Example 6 for treating senile arthritis

[0133] 5 mL of the protein composition of Example 6 was subcutaneously injected into the affected area of ​​senile arthritis patients once every month for a total of 3 injections. Figure 1 A and 1B are photographs of the patient's leg joints before the first treatment and two months after treatment, respectively. Example 17: Example 8 for treating rosacea

[0134] 2.5 mL of the protein composition of Example 8 was injected into the affected area of ​​rosacea patients through the dermis, once every month, for a total of 3 injections. Figure 2 A and 2B are facial photographs of the patient before the first injection and two months after treatment, respectively. Example 18: Example 8 is used to treat alopecia areata

[0135] 5 mL of the protein composition of Example 8 was subcutaneously injected into the affected area of ​​alopecia areata patients once every month. Figure 3 The head photographs of the patient in A-3E were taken before the first injection, at the third month of treatment, at the fourth month of treatment, at the sixth month of treatment, and at the eleventh month of treatment. Example 19: Example 8 for treating allergic rhinitis

[0136] 2.5 mL of the protein composition of Example 8 was injected into the affected area of ​​the allergic rhinitis patient through the mucosa once every one month. Figure 4 A and 4B are photos of the inferior turbinate of the patient before the first injection and after two treatments, respectively. It can be seen that the paleness of the inferior turbinate mucosa has improved and the swelling has decreased. Example 20: Example 9 for the treatment of rheumatoid arthritis

[0137] 2.5 mL of the protein composition of Example 9 was subcutaneously injected into the affected area of ​​the rheumatoid arthritis patient once every one month for a total of 2 injections. Figure 5 A and 5B are photos of the patient's finger joints before the first injection and 40 days after treatment, respectively. It can be seen that the swelling has decreased and the patient's pain has also been alleviated. Example 21: Example 9 for treating facial hypertrophic scars

[0138] 2.5 mL of the protein composition of Example 9 was injected into the affected area of ​​facial hypertrophic scar patients through the dermis, once every month, for a total of 3 injections. Figure 6 A, 6B, 6C, and 6D are photographs of the patient's right eye before the first injection, right eye two months after treatment, left eye before the first injection, and left eye two months after treatment, respectively. Example 22: Example 10 for treating cervical filiform warts

[0139] 5 mL of the protein composition of Example 10 was injected into the affected area of ​​the filiform warts on the neck of the patient through the dermis, once every month, for a total of 3 injections. Figure 7 A and 7B are photographs of the patient's neck before the first injection and two months after treatment, respectively. Example 23: Example 10 for treating uterine prolapse

[0140] 5 mL of the protein composition of Example 10 was injected into the anterior and posterior fornix of patients with uterine prolapse via vaginal administration, once every one month, for a total of 3 injections. Figure 8 A and 8B are photographs of the patient's cervix before the first injection and two months after treatment.

[0141] Although specific embodiments of the present invention have been described and illustrated, it will be appreciated by those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the present invention. Therefore, all such changes and modifications are intended to fall within the scope of the claims of this application.

[0142] All percentages, ratios and proportions are calculated by weight of the total composition unless otherwise indicated. All temperatures are in degrees Celsius (° C.) unless otherwise indicated. All component or composition levels refer to the active level of that component or composition, and are exclusive of impurities, such as residual solvents or by-products, which may be present in commercially available sources.

[0143] It should be understood that each numerical upper limit given in this specification includes values ​​lower than it, just as if these lower values ​​are explicitly written in the application. Each numerical lower limit given in this specification includes values ​​higher than it, just as if these higher values ​​are explicitly written in the application. Each numerical range given in this specification includes narrower numerical ranges falling within the numerical range, just as if these narrower numerical ranges are explicitly written in the application.

[0144] The values ​​and dimensions disclosed herein should not be understood as being strictly limited to the exact values ​​recited. Instead, unless otherwise specified, each such value and dimension is intended to represent the recited value and a functionally equivalent range around that value. For example, a dimension disclosed as "40 millimeters" is intended to mean "about 40 millimeters."

Claims

1. A protein composition comprising a first protein or polypeptide, wherein the first protein or polypeptide is selected from the group consisting of: Type VI collagen α3 chain, Sushi, von Willebrand factor type A, EGF and valerin domain-containing protein 1, 72kDa collagenase type IV, Interstitial collagenase, and mixtures thereof, The concentration of the first protein or polypeptide in the composition is greater than 1 ug / mL.

2. The protein composition of claim 1, wherein the total protein concentration of the composition is greater than 5 mg / mL according to the BCA method, and the single protein concentration is detected by liquid chromatography-mass spectrometry.

3. The protein composition of claim 1, comprising two or more of the first proteins or polypeptides, the total protein concentration of the composition is greater than 10 mg / mL, and the sum of the concentrations of the two or more first proteins or polypeptides in the composition is greater than 3 ug / mL.

4. The protein composition of claim 1, further comprising a second protein or polypeptide different from the first protein or polypeptide, wherein the second protein or polypeptide is selected from the group consisting of: Sulfhydryl oxidase 1, Decorin, Fibronectin, Vimentin, Fibroin-1, Metalloproteinase inhibitor-1, Type I collagen alpha I chain, Type I collagen alpha II chain, and mixtures thereof.

5. The protein composition of claim 4, comprising three or more second proteins or polypeptides, and the sum of the concentrations of the three or more second proteins or polypeptides in the composition is greater than 10 ug / mL. The protein composition of claim 1 , comprising at least three proteins or polypeptides, at least one of which is collagen.

7. The protein composition of claim 1, further comprising a protein or polypeptide selected from the group consisting of: Pentraxin 3, Adipocyte enhancer binding protein 1, Type III collagen alpha I chain, Adiponectin, Complement factor H-related protein 1, Cystatin-C, Extramembrane nucleotide pyrophosphatase / phosphodiesterase family member 2, Extracellular matrix protein 1, Fetuin B, follistatin-related protein 1, Galectin-1, Glial cell-derived connexin, Histone H2B type 2-E, Histone H4, Immunoglobulin Kappa variable 3D-15, Laminin subunit β1, Latent transforming growth factor-beta binding protein 1, Microfibril-associated glycoprotein 4, Plasminogen activator inhibitor-1, Lamin A / C, Procollagen C endopeptidase enhancer 1, Sex hormone binding globulin, SH3 domain-binding glutamate-rich protein 3, Calcium-binding protein S100A6, Cathepsin B, Serine protease HTRA1, Transforming growth factor-β-induced protein IG-H3, Apolipoprotein CIII, and mixtures thereof.

8. The protein composition of any one of claims 1 to 7, comprising a first protein or polypeptide and a second protein or polypeptide different from the first protein or polypeptide, wherein the second protein or polypeptide is selected from the group consisting of: Sulfhydryl oxidase 1, Decorin, Fibronectin, Vimentin, Fibroin-1, Metalloproteinase inhibitor-1, Type I collagen alpha I chain, Type I collagen alpha II chain, and mixtures thereof. The total protein concentration of the composition is greater than 12 mg / mL, the concentration of the first protein or polypeptide in the composition is greater than 3 ug / mL, and the concentration of the second protein or polypeptide in the composition is greater than 10 ug / mL.

9. A method for preparing a protein composition as claimed in any one of claims 1 to 8, comprising: a) obtaining human fibroblasts from human skin tissue by using tissue blocks or digestion method; b) The human fibroblasts were cultured in vitro at 37°C and 3-10% CO 2 Under culture conditions, in vitro expansion was performed in the initial culture medium; c) obtaining a large amount of cell culture supernatant while expanding in vitro and collecting it; and d) filtering the cell culture supernatant using a 0.1-0.3 μm microporous filter membrane or filter element to obtain a sterile filtrate, The obtained sterile filtrate is the protein composition.

10. The preparation method according to claim 9, further comprising: e) the sterile filtered liquid obtained in step d) is ultrafiltered and concentrated through an ultrafiltration membrane to obtain an ultrafiltration concentrate, in: When the initial culture medium contains fetal bovine serum, the molecular weight of the ultrafiltration membrane is ≥80KD; when the initial culture medium contains serum-free additives, the molecular weight of the ultrafiltration membrane is ≥1KD; and f) the ultrafiltration concentrate obtained in step e) is sterilized by heating and / or radiation sterilization to inactivate bacteria / viruses, Wherein: the radiation sterilization method is 60Co-r rays ≥ 25KGy, the heating temperature of the heating method is 60±0.5℃, and the continuous duration is 10 hours.

11. The preparation method according to claim 9, wherein the concentration of the first protein or polypeptide in the initial culture medium does not exceed 0.1 ug / mL.

12. A method for preparing a protein composition as claimed in any one of claims 4, 5 and 8, comprising: a) obtaining human fibroblasts from human skin tissue by using tissue blocks or digestion method; b) The human fibroblasts were cultured in vitro at 37°C and 3-10% CO 2 Under culture conditions, in vitro expansion is performed in an initial culture medium containing fetal bovine serum or a serum-free supplement; c) obtaining and collecting a large amount of cell culture supernatant during in vitro expansion; d) filtering the cell culture supernatant using a 0.1-0.3 μm microporous filter membrane or filter element to obtain a sterile filtrate; and e) the sterile filtered liquid obtained in step d) is ultrafiltered and concentrated through an ultrafiltration membrane to obtain an ultrafiltration concentrate, in: When the initial culture medium contains fetal bovine serum, the molecular weight of the ultrafiltration membrane is ≥80KD; when the initial culture medium contains serum-free additives, the molecular weight of the ultrafiltration membrane is ≥1KD. The obtained ultrafiltration concentrate is the protein composition, wherein the concentration of the second protein or polypeptide in the protein composition increases by more than 5 times relative to its concentration in the initial culture medium of the control group.

13. A medical device comprising an effective amount of the protein composition according to any one of claims 1 to 8.

14. A pharmaceutical composition comprising an effective amount of the protein composition according to any one of claims 1 to 8.

15. Use of the protein composition according to any one of claims 1 to 8 for preparing medical implants, medical devices or medicines.

16. Use of the protein composition according to any one of claims 1 to 8 for preparing medical devices or medicines for treating inflammatory diseases.

17. The use according to claim 16, wherein the inflammatory disease is selected from inflammatory skin diseases, autoimmune diseases, sterile inflammation, or a combination thereof.

18. The use of claim 17, wherein the inflammatory skin disease is selected from dermatitis, acne, sensitive skin, rosacea, melasma, lichen planus, lichen luster, lichen sclerosus, panniculitis, hidradenitis suppurativa, or a combination thereof.

19. The use of claim 17, wherein the autoimmune disease is selected from ankylosing spondylitis, allergic rhinitis, rheumatoid arthritis, ulcerative colitis, Crohn's disease, systemic lupus erythematosus, dermatomyositis, multiple sclerosis, Sjögren's syndrome, vasculitis, psoriasis, vitiligo, or a combination thereof.

20. The use of claim 17, wherein the sterile inflammation is selected from migraine, finger tenosynovitis, facial paralysis, scapulohumeral periarthritis, or a combination thereof.

21. Use of the protein composition according to any one of claims 1 to 8 for preparing medical devices or medicines for treating mycoplasma infection-related diseases.

22. The use according to claim 21, wherein the mycoplasma infection-related disease is selected from cervical hypertrophy, uterine prolapse, hemorrhoids, or a combination thereof.

23. Use of the protein composition according to any one of claims 1 to 8 for preparing medical devices or medicines for treating viral infections on the skin and mucous membrane surfaces.

24. The use according to claim 23, wherein the skin and mucosal surface viral infection diseases are selected from flat warts, filiform warts on the neck, HPV infection of the female genital tract, or a combination thereof.

25. Use of the protein composition according to any one of claims 1 to 8 for preparing medical devices or medicines for treating diseases caused by functional lubricating fluid deficiency.

26. The use according to claim 25, wherein the functional lubricating fluid deficiency disease is selected from senile arthritis, dry eye, or a combination thereof.

27. Use of the protein composition according to any one of claims 1 to 8 for preparing a medical device or medicine for treating hypertrophic scars.

28. The use of claim 27, wherein the hypertrophic scar is selected from thyroid hyperplasia, breast hyperplasia, prostatic hyperplasia, uterine fibroids, facial hypertrophic scars, or a combination thereof.

29. Use of the protein composition according to any one of claims 1 to 8 for preparing a medical device or medicine for treating alopecia.

30. The use of claim 29, wherein the hair loss disease is selected from alopecia areata, diffuse alopecia, scarring alopecia, or a combination thereof.

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