Protein composition, preparation method therefor, and use thereof

Abstract

Provided is a protein composition, comprising a first protein or polypeptide selected from a specific group. The protein composition exhibits therapeutic efficacy against inflammatory diseases. Further provided are a preparation method for the protein composition and use of the protein composition in the preparation of a medical implant, a medical device, or a medicine.

Description

A protein composition, its preparation method and application Technical Field

[0001] This application belongs to the field of biology, specifically relating to a protein composition and a method for preparing the same. This application also relates to the use of this protein composition in the preparation of medical implants, medical devices, or pharmaceuticals. Background Technology

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

[0003] Immune system disorders often lead to abnormal inflammation. Many inflammatory diseases arise when the immune system mistakenly triggers an inflammatory response in the absence of infection. Immune-mediated inflammatory diseases are the leading cause of disease death worldwide. Although the clinical symptoms of these diseases may appear different, they share many similarities in genetic background and pathophysiological pathways. Regardless of their complex etiologies, the pathogenesis of these diseases mostly involves inflammation and immunity. For example, inflammatory skin diseases are a common skin condition mediated by both immune cells and keratinocytes. Abnormal responses of the innate immune system, abnormal activation of T lymphocytes, and various inflammatory cytokines and their target cells (keratinocytes) play important roles in the pathogenesis of inflammatory skin diseases. Disruption of the immune balance among cell subsets leads to abnormal secretion of inflammatory cytokines such as interleukins, interferons, and tumor necrosis factor. These inflammatory cytokines further act on keratinocytes to induce damage to the skin barrier, resulting in inflammatory skin diseases.

[0004] For inflammatory diseases, immunosuppression or immunomodulation is currently the primary treatment approach. However, almost all immunosuppressants have significant and serious side effects. For example, widely used oral immunosuppressants, such as cyclosporine A, methotrexate, and azathioprine, used for severe inflammatory skin diseases, can lead to toxic blood levels and organ damage, particularly in the liver and kidneys. Furthermore, the rapid development of biopharmaceuticals in recent years has provided an increasing number of options, including monoclonal antibodies, immunoglobulins, and biologics specifically targeting immune molecules. While their efficacy is certain, they are expensive, systemic adverse reactions are not negligible, and the safety of long-term use is unclear. Although some inflammatory diseases involve multi-systemic systemic diseases, most inflammatory diseases manifest as localized diseases, such as allergic rhinitis and rheumatoid arthritis. For these localized inflammatory diseases, systemic administration methods such as intravenous or oral administration are often used clinically, often resulting in various drug side effects.

[0005] Furthermore, with the development of biomaterials and cell therapy, hematopoietic stem cell transplantation, autologous somatic cell or stem cell therapy, and collagen have all been applied clinically. It is worth mentioning the extracellular matrix (ECM), a fibrous gel network structure secreted by cells into the extracellular space, whose main components include collagen, fibronectin, elastin, and polysaccharides. (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.) The ECM provides support, protection, and nutrition to cells and is closely related to physiological activities such as cell metabolism, proliferation, and tissue wound repair. However, although the ECM occupies at least one-third of the tissue structure, its role in immunology has not been fully studied due to its complex and constantly changing structure, and there are no reports of systemic use of the ECM for the treatment of immune-mediated inflammatory diseases.

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

[0007] Furthermore, the present invention provides a protein composition that has a wide range of therapeutic effects not only on inflammatory diseases, but also on other related diseases, including but not limited to mycoplasma infections, viral infections of the skin and mucous membranes, diseases with functional lubrication deficiency, hypertrophic scars, and hair loss.

[0008] Another advantage of this invention is that it provides an efficient, large-scale preparation process for producing protein compositions using massive expansion of fibroblasts. (Invention Summary)

[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 A, protein containing EGF and pentaneine domain 1, 72kDa type IV collagenase, interstitial collagenase, and mixtures thereof, wherein the concentration of the first protein or polypeptide in the composition is greater than 1ug / mL.

[0010] In some embodiments, the protein composition further comprises a second protein or polypeptide different from the first protein or polypeptide, the second protein or polypeptide being selected from the group consisting of: thiol oxidase 1, core proteoglycan, fibronectin, vimentin, peronein-1, inhibitor of metalloproteinase-1, type I collagen αI chain, type I collagen αII chain, and mixtures thereof.

[0011] In some embodiments, the total protein concentration is detected by the BCA method, and the concentration of a single protein (such as the concentration of a first protein or polypeptide, or the concentration of a 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 mammalian fibroblasts from mammalian skin tissue using a tissue block or digestion method; b) in vitro incubating the mammalian fibroblasts at 37°C and 3-10% CO2. 2 Under culture conditions, in vitro amplification is carried out in the initial culture medium; c) a large amount of cell culture supernatant is obtained and collected during in vitro amplification; and d) the collected cell culture supernatant is filtered through a 0.1-0.3 μm microporous membrane or filter cartridge to obtain a sterile filtrate, which is the protein composition.

[0013] In some embodiments, the present invention provides a method for preparing the protein composition, the method comprising: a) obtaining human fibroblasts from human skin tissue using a tissue block or digestion method; b) in vitro incubating the human fibroblasts at 37°C and 3-10% CO2. 2 Under culture conditions, in vitro amplification is carried out in the initial culture medium; c) a large amount of cell culture supernatant is obtained and collected during in vitro amplification; and d) the collected cell culture supernatant is filtered through a 0.1-0.3 μm microporous membrane or filter cartridge to obtain a sterile filtrate, which is the protein composition.

[0014] In some embodiments, the present invention provides a method for preparing a protein composition comprising the aforementioned first protein or polypeptide and a second protein or polypeptide, the method comprising: a) obtaining human fibroblasts from human skin tissue using a tissue block or digestion method; b) in vitro incubating the human fibroblasts at 37°C and 3-10% CO2. 2 Under culture conditions, in vitro amplification is performed in an initial culture medium containing fetal bovine serum or serum-free additives; c) A large amount of cell culture supernatant is obtained and collected during in vitro amplification; d) The collected cell culture supernatant is filtered using a 0.1-0.3 μm microporous membrane or filter cartridge to obtain a sterile filtrate; and e) The sterile filtrate obtained in step d) is ultrafiltered and concentrated to obtain an ultrafiltration concentrate, wherein: when the initial culture medium contains fetal bovine serum, the molecular weight of the ultrafiltration membrane is ≥80 KD; when the initial culture medium contains serum-free additives, the molecular weight of the ultrafiltration membrane is ≥1 KD. The obtained ultrafiltration concentrate is the protein composition, wherein: the concentration of the second protein or polypeptide in the protein composition is more than 5 times higher than its concentration in the initial culture medium of the control group.

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

[0016] In another aspect, the present invention provides a medical device comprising an effective amount of the aforementioned protein composition.

[0017] In another aspect, the present invention provides the use of the aforementioned protein composition in the preparation of medical implants, medical devices or pharmaceuticals.

[0018] In another aspect, the present invention provides the aforementioned protein composition for the preparation of medical devices or pharmaceuticals for treating inflammatory diseases. In another aspect, the present invention provides the aforementioned protein composition for treating inflammatory diseases. In another aspect, the present invention provides a method for treating inflammatory diseases using the aforementioned protein composition. The inflammatory disease is preferably a localized inflammatory disease. In some embodiments, the inflammatory disease is selected from inflammatory skin diseases, autoimmune diseases, aseptic inflammation, or combinations thereof.

[0019] In another aspect, the present invention provides the aforementioned protein composition for the preparation of medical devices or pharmaceutical products for treating mycoplasma infections. In another aspect, the present invention provides the aforementioned protein composition for treating mycoplasma infections. In another aspect, the present invention provides a method for treating mycoplasma infections using the aforementioned protein composition.

[0020] In another aspect, the present invention provides the aforementioned protein composition for the preparation of medical devices or pharmaceutical products 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.

[0021] In another aspect, the present invention provides the aforementioned protein composition for the preparation of medical devices or pharmaceutical products for treating diseases of functional lubricant deficiency. In another aspect, the present invention provides the aforementioned protein composition for treating diseases of functional lubricant deficiency. In another aspect, the present invention provides a method for treating diseases of functional lubricant deficiency using the aforementioned protein composition.

[0022] In another aspect, the present invention provides the aforementioned protein composition for the preparation of medical devices or pharmaceutical products for treating hypertrophic scars. In another aspect, the present invention provides the aforementioned protein composition for treating hypertrophic scars. In another aspect, the present invention provides a method for treating hypertrophic scars using the aforementioned protein composition.

[0023] In another aspect, the present invention provides the aforementioned protein composition for the preparation of medical devices or pharmaceutical products for treating hair loss. In another aspect, the present invention provides the aforementioned protein composition for treating hair loss. In another aspect, the present invention provides a method for treating hair loss using the aforementioned protein composition. Attached Figure Description

[0024] Figure 1 is a photograph of a patient treated with senile arthritis according to an embodiment of the present invention. 1A - before the first treatment; 1B - two months after treatment.

[0025] Figure 2 shows photographs of patients treated with rosacea according to another embodiment of the present invention. 2A - Before the first treatment; 2B - Two months after treatment.

[0026] Figure 3 shows photographs of patients treated with alopecia areata according to another embodiment of the present invention. 3A - before the first treatment, 2B - third month of treatment, 3C - fourth month of treatment, 3D - sixth month of treatment, and 3E - eleventh month of treatment.

[0027] Figure 4 is a photograph of a patient treated with allergic rhinitis according to another embodiment of the present invention. 4A - before the first treatment; 4B - after two treatments.

[0028] Figure 5 is a photograph of a patient used to treat rheumatoid arthritis according to another embodiment of the present invention. 5A - Before the first treatment; 5B - 40 days after treatment.

[0029] Figure 6 is a patient photograph used for treating facial hypertrophic scars according to another embodiment of the present invention. 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.

[0030] Figure 7 is a photograph of a patient treated with filiform warts of the neck according to another embodiment of the present invention. 7A - Before the first treatment; 7B - Two months after treatment.

[0031] Figure 8 is a photograph of a patient treated with uterine prolapse according to another embodiment of the present invention. 8A - Before the first treatment; 8B - Two months after treatment. Detailed Description of the Invention

[0032] The protein composition of this invention comprises a first protein or polypeptide selected from a specific group and must reach a certain concentration. The applicant has surprisingly discovered that the protein composition of this invention, when applied to the human body, exhibits broad-spectrum and highly effective therapeutic effects on inflammatory diseases. Unconstrained by theory, the applicant believes that the therapeutic effect of the protein composition of this invention on inflammatory diseases and related diseases mainly lies in its effect on human immune cells, body fluids, and / or in situ cells. Specifically, due to its specific components and protein concentrations, after application to the human body, the protein composition of this invention can promote the migration and chemotaxis of immune cells, thereby facilitating and enhancing cellular immunity. Generally, whether cells migrate in the extracellular matrix and the speed of migration depend on the level and interaction of integrins and their corresponding ligands. Integrins are receptors located on the cell membrane surface, while the protein composition of this invention, particularly the proteins or polypeptides contained therein, are equivalent to ligands; their binding and interaction are important mechanisms determining cell adhesion and migration. Therefore, after application to the human body, this invention increases the anchoring and migration of immune cells, facilitating their function and thereby enhancing cellular immunity. Simultaneously, the injection of the protein composition of this invention increases extracellular fluid content and accelerates circulation, which is beneficial for enhancing humoral immune function. It is worth mentioning that, compared to the systemic administration of traditional immunotherapy, the protein composition of this invention can be administered locally, inducing the aggregation of immune cells locally, thereby effectively treating local inflammatory diseases and reducing and avoiding the side effects caused by systemic administration.

[0033] Furthermore, when applied to the human body, the proteins or peptides contained in this invention are absorbed by in situ cells, namely dermal fibroblasts, through collagen receptors (such as matrix metalloproteinase 14 (MT-MMP1) and mannose receptor type C-2 (MRC2)), 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 lubricants.

[0034] Based on the above mechanism, the applicant has discovered that the protein composition of the present invention can not only be used to treat inflammatory diseases, but also has broad therapeutic effects on other related diseases, including but not limited to mycoplasma infections, viral infections of the skin and mucous membranes, diseases caused by loss of functional lubrication, hypertrophic scars, and alopecia. Definition

[0035] As used in this application, the term "protein" refers to a large molecule composed of one or more long-chain amino acids, typically containing 50 or more amino acids. The term "peptide" refers to a linear amino acid chain, typically containing 2-50 amino acids. Proteins or peptides in this application may be found in living organisms, naturally secreted, or artificially recombinant.

[0036] As used in this application, "collagen" refers to the main structural protein in the extracellular matrix of various connective tissues in mammals. Amino acids bind together to form a long, thin, triple helix called the collagen helix. Collagen is the most abundant protein in mammals, including the human body, and is primarily found in connective tissues such as cartilage, bone, tendons, ligaments, and skin.

[0037] As used in this application, the term "fibroblast" refers to the most common cells in animal connective tissue, a cell type that synthesizes the extracellular matrix and collagen and plays a crucial role in wound healing. Fibroblasts typically have branched cytoplasm surrounding an oval, speckled nucleus with two or more nucleoli. Fibroblasts are derived from animal connective tissues such as skin.

[0038] The term "culture medium" as used in this application refers to the substance that supplies nutrients to cultured cells and promotes cell proliferation, and is also the living environment for cell growth and reproduction. Culture media include basal culture media and complete culture media. Complete culture media is prepared by adding animal-derived serum, such as fetal bovine serum, or serum-free additives (also known as "serum substitutes") to basal culture media. Basal culture media typically consists of amino acids, vitamins, carbohydrates, inorganic ions, etc., while additives such as fetal bovine serum or serum-free additives contain proteins required for cell growth and reproduction. The two are mixed to obtain complete culture media. The "initial culture medium" referred to in this application is actually the initial complete culture medium, that is, the complete culture medium before the addition of fibroblasts. The "control group initial culture medium" referred to in this application refers to the same initial culture medium as the one in the preferred preparation method of this invention, and which has undergone the same subsequent steps (including but not limited to culturing, refrigeration, filtration, concentration, bacterial / viral inactivation, etc.), as a control group for the protein composition of this invention.

[0039] As used in this application, the term "Fibroblast Conditioned Medium" refers to the cell culture supernatant produced during the in vitro culture, expansion, and preparation of cell suspensions of mammalian fibroblasts. This supernatant contains proteins or polypeptides secreted by fibroblasts, and is specifically the culture supernatant produced by in vitro expanded mammalian fibroblasts. The fibroblast conditioned medium referred to in this application is preferably human fibroblast conditioned medium.

[0040] As used in this application, the term "medical implant" refers to a device or tissue placed inside or on the surface of the body. Many implants are prostheses used to replace missing body parts; other types of implants are used to deliver drugs, monitor bodily functions, or provide support to organs and tissues.

[0041] As used in this application, the term "medical device" refers to an article, instrument, device, or machine used to prevent, diagnose, or treat disease, or to detect, measure, restore, correct, or modify bodily structure or function to achieve certain health purposes. Typically, the purpose of a medical device is not achieved through pharmacological, immunological, or metabolic means.

[0042] As used in this application, the term "medicine" refers to a composition or compound intended to treat or prevent disease, relieve symptoms, or aid in the diagnosis of disease. The term "medicine composition" refers to a composition comprising an effective amount of one or more active pharmaceutical ingredients, and pharmaceutically acceptable excipients, carriers, or diluents. Typically, the therapeutic or preventative effects of medicines are achieved through pharmacological, immunological, or metabolic means.

[0043] As used herein, the term "treatment" refers to any application of a therapeutic agent through a therapeutic regimen that achieves a desired effect, namely, partial or complete reduction, improvement, relief, suppression, delay of onset, reduction of severity, and / or reduction of 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 relevant disease, disorder, and / or condition and / or to subjects exhibiting only early signs of the disease, disorder, and / or condition, or to subjects exhibiting one or more identified signs of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be directed to subjects who have been diagnosed with the relevant 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 developing the relevant disease, disorder, and / or condition.

[0044] As used herein, the terms “effective amount,” “therapeutic effective amount,” or “pharmaceutical effective amount” refer to the amount of a therapeutic agent that imparts a therapeutic effect to a patient with a reasonable benefit / risk ratio suitable for any drug treatment. Such therapeutic effects can be objective (i.e., measurable by some test or label) or subjective (i.e., indicated or felt by the subject). In some embodiments, a “therapeutic effective amount” refers to the amount of a therapeutic agent or composition that effectively treats, improves, or prevents (e.g., delays onset) an associated 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.

[0045] As used in this application, the term "mixture" refers to adding ingredients together to achieve homogeneity, and the term "mixture" refers to a homogeneous mixture of ingredients.

[0046] In this application, when a composition is "substantially free" of a specific ingredient, it means that the composition contains less than trace amounts, or less than 0.1%, or less than 0.01%, or less than 0.001%, of the specific ingredient by weight of the composition.

[0047] In this application, unless the order is specifically specified, the terms “a)”, “b)”, and “c)” used to refer to steps in the preparation method do not indicate the order. For example, step a) may occur before or after step b), or steps b) and c) may occur simultaneously, or step b) may occur after step c).

[0048] The terms “comprising,” “including,” “containing,” “including,” “comprising,” “including,” “containing,” “containing,” and “containing” as used in this application are intended to be non-limiting, meaning that other ingredients and other steps may be added without affecting the final result. Protein composition

[0049] 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 μg / mL. The protein composition may be in any suitable form such as a liquid or gel, but is preferably a liquid.

[0050] With regard to its preferred preparation method (but not limited to this method), the protein composition of the present invention is essentially a fibroblast conditioned culture medium, particularly a human fibroblast conditioned culture medium, containing one or more proteins or polypeptides secreted by fibroblasts during in vitro expansion. As previously stated, the applicant has found that specific protein components (i.e., the first protein or polypeptide) and their concentrations 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, when applied to the human body, diffuses more easily through intercellular spaces, thus resulting in better clinical therapeutic effects.

[0051] 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, and preferably detected according to the BCA method. When containing two or more proteins or peptides, the total protein concentration of the protein composition of the present invention refers to the sum of the protein concentrations of each protein or peptide contained therein. The method for detecting protein concentration is described below.

[0052] The protein composition of the present invention may comprise one, two, or more proteins or peptides. In some embodiments, the protein composition of the present invention comprises at least two proteins or peptides, preferably at least three proteins or peptides, preferably at least four proteins or peptides, preferably at least five proteins or peptides, preferably at least eight proteins or peptides, and preferably at least ten proteins or peptides. Preferably, at least one of them is collagen, preferably at least two, preferably at least three, preferably at least four, preferably at least five, preferably at least eight, and preferably at least ten. For clarity, the aforementioned number of proteins or peptides includes the first protein or peptide.

[0053] In other embodiments, the protein composition of the present invention comprises at least three proteins or polypeptides, wherein at least one of them is collagen; preferably, it comprises at least four proteins or polypeptides, wherein at least two of them are collagen; and more preferably, it comprises at least five proteins or polypeptides, wherein at least three of them are collagen. The first protein or polypeptide

[0054] The protein composition of the present invention comprises a first protein or polypeptide 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.

[0055] In one embodiment, the first protein or polypeptide of the present invention is a type VI collagen α3 chain. In another embodiment, the first protein or polypeptide is Sushi, von Willebrand factor A, containing EGF and a pentane protein domain 1. In another embodiment, the first protein or polypeptide is a 72kDa type IV collagenase. In yet another embodiment, the first protein or polypeptide is an interstitial collagenase. Preferably, the first protein or polypeptide of the present invention is a mixture of two or more of the aforementioned proteins or polypeptides, such as a type VI collagen α3 chain and Sushi, von Willebrand factor A, containing EGF and a pentane protein domain 1, a type VI collagen α3 chain and a 72kDa type IV collagenase, or a 72kDa type IV collagenase and an interstitial collagenase. More preferably, the present invention comprises a mixture of the aforementioned four proteins or polypeptides: a type VI collagen α3 chain, Sushi, von Willebrand factor A, containing EGF and a pentane protein domain 1, a 72kDa type IV collagenase, and an interstitial collagenase.

[0056] With regard to the preferred preparation method (but not limited to this method), the first protein or polypeptide of the present invention is a new protein or polypeptide secreted during the in vitro expansion of fibroblasts, especially human fibroblasts, and is not present in the initial culture medium (details will be described in detail in the "Preparation Method" section below). In other words, the first protein or polypeptide constitutes the difference between the fibroblast conditioned culture 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 achieving the aforementioned therapeutic effects. Without being bound by theory, the applicant believes that, after being applied to the human body, the aforementioned first protein or polypeptide can be reused by fibroblasts, undergoing endocytosis and exocytosis, and then reassembled intracellularly to secrete an active new protein, thereby achieving a specific therapeutic effect.

[0057] In this invention, the concentration of the first protein or polypeptide in the protein composition is greater than 1 μg / mL, preferably greater than 1.5 μg / mL, preferably greater than 2 μg / mL, preferably greater than 3 μg / mL, preferably greater than 5 μg / mL, preferably greater than 8 μg / mL, preferably greater than 10 μg / mL, and preferably greater than 12 μg / mL. When two or more first proteins or polypeptides are included, the "concentration" of the first protein or polypeptide referred to in this application means the sum of the concentrations of the two or more first proteins or polypeptides in the protein composition. Furthermore, 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 in vivo diffusion range, and the better the therapeutic effect of the protein composition. Therefore, this invention does not require an upper limit on the concentration of the first protein or polypeptide.

[0058] In some embodiments, the first protein or polypeptide of the present invention is a type VI collagen α3 chain, and its concentration in the protein composition is greater than 2 μg / mL, preferably greater than 3 μg / mL, and more preferably greater than 4 μg / mL. In other embodiments, the first protein or polypeptide of the present invention is Sushi, von Willebrand factor A, containing EGF and a pentaneine protein domain 1, and its concentration in the protein composition is greater than 3 μg / mL, preferably greater than 4 μg / mL, and more preferably greater than 5 μg / mL. In other embodiments, the first protein or polypeptide of the present invention is a 72 kDa type IV collagenase, and its concentration in the protein composition is greater than 1.5 μg / mL, preferably greater than 2 μg / mL, and more preferably greater than 2.5 μg / 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 1 μg / mL, preferably greater than 1.5 μg / mL, and more preferably greater than 2 μg / mL.

[0059] The protein composition of the present invention may comprise any mixture of the aforementioned first proteins or polypeptides, provided that the sum of their concentrations in the protein composition is greater than 1 μg / mL; of course, when two or more of the aforementioned first proteins or polypeptides are included, their concentrations in the protein composition may be higher, preferably greater than 3 μg / mL, more preferably greater than 5 μg / mL, and more preferably greater than 7 μg / mL. In some embodiments, the present invention comprises two first proteins or polypeptides: type VI collagen α3 chain and Sushi, von Willebrand factor A, containing EGF and a pentaneine protein domain 1, and the sum of the concentrations of the two first proteins or polypeptides in the protein composition is greater than 5 μg / mL, preferably greater than 6 μg / mL, and more preferably greater than 8 μg / mL. In other embodiments, the present invention comprises two first proteins or polypeptides: type VI collagen α3 chain and 72 kDa type IV collagenase, and the sum of the concentrations of the two first proteins or polypeptides in the protein composition is greater than 3 μg / mL, preferably greater than 4 μg / mL, and more preferably greater than 5 μg / mL. In other embodiments, the present invention comprises two first proteins or polypeptides: a 72kDa type IV collagenase and Sushi, von Willebrand factor A, containing EGF and a pentaneine 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, and more preferably greater than 7ug / mL.

[0060] In some preferred embodiments, the present invention comprises a mixture of the aforementioned four proteins or peptides: type VI collagen α3 chain, Sushi, von Willebrand factor A, a protein containing EGF and a pentaneine domain 1, a 72 kDa type IV collagenase, and interstitial collagenase, wherein the sum of the concentrations of the four proteins or peptides in the protein composition is greater than 8 μg / mL, preferably greater than 10 μg / mL, and more preferably greater than 12 μg / mL. A second protein or peptide...

[0061] 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), core proteoglycan (DCN), fibronectin (FN), vimentin, fibulin-1 (FBLN1), metalloproteinase inhibitor 1 (TIMP1), type I collagen alpha-1(I) chain (COL1A1), type I collagen alpha-2(I) chain (COL1A2), and mixtures thereof.

[0062] In one embodiment, the second protein or polypeptide of the present invention is thiol oxidase 1. In another embodiment, the second protein or polypeptide is core proteoglycan. In another embodiment, the second protein or polypeptide is a 72 kDa type IV collagenase. In another embodiment, the second protein or polypeptide is fibronectin. In another embodiment, the second protein or polypeptide is peroneal protein-1. In another embodiment, the second protein or polypeptide is a type I collagen αI chain. In another embodiment, the second protein or polypeptide is a type I collagen αII chain. Preferably, the second protein or polypeptide of the present invention is a mixture of two or more of the aforementioned proteins or polypeptides. The mixture can be any combination of the listed proteins or polypeptides, such as thiol oxidase 1 and core proteoglycan, thiol oxidase 1 and fibronectin, peroneal protein-1 and type I collagen αII chain, thiol oxidase 1, core proteoglycan and fibronectin, or thiol oxidase 1, fibronectin and type I collagen αII chain. In some preferred embodiments, the present invention comprises a mixture of three of the aforementioned proteins or peptides, preferably a mixture of five of the aforementioned proteins or peptides, and more preferably a mixture comprising all eight of the aforementioned proteins or peptides.

[0063] With regard to the preferred preparation method (but not limited to this method), the second protein or polypeptide of the present invention is a protein or polypeptide that rapidly proliferates during the in vitro expansion of mammalian fibroblasts, especially human fibroblasts, even though it is also present in the initial culture medium. Its content increases by tens or even hundreds of times during cell expansion (details will be described 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 distinction between the fibroblast conditioned culture medium obtained according to the preferred preparation method and the initial culture medium. Based on this, the preferred protein composition has a superior therapeutic effect on inflammatory diseases. Without being bound by theory, the applicant believes that, similar to the role of 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, re-secreting active new proteins, thereby further enhancing the therapeutic effect.

[0064] In this invention, the concentration of the second protein or polypeptide in the protein composition is greater than 5 μg / mL, preferably greater than 10 μg / mL, more preferably greater than 20 μg / mL, more preferably greater than 30 μg / mL, and more preferably greater than 50 μg / mL. When two or more second proteins or polypeptides are included, the "concentration" of the second protein or polypeptide referred to in this application means the sum of the concentrations of the two or more second proteins or polypeptides in the protein composition. Furthermore, 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, this invention does not impose an upper limit on the concentration of the second protein or polypeptide.

[0065] In some embodiments, the second protein or polypeptide of the present invention is thiol oxidase 1, and its concentration in the protein composition is greater than 5 μg / mL, preferably greater than 10 μg / mL, and more preferably greater than 15 μg / mL. In other embodiments, the second protein or polypeptide of the present invention is a core proteoglycan, and its concentration in the protein composition is greater than 5 μg / mL, preferably greater than 7 μg / mL, and more preferably greater than 10 μg / mL. In other embodiments, the second protein or polypeptide of the present invention is a fibronectin, and its concentration in the protein composition is greater than 50 μg / mL, preferably greater than 100 μg / mL, more preferably greater than 300 μg / mL, more preferably greater than 500 μg / mL, more preferably greater than 700 μg / mL, and more preferably greater than 900 μg / 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 3 μg / mL, preferably greater than 5 μg / mL, and more preferably greater than 7 μg / 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 5 μg / mL, preferably greater than 8 μg / mL, and more preferably greater than 10 μg / mL. In some embodiments, the second protein or polypeptide of the present invention is metalloproteinase inhibitor-1, and its concentration in the protein composition is greater than 0.5 μg / mL, preferably greater than 1 μg / mL, and more preferably greater than 1.2 μg / mL. In some embodiments, the second protein or polypeptide of the present invention is a type I collagen αI chain, and its concentration in the protein composition is greater than 7 μg / mL, preferably greater than 10 μg / mL, and more preferably greater than 13 μg / mL. In some embodiments, the second protein or polypeptide of the present invention is a type I collagen αII chain, and its concentration in the protein composition is greater than 10 μg / mL, preferably greater than 15 μg / mL, more preferably greater than 20 μg / mL, and more preferably greater than 25 μg / mL.

[0066] The protein composition of the present invention may comprise any mixture of the aforementioned second proteins or polypeptides. When two or more of the aforementioned second proteins or polypeptides are included, their concentrations in the protein composition may be higher, preferably greater than 10 μg / mL, preferably greater than 15 μg / mL, preferably greater than 20 μg / mL, preferably greater than 30 μg / mL, preferably greater than 50 μg / mL, and preferably greater than 100 μg / mL. In some embodiments, the present invention comprises two second proteins or polypeptides: thiol oxidase 1 and core proteoglycan, and the sum of the concentrations of the two second proteins or polypeptides in the protein composition is greater than 10 μg / mL, preferably greater than 13 μg / mL, and preferably greater than 18 μg / mL. In other embodiments, the present invention comprises two second proteins or polypeptides: thiol oxidase 1 and fibronectin, and the sum of the concentrations of the two second proteins or polypeptides in the protein composition is greater than 500 μg / mL, preferably greater than 700 μg / mL, and preferably greater than 900 μg / mL. In some embodiments, the present invention comprises two second proteins or polypeptides: fibular 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 10 μg / mL, preferably greater than 15 μg / mL, and more preferably greater than 20 μg / mL. In other embodiments, the present invention comprises two second proteins or polypeptides: fibular 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 10 μg / mL, preferably greater than 20 μg / mL, and more preferably greater than 30 μg / mL.

[0067] In some preferred embodiments, the present invention comprises three or more of the second proteins or peptides, wherein the sum of the concentrations of the three or more second proteins or peptides in the composition is greater than 10 μg / mL. More preferably, the present invention comprises five or more of the second proteins or peptides, wherein the sum of the concentrations of the five or more second proteins or peptides in the composition is greater than 20 μg / mL. More preferably, the present invention comprises a mixture of the aforementioned eight proteins or peptides: thiol oxidase 1, core proteoglycan, fibronectin, vimentin, peroneal protein-1, inhibitor of metalloproteinase-1, type I collagen αI chain, and type I collagen αII chain, and the sum of the concentrations of the eight proteins or peptides in the protein composition is greater than 300 μg / mL, preferably greater than 500 μg / mL, and more preferably greater than 900 μg / mL.

[0068] In a preferred embodiment, the present invention comprises the aforementioned first protein or polypeptide and 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 μg / mL, and the concentration of the second protein or polypeptide in the composition is greater than 10 μg / mL.

[0069] In a preferred embodiment, the present invention comprises the aforementioned first protein or polypeptide and second protein or polypeptide, wherein the first protein or polypeptide is a type VI collagen α3 chain, and the concentration in the composition is greater than 3 μg / mL, preferably greater than 4 μg / mL; the second protein is fibronectin and / or type I collagen αII chain, and the concentration in the composition is greater than 20 μg / mL, preferably greater than 25 μg / mL; and the total protein concentration of the protein composition is greater than 12 mg / mL. Other components

[0070] In addition to the aforementioned first protein or polypeptide and second protein or polypeptide, the protein composition of the present invention may also contain other components. In some embodiments, the protein composition further comprises a protein or polypeptide selected from the group consisting of: 3-Pentraxin-related protein PTX3, adipocyte enhancer-binding protein 1, collagen alpha-1(III) chain, adiponectin, complement factor H-related protein 1, cysteine ​​protease inhibitor-C, ectonucleotide pyrophosphatase / phosphodiesterase family member 2, extracellular matrix protein 1, fetuin-B, follistatin-related protein 1, and 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, and Sex hormone-binding globulin. globulin),The SH3 domain binds to glutamic acid-rich-like protein 3, protein S100-A6, cathepsin B, serine protease HTRA1, transforming growth factor-beta-induced protein ig-h3, apolipoprotein C-III, and mixtures thereof.

[0071] With regard to its preferred preparation method (but not limited to this method), the protein composition of the present invention is essentially a fibroblast conditioned culture 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 a serum-free additive. Therefore, the protein composition may also contain one or more components found in conventional basal culture media, fetal bovine serum, or serum-free additives. Such components include, but are not limited to: growth factors such as natural or synthetic growth factors, amino acids such as glutamine, leucine, lysine, and 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, while in other embodiments it may also contain proteins, lipids, etc., derived from serum-free additives. Preparation Method

[0072] The protein compositions 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 at a specific concentration. For example, one or more recombinant proteins or polypeptides, such as recombinant fibronectin, can be commercially available and mixed with deionized water in a specific ratio to obtain the protein compositions of the present invention. Those skilled in the art will understand that even if certain proteins or polypeptides are difficult to obtain directly, the desired proteins or polypeptides can be obtained indirectly by purchasing the corresponding raw materials. For example, type VI collagen α3 chains can be obtained by recombinant type VI collagen, and type I collagen αI chains can be obtained by recombinant type I collagen.

[0073] Preferably, the protein composition of the present invention is a fibroblast conditioned culture medium, that is, a culture supernatant produced by in vitro expanded mammalian fibroblasts, especially human fibroblasts, under specific culture conditions. Compared with the aforementioned conventional preparation methods, this method has low production costs and can achieve efficient and large-scale preparation of the protein composition of the present invention.

[0074] 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 using a tissue block or digestion method; b) in vitro incubating the human fibroblasts at 37°C and 3-10% CO2. 2 Under culture conditions, in vitro amplification is carried out in the initial culture medium; c) a large amount of cell culture supernatant is obtained and collected during in vitro amplification; and d) the collected cell culture supernatant is filtered through a 0.1-0.3 μm microporous membrane or filter cartridge to obtain a sterile filtrate, which is the protein composition.

[0075] Step a) above refers to primary cell culture, which is the process of obtaining target cells from a tissue suspension after dispersing the tissue, typically using the tissue block method or digestion method. In the preferred preparation method of this invention, the tissue is human skin tissue, and the target cells are human fibroblasts, i.e., step a) is primary human fibroblast culture. Preferably, the aforementioned human skin tissue is obtained after routine serological testing for HIV, HBV, HCV, and syphilis has been negative; more preferably, it has been negative for mycoplasma and bacteria, obtained by aseptically harvesting skin separately or by harvesting discarded or intentionally harvested skin from surgeries such as blepharoplasty, double eyelid surgery, wrinkle removal, and circumcision. Preferably, the obtained human skin tissue is cultured into human fibroblasts using the conventional tissue block method or enzymatic digestion method.

[0076] Step b) above refers to in vitro cell expansion, which can be performed using any conventional method, such as using ordinary cell culture flasks, or using roller bottles or hollow fiber tube cell culture systems. The primary human fibroblasts obtained in step a) are then expanded in vitro in the initial culture medium at 37°C and 3-10% CO2. Preferably, the culture conditions are 37°C and 4-8% CO2, and more preferably 37°C and 5% CO2.

[0077] The initial culture medium provides the basal environment necessary for cell growth, proliferation, and functional maintenance. It can be selected from any conventional initial culture medium and is commercially available, such as those purchased from Sigma-Aldrich or Thermo Fisher Scientific. In some embodiments, the initial culture medium can be prepared by adding animal-derived serum, serum-free additives, or mixtures thereof to a basal culture medium. Typically, the basal culture medium and its serum or serum-free additive are available together. 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, RPMI 1640, Glasgow's Minimal Essential Medium (GMEM), α Minimal Essential Medium (αMEM), Iscove's Modified Dulbecco's Medium, or M199. In some preferred embodiments, the animal-derived serum 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 has no serum additives, preferably fetal bovine serum.

[0078] In step c) above, a large amount of cell culture supernatant is obtained and collected during the in vitro expansion of fibroblasts. In some embodiments, the fibroblast supernatant is collected when the cell coverage is ≥90%. Preferably, the collected fibroblast supernatant is stored in a refrigerator at 4°C. Storage can be done by storing fibroblast supernatant collected from a single culture separately, or by storing a mixture of supernatants collected from multiple cultures, or by storing a mixture of supernatants from multiple generations of fibroblasts.

[0079] In step d) above, the collected cell culture supernatant is filtered using a 0.1-0.3 μm microporous membrane or filter cartridge to obtain a sterile filtrate. Preferably, the microporous membrane has a pore size of 0.1-0.25 μm, more 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 after step d) is the protein composition. However, in some preferred embodiments, the sterile filtrate may undergo further processing steps (detailed below) to obtain a more concentrated or sterile protein composition.

[0080] In some preferred embodiments, the aforementioned preparation method further includes: e) the sterile filtrate obtained in step d) is concentrated by ultrafiltration through an ultrafiltration membrane to obtain an ultrafiltration concentrate.

[0081] Step e) above, following step d), primarily aims 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 different pore sizes may be selected depending on the initial culture medium. When the initial culture medium contains fetal bovine serum, the preferred molecular weight of the ultrafiltration membrane is ≥80 KD, more preferably ≥90 KD, and even more preferably ≥100 KD. When the initial culture medium contains serum-free additives, the molecular weight of the ultrafiltration membrane is ≥1 KD, more preferably ≥3 KD, and even more preferably ≥5 KD.

[0082] In some preferred embodiments, the aforementioned preparation method further includes: f) inactivating bacteria / viruses in the obtained sterile filtrate or ultrafiltration concentrate.

[0083] Step f) above can be performed after step d) above, i.e., bacterial / viral inactivation of the obtained sterile filtrate, preferably after step e) above, i.e., bacterial / viral inactivation of the obtained ultrafiltration concentrate. Sterilization can be performed using any conventional sterilization method, preferably using heating and / or radiation sterilization, more preferably using both heating and radiation sterilization, and even more preferably using heating first, followed by radiation sterilization. The heating method is preferably a constant temperature water bath, preferably at a temperature of 60±5℃ for a continuous duration of 8-12 hours, more preferably at a temperature of 60±0.5℃ for a continuous duration of 10 hours. The radiation sterilization method is preferably using 60Co-r rays ≥25KGy.

[0084] Preferably, some or all of the steps of the aforementioned preparation method are performed in a clean bench, preferably a Class 100 clean bench.

[0085] As described above, in conjunction with the aforementioned preparation method, the first protein or polypeptide of the present invention is a novel protein or polypeptide secreted during the in vitro expansion of mammalian fibroblasts, especially human fibroblasts. It is substantially absent from the initial culture medium or the initial culture medium of the control group, or has only extremely low concentrations or is undetectable 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 μg / mL, preferably not more than 0.01 μg / mL, and most preferably not more than 0.001 μg / mL.

[0086] On the other hand, in conjunction with the aforementioned preparation method, the second protein or polypeptide of the present invention is a protein or polypeptide that rapidly increases or even doubles during in vitro expansion of mammalian fibroblasts, especially human fibroblasts, even though it is also present in the initial culture medium; its content increases by tens or even hundreds of times during cell expansion. Preferably, in the method for preparing a protein composition comprising the first protein or polypeptide and the 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, and 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 exhibits a relatively high concentration in the obtained protein composition, preferably greater than 5 μg / mL, preferably greater than 10 μg / mL, preferably greater than 20 μg / mL, preferably greater than 30 μg / mL, and preferably greater than 50 μg / mL. Uses and Indications

[0087] The protein compositions of the present invention have broad therapeutic applications for inflammatory diseases and other related diseases. In one aspect, the protein compositions of the present invention are used in the preparation of medical implants, medical devices, or pharmaceuticals, preferably in the preparation of medical devices or pharmaceuticals. The protein compositions of the present invention can also be used in cosmetics. In some embodiments, the protein composition is the sterile filtrate obtained in step d) of the aforementioned preparation method, preferably the ultrafiltration concentrate obtained in step e), and more preferably the sterile concentrate obtained in step f).

[0088] When used for the foregoing purposes, the protein composition of the present invention can be directly administered to a patient. In another embodiment, the medical device, pharmaceutical product, or drug composition used for the foregoing purposes comprises an effective amount of the protein composition of the present invention, as well as a pharmaceutically acceptable excipient, carrier, or diluent. This “pharmaceutically acceptable excipient, carrier, or diluent” includes, but is not limited to, any adjuvant, carrier, excipient, flow aid, sweetener, diluent, preservative, dye / coloring agent, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier permitted by the relevant governmental regulatory authority for acceptable use in humans or livestock.

[0089] The protein compositions of the present invention can be administered to patients via a variety of routes, such as transdermal, dermal, subcutaneous, intranasal, intravenous, intramuscular, intrathecal, regional, or local (e.g., mucosal). The most suitable route of administration in any given situation will depend on the subject, the nature and severity of the disease, and the subject's physical condition, etc. Accordingly, the pharmaceuticals of the present invention can be prepared in different dosage forms depending on the different methods of administration. For example, in one embodiment, the medical device or pharmaceutical product comprising an effective amount of the protein composition of the present invention can be prepared as an injection, gel, etc. Preferably, the protein compositions of the present invention are administered to the affected area via subcutaneous or dermal injection.

[0090] In one aspect, the protein composition of the present invention is used to prepare medical devices or pharmaceuticals for treating inflammatory diseases. In another aspect, the protein composition of the present invention is used to treat inflammatory diseases. In yet another aspect, the present invention provides a method for treating inflammatory diseases using the aforementioned protein composition. Preferably, the inflammatory disease is a localized inflammatory disease. In some embodiments, the inflammatory disease is selected from inflammatory skin diseases, autoimmune diseases, aseptic inflammation, or combinations thereof.

[0091] The aforementioned inflammatory skin disease can be any inflammatory skin disease, preferably an immune-related inflammatory skin disease. Preferably, the inflammatory skin disease is selected from dermatitis, acne, sensitive skin, rosacea, melasma, lichen planus, lichen sclerosus, lichen sclerosus, panniculitis, hidradenitis suppurativa, or a combination thereof. 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, melasma, 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 inflammatory skin diseases mainly lies in its effect on in situ cells in the human body. Specifically, when applied to the human body, for example, subcutaneously or via dermal injection, the proteins or polypeptides contained in the protein composition are absorbed by dermal fibroblasts in vivo and reassembled into collagen, thus increasing local tissue elasticity and repairing skin damage.

[0092] Autoimmune diseases refer to disease states caused by the body's immune system's immune response to its own components, leading to damage to its own tissues. The autoimmune diseases to which this invention applies can be any type of autoimmune disease. 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 combinations thereof. More preferably, the autoimmune diseases are selected from ankylosing spondylitis, allergic rhinitis, rheumatoid arthritis, or combinations thereof. Without being bound by theory, the applicant believes that the therapeutic effect of the protein composition of this invention on autoimmune diseases mainly lies in its effect on human immune cells and humoral processes. When applied to the human body, the protein composition of this invention, particularly the proteins or polypeptides contained therein, increases the anchoring and migration of immune cells and promotes the circulation of local extracellular fluid, thereby enhancing humoral and cellular immune function and providing relief and treatment for autoimmune diseases.

[0093] Specifically, the pathogenesis of ankylosing spondylitis (AS) primarily stems from genetic factors, chronic inflammation, and the periodic tension generated by upright walking. Under the combined influence of these factors, the soft tissues surrounding the damaged spine continuously secrete type I collagen to achieve mechanical balance, invading the intervertebral joints to form scar tissue in an attempt to stabilize the joints. Therefore, ankylosing spondylitis progresses upwards, eventually leading to a bamboo spine appearance. Surprisingly, the applicant discovered that the implantation of the protein composition of this invention not only enhances humoral and cellular immune function but also generates new matrix metalloproteinases (MMPs) that degrade collagen, reducing type I collagen production and increasing degradation, thus reducing scarring in various spinal segments, thereby alleviating pain and symptom relief.

[0094] Aseptic inflammation refers to an inflammatory response caused by various non-biological factors or unknown reasons. The aseptic inflammation applicable to this invention can be any type of aseptic inflammation. Preferably, the aseptic inflammation is selected from migraine, finger tenosynovitis, facial paralysis, frozen shoulder, or combinations thereof. Without being bound by theory, the applicant believes that the therapeutic effect of the protein composition of this invention on aseptic inflammation is similar to the aforementioned therapeutic effect on autoimmune diseases, mainly due to its effect on human immune cells and body fluids.

[0095] In another aspect, the present invention provides the aforementioned protein composition for the preparation of medical devices or pharmaceuticals for treating mycoplasma infections. In another aspect, the present invention provides the aforementioned protein composition for treating mycoplasma infections. In another aspect, the present invention provides a method for treating mycoplasma infections using the aforementioned protein composition. Preferably, the mycoplasma infection is selected from cervical hypertrophy, uterine prolapse, hemorrhoids, or combinations thereof. Without being bound by theory, the applicant believes that, in addition to local swelling caused by mycoplasma infection, the aforementioned diseases are further aggravated by the postural gravity of upright walking, which leads to a continuous worsening of the original swelling and a slowing of extracellular fluid circulation, thus further aggravating the symptoms. However, by injecting the protein composition of the present invention, the extracellular fluid circulation at the aforementioned sites is accelerated, humoral and cellular immunity are enhanced, which is beneficial for clearing mycoplasma infection and reducing swelling.

[0096] In another aspect, the present invention provides the aforementioned protein composition for the preparation of medical devices or pharmaceuticals 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. Generally, the aforementioned viral infections of the skin and mucous membranes are caused by different subtypes of human papillomavirus and / or other viral infections. Preferably, the aforementioned viral infections of the skin and mucous membranes are selected from flat warts, filiform warts of the neck, female genital tract HPV infection, or combinations thereof. Without being bound by theory, the applicant believes that the protein composition of the present invention, by enhancing humoral and cellular immunity, can help or achieve the effect of clearing such viruses.

[0097] In another aspect, the present invention provides the aforementioned protein composition for the preparation of medical devices or pharmaceuticals for treating diseases of functional synovial fluid deficiency. In another aspect, the present invention provides the aforementioned protein composition for treating diseases of functional synovial fluid deficiency. In another aspect, the present invention provides a method for treating diseases of functional synovial fluid deficiency using the aforementioned protein composition. The functional synovial fluid deficiency diseases to which the present invention applies can be any known such diseases; preferably, functional synovial fluid deficiency diseases are selected from senile arthritis, dry eye syndrome, or combinations thereof. When used to treat senile arthritis, the protein composition of the present invention is preferably injected into the joint capsule (rather than the joint cavity). Without being bound by theory, the pathogenesis of senile arthritis lies in the inability of the synovial cells of the joint capsule wall to produce sufficient synovial fluid to lubricate the articular cartilage. The applicant has discovered that after the protein composition of the present invention is injected around the joint capsule, it increases extracellular fluid and accelerates blood circulation, thereby enabling the synovial cells to produce sufficient synovial fluid, thereby achieving the purpose of nourishing the articular cartilage and treating senile arthritis.

[0098] In another aspect, the present invention provides the aforementioned protein composition for the preparation of medical devices or pharmaceuticals for treating hypertrophic scars. In another aspect, the present invention provides the aforementioned protein composition for treating hypertrophic scars. In another aspect, 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 can be any known such disease; preferably, the hypertrophic scars are selected from thyroid hyperplasia, breast hyperplasia, prostatic hyperplasia, uterine fibroids, facial hypertrophic scars, or combinations thereof. Without being bound by theory, research suggests 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 induce the regeneration of matrix metalloproteinases, reduce the production of type I collagen, and increase its degradation, thereby alleviating hypertrophic scars.

[0099] In another aspect, the present invention provides the aforementioned protein composition for the preparation of medical devices or pharmaceuticals for treating hair loss diseases. In another aspect, the present invention provides the aforementioned protein composition for treating hair loss diseases. In another aspect, the present invention provides a method for treating hair loss diseases using the aforementioned protein composition. The hair loss diseases to which the present invention is applicable can be any known such diseases, or the present invention can be used for the purpose of hair growth (e.g., eyelashes) on any part of the human body. Preferably, the hair loss disease is selected from alopecia areata, diffuse hair loss, cicatricial alopecia, or a combination thereof. Without being bound by theory, the applicant believes that the proteins or polypeptides contained in the present invention are reassembled into collagen in situ by cells in vivo, thereby improving the hair follicle growth environment and enabling resting hair follicles to regrow hair. Methods for detecting protein concentration.

[0100] In this application, the total protein concentration and individual protein concentration are detected using different methods, namely the BCA method and liquid chromatography-mass spectrometry, which are detailed below. 1. Determination of total protein concentration using the BCA method

[0101] The total protein concentration of the protein composition described in this application can be determined using any conventional BCA method. The experimental procedure is as follows: Dilute the sample 50-fold with PBS diluent, then perform a standard curve using BSA protein standards (range: 0.05-0.5 mg / mL), and determine the total protein concentration of the sample. The BCA protein concentration assay kit used is available from Solarbio. 2. Determination of individual protein concentration using liquid chromatography-mass spectrometry.

[0102] The concentration of individual proteins or peptides (such as the first protein or peptide, the second protein or peptide) in the protein composition of this application can be determined using any conventional liquid chromatography-mass spectrometry method, with appropriate instruments selected. In some embodiments, the following instruments may be used: electronic balance (Mettler Toledo Instruments (Shanghai) Co., Ltd., model: XPE205), microplate reader (Molecular devices, model: Spectramax M5), and combined high-resolution mass spectrometer (Thermo Scientific, model: Exploris 480).

[0103] The experimental and analytical process is as follows:

[0104] 1) Sample processing

[0105] The sample was diluted with 50 mM NH4HCO3 solution, and standard proteins were added as internal standards. Reduction-alkylation was performed using DL-dithiothreitol and iodoacetamide. After trypsin digestion, the supernatant was collected for liquid chromatography-mass spectrometry analysis. The standard proteins were Protein_AG, Thioredoxin, Klenow, Carbonic, IGF-I_LR3, and Protein_G. DL-dithiothreitol and iodoacetamide were available from Sigma-Aldrich.

[0106] 2) Liquid Chromatography Conditions: Column: BEH C18 2.1 mm × 150 mm, 1.7 μm; Column temperature: 60 °C; Mobile phase: A: 0.1% formic acid-water; B: 0.1% formic acid-acetonitrile; Flow rate: 0.2 mL / min;

[0107] 3) Mass spectrometry conditions: Detection mode: positive ion; Spray voltage: 3.8kV; Capillary temperature: 320℃; Evaporation temperature: 350℃; Resolution: Level 1 60,000@m / z 200; Level 2 15,000@m / z 200; Precursor ion scan range: m / z 300-1600.

[0108] 4) Qualitative and quantitative analysis of protein components

[0109] The experimental mass spectrometry data were retrieved using Proteome Discoverer 2.5 (Thermo Fisher Scientific) software. The database used was the uniprot database downloaded on November 27, 2023 (uniprotkb_taxonomy_id_9606_reviewed_2023_11_27, containing 20429 proteins) plus the sequences of Thermo standards. Search parameters were set as follows: trypsin digestion with high specificity and a maximum missed cleavage number of 2; minimum peptide length of 6 amino acid residues; primary precursor ion mass error tolerance of 10 ppm and secondary fragment ion mass error tolerance of 0.02 Da. Variable peptide modifications included N-terminal acetylation, methionine oxidation, and asparagine and glutamine deamidation. Cysteine ​​alkylation was set as a fixed modification.

[0110] Based on the total ion chromatogram, base peak chromatogram, and secondary mass spectrometry (LC-MS) chromatogram of the sample, qualitative and quantitative analysis of protein components can be performed. The detection of two or more characteristic peptides in a protein / protein peptide chain, with a high abundance in the sample, confirms detection. 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 peak areas of the standard peptides and the sample peptides at 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 aforementioned six standard proteins is taken as the protein content of the sample (Formula 3), and the protein concentration in the sample is calculated according to Formula 4. Detailed Implementation

[0111] The embodiments described herein are intended to illustrate the invention, but are not intended to limit or otherwise define the scope of the invention.

[0112] 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 on the therapeutic effects of Examples 6 and 8-10. Examples 1-6: Protein Compositions

[0113] The protein compositions shown in Table 1 consist of the listed ingredients at the listed concentrations (ug / mL). Table 1 Example 7: Preparation method of protein compositions from Examples 1-6

[0114] The first or second protein or polypeptide listed in Table 1 was purchased through commercial channels and mixed with deionized water in a specific ratio to achieve the protein concentrations listed in the table. Among them, type VI collagen α3 chain is derived from recombinant type VI collagen protein and is available from MyBioSource.com; Sushi, von Willebrand factor A, containing EGF and pentane protein domain 1, is a recombinant human SVEP1 control fragment and is available from Sigma-Aldrich; 72kDa type IV collagenase is available from Sigma-Aldrich, MDL number MFCD01324322; interstitial collagenase is available from MyBioSource.com; thiol oxidase 1 recombinant protein is available from MyBioSource.com; core proteoglycan is available from Sigma-Aldrich, MDL number MFCD00283027; fibronectin is available from Sigma-Aldrich, MDL number MFCD00131062; vimentin is available from MedChemExpress; and fibular protein-1 is recombinant human fibular 1C protein and is available from R&D. Systems, Inc.; Inhibitor of metalloproteinases-1 (IMF-1) was available from MedChemExpress; Type I collagen αI and αII chains were derived from recombinant Type I collagen and were available from MyBioSource.com. Examples 8-11: Protein Compositions

[0115] The protein compositions shown in Table 2 comprise the listed components and water at the listed concentrations (ug / mL), and may also contain other proteins or peptides. Protein compositions 8-10 are the protein compositions of the present invention, while composition 11 is the control group. Compared to control group 11, the protein compositions of the present invention contain specific proteins or peptides, and the content of certain proteins or peptides in the protein compositions of the present invention is significantly higher than their content in the control group. The concentrations of the listed components were determined by the aforementioned liquid chromatography-mass spectrometry detection method.

[0116] According to the aforementioned BCA method, the total protein concentrations in 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

[0117] The preparation method of the protein composition in Example 8 is as follows:

[0118] 1. Before receiving treatment, the subjects routinely undergo serological tests for HIV, HBV, HCV, and syphilis. Samples are collected from those who test negative and discarded from those who test positive.

[0119] 2. Tissues obtained by aseptically harvesting skin separately or by using discarded or intentionally retained skin from surgeries such as double eyelid surgery, eye bag removal, wrinkle removal, and circumcision for in vitro cell culture;

[0120] 3. Primary fibroblasts were cultured using conventional tissue blocks or enzymatic digestion methods 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.

[0121] 4. In vitro cell expansion was performed using a roller bottle method;

[0122] 5. Once the cell culture coverage reaches ≥90%, passage the cells and collect the supernatant from passage 6. Store the collected cell culture supernatant at 4°C.

[0123] 6. The preserved cell culture supernatant was filtered through a 0.22 μm microporous membrane to obtain sterile filtrate;

[0124] 7. The obtained sterile filtrate is subjected to protein concentration through an ultrafiltration membrane of ≥80KD to obtain ultrafiltration concentrate;

[0125] 8. The obtained ultrafiltration concentrate was first heated and then sterilized by radiation to inactivate bacteria / viruses, thus obtaining the protein composition of Example 7. The heating conditions for the heating method were 60±0.5℃ for a continuous duration of about 10 hours, and the radiation sterilization method used 60Co-r rays ≥25KGy.

[0126] Some or all of the above steps were performed in a Class 100 clean bench. Example 13: Preparation method of the protein composition from Example 9

[0127] Example 9: The preparation method of the protein composition is the same as in Example 12 above, except that in step 5, the supernatant of 8th generation fibroblasts is collected. Example 14: Preparation method of the protein composition from Example 10

[0128] Example 10: The preparation method of the protein composition is the same as that in Example 12 above, except that in step 5, the supernatant of 11 generations of fibroblasts is collected. Example 15: Preparation method of control group 11

[0129] The preparation method of control group 11 is as follows:

[0130] 1. Take the same initial culture medium as in Examples 12-14, i.e., DMEM:F12 at a ratio of 1:1 and add 10% fetal bovine serum (but do not add fibroblasts), and incubate at 37°C in a 5% CO2 incubator. Then refrigerate for the same period as in Examples 12-14.

[0131] 2. The initial culture medium, which was kept under refrigeration, was filtered through a 0.22 μm microporous membrane to obtain a sterile filtrate;

[0132] 2. The obtained sterile filtrate is subjected to protein concentration through an ultrafiltration membrane of ≥80KD to obtain ultrafiltration concentrate;

[0133] 3. The obtained ultrafiltration concentrate was first subjected to heating, followed by radiation sterilization to inactivate bacteria / viruses, yielding the control group protein composition of Example 11. The heating conditions for the heating method were 60±0.5℃ for approximately 10 hours, and the radiation sterilization method used 60Co-r rays ≥25KGy. Example 16: Example 6 was used to treat senile arthritis.

[0134] 5 mL of the protein composition from Example 6 was injected subcutaneously into the affected area of ​​a patient with senile arthritis, once a month, for a total of 3 injections. Figures 1A 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 the treatment of rosacea

[0135] 2.5 mL of the protein composition from Example 8 was injected dermally into the affected area of ​​a patient with rosacea, once a month, for a total of 3 injections. Figures 2A and 2B are facial photographs of the patient before the first injection and two months after treatment, respectively. Example 18: Example 8 used to treat alopecia areata.

[0136] 5 mL of the protein composition from Example 8 was injected subcutaneously into the affected area of ​​patients with alopecia areata, once a month. The head photographs in Figures 3A-3E were taken before the first injection, at the third month of treatment, the fourth month of treatment, the sixth month of treatment, and the eleventh month of treatment, respectively. Example 19: Example 8 was used to treat allergic rhinitis.

[0137] 2.5 mL of the protein composition from Example 8 was injected via mucosal injection into the affected area of ​​a patient with allergic rhinitis, once a month. Figures 4A and 4B are photographs of the inferior turbinate before the first injection and after two treatments, respectively, showing improvement in the paleness of the inferior turbinate mucosa and reduction in swelling. Example 20: Example 9 was used to treat rheumatoid arthritis.

[0138] 2.5 mL of the protein composition from Example 9 was injected subcutaneously into the affected area of ​​a patient with rheumatoid arthritis, once a month, for a total of two injections. Figures 5A and 5B are photographs of the patient's finger joints before the first injection and 40 days after treatment, respectively, showing reduced swelling and less pain. Example 21: Example 9 used to treat facial hypertrophic scars

[0139] 2.5 mL of the protein composition from Example 9 was injected dermally into the affected area of ​​a patient with facial hypertrophic scars, once a month, for a total of 3 injections. Figures 6A, 6B, 6C, and 6D are photographs of the patient's right eye before the first injection, the right eye two months after treatment, the left eye before the first injection, and the left eye two months after treatment, respectively. Example 22: Example 10 used to treat filiform warts on the neck.

[0140] 5 mL of the protein composition from Example 10 was injected dermally into the affected area of ​​a patient with filiform warts on the neck, once a month, for a total of 3 injections. Figures 7A and 7B are photographs of the patient's neck before the first injection and two months after treatment, respectively. Example 23: Example 10 used to treat uterine prolapse

[0141] Five mL of the protein composition from Example 10 was injected vaginally into the anterior and posterior fornix of the patient with uterine prolapse, once every month for a total of three injections. Figures 8A and 8B show photographs of the cervix of the patient before the first injection and two months after treatment.

[0142] While specific embodiments of the invention have been described and illustrated, those skilled in the art will understand that various other changes and modifications can be made without departing from the spirit and scope of the invention. Therefore, all such changes and modifications fall within the scope of the claims of this application.

[0143] Unless otherwise stated, all percentages, ratios, and proportions are based on the total weight of the composition. Unless otherwise stated, all temperatures are in degrees Celsius (°C). All component or composition levels refer to the activity level of that component or composition and do not include impurities that may be present in commercially available sources, such as residual solvents or byproducts.

[0144] It should be understood that each upper limit of a value given in this specification includes lower values ​​as if those lower values ​​were explicitly stated in the application. Each lower limit of a value given in this specification includes higher values ​​as if those higher values ​​were explicitly stated in the application. Each range of values ​​given in this specification includes a narrower range of values ​​falling within that range as if those narrower ranges were explicitly stated in the application.

[0145] The numerical values ​​and dimensions disclosed herein should not be construed as strictly limited to the precise values ​​stated. Rather, unless otherwise stated, each such numerical value and dimension is intended to represent the value and a functionally equivalent range around that value. For example, a dimension disclosed as “40 mm” is intended to represent “approximately 40 mm”.

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 A, contains EGF and a pentaneine protein domain 1. 72 kDa type IV collagenase, Interstitial collagenases and their mixtures The concentration of the first protein or polypeptide in the composition is greater than 1 μg / 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 concentration of the individual protein is detected by liquid chromatography-mass spectrometry.

3. The protein composition of claim 1, wherein the composition comprises two or more of the first protein or polypeptide, 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 μg / 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: Thiol oxidase 1, Core proteoglycan, Fibronectin, vimentin, Peronein-1, Inhibitor of metalloproteinases-1, Type I collagen αI chain, Type I collagen αII chains and their mixtures.

5. The protein composition of claim 4, wherein the composition comprises three or more of the second protein or polypeptide, and the sum of the concentrations of the three or more second proteins or polypeptides in the composition is greater than 10 μg / mL.

6. The protein composition of claim 1, comprising at least three proteins or polypeptides, wherein at least one of them is collagen.

7. The protein composition of claim 1 further comprises a protein selected from the group consisting of: Pentanoic acid protein 3, Adipocyte enhancer-binding protein 1, Type III collagen αI chain Adiponectin, Complement factor H-related protein 1, Cysteine ​​protease inhibitor-C, Extracellular nucleotide pyrophosphatase / phosphodiesterase family member 2, Extracellular matrix protein 1, Fetoprotein B, Follicle-associated protein 1 (FOMT1) Gastrin-1, Glial cell-derived junction proteins, Histone H2B 2-E type, Histone H4, Immunoglobulin Kappa variable 3D-15, Laminin subunit β1, Potential transforming growth factor β-binding protein 1, Microfibril-associated glycoprotein 4, Plasminogen activator inhibitor-1, Lamin A / C, Procollagen C endopeptidase enhancer 1, Sex hormone-binding globulin, The SH3 domain binds to glutamate-rich protein 3. Calcium-binding protein S100A6, cathepsin B, Serine protease HTRA1, Transforming growth factor-β inducer protein IG-H3, Apolipoprotein CIII and its mixtures.

8. The protein composition of any one of claims 1-7, 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: Thiol oxidase 1, Core proteoglycan, Fibronectin, vimentin, Peronein-1, Inhibitor of metalloproteinases-1, Type I collagen αI chain, Type I collagen αII chains and their mixtures. 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 μg / mL, and the concentration of the second protein or polypeptide in the composition is greater than 10 μg / mL.

9. A method for preparing the protein composition according to any one of claims 1-8, comprising: a) Obtain human fibroblasts from human skin tissue using tissue blocks or digestion methods; b) The human fibroblasts were subjected to in vitro treatment at 37°C and 3-10% CO2. 2 Under culture conditions, in vitro amplification was carried out in the initial culture medium; c) Obtain and collect a large amount of cell culture supernatant during in vitro expansion; and d) Filter the cell culture supernatant using a 0.1-0.3 μm microporous membrane or filter cartridge 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 filtrate obtained in step d) is concentrated by ultrafiltration 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 ≥80 kDa; when the initial culture medium contains no serum additives, the molecular weight of the ultrafiltration membrane is ≥1 kDa; and f) The ultrafiltration concentrate obtained in step e) is sterilized by heating and / or radiation to inactivate bacteria / viruses. Wherein: the radiation sterilization method uses 60Co-r rays ≥25KGy, the heating method has a heating temperature of 60±0.5℃, and a continuous duration of 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 the protein composition according to any one of claims 4, 5, and 8, comprising: a) Obtain human fibroblasts from human skin tissue using tissue blocks or digestion methods; b) The human fibroblasts were subjected to in vitro treatment at 37°C and 3-10% CO2. 2 Under culture conditions, in vitro amplification was carried out in an initial culture medium containing fetal bovine serum or without serum additives; c) Obtain and collect a large amount of cell culture supernatant during in vitro expansion; d) Filter the cell culture supernatant using a 0.1-0.3 μm microporous membrane or filter cartridge to obtain a sterile filtrate; and e) The sterile filtrate obtained in step d) is concentrated by ultrafiltration 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 no serum 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 is increased 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 1-8.

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

15. Use of the protein composition according to any one of claims 1-8 in the preparation of medical implants, medical devices or pharmaceuticals.

16. Use of the protein composition according to any one of claims 1-8 in the preparation of a medical device or pharmaceutical product for treating inflammatory diseases.

17. The use as described in claim 16, wherein the inflammatory disease is selected from inflammatory skin diseases, autoimmune diseases, aseptic inflammation, or combinations thereof.

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

19. The use as described in 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 according to claim 17, wherein the aseptic inflammation is selected from migraine, finger tenosynovitis, facial paralysis, frozen shoulder, or a combination thereof.

21. Use of the protein composition according to any one of claims 1-8 in the preparation of a medical device or pharmaceutical product for treating mycoplasma infection diseases.

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

23. Use of the protein composition according to any one of claims 1-8 in the preparation of a medical device or pharmaceutical product for treating viral infections of the skin and mucous membranes.

24. The use as described in claim 23, wherein the viral infection of the skin and mucous membrane surface is selected from flat warts, filiform warts of 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 1-8 in the preparation of a medical device or pharmaceutical product for treating diseases of functional lubrication deficiency.

26. The use as described in claim 25, wherein the functional lubricant deficiency disease is selected from senile arthritis, dry eye syndrome, or a combination thereof.

27. Use of the protein composition according to any one of claims 1-8 in the preparation of a medical device or pharmaceutical product for treating hypertrophic scars.

28. The use according to claim 27, wherein the hypertrophic scar is selected from thyroid hyperplasia, breast hyperplasia, benign prostatic hyperplasia, uterine fibroids, facial hypertrophic scars, or combinations thereof.

29. Use of the protein composition according to any one of claims 1-8 in the preparation of a medical device or pharmaceutical product for treating hair loss.

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

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