CN116769037A - Anti-aging composition containing collagen and application thereof in premature senility - Google Patents
Anti-aging composition containing collagen and application thereof in premature senility Download PDFInfo
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- CN116769037A CN116769037A CN202211113202.8A CN202211113202A CN116769037A CN 116769037 A CN116769037 A CN 116769037A CN 202211113202 A CN202211113202 A CN 202211113202A CN 116769037 A CN116769037 A CN 116769037A
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- collagen peptide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
- A61K38/014—Hydrolysed proteins; Derivatives thereof from animals from connective tissue peptides, e.g. gelatin, collagen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/18—Antioxidants, e.g. antiradicals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
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- Public Health (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- Zoology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
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- Immunology (AREA)
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- Endocrinology (AREA)
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Abstract
The present application relates to an anti-aging composition comprising collagen and its use in premature aging. According to the application, the collagen peptide with the functions of resisting oxidation and aging is prepared by separating the Engraulis japonicus Temminck et Schlegel skin, and after the collagen peptide is used together with the prepared PTEN monoclonal antibody, the aging of endothelial cells in a high-sugar environment can be effectively inhibited, so that the collagen peptide has a better application for resisting aging of blood vessels of diabetics. Has excellent application value.
Description
Technical Field
The application relates to the field of biology, in particular to an anti-aging composition containing collagen and application thereof in premature senility.
Background
Currently, the incidence of cardiovascular and cerebrovascular diseases increases year by year, and these diseases add up to about 39.6% of age-related diseases. There is growing evidence that age is a major risk factor for the development and progression of cardiovascular and cerebrovascular diseases, and vascular aging plays an important role therein, with vascular endothelial cell aging being a major cause.
The vascular endothelial cells VECs are flat squamous epithelial cells which are lined on the surface of the vascular cavity and are longitudinally arranged in a single layer, and are the largest tissues of a human body; the biological functions are various, and the biological functional composite can form a selective permeability barrier between blood flow and blood vessel walls, has the functions of regulating blood vessel permeability and blood vessel tension, maintaining blood coagulation function balance, sensing vascular stress, reacting to cell injury, secreting various active substances and the like. VEC senescence mainly involves 2 ways of cell replicative senescence and stress-induced premature cell senescence. The cessation of cell division due to cellular genetic abnormalities (e.g., DNA damage of various types) is referred to as cellular replicative senescence; cells cultured in vitro gradually senesce, i.e. replicative senescence, after a series of passages without any stress and injury. Cells are affected by some external factors to cause permanent and irreversible proliferation arrest, which is called premature senility; the cells cultured in vitro are treated by high sugar, tumor necrosis factor and the like, so that the premature senility of the cells can be induced.
At present, many systemic diseases, such as hypertension, coronary heart disease, diabetes and the like, are considered to be related to VEC aging and injury; some stimulus factors, such as smoking, radiation injury, etc., can cause aging changes in endothelial cells, and although the mechanism of action of the different factors on endothelial cells is different, they all eventually enter the common pathway of cell aging. The research shows that the PI3K/Akt signal path can influence the activation states of various downstream effector molecules, plays roles in inhibiting apoptosis and promoting proliferation, and is one of important signal transduction paths in cells. The results of culturing human umbilical vein endothelial cells outside a culture medium containing angiotensin 2 for 1 week by Li and the like show that the angiotensin 2 is dose-dependent, depolarizes mitochondrial membrane potential, increases ROs production, remarkably increases the expression level of proteins such as TERT, uncoupling protein2 (UCP 2), akt, p-Akt, p53 and the like, and accelerates the cell aging degree. PI3K inhibitor LY294002 can slow down endothelial cell aging and apoptosis induced by it. It was shown that angiotensin 2 induces VEC aging through the PI3K/Akt/UCP2 pathway.
PTEN, a cancer suppressor gene, has received much attention since its discovery in 1997, primarily regulating the balance of phosphatidylinositol 3kinase (PI 3K)/protein kinase B (AKT) signaling pathways. Abnormal expression and function of PTEN gene exists in various tumors such as glioma, ovarian cancer, endometrial cancer, prostate cancer, melanoma in humans. After PTEN protein has lost function, negative regulation of PI3K/AKT is lost, so that AKT is excessively activated, and proliferation, metastasis, invasion, angiogenesis and self-renewal of stem cells of cancer cells are promoted.
The activity of PTEN gene encoding protein-lipid phosphatase plays an important role in regulating apoptosis, and can quickly block the cell cycle in the G1 phase, thereby promoting apoptosis. The PTEN gene allows for lower levels of phosphatidylinositol (3, 4, 5) -triphosphate (ph 0sphatidylinositol (3, 4, 5) -trisphosphate, PIP 3), thereby inhibiting the PI3K/AKT pathway, inducing cell cycle arrest and apoptosis. Once PTEN gene function is lost, loss of negative regulation of PI3K/AKT causes AKT to be overactivated, which necessarily results in acceleration of cell cycle and increase of cell growth and proliferation. Activation of the PI3K/AKT pathway reduces the activity of apoptotic factors and may increase the activity of anti-apoptotic proteins. Many matrices downstream of the PTEN-PI3K/AKT signaling pathway have been found to play an important role in regulating cell proliferation and apoptosis. The hyperactivated AKT may also promote cell proliferation by down-regulating the cyclin-dependent hormone inhibitor P27 and up-regulating cyclin D1. The PTEN-PI3K/AKT signaling pathway is found to regulate proliferation of oral squamous cell carcinoma cells. It was also found by studies on tongue cancer that overexpression of the PTEN gene induced apoptosis in tongue cancer cells by negatively regulating PI3K/AKT pathway.
Studies show that high sugar induces endothelial cell apoptosis by up-regulating PTEN, PIC is an inhibitor of PTEN and an inhibitor of protein tyrosine phosphatase, and PTEN can be inhibited by a small dosage, and PI3K/AKT signal pathway is regulated. PTEN inhibitors may reduce high sugar-induced endothelial cell aging. However, at present, there is not enough research on PTEN inhibitors, and in particular, on monoclonal antibodies to PTEN.
Collagen belongs to structural protein, is a polysaccharide protein, has white character, contains a small amount of galactose and glucose, is the most main component of extracellular matrix, and is the most content in the dairy animals in the end. Collagen is found everywhere in the tissues and organs of the human body, such as skin, fascia, ligament, hair, bone, muscle, joint, cartilage and the like, wherein the dermis content in the skin is the largest, and has three anti-aging effects of supporting, repairing and protecting. Collagen in the dermis is synthesized and secreted by fibroblasts, and sufficient collagen can maintain skin elasticity, reduce water loss, delay wrinkle generation and pigmentation. Research shows that the collagen polypeptide can promote the proliferation of vascular endothelial cell with oxidation stress damage, inhibit the apoptosis of endothelial cell and protect vascular endothelial cell. However, the current research on protecting vascular endothelial cells by collagen is insufficient, and the types of the provided collagen are insufficient.
Disclosure of Invention
In one aspect, the application provides a monoclonal antibody specific for PTEN.
In one aspect, the monoclonal antibody of PTEN is P2G9, and the light chain variable region sequence thereof is SEQ ID NO:1, the heavy chain variable region sequence of which is SEQ ID NO: 2.
Furthermore, the monoclonal antibodies of the application may be substituted with appropriate conserved amino acids, which substituted antibodies still retain the corresponding activity.
In the monoclonal antibody of the present application, the number of amino acids after modification is preferably 10 amino acids or less, more preferably 5 amino acids or less, more preferably 3 amino acids or less (e.g., 2 amino acids or less, 1 amino acid), that is, in the present specification, "a plurality of amino acids" is preferably 10 or less, more preferably 5 or less, more preferably 3 or less, more preferably 2 or less. For example, among acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine, arginine, histidine), neutral amino acids, amino acids having a hydrocarbon chain (glycine, alanine, barren, luo Yixin, isoleucine, proline), amino acids having a hydroxyl group (serine, tertiaryamino acid), and amino acids containing sulfur (cysteine, methionine).
Further, the monoclonal antibody of the application may be found in the light chain variable region sequence SEQ ID NO:1 maintains 99% sequence identity on a 1-basis.
Further, the monoclonal antibody of the application may be found in the light chain variable region sequence SEQ ID NO:1 maintains 98% sequence identity on a 1-basis.
Further, the monoclonal antibody of the application may be found in the light chain variable region sequence SEQ ID NO:1 maintains 97% sequence identity on a 1-basis.
Further, the monoclonal antibody of the application may be found in the light chain variable region sequence SEQ ID NO:1 maintains 96% sequence identity on a 1-basis.
Further, the monoclonal antibody of the application may be found in the light chain variable region sequence SEQ ID NO:1 maintains 95% sequence identity on a 1-basis.
Further, the use of the monoclonal antibodies of the application in the preparation of an agent for inhibiting PTEN expression.
Further, the use of the monoclonal antibodies of the application in the manufacture of a medicament for the treatment of diseases associated with PTEN overexpression.
Further, the use of the monoclonal antibody of the application for the preparation of a medicament for treating endothelial cell senescence in a high sugar environment.
Further, the use of the monoclonal antibody of the application in the preparation of a medicament for treating vascular aging in a diabetic patient.
Furthermore, the application also provides a collagen peptide with the functions of resisting oxidation and aging.
Further, the collagen peptide is obtained by extraction of Engraulis japonicus Temminck et Schlegel skin.
Further, the extraction method of the collagen peptide comprises the steps of cleaning and peeling purchased Engraulis japonicus Temminck et Schlegel to obtain clean Engraulis japonicus Temminck et Schlegel skin. Soaking Engraulis japonicus Temminck et Schlegel skin in diethyl ether, removing fat from Engraulis japonicus Temminck et Schlegel skin, washing with clear water for several times, and air drying. Chopping air-dried Engraulis japonicus Temminck et Schlegel skin to obtain Engraulis japonicus Temminck et Schlegel skin powder, placing in an electronic balance, adding water, placing in a water bath at 100deg.C, and regulating pH. Enzymolysis: respectively and sequentially adding 0.2% of alkaline protease and 0.3% of neutral protease, adjusting the temperature of the water bath kettle to 45 ℃, and sequentially and respectively carrying out enzymolysis for 3 hours. And (3) inactivation: taking out after enzymolysis, regulating the temperature of the water bath kettle to 100 ℃, and placing the water bath kettle into the water bath kettle for inactivation for 10min. And (3) centrifugal filtration: and centrifuging and filtering the inactivated enzymolysis liquid to obtain supernatant. The obtained supernatant was freeze-dried to obtain collagen peptide (white powder).
Furthermore, the application also provides application of the monoclonal antibody and collagen peptide in preparing a medicine box for treating endothelial cell aging in a high-sugar environment.
Further, the medicine box contains a pharmaceutically acceptable carrier.
Further, the kit and the medicament of the application can comprise pharmaceutically acceptable carriers. Examples of pharmaceutically acceptable carriers suitable for oral administration include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes and perfumes. For injectable formulations, buffers, preservatives, analgesics, solubilizers, isotonic agents and stabilizers may be used in combination. For topical administration, the composition may also contain bases, excipients, lubricants and/or preservatives. The pharmaceutical composition of the application can be formulated into various dosage forms with pharmaceutically acceptable carriers. For example, the composition may be in the form of a tablet, capsule, elixir, suspension, syrup or wafer oral dosage form. For injection, the composition may be formulated in unit-dose ampoule form or in multi-dose ampoule form. Furthermore, the composition may be applied in solution, suspension, tablet, pill, capsule and suspension release forms. Examples of carriers, excipients, or diluents suitable for use in the formulation of the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polypyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. Fillers, anticoagulants, lubricants, wetting agents, fragrances and/or preservatives may additionally be used. The effective dose of the pharmaceutical composition according to the present application depends on various factors including the kind of cancer, the administration route, the age, sex and weight of the patient, and the severity of the disease, etc.
In a preferred embodiment, the composition of the application may further comprise a therapeutic agent that may be conjugated to a human monoclonal antibody of the application. The therapeutic agent is selected from the group consisting of radionuclides, drugs, lymphokines, toxins and bispecific antibodies. Examples of drugs or toxins include etoposide, teniposide, doxorubicin, daunorubicin, carbo Mi Mei, aminopterin, actinomycin, mitomycin, cisplatin and cisplatin analogs, bleomycin, epothilone, 5-fluorouracil, melphalan and nitrogen mustard, or other drugs for treating vascular aging and anti-aging, but are not limited thereto.
In compositions and kits for inhibiting the growth and metastasis of cancer associated with the overexpression of PTEN or for treating or preventing cancer, PTEN-specific human monoclonal antibodies or functional fragments thereof are used to inhibit the function of PTEN in various cancers, thereby inhibiting the growth and metastasis of cancer, thereby killing tumor cells. Preferably, the treatment or prevention of cancer is achieved by modulating proliferation, migration or invasion of cancer. Preferably, the cancer is pancreatic cancer, gastric cancer, ovarian cancer or colorectal cancer.
In more detail, the method of the application comprises administering the pharmaceutical composition to the body in a pharmaceutically effective dose. The pharmaceutical composition may be administered parenterally, subcutaneously, intrapulmonary or intranasally. For topical immunosuppressive treatment, the composition can be administered, if desired, using suitable methods, including intratumoral administration. Parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal and subcutaneous routes. Preferred modes of administration include intravenous, subcutaneous, intradermal, intramuscular and instillation injection.
Advantageous effects
According to the application, the collagen peptide with the functions of resisting oxidation and aging is prepared by separating the Engraulis japonicus Temminck et Schlegel skin, and after the collagen peptide is used together with the prepared PTEN monoclonal antibody, the aging of endothelial cells in a high-sugar environment can be effectively inhibited, so that the collagen peptide has a better application for resisting aging of blood vessels of diabetics. Has excellent application value.
Drawings
FIG. 1 is a graph showing the results of identifying the antioxidant capacity of collagen peptides
FIG. 2 is a graph showing the results of anti-aging of endothelial cells with the drug
FIG. 3 results of effects of drugs on PTEN expression
Detailed Description
The application may be understood more readily by reference to the following detailed description of some embodiments of the application and the examples included therein. Before the present application is further described, it is to be understood that this application is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present application will be limited only by the appended claims.
EXAMPLE 1 preparation of collagen polypeptide
(1) And cleaning and peeling the purchased Engraulis japonicus Temminck et Schlegel to obtain clean Engraulis japonicus Temminck et Schlegel skin. (2) Soaking Engraulis japonicus Temminck et Schlegel skin in diethyl ether for 18min, removing fat from Engraulis japonicus Temminck et Schlegel skin, washing with clear water for several times, and air drying. (3) Chopping air-dried Engraulis japonicus Temminck et Schlegel skin to 1mm to obtain Engraulis japonicus Temminck et Schlegel skin powder.
(4) Preparation of Engraulis japonicus Temminck et Schlegel skin collagen peptide: weighing: taking a dry and clean beaker, placing on an electronic balance, taking 10g of Engraulis japonicus Temminck et Schlegel skin, adding 30ml of water, placing under a water bath at 100 ℃ for 10min, and then adjusting the pH to 9.0. Enzymolysis: respectively and sequentially adding 0.2% of alkaline protease and 0.3% of neutral protease, adjusting the temperature of the water bath kettle to 45 ℃, and sequentially and respectively carrying out enzymolysis for 3 hours. And (3) inactivation: taking out after enzymolysis, regulating the temperature of the water bath kettle to 100 ℃, and placing the water bath kettle into the water bath kettle for inactivation for 10min. And (3) centrifugal filtration: and centrifuging and filtering the inactivated enzymolysis liquid to obtain supernatant. The obtained supernatant was freeze-dried to obtain collagen peptide (white powder). Collagen peptide extraction = M1/m2×100%, where: m1 is the mass g of collagen peptide powder; m2 is the mass g of fresh Engraulis japonicus Temminck et Schlegel skin. The results show that the extraction amount of the collagen peptide reaches 18.23%.
Measurement of collagen content: the hydrochloric acid solution dissolves collagen peptide to produce released hydroxyproline, which is oxidized by chloramine T as oxidant, and may react with p-dimethylaminobenzaldehyde solution to produce red complex with maximum absorption peak at 560nm wavelength, so that it may be quantitatively determined by colorimetry. The results showed a collagen content of 88.47%.
Determination of hydroxyproline content: according to methods known in the art. When absorbance is measured, a wave with a wavelength of 560nm is selected for measurement, and the hydroxyproline content in the collagen peptide powder sample is calculated to be 9.82% according to a hydroxyproline standard curve. From the results, the collagen peptide prepared by the application has better purity and content.
Example 2 identification of antioxidant Capacity of collagen peptides
The DPPH method enables a more comprehensive evaluation of the antioxidant activity of collagen peptides. First, preparing a DPPH-ethanol solution with the concentration of 0.04mg/mL, sucking 1.5mL of the solution, preparing a 95% ethanol solution, adding a proper amount of an anchovy skin collagen peptide extract, and then titrating the solution to 3mL with the 95% ethanol solution. The prepared solution is kept stand for 30min in dark, and then the absorbance is measured at 517nm by using an ultraviolet spectrophotometer, and is zeroed by using 95% ethanol solution. Vitamin E was also used as a positive control. The DPPH clearance is expressed by the formula (calculated. Clearance= [1- (A1-A2)/A0 ]. Times.100% (2) wherein A1 is absorbance of DPPH solution after adding the extract, A2 is absorbance of extract, and A0 is absorbance of DPPH solution without adding the extract. The result is shown in figure 1.
As shown in FIG. 1, the removal ability of VE and collagen peptide from DPPH is continuously enhanced with the increase of the concentration of the collagen peptide solution. The clearance of the collagen peptide to DPPH is obviously enhanced compared with VE between 1 and 5 mg/mL. In general, the clearance of the collagen peptide is stronger than that of VE on DPPH, and the clearance of the collagen peptide on DPPH reaches 53.2 at the concentration of 5mg/mL, so that the collagen peptide has better effect.
EXAMPLE 3 preparation of PTEN monoclonal antibodies
PTEN recombinant protein was purchased from Hzbscience, cat: ZY822Hu015. Taking the recombinant protein as an immunogen, taking a BALB/c female mouse with the age of 6 weeks, and performing subcutaneous multipoint immunization, wherein the immunization dose is 50 mug/mouse. And finally screening out 2 positive cell strains which are respectively named as P2A3 and P2G9 by adopting a conventional cell fusion and screening method, preparing a abdominal water type antibody by adopting a mouse in-vivo induction method, and purifying by adopting a Protein G affinity chromatographic column for later use.
The screened antibody Ig class and subclass was assayed using the SBAClonotyping System-HRP kit, all procedures strictly followed the kit instructions. The results showed that both P2A3 and P2G9 were IgG1.
Identification of antibody specificity: BSA, PTEN, and human PD-1 protein were diluted to 1. Mu.g/ml, 100. Mu.l per well was added to the ELISA plate and incubated overnight at 4 ℃. The cells were washed 3 times with PBST containing 0.05% Tween for 3min each, blocked with 5% skimmed milk powder at 37deg.C for 1h, and washed 3 times with PBST for 3min each. The purified two kinds of antibodies were subjected to dilution at a ratio of 1:1000. Mu.l of each well was added and incubated at 37℃for 1h. PBST was washed 3 times, 3min each, and 100 μl of 8000-fold dilution of HRP-labeled goat anti-mouse enzyme-labeled secondary antibody was added and incubated at 37deg.C for 40min. PBST is washed for 5 times, TMB single-component color development liquid is added, after reaction is carried out for 10min at room temperature and light is prevented, 50 μl of concentrated sulfuric acid stop solution is added for stopping the reaction, an OD450 value is read by an enzyme-labeling instrument, and an experimental OD450 value/a control OD450 value (p/n) is taken as a positive result, and the result is shown in the following table 1.
TABLE 1 Cross-reaction of antibodies
| Each group of | P2A3 monoclonal antibody | P2G9 monoclonal antibody |
| BSA | - | - |
| PTEN | + | + |
| Human PD-1 proteins | - | - |
+ indicates positive, -indicates negative
EXAMPLE 3 determination of affinity of PTEN monoclonal antibody P2G9
An indirect ELISA method is used, an ELISA plate is coated with PTEN recombinant protein at the concentration of 1 mug/mL, the ELISA plate is closed, purified monoclonal antibody diluted in a doubling ratio is added for incubation, goat anti-mouse IgG marked by HRP is used as a secondary antibody, and an OD450nm absorbance value is read by an ELISA. The OD450nm readings of several dilutions in succession are regarded as 100% binding of antigen-antibody when no longer increased, the ordinate is the absorbance at OD450nm, and the ordinate is the scatter plot, and the 50% binding of antigen-antibody when half of the maximum value of the readings is used to generate a logarithmic trend line and formula. Half of the OD450nm maximum was substituted into the formula, and the concentration of the antibody at this time was determined as affinity dissociation constant (Kd). The result shows that the Kd value of the P2G9 monoclonal antibody is 8.46nM, and the binding capacity is stronger.
EXAMPLE 4 Effect of PTEN monoclonal antibody P2G9 on endothelial cell aging
Vascular endothelial cells (stock BH-0111227, bohu organism) were cultured in RPMI-1640 medium at a temperature of 37 ℃ with carbon dioxide 5% and in an incubator at a humidity of 70% -80%.
Then, the experiment is divided into a normal group, a model group and a positive control group, wherein the monoclonal antibody P2G9 group and the monoclonal antibody combined collagen peptide treatment group, and the basic culture mediums are RPMI-1640 culture mediums;
the sugar concentration of the normal group A is 5.6mmol/L;
model group B sugar concentration was 30mmol/L;
adding 100 mug/mL vanadium peroxide positive control medicament under the condition that the sugar concentration of the positive control group C is 30mmol/L;
adding 100 mug/mL monoclonal antibody P2G9 under the condition that the sugar concentration of the monoclonal antibody group D is 30mmol/L;
2mg/mL of monoclonal antibody P2G9 and 100 mug/mL of the collagen peptide prepared in example 1 are added under the condition that the concentration of the monoclonal antibody combined with the collagen peptide group E sugar is 30mmol/L;
2mg/mL of the collagen peptide prepared in example 1 is added under the condition that the concentration of the collagen peptide group F saccharide is 30mmol/L;
the duration of action was 5d for each group and the experiment was repeated 3 times.
Procedures were performed with reference to the Annexin V-FLUOS staining kit instructions: after cell grouping stem pretreatment, cells were treated by digestion with pancreatin (0.25% EDTA-free), centrifuged at 1000r/min for 5min, cell pellet was collected, washed 3 times with PBS, resuspended in 500. Mu.L of FITC/PI kit buffer, 300. Mu.L of suspension was taken in flow tubes, FITC 3. Mu. L, PI 3. Mu.L was added, respectively, and after 15min of screening from light, detection was performed on the machine.
As shown in FIG. 2, the apoptosis rate of the cells increases after the high sugar acts on endothelial cells relative to the low sugar group, while under the high sugar condition, the addition of vanadium peroxide, monoclonal antibody or collagen peptide bacteria can reduce the apoptosis induced by the high sugar, and P is less than 0.01 compared with the high sugar non-administration group. In particular, after the monoclonal antibody is combined with the collagen peptide for combined administration, the apoptosis rate is reduced to (10.19+/-0.48)%, and the apoptosis rate is well inhibited under the condition of high sugar.
Collecting each group of cells, lysing the lysate, centrifuging at 4 ℃ for 10min at 12000r/min, collecting the supernatant, measuring the protein concentration by a BCA method, diluting each sample protein by a loading buffer solution, and loading after denaturation at 95 ℃ for 5 min. After electrophoresis, membrane transfer and overnight sealing, PTEN primary antibody is added respectively, oscillation is carried out at 4 ℃ for 5min multiplied by 3 times, TBST is carried out, HRP-marked secondary antibody is added for incubation for 1h at normal temperature, ECL method development is carried out, and after the gray level of protein strips is measured by using a gel imaging analyzer, the ratio of each protein strip to internal reference beta-actin is calculated. The results are shown in FIG. 3.
As can be seen from the results of fig. 3, PTEN protein expression was increased and significantly, P <0.01, in the high sugar B group relative to the low sugar a group. The positive control group C, the monoclonal antibody and the monoclonal antibody combined collagen peptide can obviously reduce the expression of PTEN, particularly the result of the group E is only (0.28+/-0.04), and the positive control group C, the monoclonal antibody and the monoclonal antibody combined collagen peptide have obvious effect of reducing the expression of PTEN, but the collagen peptide does not have obvious effect of reducing the expression of PTEN.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.
Claims (6)
1. A monoclonal antibody of PTEN is P2G9, characterized in that the light chain variable region sequence is SEQ ID NO:1, the heavy chain variable region sequence of which is SEQ ID NO: 2.
2. Use of PTEN monoclonal antibody P2G9 as defined in claim 1 in the manufacture of a medicament for inhibiting PTEN overexpression.
3. Use of PTEN monoclonal antibody P2G9 and collagen peptide in preparing a kit for endothelial cell aging resistance in high sugar environment, wherein the light chain variable region sequence of PTEN monoclonal antibody P2G9 is SEQ ID NO:1, the heavy chain variable region sequence of which is SEQ ID NO:2, wherein the high sugar environment is 30mmol/L; the collagen peptide is prepared by the following method: cleaning and peeling purchased Engraulis japonicus Temminck et Schlegel to obtain clean Engraulis japonicus Temminck et Schlegel skin; soaking Engraulis japonicus Temminck et Schlegel skin in diethyl ether for 18min, removing fat from Engraulis japonicus Temminck et Schlegel skin, washing with clear water for several times, and air drying; chopping the dried Engraulis japonicus Temminck et Schlegel skin to 1mm to obtain Engraulis japonicus Temminck et Schlegel skin powder; preparation of Engraulis japonicus Temminck et Schlegel skin collagen peptide: weighing: taking a dry and clean beaker, placing on an electronic balance, taking 10g of Engraulis japonicus Temminck et Schlegel skin, adding 30ml of water, placing under a water bath kettle at 100 ℃ for 10min, and then adjusting the pH to 9.0; enzymolysis: respectively and sequentially adding 0.2% of alkaline protease and 0.3% of neutral protease, adjusting the temperature of the water bath kettle to 45 ℃, and sequentially and respectively carrying out enzymolysis for 3 hours; and (3) inactivation: taking out after enzymolysis, regulating the temperature of the water bath kettle to 100 ℃, and placing the water bath kettle into the water bath kettle for inactivation for 10min; and (3) centrifugal filtration: centrifuging and filtering the inactivated enzymolysis liquid to obtain supernatant; and freeze-drying the obtained supernatant to obtain the collagen peptide.
4. A use according to claim 2 or 3, wherein the medicament or kit comprises a pharmaceutically acceptable carrier.
5. The use according to claim 4, wherein the pharmaceutically acceptable carrier comprises a binder, lubricant, disintegrant, excipient, solubilizer, dispersant, stabilizer, suspending agent, dye or perfume.
6. Use according to claim 5, wherein the excipient or diluent comprises lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate or cellulose.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211113202.8A CN116769037B (en) | 2022-09-14 | Anti-aging composition containing collagen and application thereof in premature senility |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211113202.8A CN116769037B (en) | 2022-09-14 | Anti-aging composition containing collagen and application thereof in premature senility |
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|---|---|---|---|---|
| JP2005306806A (en) * | 2004-04-23 | 2005-11-04 | Nikko Chemical Co Ltd | Promoter of collagen synthesis and cosmetic for antiaging |
| US20160340396A1 (en) * | 2013-11-04 | 2016-11-24 | Tissuegene, Inc. | Treatment of damaged nerve with pten inhibitor |
| CN112807417A (en) * | 2021-04-08 | 2021-05-18 | 北京瀚梅生物科技有限公司 | Collagen-containing skin-whitening and anti-wrinkle medicine or cosmetic |
| CN113082192A (en) * | 2021-04-29 | 2021-07-09 | 北京瀚梅生物科技有限公司 | Application of composition of collagen and adipose-derived stem cells in preparation of medicines or cosmetics |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005306806A (en) * | 2004-04-23 | 2005-11-04 | Nikko Chemical Co Ltd | Promoter of collagen synthesis and cosmetic for antiaging |
| US20160340396A1 (en) * | 2013-11-04 | 2016-11-24 | Tissuegene, Inc. | Treatment of damaged nerve with pten inhibitor |
| CN112807417A (en) * | 2021-04-08 | 2021-05-18 | 北京瀚梅生物科技有限公司 | Collagen-containing skin-whitening and anti-wrinkle medicine or cosmetic |
| CN113082192A (en) * | 2021-04-29 | 2021-07-09 | 北京瀚梅生物科技有限公司 | Application of composition of collagen and adipose-derived stem cells in preparation of medicines or cosmetics |
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| Title |
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