81 results about "Collagenase" patented technology

The invention discloses starch-collagen composite hydrogel as well as a preparation method and application thereof. Collagen is catalyzed by microbial transglutaminase to form a covalent cross-linked network, and meanwhile, hydroxypropyl starch is introduced to construct a physical interpenetrating network through molecular chain penetration and hydrogen-bond interaction; and chondroitin sulfate is further integrated to strengthen the network structure and biological activity through electrostatic interaction. The method is mild in condition and does not need a toxic chemical cross-linking agent, the obtained composite hydrogel has an interpenetrating double-network structure and has excellent mechanical properties, enzymatic degradation resistance and cell affinity, the compression modulus of the composite hydrogel is remarkably improved, the composite hydrogel can keep structural stability for a long time in a collagenase environment, cell adhesion and proliferation can be effectively promoted, and the composite hydrogel has a good application prospect. The hydrogel can be widely applied to tissue engineering scaffolds, wound dressings, drug sustained-release carriers and cartilage repair materials.

The present invention provides a composition comprising collagenase as an enzyme; calcium, histidine, and glycine as coenzymes, and a method for stabilizing enzymes, the method comprising the preparation of the composition by adding histidine and glycine to collagenase. According to the present invention, the enzyme composition and method for stabilizing enzymes can minimize or inhibit the agglutination of enzymes, maintain the concentration of freeze-dried enzymes even during long-term storage, and provide a formulation that has excellent rehydration time and hygroscopicity when restored to a liquid state, as well as superior enzyme activity.

The invention relates to a collagenase-loaded LDH (layered double hydroxide) / EDTA (ethylene diamine tetraacetic acid) layered double hydroxide nano material as well as a preparation method and application thereof. The collagenase-loaded LDH / EDTA layered double hydroxide nano material is prepared from a zinc-aluminum layered double hydroxide LDH carrier, ethylenediamine tetraacetic acid EDTA inserted between layers of the LDH carrier through anion exchange, and collagenase loaded on the surface of the LDH carrier through an electrostatic adsorption effect. The invention further discloses a preparation method of the collagenase-loaded LDH / EDTA layered double hydroxide nano material for preparing the collagenase-loaded LDH / EDTA layered double hydroxide nano material for preparing the collagenase-loaded LDH / EDTA layered double hydroxide nano material.

The present application relates to a kind of anti-aging composition and its application of brightening and shrinking pores.The composition includes the following components: halocyprid extract, purple magnolia extract, desert rose leaf cell extract, rain lily labiatae flower extract and white lily extract, and the mass ratio of the halocyprid extract, purple magnolia extract, desert rose leaf cell extract, rain lily labiatae flower extract and white lily extract is (0.2-2):(0.001-0.1):(0.001-0.1):(0.001-0.1):(0.001-0.1).The composition of the present application can effectively improve the clearance rate of collagenase, maintain the elasticity and tightness of skin, have good anti-aging and pore-shrinking effect, and can also effectively remove hydroxyl radicals, have good whitening and brightening effect.

The invention relates to a low-temperature gradient enzymolysis technology for collagen peptide in the body wall of a sea cucumber, and discloses mild decomposition and efficient extraction of collagen in the body wall of the sea cucumber by virtue of a multi-section temperature zone reaction, enzyme seed compounding control and on-line regulation system. And the collagen peptide with concentrated molecular weight, strong biological activity and high terminal product quality is prepared by the low-temperature gradient enzymolysis technology. The method is characterized by comprising the following steps: cutting the body wall of the sea cucumber to obtain a particle size suitable for enzymolysis, adding the cut body wall of the sea cucumber into a neutral buffer solution, stirring at normal temperature, and adjusting the pH value to 6.5-7.5, so that the tissue of the body wall of the sea cucumber is fully expanded and uniformly dispersed to be more suitable for the subsequent enzymolysis process; the method comprises the following steps: carrying out staged reaction on the body wall of the sea cucumber and an enzyme at different temperatures, carrying out fine separation and terminal treatment on an enzymatic hydrolysate, and immediately cooling a system to 4 DEG C after the reaction of the enzymatic hydrolysate is finished.

The application provides a preparation method of non-denatured type II collagen for increasing bone density and improving bone elasticity, and belongs to the technical field of biotechnology. The application adopts EDTA-citric acid buffer containing beta-mercaptoethanol to decalcify cartilage, adopts supercritical CO2 extraction technology to defat, adopts alkaline protease and collagenase II to perform matrix directional enzymolysis, and performs gradient digestion; then three-phase centrifugal separation is adopted to separate non-denatured type II collagen, and biomimetic H column chromatography purification is adopted, so that non-denatured type II collagen with high extraction rate, high purity and high structural integrity can be obtained.

The present application relates to cell preparation process in the field of bioengineering, disclose a kind of high-efficiency preparation mouse primary hepatocyte method, comprising: perfusion needle is placed in reverse direction to liver direction through inferior vena cava, utilize the blood vessel clamp with elastic micro-tooth damping surface to lock the perfusion needle and inferior vena cava wall;The overflow damping of hepatic portal vein outflow end is dynamically regulated by blood vessel clamp, to construct controlled positive pressure permeation environment in liver vascular bed;Collagenase solution is radially permeated to liver parenchyma using the environment, the present application makes enzyme solution radial deflection and cross blood sinus barrier into di's cavity by constructing micro-pressure environment, eliminate the digestion dead zone originally due to flow path preference, ensure that enzyme molecule and extracellular matrix are uniformly contacted in global dimension, realize the collaborative promotion of tissue cutting efficiency and total amount of cell release, ensure that the output cell population has highly consistent physiological state.

The invention relates to an anti-aging, brightening and pore-shrinking composition and application thereof. The composition comprises the following components: an artemia extract, a magnolia liliiflora extract, a desert rose leaf cell extract, a lilium lanceolarium extract and a lilium fortunei extract. The mass ratio of the artemia extract to the magnolia liliiflora extract to the rosa deserticola leaf cell extract to the lilachium lanceolatum extract to the lilium fortunei extract is (0.2 to 2) to (0.001 to 0.1) to (0.001 to 0.1) to (0.001 to 0.1) to (0.001 to 0.1) to (0.001 to 0.1). The composition provided by the invention can effectively improve the collagenase clearance rate, maintain the elasticity and compactness of the skin, achieve good anti-aging and pore-shrinking effects, also can effectively remove hydroxyl radicals, and has good whitening and brightening effects.

Compositions and methods are provided of a therapeutic nanoparticle composed of collagenase IV linked via a linker (e.g. cathepsin B cleavable linker) to ferumoxytol (iron oxide). The collagenase IV is key in the composition intended for the breakdown of the tumor wall as the collagenase. Such compositions and methods are aimed at solving at the same time two of the main challenges of current approaches in the treatment of glioblastoma multiforme (GBM) which are low specificity and poor uptake.

The invention belongs to the technical field of collagen peptide preparation, and particularly relates to a proline aminopeptidase mutant and application thereof in collagen peptide preparation. The amino acid sequence of the proline aminopeptidase mutant is as shown in SEQ ID NO. 2. The proline aminopeptidase mutant provided by the invention has the advantages of high enzyme activity, wide temperature and pH adaptation range, capability of adapting to a long-time high-temperature environment of collagen enzymolysis and the like, and is suitable for deep hydrolysis of collagen. Through cooperation with neutral protease, papain and collagenase, collagen raw materials from fish skin can be effectively degraded, the yield of low-molecular-weight peptide is increased, and collagen peptide with the molecular weight smaller than 1000 Da and accounting for 90% or above, collagen tripeptide accounting for 39% or above, collagen dipeptide accounting for 2% or above and the content of characteristic dipeptide Hyp-Gly being remarkably increased is generated. The method has important application value in the field of oriented preparation of collagen dipeptide.

The invention belongs to the technical field of biology, and particularly relates to a nucleus pulposus cell separation and culture method. According to the method, a step-by-step enzyme digestion method is adopted, NB6 collagenase is firstly used for primary digestion, and then compound enzyme liquid composed of trypsin and hyaluronidase is adopted for subsequent digestion; and culturing in combination with a culture vessel coated with a specific concentration of extracellular matrix component and a special culture medium. According to the method, on the premise of not depending on an immortalization technology, the primary nucleus pulposus cells with good activity can be efficiently obtained, stable amplification and long-term passage of the primary nucleus pulposus cells in vitro are realized, and specific marker expression and biological functions of the nucleus pulposus cells can be effectively maintained. The invention provides a stable cell source and technical platform for the development of cell therapy products and the research of spinal degenerative diseases.

The invention discloses brown cattle bone marrow collagen polypeptide with an immunoregulation effect as well as a preparation method and application of the brown cattle bone marrow collagen polypeptide, and belongs to the technical field of animal-derived food processing. The preparation method comprises the following steps: pretreating brown cattle bone marrow; the method comprises the following steps: dissolving pretreated brown cattle bone marrow in water, firstly adding alkaline protease to carry out enzymolysis, then simultaneously adding collagenase U and basophilic protease into a system, carrying out coupled enzymolysis under the protection of nitrogen, then cooling the system to 0-4 DEG C, regulating the pH value to 3-4, then carrying out autolysis for 10-30 minutes, and finally carrying out enzyme deactivation; centrifuging an enzyme deactivation product, taking supernatant, adjusting the pH value to be neutral, concentrating, dialyzing and freeze-drying to obtain crude collagen peptide; and carrying out primary purification and secondary refining on the crude collagen peptide to obtain the collagen peptide. The preparation process is simple, the molecular weight of the prepared polypeptide is 366-1514 Da, and the polypeptide has immunoregulation and antioxidant activity, can be used for preparing food, health care products or medicines for improving immunity and resisting oxidation, and realizes high-value utilization of livestock and poultry byproducts.

The application provides king salmon bone collagen peptide for promoting longitudinal growth of bone, a preparation method and application, and relates to the technical field of collagen peptides. The preparation method comprises the following steps: (1) treating king salmon bone raw materials by steam explosion to obtain pretreated king salmon bone; then mixing the pretreated king salmon bone with pepsin and performing first enzymolysis under pressure to obtain a first enzymolysis mixture; mixing the first enzymolysis mixture with collagenase and ficin, performing second enzymolysis under pressure, collecting an enzymolysis liquid, and performing ultrafiltration on the enzymolysis liquid to obtain collagen peptide. In the application, pepsin is used for first enzymolysis under pressure, then collagenase and ficin are used for compounding, the synergistic effect of the three kinds of proteases makes the collagen be fully enzymolyzed, the diversity of the cleavage site is improved, the molecular weight of the polypeptide is reduced, the activity of the polypeptide is improved, the longitudinal growth of the bone is promoted, and the bone density is improved.

The invention discloses a preparation method and application of a prostate cancer tissue single-cell suspension. The preparation method of the prostate cancer tissue single-cell suspension comprises the following steps: S1, tissue pretreatment: putting a prostate cancer tissue sample into a low-temperature pretreatment buffer solution, and cutting the prostate cancer tissue sample into tissue fragments; s2, circular enzymolysis: mixing the tissue fragments with a composite enzymatic hydrolysate, and performing digestion for multiple rounds under an oscillation condition; after each round of digestion, collecting supernate into a stop solution containing serum, repeatedly digesting the remaining tissues for multiple times, and combining the supernate to obtain a cell crude suspension; wherein the composite enzymatic hydrolysate contains collagenase, protease, nuclease, an androgen receptor stimulant and / or a ROCK inhibitor; and S3, sequentially carrying out separation and purification on the cell crude suspension through filtration and density gradient centrifugation to obtain the prostate cancer tissue single-cell suspension.

This invention relates to the medical field, specifically to a cell digestion solution, a method for isolating γδT cells, and its application. The cell digestion solution comprises: collagenase, mammalian serum, antibiotics, a buffer, and a cell culture medium; wherein the collagenase content is 1-4 mg / ml, the mammalian serum content is 5 vol%-15 vol%, the antibiotic content is 0.5 vol%-2 vol%, and the buffer content is 5-15 mM / L. Through the synergistic effect and optimized content of components such as collagenase, mammalian serum, and buffer, the cell digestion solution effectively protects cell viability and surface antigen integrity while efficiently dissociating skin tissue, providing a high-quality cell source for subsequent isolation of rare immune cells.

The application provides a fish collagen peptide with high GPH content, a functional food and a method for preparing the fish collagen peptide by using collagenase colG. The method comprises the following steps: a) homogenizing fish scales; b) performing first-step enzymolysis on the homogenate by using alkaline protease; and c) performing second-step enzymolysis on the homogenate by using collagenase colG. The process parameters of the first-step enzymolysis are as follows: the pH is 8-10, the enzymolysis temperature is 45-55 DEG C, the dosage of alkaline protease is 0.5-2% of the mass of the fish scales, and the enzymolysis time is 0.5-3 h. The process parameters of the second-step enzymolysis are as follows: the pH is 7-8, the enzymolysis temperature is 35-45 DEG C, the dosage of collagenase colG is 1-2.5% of the mass of the fish scales, and the enzymolysis time is 4-8 h. The content of the characteristic fragment Gly-Pro-Hyp in the fish collagen peptide prepared by the method can be up to 6.1%.

This invention discloses a multi-stage separation and purification device for collagen peptides, relating to the field of collagen processing technology. It aims to solve the technical problem of difficulty in separating and purifying collagenase raw materials due to their diversity, as demonstrated by existing equipment. The device includes an enzymatic hydrolysis tank with a multi-stage separation mechanism at its bottom. This mechanism comprises a drive rod assembly, a fixed cylinder assembly, and a centrifugal inner ball assembly. The drive rod assembly drives the centrifugal inner ball assembly to rotate within the fixed cylinder assembly. The centrifugal inner ball assembly expands radially due to centrifugal force, forming a variable-gap extrusion and grinding chamber with the inner wall of the fixed cylinder assembly. It has two working states: bone fragment crushing and fiber chopping. The centrifugal ball segments have grooves, within which arc blades are movably inserted. These arc blades automatically extend and retract according to the rotational speed under the control of an elastic telescopic rod. A linked defoaming mechanism includes a guide plate and a defoaming disc, using rotational power to guide the foam to the vortex blades for rupture. This invention can simultaneously achieve bone fragment crushing, fiber depolymerization, and multi-stage filtration and defoaming, significantly improving raw material utilization and purification efficiency.

The application discloses an integrin-targeting peptide-collagenase drug combination and a drug composition constructed by the integrin-targeting peptide-collagenase drug combination. The drug combination can effectively block mechanical signals and biochemical signals mediating fibrosis, efficiently inhibit fibroblast activation, reduce the secretion of extracellular matrix proteins, and reshape the fibrotic microenvironment. Efficient reduction of fibrosis can promote the penetration of subsequent therapeutic drugs, and has a cascade penetration effect. The constructed drug composition can synergistically deliver integrin-targeting peptides and collagenase, and after release, the integrin-targeting peptides and collagenase can synergistically act in the form of free molecules in fibrotic lesions, thereby providing a new strategy for efficient delivery of fibrosis treatment drugs.

The invention relates to the technical field of nano biological medicines, and discloses a photo-thermal-enzyme catalysis nano-particle as well as a preparation method and application thereof. The photo-thermal-enzyme catalysis nano-particles are constructed by crosslinking water-soluble self-doped polyaniline nano-particles, human serum albumin, lactate oxidase and collagenase through a thioacetal linker capable of being split by active oxygen. Collagenase and lactate oxidase (LOx) are integrated into the same nanoparticle system through a thioacetal (TK) linker, the TK linker is split under the action of tumor microenvironment active oxygen (ROS) and H2O2 generated by catalysis of the LOx, the activity of the two enzymes is synchronously activated, a collagen matrix (physical barrier) is degraded, lactic acid (chemical barrier) is removed, the tumor microenvironment is efficiently remodeled, and the tumor microenvironment can be effectively inhibited. The problems that the regulation effect of a single barrier is limited, and the tumor microenvironment is difficult to improve are solved.

The invention relates to the technical field of collagen material degradation, and particularly discloses an in-vitro degradation method of a collagen biological material, which comprises the following steps: pretreating the collagen biological material to obtain a to-be-degraded sample; adding the enzymatic hydrolysate into a to-be-degraded sample, sealing, and degrading at 37 + / -2 DEG C to obtain a degradation solution; the enzymatic hydrolysate is prepared from the following components: 10 to 20 mg / L of collagenase, 3 to 6 mg / L of sorbitol, 1 to 2 mg / L of calcium chloride, 0.3 to 0.8 mg / L of dithiothreitol and the balance of a Tris-HCl buffer solution with the pH value of 7 to 8. The in-vitro degradation method of the collagen biological material provided by the invention has the advantages of good degradation stability, small batch-to-batch difference and the like, and can be used for investigating the degradation behavior of the collagen biological medical device against collagenase in vivo, so that research and development and quality control of the collagen biological medical device are realized.

The application relates to a method for separating and primary culturing of monopterus albus liver cells. The method comprises the following steps: obtaining monopterus albus liver tissue, perfusing the liver tissue with HBSS buffer solution without calcium and magnesium until blood is completely drained; removing the capsule, blood clots and connective tissue after rinsing, and cutting the liver tissue; sequentially using 0.02% EDTA solution, 0.25% trypsin solution and 50 U / mL collagenase IV working solution to perform three-step digestion treatment on the liver tissue fragments; obtaining a cell suspension by blowing and filtering after the digestion is completed, and then performing gradient centrifugal purification of the cells at rotating speeds of 600 rpm, 250 rpm and 80 rpm in sequence; and finally resuspending the cells in DMEM / F12 culture medium containing 10% FBS and inoculating and culturing the cells. The monopterus albus primary liver cells obtained by the method have the advantages of large quantity, high activity and high purity, and provide a reliable in-vitro model for monopterus albus related research.

The invention belongs to the technical field of microorganisms, and particularly discloses an anesthesia coating spore as well as a preparation method and application thereof. The coating spore comprises a spore and a ferric iron-propofol coating attached to the body surface of the spore, and the coating is formed through metal-phenol complexing and pi-pi stacking. The spores are dormant bodies of collagenase-producing bacteria. The coating spores can be selectively germinated and colonized in a tumor hypoxic microenvironment, Fe < 3 + > can promote bacterial proliferation, loaded propofol inhibits tumor metastasis by inhibiting migration and invasion of tumor cells, generated collagenase degrades tumor collagen, meanwhile, Fe < 3 + > is reduced into Fe < 2 + > by glutathione in the cells, and the coating spores can be selectively colonized in the tumor hypoxic microenvironment. Therefore, a powerful Fenton reaction is induced to trigger lipid peroxidation, and ferroptosis of tumor cells is initiated. The injection of the anesthetic coating spores in the single tumor not only effectively ablates the primary tumor and inhibits the growth of the tumor, but also significantly inhibits the distal pulmonary metastasis of the in-situ tumor model.

This invention belongs to the field of genetic engineering technology and discloses a high-efficiency expression method for recombinant collagenase and its application in the preparation of bovine bone collagen peptides. The collagenase gene derived from Bacillus sp. HUB-I-004 strain (accession number J9CTX3) was optimized using E. coli-preferred codons. A recombinant expression vector was constructed and transformed into E. coli BL21(DE3). After optimization of induction conditions and Ni-NTA affinity purification, a highly active recombinant collagenase with a specific activity of 231.05 U / mg was obtained. Bovine bone collagen was extracted using a high-temperature, high-pressure method. Bovine bone collagen peptides were prepared under optimal conditions of a substrate concentration of 10 mg / mL, an enzyme addition of 20%, and enzymatic hydrolysis at 37℃ for 8 h, significantly improving peptide yield. The obtained product is mainly composed of small molecule peptides and exhibits significant osteoblast proliferation-promoting activity, making it widely applicable in the preparation of health foods and pharmaceuticals.

The application discloses collagen tripeptide and a preparation method and application thereof, and belongs to the technical field of bioengineering. The preparation method comprises the following steps: obtaining bacillus velezensis with high collagenase yield through separation and identification, the bacillus velezensis belongs to probiotics, and high-activity collagenase is obtained through fermentation culture and purification; taking shark cartilage or blue fish skin as raw materials, and obtaining collagen tripeptide through the following steps of combined enzymolysis of alkaline protease and collagenase after pretreatment, inactivation, centrifugal separation and freeze-drying. The collagen tripeptide prepared by the application has a molecular weight of less than 500 Dalton, contains active components such as GHK tripeptide, is easy to absorb and has high safety, and can be used for preparing antioxidant products, anti-inflammatory products, memory-enhancing products or immunity-enhancing products. The application solves the problems of unsafe collagenase source and low collagen tripeptide preparation efficiency, and has a good industrial application prospect.

This invention relates to the field of cell engineering technology, and more particularly to a method for preparing immunomodulatory stem cells for treating graft-versus-host disease (GVHD). The invention provides a method for preparing immunomodulatory stem cells for treating GVHD, comprising the following steps: (1) washing human fat aspirate and digesting it with collagenase to obtain monocytes; (2) inoculating the monocytes obtained in step (1) into differentiation medium and culturing for 2–3 days, then continuing to culture the adherent cells for 18–22 days to obtain mesenchymal stem cells; (3) inoculating the mesenchymal stem cells obtained in step (2) into induction medium for induction culture, and after screening, obtaining immunomodulatory stem cells. The preparation method provided by this invention solves the technical problems of unstable sources, functional decline, and poor therapeutic effects of immunomodulatory stem cells in the prior art, providing a new, efficient, safe, and standardized solution for the treatment of GVHD.

Disclosed herein are methods of treating plantar fasciitis (PFA) in a subject comprising injecting a pharmaceutical formulation comprising collagenase into one or more plantar fasciitis affected areas in the subject's foot to thereby treat the plantar fasciitis.

The invention belongs to the technical field of cell separation, and particularly relates to a method for separating kidney immune cells, which comprises the following steps: digesting an obtained mouse kidney tissue block in an RPMI 1640 culture medium of fetal calf serum containing collagenase IV and DNAaseI, collecting an upper-layer single-cell suspension after digestion is completed, washing, centrifuging, and discarding supernate, thereby obtaining the kidney immune cells. And centrifuging by a discontinuous Percoll density gradient centrifugation method, and sucking cells of a high interface layer of 40-80% to obtain the mouse kidney immune cells. According to the technical scheme, a three-step optimization scheme of enzyme digestion, room-temperature sedimentation and density gradient separation is adopted, so that when tissues are dissociated in a warm manner, cell damage is greatly reduced, and antigen loss or degradation caused by enzyme activity and metabolism is effectively reduced.

This invention belongs to the field of stem cell sorting technology and discloses a method for preparing canine adipose-derived MUSE cells using magnetic beads and its application. The method first involves digesting canine adipose tissue with collagenase and then expanding it by adhesion. Subsequently, FITC-labeled SSEA-3 antibody is combined with anti-FITC magnetic beads, and SSEA-3 positive cells are captured using EasySep™ column-free magnetic bead sorting technology. The sorted positive cells are then seeded into a microsphere culture apparatus containing 81 microwells for induction culture. Experiments show that the purity of the MUSE cells obtained by this method is over 90%, and the spontaneously reconstructed colonies (M-clusters) in the microwell system exhibit strong SSEA-3 fluorescent expression. This invention effectively solves the problems of low initial content of canine MUSE cells and significant damage during flow cytometry sorting, providing a high-quality source of seed cells for canine regenerative medicine treatment.

Provided herein compositions including collagenase, and uses thereof in orthodontic procedures, wherein the compositions may include recombinant collagenase and / or modified forms of recombinant collagenase having amino acid(s) truncation or substitutions.

The invention discloses application of a hydrogel microneedle capable of being implanted into an abdominal cavity in colorectal cancer abdominal wall infiltration focus, and relates to the field of medicine. The traditional Chinese medicine composition is applied to clinical treatment of rectal cancer abdominal wall infiltration lesions, and a mouse subcutaneous tumor model and the abdominal wall infiltration lesions are verified, and the specific application method comprises the following steps that 1, after a mouse is subjected to general anesthesia, the abdomen of the mouse is opened, and the position of the abdominal wall infiltration lesions is detected and determined; step 2, clamping the sterilized micro-needle sheet by using noninvasive forceps and feeding the sterilized micro-needle sheet into an abdominal cavity; 3, attaching the microneedle substrate to the surface of a focus under direct view, and pressing to enable the needle body to penetrate through the peritoneum; and 4, flushing with normal saline to confirm that the microneedle is fixed, withdrawing the instrument, and suturing the incision. The microneedle physically penetrates through the surface layer of the peritoneum, the medicine is directly delivered to the deep layer of an infiltrating focus, the problem of low efficiency caused by the peritoneum barrier in the traditional therapy is solved, and the GelMA / hyaluronic acid hydrogel is gradually swelled and degraded in body fluid, so that collagenase, a macrophage scavenger and an anti-tumor medicine are continuously released, and the local effective concentration can be maintained for more than two weeks.