CN110192989B - Composition for promoting regeneration of skin stem cells and collagen blasts and preparation method thereof - Google Patents
Composition for promoting regeneration of skin stem cells and collagen blasts and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a composition for promoting regeneration of skin stem cells and collagen blasts, which comprises 20-30% of carboxymethyl cellulose, 30-40% of PBS buffer solution, 5-10% of glycerol and 25-35% of polycaprolactone-protein conjugate; wherein the polycaprolactone-protein conjugate is obtained by combining polycaprolactone with active protein of human skin fibroblasts according to the mass ratio of 2-5. The composition for promoting the regeneration of the skin stem cells and the collagen blasts takes the active protein of the skin fibroblasts as a repairing functional component, has direct effect on one hand compared with the effect of adopting growth factors, does not greatly influence the metabolism of facial cells after being injected into the face like the growth factors, avoids the problem of changing the metabolism level of the cells by exogenous stimulation, and can gradually take effect in a sustained-release mode.
Description
Technical Field
The invention relates to the technical field of preparation bioengineering, in particular to a composition for promoting regeneration of skin stem cells and collagen blasts and a preparation method thereof.
Background
The skin is the outermost tissue of the human body and plays a role in protecting the human body from external irritation or injury. With aging or trauma, the skin undergoes a marked decrease in its physiological activity or metabolic capacity in visual appearance, physical properties and physiological function until it gradually declines or dies, so that repair and cosmetology are considered to be the most important sections of cosmetology.
In the existing cosmetic treatment of skin tissues which have been subjected to trauma or aging, one approach is to inject skin filler or collagen stimulator into the face to fill or compensate the scar, and simultaneously, to promote the growth and transformation of facial cells, and to regulate the physiological functions and metabolic activities of facial cells. Or, in another milder way, the growth factor with the promoting function is added into the cosmetics at a certain effective concentration, and can effectively act with skin cells, promote the nutrition metabolism of epithelial cells, effectively promote the functions of subcutaneous collagen cells, accelerate the growth of the skin collagen cells, accelerate the cells to secrete collagen, thereby achieving the functions of repairing and beautifying.
However, the above functional components of growth factors with real effects are usually complicated in preparation method, high in cost and low in concentration of the obtained growth factors, so that the products with real effects are very expensive in price.
Based on the above situation, patent No. CN201811367770.4 proposes a method of cracking stem cells after culturing, preparing stem cell activator, injecting the stem cell activator into the face, and promoting epithelial cell metabolism by the stem cell activator, which can partially play a role in preventing and curing skin damage caused by various reasons, accelerating collagen secretion of cells, and the like.
However, after the activin product is used, particularly injected into the faces of different patients, on one hand, the dosage is extremely difficult to control, and rejection reaction is easily caused; on the other hand, activin is metabolized soon after injection, the half-life is short, and the real effect is not very stable.
Disclosure of Invention
The invention mainly aims to provide a protein stimulation injection composition for facial repair and beauty with slow release and low rejection reaction, aiming at leading the facial repair and beauty composition to exert the effect stably and simultaneously to be produced and prepared in a large scale and reduce the product price.
In order to achieve the aim, the composition for promoting the regeneration of skin stem cells and collagen blasts provided by the invention comprises 20-30% of carboxymethyl cellulose, 30-40% of PBS buffer solution, 5-10% of glycerol and 25-35% of polycaprolactone-protein conjugate by mass percent; wherein the content of the first and second substances,
the polycaprolactone-protein conjugate is obtained by combining polycaprolactone with active protein of human skin fibroblasts according to the mass ratio of 2-5; and the active protein of the human skin fibroblast is prepared by the following steps:
obtaining a human skin fibroblast sample and culturing to obtain the human skin fibroblast.
Carrying out cell disruption on the human skin fibroblasts obtained by the culture, and taking human skin fibroblast proteins;
taking rabbit skin wound fibroblasts and carrying out subculture;
and (3) carrying out cell disruption on the cultured rabbit skin wound fibroblasts, extracting protein, and immunizing a rat by using the extracted protein as an antigen to obtain the polyclonal antibody.
Preparing an affinity chromatographic column by using the polyclonal antibody as a chromatographic medium;
and (3) carrying out chromatography column chromatography on the human skin fibroblast protein in the affinity chromatography column of the polyclonal antibody to obtain the active protein of the human skin fibroblast.
Preferably, the pH of the PBS buffer is 7.0-7.5.
Preferably, in the step of obtaining a human skin fibroblast sample and culturing, FGF and EGF are used for induction culture.
Preferably, during the induction culture, the concentration of FGF in the culture medium is controlled to be 0.1-0.2 ng/ml, and the concentration of EGF in the culture medium is controlled to be 0.5-0.8 ng/ml.
Preferably, the polycaprolactone-protein conjugate is obtained by modifying and grafting polycaprolactone by active protein of human skin fibroblasts.
Preferably, the polycaprolactone-protein conjugate is prepared by the following steps:
reacting the active protein of the human skin fibroblast with glucose and sodium cyanoborohydride in a boric acid buffer solution to obtain glycosylated protein;
uniformly mixing a caprolactone monomer and glycosylated protein according to the mass ratio of 2-5, reacting under the condition of porcine pancreatic lipase, stopping the reaction of dichloromethane in the product, and completely dissolving the product; and filtering out lipase and purifying to obtain the polycaprolactone-protein conjugate.
Preferably, the solid phase matrix of the affinity chromatography column is Sepharose CL 4B.
Preferably, the sum of the mass fractions of the carboxymethylcellulose, the PBS buffer and the glycerol in the composition is 70%, and the mass fraction of the polycaprolactone-protein conjugate is 30%.
The invention further provides a preparation method of more than one composition for promoting the regeneration of skin stem cells and collagen blasts, which comprises the following steps:
uniformly mixing carboxymethyl cellulose, PBS buffer solution and glycerol, and fully dissolving to form gel solution;
and uniformly suspending the polycaprolactone-protein conjugate in the gel solution to obtain the protein stimulation injection composition for facial repair and beauty.
According to the preparation method, active protein close to human skin is extracted from rabbit wound skin fibroblasts with active expression of cell protein, and the active protein is used as an antigen to carry out immunoreaction on a rat to obtain a polyclonal antibody; then separating the polyclonal antibody from human skin fibroblasts obtained by mass culture to obtain a protein component with active function, forming a binding product with polycaprolactone, and finally preparing the injection composition with other components. Compared with the function and effect of slow release, because the protein component is weaker than the cell body of the directly transplanted allogeneic skin cells/stem cells in rejection reaction, and the proteins are derived from fibroblasts and have lower self-antigenicity, the protein has great advantages in both effectiveness and safety. Meanwhile, compared with direct injection of activin/factors and the like on the face, the effect on the metabolism level of normal cells is lower, and the effect is better.
Drawings
FIG. 1 is a schematic diagram of an electrophoresis strip of active proteins from human skin fibroblasts according to an embodiment of the present invention;
FIG. 2 is a microscopic scan of a polycaprolactone-protein conjugate in accordance with one embodiment of the invention;
FIG. 3 is a photomicrograph of PSR stained tissue with unpolarized light at 9 months of injection of an injectable composition in accordance with one embodiment of the present invention;
fig. 4 is a photomicrograph of polarized PSR stained tissue at 9 months of injection of an injectable composition according to one embodiment of the invention.
Detailed Description
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides a composition for promoting regeneration of skin stem cells and collagen blasts, which comprises the following components of 20-30% of carboxymethyl cellulose, 30-40% of PBS buffer solution, 5-10% of glycerol and 25-35% of polycaprolactone-protein conjugate by mass percent; wherein, the first and the second end of the pipe are connected with each other,
PBS (phosphate) buffer is a solvent medium, the pH value is preferably 7.0-7.5 in combination with the acid-base property of the final injection composition, carboxymethyl cellulose and PBS buffer form a gel carrier, and glycerol enhances the solubility of the carboxymethyl cellulose;
carboxymethyl cellulose is a polysaccharide cellulose derivative, and carboxymethyl (-CH) 2 -COOH) binds to certain hydroxyl groups of the glucose pyranose monomer to form a cellulose chain; the injection composition has hygroscopicity, forms gel with water, is mainly used as a component with filling function in the injection composition on one hand, and repairs tissue loss caused by physical damage of the face; on the other hand, it can be used as a guide for the function of the polycaprolactone-protein conjugate.
The polycaprolactone-protein conjugate is a product obtained by combining nanoparticles of polycaprolactone with active protein from skin fibroblasts, and on one hand, the polycaprolactone is aliphatic polyester with poly-alpha-hydroxy acid groups and is a synthetic polymer with biocompatibility and bioabsorbability; on the other hand, the sustained-release tablet has biocompatibility with various active ingredients from skin fibroblasts, is beneficial to combination and maintenance of the various active ingredients, and promotes the sustained-release effect.
In biology, polycaprolactone (PCL) is a natural polymer and is used as a biomolecule carrier and a support, and partial functions are applied to biomolecule and cell transmission of tissue engineering, so that collagen mother cell regeneration can be stimulated, cell immune expression can be recovered, and skin self-repair and energy regeneration can be enhanced.
Meanwhile, compared with the method of adopting the growth factor to take effect directly on one hand, the method takes effect gradually by taking the composite active protein of the skin fibroblast as a repairing functional component, does not influence the metabolism of the facial cells after being injected into the face like the growth factor, avoids the problem of changing the metabolic level of the cells by exogenous stimulation, and can take effect gradually in a slow-release mode.
In another direction, skin fibroblasts are terminally differentiated cells, but have the properties of a variety of stem cells and are also capable of differentiating into other functional cells of the skin tissue. Many protein components contained in the facial mask belong to proteins and enzymes with stem cell function, influence the properties of facial tissue cells, enhance the stem cell performance of the facial tissue cells and improve the self-repairing capability of the facial tissue cells.
The preparation of the active protein of the skin fibroblast is carried out in large quantity by adopting the following steps:
and S10, obtaining a human skin fibroblast sample, and carrying out mass culture to obtain the human skin fibroblasts.
S20, carrying out cell disruption on the human skin fibroblasts cultured in the step S10, and taking active protein components.
And S30, taking the rabbit skin wound fibroblasts and carrying out subculture.
And S40, extracting protein from the rabbit skin wound fibroblasts cultured in the step S30, and immunizing a rat to obtain the polyclonal antibody.
S50, preparing an affinity chromatographic column by using the polyclonal antibody obtained in the step S40 as a chromatographic medium;
and S60, carrying out sample loading and column passing on the active protein of the human face skin fibroblasts prepared in the step S20 by using a chromatography column of a polyclonal antibody of the rabbit skin wound fibroblast active protein, and collecting eluted components to obtain the required active protein prepared in large quantity.
In the process of culturing the human skin fibroblasts in the step S10, on one hand, the expression level of the protein can be improved, and the purpose of obtaining a large amount of protein with repairing activity is achieved; on the other hand, the cell characteristics of the skin fibroblasts are maintained at a required degree and cannot be differentiated or dedifferentiated, and a low-concentration inducer can be adopted to assist the culture in the culture process. The inducer component is added with 0.1-0.2 ng/ml FGF (fibroblast growth factor) and 0.5-0.8 ng/ml EGF (epidermal growth factor) for induction
After the above components of each part of the present invention are completely obtained, a composition for promoting regeneration of skin stem cells and collagen blasts is completely prepared as follows. Wherein the active protein fraction is obtained by the above preparation, and the other components are directly purchased. The preparation process of the protein-stimulated injection composition for facial repair and beauty comprises the following steps:
s100, reacting the active protein obtained in the step S60 with polycaprolactone to generate an active protein modified polycaprolactone product;
the mode of modifying polycaprolactone with protein usually includes graft reaction, covalent bonding, anchoring, etc., and graft copolymerization is the most common method among the commonly adopted modification modes, and the skilled person can perform experimental operation according to the modes. In the present invention, based on the stability and activity of the protein after modified grafting, a glycosylation modification mode that is relatively popular in about two years is preferably adopted in the implementation, and specifically, the following steps can be adopted:
s110, dissolving the active protein obtained in the step S64, glucose and sodium cyanoborohydride in a boric acid buffer solution, carrying out water bath reaction at about 37 ℃ for 2-4 days, separating and purifying by a glucan G-50 column, and carrying out freeze drying to obtain glycosylated protein;
s120, uniformly mixing a caprolactone monomer and glycosylated protein in a mass ratio of 2-5, adding porcine pancreatic lipase accounting for 10% of the mass of the caprolactone monomer, introducing nitrogen, sealing, and reacting in a constant-temperature oscillation reactor at the temperature of 30 ℃ for about 2 days;
s130, after the reaction in the step S120 is finished, adding dichloromethane with 2 times of the volume of the product into the product to completely dissolve the product, filtering out lipase by using filter paper, adding cold methanol with 10 times of the volume of the filtrate at the temperature of-20 ℃, standing in a refrigerator at the temperature of-20 ℃ for 1 day, and filtering to obtain a crude product;
and S140, extracting the crude product obtained in the step S130 for 2 days by using acetone as a solvent to obtain a glycosylated protein modified polycaprolactone product.
S200, obtaining materials according to the mass percentage of 20-30% of carboxymethyl cellulose, 30-40% of PBS buffer solution, 5-10% of glycerol and 25-35% of polycaprolactone-protein conjugate;
s300, uniformly mixing carboxymethyl cellulose, PBS buffer solution and glycerol, fully dissolving to form hydrogel-state solution, and then uniformly suspending the polycaprolactone-protein conjugate in the solution to obtain the protein-stimulated injection composition for facial repair and beauty.
The preparation method of the invention is characterized in that a large amount of active protein with substantial damage repair function is prepared in skin fibroblasts, active protein close to human skin is extracted from rabbit wound skin fibroblasts with active cell protein expression, and the active protein is used as an antigen to carry out immunoreaction to rats to obtain polyclonal antibodies; then separating the polyclonal antibody from human skin fibroblasts obtained by mass culture to obtain a protein component with active function, forming a binding product with polycaprolactone, and finally preparing the injection composition with other components. Compared with the function and effect of slow release, because the protein component is weaker than the cell body of the directly transplanted allogeneic skin cells/stem cells in rejection reaction, and the proteins are derived from fibroblasts and have lower self-antigenicity, the protein has great advantages in both effectiveness and safety. Meanwhile, compared with direct injection of activin/factors and the like on the face, the effect on the metabolism level of normal cells is lower, and the effect is better.
In the implementation, the weight percentage of the carboxymethyl cellulose, the PBS buffer solution and the glycerol in the injection composition is preferably 70%, and the weight percentage of the polycaprolactone-protein conjugate in the injection composition is preferably 30%; the form of the aqueous gel suspension formed is best.
In order to truly reflect the progressive effects of the above protein-stimulated injection composition for facial repair and beauty of the present invention, and to verify the above process, it will be described below with reference to specific examples.
S10, obtaining human skin fibroblasts, carrying out mass culture, carrying out the specific process according to the following experimental steps, and finally collecting the cultured human skin fibroblasts.
S11, obtaining a human face skin tissue specimen, and cutting 2-3 cm in the operation of the sixth people hospital in Shenzhen city 2 The skin tissue of (1) is washed 3 to 5 times with a PBS phosphate buffer solution of pH7.5 containing 100U/ml ampicillin, and then cut off the fat with a blade and finely pulverized to about 1mm 3 The small blocks are uniformly crushed to the greatest extent;
s12, placing the finely-crushed skin fine tissues obtained in the step S11 in a DEME/F12 culture medium containing 200U/ml of collagenase and hyaluronidase in a refrigerator overnight;
s13, adding a PBS solution containing 0.25% of trypsin-EDTA into the culture medium for digestion, and after shaking digestion in a shaking table for 30min, stopping digestion of the serum; further, the cells were collected by centrifugation, washed 2 to 3 times with DEME/F12 medium, and then inoculated into DMEM/F12 medium containing 8 to 12% FBS at an inoculation density of 2 to 3X 10 5 /ml, cultured in an incubator at 37 ℃ and 5% by volume in CO2;
s14, after culturing to a cell monolayer, collecting a PBS (phosphate buffer solution) of the cells, washing the cells, digesting the cells by using 0.25 mass percent of trypsin-EDTA (ethylene diamine tetraacetic acid), and centrifugally collecting the cells after terminating digestion; according to the following steps of 1:8, subculture at a passage ratio, wherein the culture conditions are controlled to 37 ℃ and 5% CO by using a DMEM/F12 medium containing 8 to 12% FBS during the culture 2 Culturing; and 0.1ng/ml FGF (fibroblast) was added to the medium during subcultureA fibroblast growth factor), and EGF (epidermal growth factor) at 0.5ng/ml were induced, and the cells were collected after culturing.
S20, breaking the cells collected in the step S14, taking active protein components: centrifuging the skin fibroblasts obtained by subculture in the step S14, washing with PBS or normal saline for 2-3 times, discarding the supernatant, digesting the cells, centrifuging, and adding deionized H 2 And (4) cracking the O. Cell size per 2 flasks (about 5X 10) 6 Cell) 2ml of ion H was added 2 Cracking O, and then centrifuging at low temperature; adding a large amount of salt into the supernatant to carry out salting-out precipitation, and then separating the precipitate to obtain primarily separated protein for later use.
S30, taking rabbit skin wound fibroblasts and carrying out subculture, wherein the detailed steps of the culture process can be referred to as 'Wang Yanzhuang'; xuerzhen; rong Qirong; subculturing rabbit skin wound fibroblasts [ J ]; chinese trauma journal; in 1993 stage 02 ", and following the experimental procedure described therein, rabbit skin wound fibroblasts were obtained.
S40, extracting protein from the rabbit skin wound fibroblasts cultured in the step S30, immunizing a rat, and obtaining a polyclonal antibody, wherein the step can be carried out according to the detailed steps of an immunology laboratory manual, and the following specific steps can be referred to:
s41, adding deionized H into the rabbit skin wound fibroblasts obtained by culturing in the step S30 2 And (4) cracking the O. Cell size per 2 flasks (about 5X 10) 6 Cell) 2ml of ion H was added 2 Cracking O, and then centrifuging at low temperature; adding a large amount of salt into the supernatant to carry out salting-out precipitation, then separating the precipitate to obtain primarily separated protein for later use;
s42, basic immunization: taking 3mL of the rabbit skin wound surface fibroblast active protein extracted in the step S41, mixing the rabbit skin wound surface fibroblast active protein with 2mL of Freund' S complete adjuvant, completely emulsifying by ultrasonic waves, then carrying out subcutaneous multipoint injection on 3 white rats, and taking blood samples from auricular root veins for storage;
s43, boost: after about 15 days, taking 3mL of the rabbit skin wound surface fibroblast active protein extracted in the step S41, mixing the rabbit skin wound surface fibroblast active protein with 2mL of Freund incomplete adjuvant, completely emulsifying by ultrasonic waves, then carrying out subcutaneous multipoint injection on 3 white rats, and taking blood samples from the ear root veins for storage;
s44, measuring the specific adsorption condition of the generated polyclonal antibody to the extracted rabbit skin wound fibroblast active protein by using the rat blood sample obtained in the step S43 through an ELISA (enzyme-Linked immuno sorbent assay) experiment; when the blood drop of the rabbit for detecting immunity by ELISA test reaches 10 4 ~10 5 At that time, the rats were sacrificed for blood collection. If the ELISA test result does not meet the standard, continuing repeating the step S43 to carry out the enhanced immunity on the white rat until the standard is met, and killing the white rat to take blood;
s45, separating plasma from the blood sample of the dead rat in the step S44, putting the obtained blood into a container filled with an anticoagulant in advance, fully and uniformly mixing, centrifuging at 1000rpm and 4 ℃ for 5min, and taking out supernatant to obtain plasma;
s46, extracting polyclonal antibodies from the plasma of step S45:
diluting 5 times with PBS buffer solution with pH7.0; taking a Protein G column (1 ml), and firstly washing the column with PBS buffer solution with pH7.0 until the data displayed by the Protein nucleic acid detector reach a baseline; taking out rat plasma solution diluted by PBS buffer solution with pH7.0, loading on Protein G column at flow rate of 0.5ml/min, washing with PBS buffer solution with pH7.0 to baseline after loading, and collecting flow-through solution;
washing the medium with pH2.5Gly-HCl buffer solution, receiving eluted protein solution, namely polyclonal antibody aiming at the rabbit skin wound fibroblast active protein, and immediately adjusting the pH value to be neutral; measuring A280nm and A260nm, and estimating the protein concentration; the eluted protein solution was subjected to SDS-PAGE analysis.
S50, preparing an affinity chromatographic column by using the polyclonal antibody obtained in the step S40 as a chromatographic medium; the process of preparing the chromatographic column is carried out by adopting the steps of a laboratory manual of a biological laboratory:
s51, obtaining a Sepharose CL 4B medium;
s52, washing the Sepharose CL 4B medium obtained in the step S51 by using 500mL of deionized water, pumping the washed medium to be in a wet cake shape (the sedimentation volume is about 45 mL), and transferring the wet cake shape to a 200 mL beaker; suspending the gel in 100 mL of deionized water, and gently stirring by using a magnetic stirring rod;
s53, dissolving 4g of CNBr in 50 ml of HPLC-grade acetonitrile, adding the solution into the gel suspension, putting a pH probe, maintaining the pH of the reaction mixture at 11 by using 20% NaOH, and maintaining the temperature at 0 ℃ in the whole system in an ice bath; after 10 to 15min of the reaction, whether all CNBr was dissolved was checked. At this time, the NaOH consumption rate will decrease; after the pH stabilized at 11.0, the reaction mixture was poured into a pre-cooled glass funnel of a sand core and washed with 1L of ice-cold deionized water and 500mL of ice-cold coupling buffer (pH 8.5,0.1M NaHCO) 3 ) The gel is washed rapidly, and must be immediately coupled with proteins or ligands due to the instability of the activated support;
the filtrate is treated with 500 mL0.1M FeSO 4 Neutralizing unreacted CNBr, and pouring into a waste liquid tank;
s54, with 0.1M NaHCO, pH8.5 3 Dialyzing the eluted protein solution prepared in step S46 (polyclonal antibody of rabbit skin wound fibroblast-derived active protein separated in the immunization step, mixing with the medium, uniformly mixing at 4 deg.C for 35h, adding 0.5ml of ethanolamine, shaking at 4 deg.C for 5h, washing the medium with PBS (pH7.0, 0.01M), and packing.
S60, carrying out sample loading and column passing on the active protein of the human face skin fibroblast prepared in the step S20 by using a chromatography column of a polyclonal antibody of the rabbit skin wound fibroblast active protein:
s61, taking 20 muL of the prepared protein sample in the step S20, and adding 980 muL of 10mM PBS; using the formula: c (mg/ml) =1.45 × a280nm-0.74 × a260nm estimation of protein concentration of the sample; taking about 1mg of sample to be detected according to the estimated concentration for later use;
s62, preparing a blank chromatographic column by taking a Sepharose CL 4B medium, and washing 2 volume of flow-through fluid by using PBS after the blank chromatographic column is balanced by 10mM PBS on the sample to be detected prepared in the step S61;
s63, the flow-through liquid collected after the preliminary filtration by the blank chromatographic column in the step S62 is applied to the chromatographic column prepared in the step S50 and balanced by 10mM PBS; washing the medium with PBS for 3-4 column volumes, and receiving each flow-through solution (the flow-through solution can pass through the column again and repeat for 2-3 times);
and S64, eluting the affinity chromatography column after the column is passed through the step S63 by using imidazole eluent, and collecting eluted components.
In order to verify the product separated in step S64, the result of SDS-PAGE electrophoresis of the eluate and photographing the electrophoretic gel staining is shown in FIG. 1, which indicates that the active protein of human fibroblasts that can be affinity-adsorbed by the antibody of rabbit skin fibroblasts is indeed obtained. Furthermore, as can be seen from the bands, each of the loaded bands contains multiple western blots, which indicates that the protein contains multiple proteins with different molecular weights, rather than a single protein.
Further preparation of injection compositions:
s110, taking 20 mu L of the active protein sample obtained in the step S64, and adding 980 mu L of 10mM PBS; using the formula: c (mg/ml) =1.45 × a280nm-0.74 × a260nm estimation of protein concentration of the sample; if the concentration of the sample is too low due to large amount of the eluent, the subsequent steps can be carried out after the corresponding concentration treatment is carried out to reach a certain concentration;
measuring an active protein sample according to the concentration and the weight of the active protein of about 500mg, dissolving the active protein sample with 1200mg of glucose and 1200mg of sodium cyanoborohydride in 0.2mol \ L boric acid buffer solution (pH 9), carrying out water bath reaction at about 37 ℃ for 2-4 days, separating and purifying by a glucan G-50 column, and carrying out freeze drying to obtain glycosylated protein;
s120, uniformly mixing a caprolactone monomer and glycosylated protein according to the mass ratio of 4;
s130, after the reaction in the step S120 is finished, adding dichloromethane with 2 times of the volume of the product into the product to completely dissolve the product, then filtering out lipase by using filter paper, adding cold methanol with 10 times of the volume of the filtrate at the temperature of 20 ℃ below zero into the filtrate, standing for 1 day in a refrigerator at the temperature of 20 ℃ below zero, and filtering to obtain a crude product;
and S140, extracting the crude product obtained in the step S130 for 2 days by using acetone as a solvent to obtain a glycosylated protein modified polycaprolactone product.
In order to verify the condition of the product, the result of the electron microscope scanning of the product is shown in fig. 2, and a plurality of large and small spherical binders with different sizes exist, one part is a protein component containing a plurality of different molecular weights, and the other part is the formed polycaprolactone particles.
S200, obtaining materials according to the material proportion of 20-30% of carboxymethyl cellulose, 30-40% of PBS buffer solution, 5-10% of glycerol and 25-35% of polycaprolactone-protein conjugate by mass;
s300, uniformly mixing carboxymethyl cellulose, PBS buffer solution (10 mM pH7.2) and glycerol, fully dissolving to form hydrogel-state solution, and then uniformly suspending the polycaprolactone-protein conjugate in the solution to obtain the protein stimulation injection composition for facial repair and beauty.
Further, in order to verify the injectable composition obtained in step S300, preliminary experiments were performed to verify its compatibility, toxicity, rejection, safety and repairing effect, and complete tests were required to evaluate its safety including cytotoxicity, skin irritation (intracutaneous injection of rabbits), sensitization (guinea pig-maximization method), acute systemic toxicity (intraperitoneal route mice), subchronic toxicity (28-day toxicity in sprague-dawley rats), genotoxicity (direct contact eimeria test), local tolerance (2 weeks after intracutaneous injection of rabbits), degradation and pyrogenicity.
In view of the fact that the content of the complete test is too large, a part of the test experiment process with definite results is selected for description:
A. injected into the back of a rabbit and the action mechanism of the rabbit on the animal body is researched. Biopsies were performed at various times after injection to examine polycaprolactone microspheres and protein. After 9 months of use, collagen III and collagen I were found, almost all being collagen I by 21 months, with microparticles appearing at 21 months. The polycaprolactone microspheres were not visible after 9 months of use.
B. Human biopsy was performed after injection in the temple area of a patient willing to undergo temple lift surgery. At month 13, collagen was found to appear in humans for the first time, confirming that the metabolism of collagen in the skin has a stimulating effect.
The key points of safety are as follows:
C. was performed by a famous dermatologist morse khaki doctor at the european medical clinic in munich, germany. A total of 40 patients received follow-up at different times at 3, 6, 9, 12, 15, 18 and 24 months post-injection. At 12 months, the efficacy results of the Wrinkle Severity Rating Scale (WSRS) and the Global Aesthetic Improvement Scale (GAIS) injected with the above compositions remained consistent, with sustained improvement in 90% and 91.4% of patients, respectively.
And after 9 months after injection, a photomicrograph of the non-polarized light PSR stained tissue was taken at the injection site, showing newly formed collagen as shown in fig. 3. The results of the PSR staining with polarized light are shown in FIG. 4, showing the presence of orange-red and green birefringence, recording the deposition of collagen fibers of type I and type III, confirming the formation of new collagen fibers by the PCL microparticles being perfectly smooth and spherical.
With respect to safety, all patients tolerated the treatment by injection of the above composition well. Some of the slight adverse effects that appeared after the primary treatment disappeared without intervention. No adverse events were observed at 1, 3, 6, 9, 12 months after treatment.
D. Part of adverse reaction data which have been presented subsequently:
the reactions of redness, edema, etc. that occur after dermal filler injection are mostly temporary. The occurrence of this phenomenon is influenced by a number of external factors: injection technique, injection site, injection times, injection depth, etc. It is also influenced by certain factors: personal intolerance, medical history, treatment history. French institute (ANSM) has detailed the side effects that appear after skin augmentation during surgery:
direct side effects (1-15 days): hematoma, erythema, edema were estimated to last 8 days;
semi-delayed side effects (15 days-3 months): infection (associated with sterile conditions), necrosis, non-specific inflammation were estimated to last for 1-6 months;
delayed type (3-24 months): allergies, erythema, pigmentation estimated to last 1-12 months;
ANSM estimates that side effects have an effect on 0.1% to 1% of injected humans.
The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by using the contents of the present specification and the accompanying drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (8)
1. A composition for promoting regeneration of skin stem cells and collagen blasts is characterized by comprising 20-30% of carboxymethyl cellulose, 30-40% of PBS buffer solution, 5-10% of glycerol and 25-35% of polycaprolactone-protein conjugate by mass percent; wherein the content of the first and second substances,
the polycaprolactone-protein conjugate is obtained by combining polycaprolactone with active protein of human skin fibroblasts according to the mass ratio of 2-5:
reacting the active protein of the human skin fibroblast with glucose and sodium cyanoborohydride in a boric acid buffer solution to obtain glycosylated protein;
uniformly mixing a caprolactone monomer and glycosylated protein according to the mass ratio of 2-5, reacting under the condition of porcine pancreatic lipase, stopping the reaction of dichloromethane in the product, and completely dissolving the product; filtering out lipase and purifying to obtain the polycaprolactone-protein conjugate;
wherein the active protein of the human skin fibroblast is prepared by the following steps:
obtaining a human skin fibroblast sample and culturing to obtain human skin fibroblasts;
carrying out cell disruption on the human skin fibroblasts obtained by the culture, and taking human skin fibroblast proteins;
taking rabbit skin wound fibroblasts and carrying out subculture;
carrying out cell disruption on cultured rabbit skin wound fibroblasts, extracting protein, and immunizing a rat by using the extracted protein as an antigen to obtain a polyclonal antibody;
preparing an affinity chromatographic column by using the polyclonal antibody as a chromatographic medium;
and (3) carrying out chromatography column chromatography on the human skin fibroblast protein in the affinity chromatography column of the polyclonal antibody to obtain the active protein of the human skin fibroblast.
2. The composition for promoting regeneration of skin stem cells and collagen blasts as claimed in claim 1, wherein said PBS buffer has a pH of 7.0 to 7.5.
3. The composition for promoting regeneration of skin stem cells and collagen blasts according to claim 1, wherein said human skin fibroblast sample is obtained and cultured by induction with FGF and EGF.
4. The composition for promoting regeneration of skin stem cells and collagen blasts according to claim 3, wherein the concentration of FGF in the culture medium is controlled to be 0.1 to 0.2ng/ml and the concentration of EGF in the culture medium is controlled to be 0.5 to 0.8ng/ml during said induction culture.
5. The composition for promoting regeneration of skin stem cells and collagen blasts as in claim 1, wherein said polycaprolactone-protein conjugate is obtained by modifying polycaprolactone with an active protein derived from human skin fibroblasts.
6. The composition for promoting regeneration of skin stem cells and collagen progenitor cells according to claim 1, wherein the solid phase matrix of the affinity chromatography column is Sepharose CL 4B.
7. The composition for promoting regeneration of skin stem cells and collagen blasts as in claim 1, wherein the sum of the mass percentages of carboxymethylcellulose, PBS buffer and glycerol in said composition is 70% and the mass percentage of polycaprolactone-protein conjugate is 30%.
8. A method for preparing the composition for promoting the regeneration of skin stem cells and collagen blasts according to any one of claims 1 to 7, comprising the steps of:
uniformly mixing carboxymethyl cellulose, PBS buffer solution and glycerol, and fully dissolving to form gel solution;
and uniformly suspending the polycaprolactone-protein conjugate in the gel solution to obtain the composition for promoting the regeneration of the skin stem cells and the collagenous progenitor cells.
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