CN115227720A - Application of human fetal dermal mesenchymal stem cell content in skin repair - Google Patents
Application of human fetal dermal mesenchymal stem cell content in skin repair Download PDFInfo
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Abstract
The invention is applicable to the technical field of bioengineering, and provides an application of human fetal dermal mesenchymal stem cell contents in skin repair, wherein the human fetal dermal mesenchymal stem cell contents comprise hFDMSCs-SCC and hFDMSCs-NSCC; mixing hFDMSCs-SCC and hFDMSCs-NSCC in different proportions, and preparing injectable or external preparations suitable for different application ranges under aseptic conditions. The invention utilizes the fact that fetal dermal mesenchymal stem cells have dermal homing characteristics, stronger proliferation capacity, differentiation potential and lower immunogenicity, and FDMSCs regulate the functional activities of dermal tissue vascular permeability, inflammatory immunoreaction, repair cells and the like through the paracrine action of secretory contents (extracellular vesicles and exosomes), promote hair follicle regeneration and dermal matrix synthesis, effectively promote acute and chronic wound repair, promote tissue regeneration, effectively inhibit scar hyperplasia, relieve tissue edema through inhibiting vascular permeability, effectively control inflammatory immunoreaction of various skin diseases, and effectively delay or reverse skin aging.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to application of human fetal dermal mesenchymal stem cell contents in skin repair.
Background
Human Fetal Dermal Mesenchymal Stem Cells (hFDMSCs) refer to a pluripotent Stem cell existing in Dermal tissue of skin of a spontaneous abortion fetus, and compared with other Mesenchymal Stem Cells (MSCs), hFDMSCs are tissue Mesenchymal Stem Cells with excellent self-renewal, multidirectional differentiation potential, dermal homing property and more primitive, and have immune regulation, anti-inflammation, anti-tumor and tissue repair effects, and in addition, can prevent progressive wound healing in early stages of burns, relieve vascular permeability and tissue edema, and relieve graft rejection, while there is almost no research on the application of hFDMSC cytoplasmic cell contents (nuclear cell contents, SCC) containing exosomes and hFDMSC non-secretory cell contents (NSFDcell contents, SCC containing nuclear membrane, DNA/RNA and nuclear protein).
The skin is the largest organ of a human body and is an important protection barrier of the body, with the arrival of the old society, the requirements of people on healing of acute and chronic wound surfaces and repairing quality, skin aging, ultraviolet injury, alopecia, inflammation and immunity related skin diseases and prolonging life are continuously enhanced, meanwhile, the quality and safety of various skin care products or external medicines are more and more emphasized, an effective regeneration repairing method needs to be searched urgently, and the problems of low survival rate, strong immunogenicity, unstable differentiation direction, possible tumorigenesis, ethical theory and the like exist in the stem cell local injection treatment.
Disclosure of Invention
The embodiment of the invention aims to provide application of human fetal dermal mesenchymal stem cell contents in skin repair, and aims to solve the problems in the background technology.
The embodiment of the invention is realized by applying the content of the human fetal dermal mesenchymal stem cells to skin repair, and is characterized in that the content of the human fetal dermal mesenchymal stem cells comprises hFDMSCs-SCC and hFDMSCs-NSCC.
Preferably, the hFDMSCs-SCC and hFDMSCs-NSCC are mixed and prepared into an injectable or external preparation under an aseptic condition, and the volume content of the mixture of the hFDMSCs-SCC and the hFDMSCs-NSCC in the preparation is 0.1-5%.
Preferably, the formulation further comprises glutathione and nicotinamide.
Preferably, the content of the glutathione is 2 to 8ug/ml based on the volume of the mixture of the hFDMSCs-SCC and the hFDMSCs-NSCC.
Preferably, the content of the nicotinamide is 2 to 8mg/ml based on the volume of the mixture of the hFDMSCs-SCC and the hFDMSCs-NSCC.
Preferably, the method for extracting the hFDMSCs-SCC comprises the following steps:
separating and purifying hFDMSCs;
obtaining an extract of hFDMSCs-SCC from the conditioned medium of cultured hFDMSCs.
Preferably, the method for extracting the hFDMSCs-NSCC comprises the following steps:
separating and purifying hFDMSCs;
and (3) crushing and centrifuging the hFDMSCs to obtain an extract of hFDMSCs-NSCC.
The application of the human fetal dermal mesenchymal stem cell content in skin repair provided by the embodiment of the invention utilizes the dermal homing property, stronger proliferation capacity, differentiation potential and lower immunogenicity of fetal dermal mesenchymal stem cells, and FDMSCs regulate the functional activities of dermal tissue vascular permeability, inflammatory immunoreaction, repair cells and the like through the paracrine action of secretory contents (extracellular vesicles and exosomes), so that tissue edema is reduced, hair follicle regeneration and dermal matrix synthesis are promoted, wound healing is accelerated, scar hyperplasia is inhibited at the same time, hFDMSCs-SCC and hFDMSCs-NSCC are mixed according to different proportions, an injectable or external preparation is obtained, the systemic and local application of tissue regeneration repair and skin disease treatment can be developed, acute and chronic wound repair is effectively promoted, tissue (blood vessels, nerves, dermal matrix and skin appendages) regeneration is promoted, scar hyperplasia is effectively inhibited, vascular permeability is inhibited, tissue edema is reduced, inflammatory immunoreaction of various skin diseases is effectively controlled, and aging of skin is effectively delayed or reversed, and anti-oxidative products are prepared by combining extracellular vesicles with extracellular vesicles and syncytial tissue regeneration.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
According to the consent of ethical committee and the informed consent of lying-in women, the early spontaneous abortion fetus of a healthy pregnant woman (without hereditary and infectious diseases) is selected through the assistance of long-term cooperative gynecologic hospitals, the hFDMSCs are separated from the skin of the early spontaneous abortion human fetus, the hFDMSCs-SCC and the hFDMSCs-NSCC extracts are sequentially prepared and mixed according to different proportions, so that an injectable or external preparation is obtained, the systemic and local application of tissue regeneration repair and skin disease treatment can be developed, and the mixture of the hFDMSCs-SCC and the hFDMSCs-NSCC is used for promoting tissue regeneration (including anti-aging) and acute and chronic wound repair, inhibiting scar hyperplasia and edema of damaged tissues and inflammatory immune reactions of different skin diseases in the modes of systemic (intravenous infusion), local injection, microneedle/laser post-local external application or dressing carrier and the like:
(1) Separating, purifying and identifying the hFDMSCs;
separation and purification: keeping thin-layer fat in the fetus in vitro for 1h, taking down the back skin of the fetus under the aseptic condition, repeatedly flushing skin tissues by PBS (phosphate buffer solution) containing 0.5-1.5% penicillin-streptomycin solution until flushing liquid is clear and free of blood stain, soaking in 0.2-0.3% chloramphenicol solution for 5-15min, and then soaking in PBS (20% -30%) with high-concentration penicillin-streptomycin solution for 5-15min; trimming to remove subcutaneous adipose tissue and obvious vellus hairs, cutting into 1.0 cm × 1.0 cm pieces, placing in 0.2-0.3% neutral protease, and standing overnight at 4 deg.C; cutting dermis into paste, and digesting in 0.12-0.13% type I collagenase at 37 deg.C for 3-5min in a constant temperature oscillator; filtering the digestive juice with 100 mesh cell filter screen, centrifuging for 3-7min at 1000rmp, removing supernatant, washing precipitate with culture medium, and suspending the cell mass at the bottom of the tube with full culture medium; inoculating into culture flask, and culturing at 37 deg.C and 5% CO 2 Culturing in a cell culture box with saturated humidity until passage;
and (3) identification:
(1) morphological observation;
(2) flow cytometry detection of cell surface markers: anti-CD14-PE-CF594, anti-CD34-PE, anti-CD44-APC, anti-CD45-FITC, anti-CD90-BV421, anti-CD105-PECY7;
(3) immunofluorescence staining detection of cell surface markers: anti-CK19, anti-vimentin, anti-SSEA-4, anti-Oct-4;
(4) detecting the differentiation capacity of osteogenesis, adipogenesis and chondrogenesis;
(2) Obtaining an extract of hFDMSCs-SCC from the conditioned medium of cultured hFDMSCs;
taking 3-5 generation FDMSCs, culturing with low-sugar complete culture medium containing 10% fetal calf serum, and extracting hFDMSCs-SCC when the cell density reaches 70-80%: firstly, sucking and discarding cell culture supernatant, performing cell starvation by using a blank culture medium without serum, after starvation is performed for 48 hours, sucking the cell culture supernatant, centrifuging at 3000rpm for 10-15min to remove cells and cell fragments, filtering the supernatant by using a 0.22 mu m filter, transferring the supernatant to a centrifugal ultrafiltration tube, concentrating the supernatant by using a centrifugal filtration method, transferring the concentrated supernatant to a 15ml centrifuge tube, adding an extraction reagent into the concentrated supernatant, adding a precipitator according to a certain proportion, placing the mixture in a refrigerator at 4 ℃ for vertical standing overnight, and taking out the centrifuge tube the next day to obtain an extract;
(3) Crushing and centrifuging the hFDMSCs to obtain an hFDMSCs-NSCC extract;
crushing hFDMSCs to obtain cell components, collecting cells, grinding the cells with a grinding rod or crushing the cells with an ultrasonic cell crusher, and centrifuging at 5000rmp for 15min to obtain an extract;
(4) Mixing the hFDMSCs-SCC and the hFDMSCs-NSCC according to different treatment mode ranges in different proportions, and preparing injectable or external preparations suitable for different application ranges under aseptic conditions, wherein the contents of the hFDMSCs-SCC and the hFDMSCs-NSCC are 0.1-5%, and the external preparations also comprise components such as glutathione (2-8 ug/ml) and nicotinamide (2-8 mg/ml);
(5) The mixture of hFDMSCs-SCC and hFDMSCs-NSCC is used for promoting tissue regeneration (including anti-aging) and acute and chronic wound repair, inhibiting scar hyperplasia and edema of damaged tissues, and inhibiting inflammatory immune reaction of different skin diseases in modes of whole body (intravenous infusion), local injection, local external application after microneedle/laser or dressing carrier and the like.
Specific implementations of the present invention are described in detail below with reference to specific embodiments.
Example 1
(1) Separating, purifying and identifying hFDMSCs;
separation and purification: keeping thin-layer fat in vitro of fetus within 1h, taking off the back skin of fetus under aseptic condition, repeatedly washing skin tissue with PBS containing 0.5% penicillin-streptomycin solution until the washing liquid is clear and free from blood contamination, soaking in 0.2% chloramphenicol solution for 5min, and soaking in high-concentration penicillin-streptomycin solution (20%) PBS for 5min; trimming to remove subcutaneous adipose tissue and obvious vellus hairs, shearing into 1.0 cm × 1.0 cm skin pieces, placing in 0.2% neutral protease, and standing overnight at 4 deg.C; shearing the dermis part into paste, and digesting in 0.125% type I collagenase at 37 deg.C for 3min in a constant temperature oscillator; filtering the digestive juice with 100 mesh cell filter screenCentrifuging at 1000rmp for 3-7min, sucking off supernatant, washing precipitate with culture medium, and suspending the cell mass at the bottom of the tube with full culture medium; inoculating into culture flask, and culturing at 37 deg.C and 5% CO 2 Culturing in a cell culture box with saturated humidity until passage;
(2) Obtaining an extract of hFDMSCs-SCC from the conditioned medium of the cultured hFDMSCs;
and (3) culturing the 3 rd generation FDMSCs, and extracting hFDMSCs-SCC when the cell density reaches 70 percent: firstly, removing cell culture supernatant by suction, performing cell starvation by using a blank culture medium, after starvation is performed for 48 hours, sucking the cell culture supernatant, centrifuging at 3000rpm for 10min to remove cells and cell fragments, filtering the supernatant by using a 0.22-micron filter, transferring the supernatant to a centrifugal ultrafiltration tube, concentrating the supernatant by using a centrifugal filtration method, transferring the concentrated supernatant to a 15ml centrifugal tube, adding an extraction reagent into the concentrated supernatant, adding a precipitator according to a certain proportion, then placing the mixture in a refrigerator at 4 ℃ for vertical standing overnight, and taking out the centrifugal tube the next day to obtain an extract;
(3) Crushing and centrifuging the hFDMSCs to obtain an hFDMSCs-NSCC extract;
crushing hFDMSCs to obtain cell components, collecting cells, grinding the cells with a grinding rod or crushing the cells with an ultrasonic cell crusher, and centrifuging at 5000rmp for 15min to obtain an extract;
(4) Mixing the hFDMSCs-SCC and hFDMSCs-NSCC, and preparing injectable or external preparations suitable for different application ranges under aseptic conditions, wherein the content of the hFDMSCs-SCC and the hFDMSCs-NSCC is 0.1%, and the external preparations also comprise components such as glutathione (2 ug/ml) and nicotinamide (2 mg/ml).
Example 2
(1) Separating, purifying and identifying the hFDMSCs;
separation and purification: keeping thin-layer fat in vitro of fetus within 1h, taking off the back skin of fetus under aseptic condition, repeatedly washing skin tissue with PBS containing 1% penicillin-streptomycin solution until the washing liquid is clear and free from blood contamination, soaking in 0.25% chloramphenicol solution for 10min, and soaking in high-concentration penicillin-streptomycin solution (20%) PBS for 10min; trimming to remove subcutaneous fatCutting the tissues and obvious vellus hairs into 1.0 cm × 1.0 cm pieces, and placing in 0.25% neutral protease at 4 deg.C overnight; shearing the dermis part into paste, and digesting in 0.125% type I collagenase at 37 deg.C for 3min in a constant temperature oscillator; filtering the digestive juice with 100 mesh cell filter screen, centrifuging for 5min at 1000rmp, removing supernatant, washing precipitate with culture medium, and suspending the cell mass at the bottom of the tube with full culture medium; inoculating into culture flask, and culturing at 37 deg.C and 5% CO 2 Culturing in a cell culture box with saturated humidity until passage;
(2) Obtaining an extract of hFDMSCs-SCC from the conditioned medium of cultured hFDMSCs;
and (3) culturing the 4 th generation of FDMSCs, and extracting hFDMSCs-SCC when the cell density reaches 70 percent: firstly, sucking and discarding cell culture supernatant, performing cell starvation by using a blank culture medium, after starvation is performed for 48 hours, sucking the cell culture supernatant, centrifuging at 3000rpm for 10min to remove cells and cell fragments, filtering the supernatant by using a 0.22 mu m filter, transferring the supernatant to a centrifugal ultrafiltration tube, concentrating the supernatant by using a centrifugal filtration method, transferring the concentrated supernatant to a 15ml centrifugal tube, adding an extraction reagent into the concentrated supernatant, adding a precipitator according to a certain proportion, placing the mixture in a refrigerator at 4 ℃ for vertical standing overnight, and taking out the centrifugal tube the next day to obtain an extract;
(3) Crushing and centrifuging the hFDMSCs to obtain an hFDMSCs-NSCC extract;
crushing hFDMSCs to obtain cell components, collecting cells, grinding the cells with a grinding rod or crushing the cells with an ultrasonic cell crusher, and centrifuging at 5000rmp for 15min to obtain an extract;
(4) Mixing the hFDMSCs-SCC and hFDMSCs-NSCC, and preparing injectable or external preparations suitable for different application ranges under aseptic conditions, wherein the content of the hFDMSCs-SCC and the hFDMSCs-NSCC is 3%, and the external preparations also comprise components such as glutathione (5 ug/ml) and nicotinamide (5 mg/ml).
Example 3
(1) Separating, purifying and identifying hFDMSCs;
separation and purification: keeping thin layer of fat in fetal in vitro for 1h, and taking offRepeatedly washing skin tissue with PBS containing 1.5% penicillin-streptomycin solution until the washing liquid is clear and free of blood stain, soaking in 0.3% chloramphenicol solution for 15min, and soaking in high concentration penicillin-streptomycin solution (20%) PBS for 15min; trimming to remove subcutaneous adipose tissue and obvious vellus hairs, shearing into 1.0 cm × 1.0 cm skin pieces, placing in 0.3% neutral protease, and standing overnight at 4 deg.C; cutting dermis into paste, and digesting in 0.125% type I collagenase at 37 deg.C for 5min in a constant temperature oscillator; filtering the digestive juice with 100 mesh cell filter screen, centrifuging for 7min at 1000rmp, removing supernatant, washing precipitate with culture medium, and suspending the cell mass at the bottom of the tube with full culture medium; inoculating into culture flask, and culturing at 37 deg.C and 5% CO 2 Culturing in a cell culture box with saturated humidity until passage;
(2) Obtaining an extract of hFDMSCs-SCC from the conditioned medium of cultured hFDMSCs;
and (3) culturing the 5 th generation FDMSCs, and extracting hFDMSCs-SCC when the cell density reaches 80 percent: firstly, sucking and discarding cell culture supernatant, performing cell starvation by using a blank culture medium, after starvation is performed for 48 hours, sucking the cell culture supernatant, centrifuging at 3000rpm for 10min to remove cells and cell fragments, filtering the supernatant by using a 0.22 mu m filter, transferring the supernatant to a centrifugal ultrafiltration tube, concentrating the supernatant by using a centrifugal filtration method, transferring the concentrated supernatant to a 15ml centrifugal tube, adding an extraction reagent into the concentrated supernatant, adding a precipitator according to a certain proportion, placing the mixture in a refrigerator at 4 ℃ for vertical standing overnight, and taking out the centrifugal tube the next day to obtain an extract;
(3) Crushing and centrifuging the hFDMSCs to obtain an hFDMSCs-NSCC extract;
crushing hFDMSCs to obtain cell components, collecting cells, grinding the cells with a grinding rod or crushing the cells with an ultrasonic cell crusher, and centrifuging at 5000rmp for 15min to obtain an extract;
(4) Mixing the hFDMSCs-SCC and hFDMSCs-NSCC, and preparing injectable or external preparations suitable for different application ranges under aseptic conditions, wherein the content of the hFDMSCs-SCC and the hFDMSCs-NSCC is 5%, and the external preparations also comprise glutathione (8 ug/ml) and nicotinamide (8 mg/ml) and other components.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (7)
1. The application of the human fetal dermal mesenchymal stem cell content in skin repair is characterized in that the human fetal dermal mesenchymal stem cell content comprises hFDMSCs-SCC and hFDMSCs-NSCC.
2. The use of the human fetal dermal mesenchymal stem cell content in skin repair according to claim 1, wherein the hFDMSCs-SCC and hFDMSCs-NSCC are mixed and then prepared into an injectable or external preparation under aseptic conditions, and the volume content of the mixture of hFDMSCs-SCC and hFDMSCs-NSCC in the preparation is 0.1 to 5%.
3. Use of human fetal dermal mesenchymal stem cell content for skin repair according to claim 2, wherein the formulation further comprises glutathione and nicotinamide.
4. The application of the human fetal dermal mesenchymal stem cell content in skin repair as claimed in claim 3, wherein the glutathione content is 2 to 8ug/ml based on the volume of the mixture of hFDMSCs-SCC and hFDMSCs-NSCC.
5. The application of the human fetal dermal mesenchymal stem cell content in skin repair according to claim 3, wherein the nicotinamide content is 2 to 8mg/ml based on the volume of the mixture of hFDMSCs-SCC and hFDMSCs-NSCC.
6. The application of the human fetal dermal mesenchymal stem cell content in skin repair according to claim 1, wherein the method for extracting the hFDMSCs-SCC comprises the following steps:
separating and purifying hFDMSCs;
obtaining an extract of hFDMSCs-SCC from the conditioned medium of the cultured hFDMSCs.
7. The use of the human fetal dermal mesenchymal stem cell content according to claim 1 for skin repair, wherein the method for extracting hFDMSCs-NSCC comprises the following steps:
separating and purifying hFDMSCs;
and (3) crushing and centrifuging the hFDMSCs to obtain an extract of hFDMSCs-NSCC.
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| CN103451153A (en) * | 2013-05-22 | 2013-12-18 | 栾佐 | Establishment method and application of clinical-grade neural stem cell line |
| CN115109752A (en) * | 2022-07-08 | 2022-09-27 | 清华大学 | Neural stem cell culture medium, freezing medium and preparation method of neural stem cells |
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| CN103451153A (en) * | 2013-05-22 | 2013-12-18 | 栾佐 | Establishment method and application of clinical-grade neural stem cell line |
| CN115109752A (en) * | 2022-07-08 | 2022-09-27 | 清华大学 | Neural stem cell culture medium, freezing medium and preparation method of neural stem cells |
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| CN118806683A (en) * | 2023-09-26 | 2024-10-22 | 天津医科大学眼科医院 | Application of intracellular nanovesicles derived from adult stem cells in anti-aging and hair follicle regeneration |
| CN118806683B (en) * | 2023-09-26 | 2024-11-22 | 天津医科大学眼科医院 | Application of adult stem cell-derived intracellular nano vesicles in anti-aging and hair follicle regeneration |
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