CN111518754A - Preparation method and application of intracellular active ingredients of human skin fibroblasts - Google Patents
Preparation method and application of intracellular active ingredients of human skin fibroblasts Download PDFInfo
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Abstract
The invention belongs to the field of skin repair, and particularly relates to a preparation method and application of intracellular active ingredients of fibroblasts of human skin, wherein the intracellular active ingredients are mixed with plant extracts to prepare freeze-dried powder, and the freeze-dried powder can be added into skin care products to achieve the effect of protecting the skin. Human skin fibroblasts are obtained by an enzyme digestion method, the cells are cracked by repeated freezing and thawing, intracellular factors of the cells are collected and plant extracts are supplemented, and the two can mutually promote, stabilize and maintain effective components, supplement components required by the skin and maintain the activity of the skin.
Description
Technical Field
The invention belongs to the technical field of skin repair, and particularly relates to an intracellular active ingredient of fibroblast of human skin, a preparation method and application thereof.
Background
Human dermal fibroblasts are the main structural components constituting the dermis of the skin, are involved in aging and repair of the skin, can synthesize and secrete effective components, and have an important role in maintaining the stability of the skin. Collagen and many cell repair factors in the skin are mostly derived from fibroblasts, and have an important role in maintaining the activity of the skin.
The lack of a single active ingredient can often maintain the balance of the skin by adding corresponding cytokines or polypeptides. However, it is often empirical how to find the missing component, which does not result in a good repair of the skin. If several are missing, the skin is more difficult to replenish. More and more researches show that the maintenance effect of the plant essence on the skin activity is necessary to protect the skin and maintain and repair the skin by combining the intracellular effective components of the fiber cells with the plant essence.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide the human-derived skin fibroblast intracellular active ingredient, and the preparation method and the application thereof.
Therefore, the invention provides the following technical scheme:
in a first aspect, the present invention provides a method for preparing an intracellular active ingredient of human skin fibroblasts, comprising the steps of:
adjusting cell concentration of human skin fibroblast to 1-3 × 10 with 0.9% physiological saline6Placing the cells/mL with a volume of 10-20mL in a refrigerator at-80 deg.C for 30min, freezing, taking out, placing in a water bath at 37 deg.C for 30min, dissolving, ultrasonically shaking for 5min, repeating for 3-5 times, completely lysing the cells, and collecting cell lysate, i.e. cell debris and cell lysate.
Centrifuging cell lysate at room temperature at 4000r/min for 10-20min, collecting supernatant, filtering with 0.22 μm filter membrane, adding 0.9% physiological saline, and adjusting to 10-20ml to obtain fibroblast intracellular effective component.
In the preparation method, the acquisition of the human skin fibroblasts comprises the following steps:
repeatedly rinsing the obtained human abdominal whole skin in PBS buffer solution containing 100U/mL penicillin and 100g/mL streptomycin for 30-50 times, and removing subcutaneous fat and redundant tissues;
cutting the skin into 1-3mm3Small pieces, attached to the dermal side of a culture flask, 2-3mL of collagenase type II 2mg/mL, placed at 37 ℃ in 5% CO2Digesting for 12-15 hours in a cell culture box;
taking out digested skin, carefully taking out epidermal tissue, repeatedly rinsing the rest part with 100U/mL penicillin and 100g/mL streptomycin-PBS buffer solution for 5-10 times, and thoroughly shearing;
then adding 3-5mL of type II collagenase of 2mg/mL, placing at 37 ℃ and 5% CO2Digesting for 4-5 hours in a cell culture box;
and filtering the digested skin by using a 100-mesh filter screen, washing the digested skin for 3-5 times by using PBS (phosphate buffer solution), and screening the digested skin by adherence to obtain fibroblasts.
In the preparation method, the collected cells are subcultured, and the cell concentration is adjusted to 1-3 × 10 by using a serum-free medium containing 100U/mL penicillin and 100g/mL streptomycin6one/mL, 5% CO at 37 ℃2And (4) culturing in a cell culture box, observing the growth condition of the cells, subculturing the cells when the cell density is about 80%, and collecting fourth generation cells for later use.
Digesting the fourth generation fibroblasts by 0.25 percent of trypsin for 3-5min, centrifuging the digested cells for 5-10min at the speed of 1000-2000r/min, and washing the precipitate for 3-5 times by 0.9 percent of normal saline.
In a second aspect, the invention provides an intracellular active ingredient of the human skin fibroblast obtained by the preparation method.
In a third aspect, the invention provides a preparation method of lyophilized powder of intracellular active ingredients of human skin fibroblasts, which comprises the steps of adding the active ingredients of the human skin fibroblasts into the lyophilized powder of the intracellular active ingredients of the human skin fibroblasts for protection, and then carrying out lyophilization.
In the preparation method, the formula of the freeze-dried powder is that each 100mL of the freeze-dried powder contains the following components: 30-50mL of intracellular active ingredients of human skin fibroblasts, 0.1-0.5mL of magnolia bark extract, 0.1-0.2mL of aloe extract, 0.1-0.2mL of cactus extract and 0.1-0.3mL of rose extract.
The conditions of the freeze-drying are as follows: the pressure is 20pa, and the temperature is sequentially at-40 deg.C for 5h, 25 deg.C for 4h, 10 deg.C for 2h, 0 deg.C for 2h, and 25 deg.C for 6 h.
In a fourth aspect, the invention provides a human-derived skin fibroblast intracellular active ingredient obtained by the preparation method, a human-derived skin fibroblast intracellular active ingredient or a human-derived skin fibroblast intracellular active ingredient freeze-dried powder obtained by the preparation method, which has an application of any one of the following:
(1) use in delaying skin aging;
(2) use in repairing skin damage;
(3) the use in the preparation of a cosmetic for delaying skin aging or repairing skin damage;
(4) the application of the composition in preparing medicines for delaying skin aging or repairing skin damage.
In a fifth aspect, the invention provides a cosmetic or a medicine for delaying skin aging or repairing skin damage, which comprises the human-derived skin fibroblast intracellular active ingredient obtained by the preparation method, the human-derived skin fibroblast intracellular active ingredient or the human-derived skin fibroblast intracellular active ingredient freeze-dried powder obtained by the preparation method.
The technical scheme of the invention has the following advantages:
1. according to the preparation method of the lyophilized powder of the intracellular active ingredients of the human skin fibroblasts, provided by the invention, the intracellular active ingredients of the human skin fibroblasts are extracted, and the plant extract is added, so that the stability of the active ingredients can be maintained, and the active ingredients can play a role on the skin together.
2. The invention provides a preparation method of intracellular active ingredients of human skin fibroblasts, which obtains a high-purity fibroblast concentrated solution through a specific culture system and a serum-free culture medium, has no other components damaging cells, and has simple operation and higher yield.
3. The lyophilized powder of the intracellular active ingredients of the human skin fibroblasts provided by the invention has stable properties, and the addition of the plant ingredients has no influence on the intracellular active ingredients with the main effects.
4. The human skin fibroblast intracellular active ingredient or the freeze-dried powder thereof provided by the invention has the following applications: use in delaying skin aging; use in repairing skin damage; the use in the preparation of a cosmetic for delaying skin aging or repairing skin damage; the application of the composition in preparing medicines for delaying skin aging or repairing skin damage.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing the effect of the addition of plant extracts on 4 cytokines in Experimental example 1 of the present invention.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The raw materials and reagents used in the present invention are commercially available.
The preparation methods of magnolia bark extract, aloe extract, cactus extract and rose extract in the following examples are as follows: the preparation method of the magnolia bark extract comprises the following steps:
(1) cleaning magnolia bark, crushing, adding into 70 volume percent ethanol solution with a solid-to-liquid ratio of 1:30, and ultrasonically soaking for 60min at the temperature of 50 ℃.
(2) Filtering the soaked bark and ethanol solution, and removing precipitate such as bark.
(3) Concentrating the filtrate at 50 deg.C under reduced pressure to obtain Magnolia bark concentrated solution.
The preparation method of the aloe extract comprises the following steps:
(1) cleaning fresh Aloe, peeling, and grinding the rest part.
(2) Adding the ground juice into 80 vol% ethanol solution at a solid-to-liquid ratio of 1:20, and ultrasonically soaking for 60min at 50 deg.C.
(3) Filtering the soaking solution to remove precipitate such as fiber.
(4) Concentrating the filtrate at 50 deg.C under reduced pressure to obtain aloe concentrated solution.
The preparation method of the cactus extract comprises the following steps:
(1) cleaning fresh radix et caulis Opuntiae Dillenii, and oven drying at 70 deg.C.
(2) Pulverizing dried radix et caulis Opuntiae Dillenii into powder, adding into 75 vol% ethanol solution at a solid-to-liquid ratio of 1:20, and ultrasonically soaking for 50min at 50 deg.C.
(3) Filtering the soaking solution, and removing the precipitate.
(4) Concentrating the filtrate at 50 deg.C under reduced pressure to obtain radix et caulis Opuntiae Dillenii concentrated solution.
The preparation method of the rose extract comprises the following steps:
(1) cleaning rose leaves, and drying in a 70 ℃ oven.
(2) Crushing the dried rose leaves into powder, then adding the powder into 90 volume percent ethanol solution with the solid-liquid ratio of 1:25, and ultrasonically soaking for 60min at the temperature of 50 ℃.
(3) Filtering the soaking solution, and removing the precipitate.
(4) Concentrating the filtrate at 50 deg.C under reduced pressure to obtain flos Rosae Rugosae concentrated solution.
Example 1 preparation of intracellular active principles of human dermal fibroblasts
The embodiment provides a preparation method of intracellular active ingredients of human skin fibroblasts, which comprises the following steps:
(1) repeatedly rinsing the obtained human abdominal whole skin in PBS buffer solution containing 100U/mL penicillin and 100g/mL streptomycin for 40 times to remove subcutaneous fat and redundant tissues;
(2) cutting the skin into 2mm3The small pieces, attached to the dermal side of the culture flask, 2.5mL of 2mg/mL collagenase II, were placed at 37 ℃ in 5% CO2Digesting for 13 hours in a cell culture box;
(3) the digested skin was removed, the epidermal tissue carefully removed, and the remaining part was repeatedly rinsed 8 times with 100U/mL penicillin and 100g/mL streptomycin in PBS buffer and thoroughly minced;
(4) then 4mL of collagenase II at 2mg/mL was added and the mixture was left at 37 ℃ with 5% CO2Digesting for 4.5 hours in a cell culture box;
(5) and filtering the digested skin by using a 100-mesh filter screen, washing the digested skin for 4 times by using PBS (phosphate buffer solution), and screening the digested skin by adherence to obtain the fibroblast.
(6) In the preparation method, the collected cells are subcultured, and the cell concentration is adjusted to 2 × 10 by using a serum-free medium containing 100U/mL penicillin and 100g/mL streptomycin6one/mL, 5% CO at 37 ℃2And (4) culturing in a cell culture box, observing the growth condition of the cells, subculturing the cells when the cell density is about 80%, and collecting fourth generation cells for later use.
(7) The fourth generation of cells was adjusted to a cell concentration of 2 × 10 with 0.9% physiological saline6Standing in 10-20 mL/mL volume, freezing in a refrigerator at-80 deg.C for 30min, taking out, and placing in 37 deg.C water bath for 30minAfter the lysis, the cell is completely lysed by ultrasonic oscillation for 5min and repeated for 4 times, and cell lysate, namely cell debris and cell lysate, is collected.
(8) Centrifuging cell lysate at room temperature at 4000r/min for 15min, collecting supernatant, filtering with 0.22 μm filter membrane, adding 0.9% physiological saline, and adjusting to 10-20ml to obtain fibroblast intracellular effective component.
Example 2 preparation of intracellular active principles of human dermal fibroblasts
The embodiment provides a preparation method of intracellular active ingredients of human skin fibroblasts, which comprises the following steps:
(1) repeatedly rinsing the obtained human abdominal full-thickness skin in PBS buffer solution containing 100U/mL penicillin and 100g/mL streptomycin for 30 times to remove subcutaneous fat and redundant tissues;
(2) cutting the skin into 1mm3The small pieces, attached to the dermal side of the culture flask, 2mg/mL collagenase II 3mL, placed at 37 ℃ in 5% CO2Digesting for 12 hours in a cell culture box;
(3) the digested skin was removed, the epidermal tissue carefully removed, and the remaining part was repeatedly rinsed 5 times with 100U/mL penicillin and 100g/mL streptomycin in PBS buffer and thoroughly minced;
(4) then 5mL of collagenase II at 2mg/mL was added and the mixture was left at 37 ℃ with 5% CO2Digesting for 4 hours in a cell culture box;
(5) and filtering the digested skin by using a 100-mesh filter screen, washing the digested skin for 3 times by using PBS (phosphate buffer solution), and screening the digested skin by adherence to obtain the fibroblast.
(6) In the preparation method, the collected cells are subcultured, and the cell concentration is adjusted to 1 × 10 by using a serum-free medium containing 100U/mL penicillin and 100g/mL streptomycin6one/mL, 5% CO at 37 ℃2And (4) culturing in a cell culture box, observing the growth condition of the cells, subculturing the cells when the cell density is about 80%, and collecting fourth generation cells for later use.
(7) Adjusting cell concentration of human skin fibroblast to 1 × 10 with 0.9% physiological saline6Placing the cell/mL with a volume of 10mL in a refrigerator at-80 deg.C for 30min for freezing, taking out, placing in a water bath at 37 deg.C for 30min, dissolving, ultrasonically shaking for 5min, repeating for 5 times, completely lysing cells, and collecting cell lysate, i.e. cell debris and cell lysate.
(8) Centrifuging cell lysate at room temperature at 4000r/min for 20min, collecting supernatant, filtering with 0.22 μm filter membrane, adding 0.9% physiological saline, and adjusting to 10ml to obtain fibroblast intracellular effective component.
Example 3 preparation of intracellular active principles of human dermal fibroblasts
The embodiment provides a preparation method of intracellular active ingredients of human skin fibroblasts, which comprises the following steps:
(1) repeatedly rinsing the obtained human abdominal full-thickness skin in PBS buffer solution containing 100U/mL penicillin and 100g/mL streptomycin for 50 times, and removing subcutaneous fat and redundant tissues;
(2) cutting the skin into 3mm3The small pieces, attached to the dermal side of the culture flask, 2mL of collagenase II at 2mg/mL, were placed at 37 ℃ in 5% CO2Digesting for 15 hours in a cell culture box;
(3) the digested skin was removed, the epidermal tissue carefully removed, and the remaining part was repeatedly rinsed 10 times with 100U/mL penicillin and 100g/mL streptomycin in PBS buffer and thoroughly minced;
(4) then 3mL of collagenase II at 2mg/mL was added and the mixture was left at 37 ℃ with 5% CO2Digesting for 5 hours in a cell culture box;
(5) and filtering the digested skin by using a 100-mesh filter screen, washing the digested skin for 5 times by using PBS (phosphate buffer solution), and screening the digested skin by adherence to obtain the fibroblast.
(6) In the preparation method, the collected cells are subcultured, and the cell concentration is adjusted to 3 × 10 by using a serum-free medium containing 100U/mL penicillin and 100g/mL streptomycin6one/mL, 5% CO at 37 ℃2And (4) culturing in a cell culture box, observing the growth condition of the cells, subculturing the cells when the cell density is about 80%, and collecting fourth generation cells for later use.
(7) Adjusting cell concentration of human skin fibroblast to 3 × 10 with 0.9% physiological saline6Placing the cells/mL with a volume of 20mL in a refrigerator at-80 deg.C for 30min for freezing, taking out, placing in a water bath at 37 deg.C for 30min, dissolving, ultrasonically shaking for 5min, repeating for 3 times, completely lysing the cells, and collecting cell lysate, i.e. cell debris and cell lysate.
(8) Centrifuging cell lysate at room temperature at 4000r/min for 10min, collecting supernatant, filtering with 0.22 μm filter membrane, adding 0.9% physiological saline, and adjusting to 20ml to obtain fibroblast intracellular effective component.
Example 4 preparation method of lyophilized powder of intracellular active ingredient of human dermal fibroblast
The embodiment provides a preparation method of lyophilized powder of intracellular active ingredients of human skin fibroblasts, which comprises the following steps:
the concentrated solution of the intracellular active ingredient of the fibroblast prepared in example 1 contains the following components: 40mL of intracellular active ingredients of human skin fibroblasts, 0.3mL of magnolia bark extract, 0.15mL of aloe extract, 0.15mL of cactus extract and 0.2mL of rose extract.
Placing the above mixture at a pressure of 20pa at-40 deg.C for 5h, at-25 deg.C for 4h, at 10 deg.C for 2h, at 0 deg.C for 2h, and at 25 deg.C for 6h to obtain lyophilized powder of human dermal fibroblast intracellular effective component.
Example 5 preparation method of lyophilized powder of intracellular active ingredient of human dermal fibroblast
The embodiment provides a preparation method of lyophilized powder of intracellular active ingredients of human skin fibroblasts, which comprises the following steps:
the concentrated solution of the intracellular active ingredient of the fibroblast prepared in example 2 contains the following components: 30mL of intracellular active ingredients of human skin fibroblasts, 0.5mL of magnolia bark extract, 0.1mL of aloe extract, 0.2mL of cactus extract and 0.1mL of rose extract.
Placing the above mixture at a pressure of 20pa at-40 deg.C for 5h, at-25 deg.C for 4h, at 10 deg.C for 2h, at 0 deg.C for 2h, and at 25 deg.C for 6h to obtain lyophilized powder of human dermal fibroblast intracellular effective component.
Example 6 preparation method of lyophilized powder of intracellular active ingredient of human dermal fibroblast
The embodiment provides a preparation method of lyophilized powder of intracellular active ingredients of human skin fibroblasts, which comprises the following steps:
the concentrated solution of the intracellular effective components of the fibroblasts prepared in example 3 contained the following components per 100 mL: human skin fibroblast intracellular effective component 50mL, Magnolia bark extract 0.1mL, Aloe extract 0.2mL, radix et caulis Opuntiae Dillenii extract 0.1mL, and flos Rosae Rugosae extract 0.3 mL.
Placing the above mixture at a pressure of 20pa at-40 deg.C for 5h, at-25 deg.C for 4h, at 10 deg.C for 2h, at 0 deg.C for 2h, and at 25 deg.C for 6h to obtain lyophilized powder of human dermal fibroblast intracellular effective component.
Experimental example 1 Effect of adding plant extracts on intracellular active ingredients of fibroblasts
By taking fibroblast lysate (prepared in example 1) without plant extracts as a blank and adding extracting solution (prepared in example 4) as a control, 4 cytokines which play a main role in skin are detected by adopting an ELISA kit according to the instruction of the kit, wherein the kits are all from R & D company, and the experimental result is shown in figure 1.
The test results are shown in FIG. 1, the variation between the two groups is small, P is greater than 0.05, and the result shows that the addition of the plant extract has no influence on 4 cytokines which play main roles in the skin.
The particular embodiments described herein are illustrative only, as the invention may be modified and supplemented by, or substituted in a similar manner by, those skilled in the art without departing from the spirit of the invention or exceeding the scope of the appended claims. Although the present invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.
Claims (10)
1. A method for preparing intracellular active ingredients of human skin fibroblasts is characterized by comprising the following steps:
adjusting cell concentration of human skin fibroblast to 1-3 × 10 with 0.9% physiological saline6Placing the cells/mL with a volume of 10-20mL in a refrigerator at-80 deg.C for 30min for freezing, taking out, placing in a water bath at 37 deg.C for 30min, dissolving, ultrasonically shaking for 5min, repeating for 3-5 times, completely lysing cells, and collecting cell lysate, i.e. cell debris and cell lysate;
centrifuging the obtained cell lysate for 10-20min at room temperature under 4000r/min, collecting supernatant, filtering with 0.22 μm filter membrane, adding 0.9% physiological saline, and adjusting to 10-20ml to obtain fibroblast intracellular effective component.
2. The method for preparing the human dermal fibroblast according to claim 1, wherein the obtaining of the human dermal fibroblast comprises the steps of:
repeatedly rinsing the obtained human abdominal whole skin in PBS buffer solution containing 100U/mL penicillin and 100g/mL streptomycin for 30-50 times, and removing subcutaneous fat and redundant tissues;
cutting the skin into 1-3mm3Attaching small pieces of collagenase type II 2-3mL to a culture flask, placing at 37 deg.C and 5% CO2Digesting for 12-15 hours in a cell culture box;
taking out digested skin, carefully taking out epidermal tissue, repeatedly rinsing the rest part with 100U/mL penicillin and 100g/mL streptomycin-PBS buffer solution for 5-10 times, and thoroughly shearing;
then adding 3-5mL of type II collagenase of 2mg/mL, placing at 37 ℃ and 5% CO2Digesting for 4-5 hours in a cell culture box;
and filtering the digested skin by using a 100-mesh filter screen, washing the digested skin for 3-5 times by using PBS (phosphate buffer solution), and screening the digested skin by adherence to obtain fibroblasts.
3. The method of claim 2The method is characterized in that the obtained fibroblasts are subcultured, and the cell concentration is adjusted to 1-3 × 10 by using a serum-free medium containing 100U/mL penicillin and 100g/mL streptomycin6one/mL, 5% CO at 37 ℃2And (4) culturing in a cell culture box, observing the growth condition of the cells, subculturing the cells when the cell density is about 80%, and collecting fourth generation cells for later use.
4. The method as set forth in claim 3, wherein the fourth-generation fibroblasts are digested with trypsin at 0.25% by mass for 3-5min, and the digested cells are centrifuged at 1000-2000r/min for 5-10min, and the resulting pellet is washed with 0.9% physiological saline for 3-5 times.
5. An intracellular active ingredient of the human dermal fibroblast obtained by the preparation method according to any one of claims 1 to 4.
6. A method for preparing lyophilized powder of intracellular active ingredients of human skin fibroblasts is characterized in that plant extracts are added into the intracellular active ingredients of the human skin fibroblasts, and then the lyophilized powder is lyophilized.
7. The preparation method of claim 6, wherein the formulation of the lyophilized powder is: 30-50mL of intracellular active ingredients of human skin fibroblasts, 0.1-0.5mL of magnolia bark extract, 0.1-0.2mL of aloe extract, 0.1-0.2mL of cactus extract and 0.1-0.3mL of rose extract.
8. The method for preparing a drug according to claim 6 or 7, wherein the conditions for lyophilization are: the pressure is 20pa, 5h at-40 ℃, 4h at-25 ℃, 2h at 10 ℃, 2h at 0 ℃ and 6h at 25 ℃.
9. The use of the active ingredient in the human dermal fibroblast cell obtained by the preparation method according to any one of claims 1 to 4, the active ingredient in the human dermal fibroblast cell according to claim 5, or the active ingredient in the human dermal fibroblast cell obtained by the preparation method according to any one of claims 6 to 8, in any one of the following:
(1) use in delaying skin aging;
(2) use in repairing skin damage;
(3) the use in the preparation of a cosmetic for delaying skin aging or repairing skin damage;
(4) the application of the composition in preparing medicines for delaying skin aging or repairing skin damage.
10. A cosmetic or a pharmaceutical for delaying skin aging or repairing skin damage, comprising the intracellular active ingredient of the human skin fibroblast obtained by the preparation method of any one of claims 1 to 4, the intracellular active ingredient of the human skin fibroblast obtained by the preparation method of claim 5 or the intracellular active ingredient freeze-dried powder of the human skin fibroblast obtained by the preparation method of any one of claims 6 to 8.
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