阅读说明：本技术 人脐带静脉内皮细胞在制备抗衰老的产品中的应用及抗衰老冻干粉和溶液 (Application of human umbilical vein endothelial cells in preparation of anti-aging product, anti-aging freeze-dried powder and anti-aging solution ) 是由 刘中民 贾文文 齐奕尧 汤红明 杜颍侠 王博闻 于 2021-08-10 设计创作，主要内容包括：本发明涉及人脐带静脉内皮细胞在制备抗衰老的产品中的应用及抗衰老冻干粉和溶液,属于抗衰老产品技术领域。人脐带静脉内皮细胞能够用于制备抗衰老的产品,可以改善衰老皮肤光泽、增加皮肤厚度、减少皮下血管数量、增加皮肤含水量和/或减少皮肤油分,将皮肤由粗糙改为柔细。(The invention relates to application of human umbilical vein endothelial cells in preparation of anti-aging products, anti-aging freeze-dried powder and anti-aging solution, and belongs to the technical field of anti-aging products. The human umbilical vein endothelial cells can be used for preparing an anti-aging product, and can improve the luster of aging skin, increase the thickness of the skin, reduce the number of subcutaneous blood vessels, increase the water content of the skin and/or reduce the oil content of the skin, and change the skin from rough to soft.)

1. Application of human umbilical vein endothelial cells in preparing antiaging product is provided.

2. Application of human umbilical vein endothelial cells in preparing products for resisting skin aging is provided.

3. Application of human umbilical vein endothelial cells in preparing products for resisting skin aging caused by D-galactose.

4. Application of human umbilical vein endothelial cells in preparing a product for improving the luster of aged skin.

5. Application of human umbilical vein endothelial cells in preparing products for increasing thickness of aged skin.

6. The application of human umbilical vein endothelial cells in preparing a product for reducing the number of subcutaneous blood vessels of aged skin.

7. Use of human umbilical vein endothelial cells for the manufacture of a product for increasing the water content and/or reducing the oil content of aged skin.

8. Application of human umbilical vein endothelial cells in preparing a product for improving the roughness of aged skin.

9. An anti-aging human umbilical vein endothelial cell freeze-dried powder, wherein the preparation method of the freeze-dried powder comprises the following steps:

and (3) repeatedly freezing and thawing the human umbilical vein endothelial cells, and freeze-drying in vacuum to obtain the human umbilical vein endothelial cell freeze-dried powder.

10. An anti-aging lyophilized human umbilical vein endothelial cell powder solution, which is prepared by mixing oligopeptide stock solution as a solvent and the lyophilized powder of claim 9 as a solute.

Technical Field

The invention relates to the technical field of anti-aging products, in particular to application of human umbilical vein endothelial cells in preparation of anti-aging products, anti-aging freeze-dried powder and anti-aging solution.

Background

Skin aging is a phenomenon of skin aging caused by various factors. Skin aging is mainly manifested by dry skin, sagging skin, appearance of wrinkles, hyperkeratosis, pigmentation, reduction in elasticity and softness, and the like. The main inducements are the natural decline of the functions of the skin accessory organs, the slow metabolism of the facial skin, and the adverse life factors (staying up, smoking and drinking) of modern people not only accelerate but also directly cause the aging of the skin. With the development of society and the improvement of living standard of people, the anti-aging treatment of skin becomes a hot point of medical cosmetology.

The conventional anti-aging skin care product mainly comprises chemical components, and is only suitable for basic water replenishing and water locking of young skin, so that the problem of skin aging cannot be improved, and the requirement of aging skin in young state is met.

Stem cells are a unique cell population with self-renewal and differentiation properties, and are widely used in clinical anti-aging studies due to their pleiotropic, easily separable, and multilineage differentiation properties. In recent years, research on the clinical use of stem cells for resisting skin aging has increased. At present, research on stem cell anti-aging is mostly focused on mesenchymal stem cells and freeze-dried powder or secreted factors thereof. The living cells injected into veins and faces are mainly used, and the fresh cells are used, so the transportation requirement is high, and the transportation cost is high; secondly, the activity of the fresh cells is difficult to completely preserve, the prepared fresh cells generally need to be used immediately within 4h, and the activity and the performance are reduced the longer the time is; through the research of using the umbilical cord mesenchymal stem cell freeze-dried powder to resist skin aging, the crowd sample is younger and mostly concentrated between 30 and 50 years old; in addition, the secretion factor for resisting skin aging can overcome the defects of inconvenient use, increased transportation and storage cost and the like, but cannot completely obtain the biological active factor of the cell.

In summary, there is a need to develop a new class of stem cell derived cosmetic products that are more suitable for the needs of aging skin youthfulness to provide a new means of anti-aging treatment of skin.

Disclosure of Invention

The invention aims to provide application of human umbilical vein endothelial cells in preparation of anti-aging products, anti-aging freeze-dried powder and anti-aging solution. The human umbilical vein endothelial cells can be used for preparing an anti-aging product, and can improve the luster of aging skin, increase the thickness of the skin, reduce the number of subcutaneous blood vessels, increase the water content of the skin and/or reduce the oil content of the skin, and change the roughness of the skin into the fineness; the prepared product has good anti-aging effect, is convenient to use, transport and store, is more suitable for the anti-aging of the skin of the aged, has less cell consumption, and can meet the young requirement of the aged skin.

The invention provides an application of human umbilical vein endothelial cells in preparing an anti-aging product.

The invention also provides application of the human umbilical vein endothelial cells in preparing products for resisting skin aging.

The invention also provides application of the human umbilical vein endothelial cells in preparing products for resisting skin aging caused by D-galactose.

The invention also provides application of the human umbilical vein endothelial cells in preparing a product for improving the luster of aged skin.

The invention also provides application of the human umbilical vein endothelial cells in preparing a product for increasing the thickness of aged skin.

The invention also provides application of the human umbilical vein endothelial cells in preparing a product for reducing the number of subcutaneous blood vessels of aged skin.

The invention also provides application of the human umbilical vein endothelial cells in preparing a product for increasing the water content of aged skin and/or reducing the oil content of the skin.

The invention also provides application of the human umbilical vein endothelial cells in preparing a product for improving the roughness of aged skin.

The invention also provides anti-aging human umbilical vein endothelial cell freeze-dried powder, and the preparation method of the freeze-dried powder comprises the following steps:

the invention also provides freeze-dried powder of the human umbilical vein endothelial cells, which is obtained by repeatedly freezing and thawing the human umbilical vein endothelial cells and freeze-drying the human umbilical vein endothelial cells in vacuum.

The invention also provides an anti-aging human umbilical vein endothelial cell freeze-dried powder solution, which is obtained by mixing oligopeptide stock solution serving as a solvent and freeze-dried powder serving as a solute according to the technical scheme.

The invention provides an application of human umbilical vein endothelial cells in preparing an anti-aging product. The human umbilical vein endothelial cell product has wide application prospect due to good anti-skin aging effect, and can be applied to 1) clinical application: medical cosmetic orientation; 2) an anti-aging skin care product. The human umbilical vein endothelial cells can be used for preparing an anti-aging product, and can improve the luster of aging skin, increase the thickness of the skin, reduce the number of subcutaneous blood vessels, increase the water content of the skin and/or reduce the oil content of the skin, and change the skin from rough to soft. The product prepared by the application of the invention has good anti-aging effect, is convenient to use, transport and store, is more suitable for the anti-aging of the skin of the aged, has less cell consumption, and can meet the young state requirement of the aged skin.

Drawings

FIG. 1 is a diagram of the identification and purity detection of clinical grade human umbilical vein endothelial cells provided by the present invention; wherein, A is the result of specific marker positive expression of Von Willebrand Factor (Von Willebrand Factor) of endothelial cells; b is the purity result of endothelial cells detected by flow detection of CD3 l antigen expression;

FIG. 2 is the change result of mouse body weight within 4 weeks of clinical grade human umbilical vein endothelial cell lyophilized powder intervention provided by the present invention;

FIG. 3 shows the results of the anti-aging effect of clinical grade lyophilized powder of human umbilical vein endothelial cells on mouse skin.

Detailed Description

The invention provides an application of human umbilical vein endothelial cells in preparing an anti-aging product. The human umbilical vein endothelial cells are preferably clinical-grade human umbilical vein endothelial cells, and the purity is preferably 99.9%. The method for separating and culturing the clinical-grade human umbilical vein endothelial cells is not particularly limited, and the conventional method for separating and culturing the human umbilical vein endothelial cells, which is well known to those skilled in the art, can be adopted. The human umbilical vein endothelial cells are applied to preparing the anti-aging product, and can play a role in anti-aging. The effect on the anti-aging improvement of aged skin is remarkable. The aging of the present invention preferably means a population aged about 60 years.

The invention also provides application of the human umbilical vein endothelial cells in preparing products for resisting skin aging. The human umbilical vein endothelial cells can improve the luster of aged skin, increase the thickness of the skin, reduce the number of subcutaneous blood vessels, increase the moisture content of the skin and/or reduce the oil content of the skin, change the skin from rough to soft and increase the elasticity of the skin.

The invention also provides application of the human umbilical vein endothelial cells in preparing products for resisting skin aging caused by D-galactose. The human umbilical vein endothelial cells can resist skin aging caused by D-galactose, improve skin luster, increase skin thickness, reduce subcutaneous blood vessel number, increase skin water content and/or reduce skin oil content, change skin from rough to soft, and increase skin elasticity.

The invention also provides application of the human umbilical vein endothelial cells in preparing a product for improving the luster of aged skin. The test result shows that the skin is bright and glossy after being treated by the endothelial cells of the umbilical vein of human.

The invention also provides application of the human umbilical vein endothelial cells in preparing a product for increasing the thickness of aged skin. The test result shows that the skin becomes thicker after being treated by the endothelial cells of the umbilical vein of the human.

The invention also provides application of the human umbilical vein endothelial cells in preparing a product for reducing the number of subcutaneous blood vessels of aged skin. Test results show that subcutaneous blood vessels are remarkably reduced after the treatment of human umbilical vein endothelial cells.

The invention also provides application of the human umbilical vein endothelial cells in preparing a product for increasing the water content of aged skin and/or reducing the oil content of the skin. Test results show that after being treated by human umbilical vein endothelial cells, the water content of the skin is increased, the oil content is reduced, and the skin elasticity is increased.

The invention also provides application of the human umbilical vein endothelial cells in preparing a product for improving the roughness of aged skin. Test results show that the skin state changes from rough to delicate after being treated by human umbilical vein endothelial cells.

The invention also provides anti-aging human umbilical vein endothelial cell freeze-dried powder, and the preparation method of the freeze-dried powder comprises the following steps:

and (3) repeatedly freezing and thawing the human umbilical vein endothelial cells, and freeze-drying in vacuum to obtain the human umbilical vein endothelial cell freeze-dried powder.

Specifically, the invention preferably expands human umbilical vein endothelial cells, and the harvested cells are resuspended in physiological saline and frozen into solid state at-80 ℃; thawing, diluting with water to obtain cell suspension, freezing at-80 deg.C, and repeating the above steps to obtain repeatedly frozen and thawed cell suspension; and (4) carrying out vacuum freeze-drying on the repeatedly frozen and thawed cells to obtain the human umbilical vein endothelial cell freeze-dried powder.

The invention expands human umbilical vein endothelial cells, and the harvested cells are resuspended in physiological saline and frozen into solid at-80 ℃. The invention preferably selects human umbilical vein endothelial cells from primary passage to P5 for expansion. The present invention preferably harvests 1000 million cells resuspended in 10mL of physiological saline. The conditions of-80 ℃ according to the invention are preferably provided by a refrigerator.

The invention is unfrozen, diluted by adding water to obtain cell suspension, frozen at-80 ℃, and the step is repeated to obtain the cells which are repeatedly frozen and thawed. According to the invention, 10 times of the volume of water is preferably added, and the water is preferably double distilled water. The-80 ℃ condition according to the invention is preferably provided by a-80 ℃ refrigerator. The freezing is preferably overnight freezing, and more preferably freezing for 8-12 h. The number of times of repeated freeze thawing is preferably 3-5, and more preferably 3.

After repeated freeze thawing, the invention carries out vacuum freeze drying on the cells subjected to repeated freeze thawing to obtain the human umbilical vein endothelial cell freeze-dried powder. The human umbilical vein endothelial cell freeze-dried powder is preferably stored at 4 ℃ after being prepared. Specifically, the vacuum freeze-drying preferably comprises the following steps: starting a vacuum freeze dryer, and freezing the cell suspension to-45 ℃; then starting a freeze dryer cold trap to refrigerate to-45 ℃, and vacuumizing to 20 Pa; opening the box body and heating to-20 deg.C, and maintaining for 240 min; continuously heating to 1 deg.C and maintaining for 240 min; continuously heating to 30 ℃ and preserving heat for 300 min; discharging at 36 ℃ after the detection is qualified; and (6) filling, inspecting and warehousing.

When the human umbilical vein endothelial cell freeze-dried powder is used, a solvent is preferably used for dilution, and after dilution, each milliliter of solution preferably contains 2000-50000 human umbilical vein endothelial cells.

The human umbilical vein endothelial cell freeze-dried powder has an anti-aging function and strong anti-skin aging activity, can improve the luster of aged skin, increase the thickness of the skin, reduce the number of subcutaneous blood vessels, increase the water content of the skin and/or reduce the oil content of the skin, and changes the roughness of the skin into the fineness of the skin. The purity of the human umbilical vein endothelial cells obtained by the preparation method is as high as 99.9 percent, and the human umbilical vein endothelial cells are clinical-grade human umbilical vein endothelial cells, have the advantages of easy obtainment, no ethical dispute and noninvasive material drawing, and are convenient to use, store and transport; at present, the clinical research of stem cell anti-aging in the market mainly takes intravenous and facial injection of living cells, and because fresh cells are used, the transportation requirement is high, and the transportation cost is increased; secondly, the viability of the fresh cells cannot be completely preserved, and the prepared fresh cells generally need to be used immediately within 4h, and the longer the time is, the viability and the performance are reduced. The invention combines a vacuum freeze-drying method, freezes the water contained in the stem cells at low temperature, and then dries the stem cells in a vacuum environment, so that the water is directly sublimated into water vapor from a solid state and is discharged from a cell product to dry all active factors of the cells, thereby effectively preventing the change of the physicochemical and biological properties of the cells, causing the damage to the biological tissues and the structures and the characteristics of the cells to be small, leading the cells to rapidly enter a dormant state, and effectively protecting the stability of the effective components of the cells and the biological efficacy of the active factors; secondly, the human umbilical vein endothelial cell freeze-dried powder is loose in shape and basically not changed in color after being dried, and can be quickly dissolved after being added with water and restore the physical and chemical properties and biological activity of the original aqueous solution. In addition, because the drying is carried out under the vacuum condition, the method has good protection effect on some substances which are easy to oxidize. Finally, the freeze-dried powder has very low water content after freeze-drying, so that the stability of the product is improved, the chance of pollution is reduced, the transportation is convenient, and the storage life of the product is prolonged.

At present, research on human umbilical vein endothelial cell anti-aging does not appear, and the research on stem cell anti-aging is mostly concentrated on mesenchymal stem cells and freeze-dried powder or secretion factors thereof, and although the secretion factors can overcome the defects of inconvenient use, increased transportation and storage costs and the like, the biological activity factors of the cells cannot be completely obtained; the research on the anti-skin aging by using the umbilical cord mesenchymal stem cell freeze-dried powder has the advantages that the population sample is younger, the population sample is mostly concentrated between 30 and 50 years old, and the required number of cells is large (100 ten thousand/mL). The lyophilized powder of human umbilical vein endothelial cells can enable the skin on the back of a 14-month-old subacute aging model (D-galactose induction) mouse (equivalent to the skin state of a human being about 60 years old) to have young state change within 15 days under the condition that the concentration is 2000-50000 cells/mL, and can remarkably delay skin aging after 30 days of treatment, maintain the water-oil balance of the skin and increase the skin elasticity. The freeze-dried powder has obvious effect on the aspect of anti-aging improvement of aged skin.

The invention also provides an anti-aging human umbilical vein endothelial cell solution, which is prepared by mixing oligopeptide stock solution serving as a solvent and the freeze-dried powder serving as a solute according to the technical scheme. In the invention, the solution preferably contains 2000-50000 personal umbilical vein endothelial cells per milliliter, and more preferably 50000 personal umbilical vein endothelial cells per milliliter. In the present invention, the raw material for preparing the oligopeptide stock solution preferably comprises the following components in percentage by mass (%): water 70, propylene glycol 1, hydrolyzed collagen 3, acetylcysteine 2, acetyl-tyrosine 1, alanine 1, aloe extract 2, witch hazel extract 2, scutellaria extract 2, sophora flavescens root extract 2, glycyrrhiza inflata root extract 2, oligopeptide-12, betaine 2, p-hydroxyacetophenone 1, 1, 2-hexanediol 1, allantoin 1, sodium hyaluronate 2, dipotassium glycyrrhizinate 1, and oat peptide 2 (the oligopeptide stock solution is made by Shanghai posture Bo Biotech Co., Ltd., product number: O-0001). The oligopeptide stock solution disclosed by the invention has no harm to the skin, is beneficial to the generation of collagen and elastin, and maintains the young elasticity of the skin. Because the oligopeptide is a micro molecular structure, the oligopeptide can easily permeate through epidermal cells and directly reach the dermis layer below the epidermal cells and can be quickly absorbed by the dermis cells, so that the oligopeptide used as the lyophilized powder solvent of the endothelial cells of the umbilical veins of the human umbilical cord can promote the subsequent absorption of the lyophilized powder of the cells to directly reach the dermis layer. The invention selects 14-month-old mice, and the D-galactose is injected subcutaneously on the back to induce the subacute aging model of the mice, so as to construct an animal model with aged skin. The lyophilized powder of human umbilical vein endothelial cells is dissolved by oligopeptide stock solution to prepare the lyophilized powder of stem cells, and the lyophilized powder of stem cells is applied to the skin on the back of a mouse, so that the gloss of aged skin can be improved, the thickness of the skin can be increased, the number of subcutaneous blood vessels can be reduced, the water content of the skin can be increased, and/or the oil content of the skin can be reduced, and the skin can be changed from rough to fine.

The application of the human umbilical vein endothelial cells in preparing the anti-aging product, the anti-aging freeze-dried powder and the anti-aging solution are further described in detail with reference to the following specific examples, and the technical scheme of the invention includes but is not limited to the following examples.

Example 1

Separation and culture of clinical-grade primary human umbilical vein endothelial cells

TABLE 1 consumable List

TABLE 2 list of reagents

Separation, preparation and passage amplification of clinical-grade primary human umbilical vein endothelial cells

1 before the experiment, the experiment environment, instruments and equipment running conditions are checked. Wiping and disinfecting the operation table top of the biological safety cabinet by 75% ethanol solution, and wiping and disinfecting instruments and supplies by high pressure or alcohol.

2 ligating two ends of the umbilical cord tissue, soaking in a physiological saline bottle containing double antibodies, and transporting to a laboratory at the temperature of 2-8 ℃. Opening the bottle in a biological safety cabinet, discarding the preservation solution in the bottle, adding physiological saline (only by immersing the umbilical cord) into the bottle, and washing by shaking.

3 after discarding the saline, the umbilical cord was poured into a sterile 100mm petri dish and the surface of the umbilical cord was sprayed with a 75% ethanol solution. And cleaning the sprayed umbilical cord with normal saline for 2-3 times, adding the normal saline into a culture dish, dividing the umbilical cord into 3-4 cm sections with sterile scissors, clamping the center of the umbilical cord with forceps, pushing the umbilical cord towards two ends, and washing off blood.

4, putting the cleaned umbilical cord into a sterile 100mm culture dish containing normal saline, finding the umbilical vein, carrying out blunt peeling off the umbilical cord from the side of the vein, removing the vein, putting the umbilical cord into the culture dish, separating umbilical cord mesenchyme (Fahrenheit gelatin), and putting the umbilical cord mesenchyme (Fahrenheit gelatin) into the culture dish. The blood vessels in the petri dishes were flushed three more times with saline and a 5mL syringe, respectively, to remove the intravascular blood.

5 clamping one end of the blood vessel by a vascular clamp, injecting collagenase by a 5ml syringe until the blood vessel is obviously swelled, and clamping the other end by the vascular clamp. Cover the petri dish and put 5% CO₂And digesting for 15min in a cell culture box at 37 ℃. The digested blood vessels were injected with physiological saline and 5mLThe container is washed for 3 times, the washed liquid is collected to a 50mL centrifuge tube and centrifuged for 5min at 1500 rpm.

6 discard the supernatant to add 10mL of complete ECM medium to the pellet to resuspend and make a cell suspension. And planting one hole of a six-hole plate according to a blood vessel of 4-5 cm. The medium was observed under a microscope and changed every 3 days.

7 when the cell fusion degree reaches 80%, adding 5mL of physiological saline to wash out residual culture medium in the bottle. After the physiological saline is discarded, 1mL of Tryple-Express is added, and the mixture is kept stand and digested for about 2min at room temperature or 37 ℃ until the cells are observed to be transparent and round by a microscope and are gently shaken and floated.

8, adding 5mL of physiological saline to stop digestion after digestion, washing residual cells on the lower bottle wall, transferring the liquid to a centrifuge tube, and centrifuging for 5min at 1500 rpm. The supernatant was discarded and the six well plates were inoculated in a ratio of 1: 3. Placing into an incubator under 5% CO₂And culturing at 37 ℃.

9 observing the cell state in the six-hole plate cell culture bottle by using a microscope, and carrying out passage when the cell fusion degree reaches 80%. Adding 10mL of physiological saline to wash off residual culture medium in the bottle, discarding the physiological saline, adding 1mL of Tryple-Express, standing and digesting for 2min at room temperature or 37 ℃, adding 5mL of physiological saline into digested cells to stop digestion, washing residual cells on the wall of the bottle, transferring the liquid to a centrifuge tube, and centrifuging for 5min at 1500 rpm.

10 discard the supernatant, add 20mL of complete medium to the pellet and resuspend to make a cell suspension. After counting 20. mu.L, the cells were inoculated into a flask at a rate of 100 ten thousand/T-175 for amplification culture. Placing into an incubator under 5% CO₂And culturing at 37 ℃.

Identification and purity detection of clinical grade human umbilical vein endothelial cells (results are shown in figure 1)

Identification of clinical grade human umbilical vein endothelial cells

1 spreading the prepared and amplified cells on a 24-well plate, washing the cells with DPBS for 3 times when the cell fusion degree is 80%, fixing the cells with 4% PFA for 15min, and washing the cells with DPBS for 3 times.

2 Primary antibody (Anti-Von Willebrand Factor antibody, cat # ab154193, abcam) was prepared in a ratio of 1:500 using 5% BSA + 0.4% Triton X-100 permeabilization solution and incubated overnight at 4 ℃.

Removing primary antibody, washing with DPBS for 3 times, and preparing secondary antibody (Anti-rabbitIgG (H + L), F (ab')2Fragment (Alexa) with the permeabilizing solution at a ratio of 1:500488Conjugate), cat No. 4412, Cell Signaling Technology, inc.) and the secondary antibody were incubated at room temperature for 1 h.

The secondary antibody was discarded 4 times, washed with DPBS 3 times, incubated with DAPI (cat. No. D9542, Sigma) at room temperature for 15min, and the staining solution of DAPI was discarded, washed with DPBS 3 times, observed with a fluorescence microscope (OLYMPUS IX51), and photographed.

5 Von Willebrand Factor (Von Willebrand Factor), a multimeric adhesion glycoprotein, synthesized and secreted by endothelial cells, is a key Factor involved in the process of angiogenesis, as shown in A in FIG. 1. The prepared cells were identified as endothelial cells by detecting positive expression of Von Willebrand Factor as 100% by immunofluorescence staining.

Purity detection of clinical grade human umbilical vein endothelial cells

1 spreading the prepared and amplified cells on a 6-hole plate (2 holes), washing residual culture medium in a bottle by using physiological saline until the cell fusion degree is 80%, discarding the physiological saline, adding 1mL of Tryple-Express, standing and digesting at room temperature or 37 ℃ for 2min, adding 5mL of physiological saline into the digested cells to stop digestion, and centrifuging for 100g for 5 min.

2 staining of cells PE Mouse Anti-Human CD31 (cat # 555446, BD Co.) and detecting the positive expression of CD31 by flow cytometry (cat # FACSCANTO II, BD Co.).

3 as shown in B in FIG. 1, endothelial cell adhesion molecule-1 (Platlet/endothelial cell adhesion molecule-1, PECAM-1 or CD31) is highly expressed at the endothelial cell-cell junction and acts as an adhesion stress response protein, which can maintain the integrity of endothelial cell junction and rapidly restore the vascular permeability barrier after inflammation or thrombosis. The expression rate of CD31 in the cells was 99.9% by detecting cell surface antigen, and the prepared cells were identified as endothelial cells with a purity of 99.9%.

Example 2

Preparation of clinical grade human umbilical vein endothelial cell freeze-dried powder

1. Taking P5 and amplifying clinical grade human umbilical vein endothelial cells qualified by quality identification, harvesting 1000 ten thousand of cells, re-suspending in 10mL of physiological saline, and placing in a refrigerator at-80 ℃ to freeze into a solid; thawing the cells, adding double distilled water with 10 times of volume to dilute the cell suspension, and then putting the cell suspension into a refrigerator with the temperature of 80 ℃ below zero for overnight freezing (repeating the step for 3 times) to obtain a cell suspension subjected to repeated freezing and thawing;

2. starting a vacuum freeze dryer, and freezing the cell suspension to-45 ℃; then starting a freeze dryer cold trap to refrigerate to-45 ℃, and vacuumizing to 20 Pa; opening the box body and heating to-20 deg.C, and maintaining for 240 min; continuously heating to 1 deg.C and maintaining for 240 min; continuously heating to 30 ℃ and preserving heat for 300 min; discharging at 36 ℃ after the detection is qualified; and (6) filling, inspecting and warehousing.

Example 3

Effect of clinical grade lyophilized powder of human umbilical vein endothelial cells on aging skin of mouse

Experimental procedure

Female Kunming mice of 9 months old were purchased from Witonglihua, raised to 12 months old, weighed, depilated on the back and subcutaneously injected with D-galactose (cat # D8310, Beijing Soilebao Tech. Co., Ltd.) at a dose of 1mg/g for 30 days, once a day, for skin aging and molding.

One week after the end of D-galactose injection, mice were randomized into 4 groups: 1) control (oligopeptide stock solution); 2)2000 cells/mL; 3)10000 cells/mL; 3)50000cells/mL for different groups of treatments (all lyophilized cells were dissolved in oligopeptide stock solution). 0.1mL of clinical grade human umbilical vein endothelial cell freeze-dried powder with different concentrations is smeared on the back of the mouse every day until the freeze-dried powder is absorbed.

The body weight of the mouse is measured once a week, the skin aging condition of the mouse is recorded by taking a picture every 15 days, the water content, the oil content and the elasticity of the back skin of the mouse are detected by a skin detector after the treatment is finished, and the anti-skin aging effect of the freeze-dried powder is evaluated.

The results are analyzed as shown in fig. 2 (change of mouse weight in 4 weeks after clinical grade human umbilical vein endothelial cell lyophilized powder intervention), as shown in fig. 2, the mouse weight is not significantly changed in four weeks after intervention treatment, which indicates that the mouse is in a normal diet state, and the weight is not significantly increased and has a small part of descending trend due to aging of the mouse.

As shown in figure 3 (clinical grade human umbilical vein endothelial cell lyophilized powder anti-mouse skin aging effect result; cell lyophilized powder is divided into three concentration gradients of 2000cells/mL, 10000cells/mL and 50000cells/mL (dissolved in oligopeptide stock solution, intervention period is four weeks)), at day0, the back skin of 4 groups of aging mice becomes thin, the skin color is dark, and subcutaneous blood vessels are obvious. After 15 days of clinical grade human umbilical vein endothelial cell freeze-dried powder treatment, compared with a control group, mice of three treatment groups with different concentrations have bright skin color, and the skin is thickened and subcutaneous blood vessels are obviously reduced by visual observation. After one month of treatment, the skin state of the mice of the cell freeze-dried powder treatment groups with three concentrations is still remarkably improved compared with that of the control group on the same day, and the skin state of the mice of the treatment group with 50000cells/mL is better.

TABLE 3 clinical grade lyophilized powder of human umbilical vein endothelial cell for resisting skin aging of mice-moisture, oil and elasticity of skin

Note: within a certain range, the water content is high, the oil content is low, the skin elasticity index is good, and the skin is proved to be more youthful; the higher the oil content and the lower the water content, the coarser the skin is.

After 4 weeks of treatment, the skin conditions of the mice treated by different groups are quantitatively evaluated, and the water content, oil content and elasticity of the skin of the mice are detected by a skin monitor. As can be seen from Table 3, the lyophilized human umbilical vein endothelial cell powder is dose-dependent in its anti-aging effect on mouse skin. Along with the increase of the concentration of the cell freeze-dried powder, the water content of the skin of the mouse is increased, the oil content is reduced, and the skin state is changed from rough to delicate.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.