CN113462641A - Umbilical cord mesenchymal stem cell extract, application and detection method - Google Patents
Umbilical cord mesenchymal stem cell extract, application and detection method Download PDFInfo
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- CN113462641A CN113462641A CN202110802323.2A CN202110802323A CN113462641A CN 113462641 A CN113462641 A CN 113462641A CN 202110802323 A CN202110802323 A CN 202110802323A CN 113462641 A CN113462641 A CN 113462641A
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Abstract
The invention provides an umbilical cord mesenchymal stem cell extract, an application and a detection method, belonging to the technical field of cosmetics, wherein the umbilical cord mesenchymal stem cell extract is obtained by the following steps of (1) separating umbilical cord mesenchymal stem cells from umbilical cord tissue blocks or umbilical cord blood; (2) directionally differentiating the umbilical cord mesenchymal stem cells obtained in the step (1) into skin stem cells; (3) collecting and cracking the skin stem cells obtained in the step (2), and enriching active small molecular substances through a molecular sieve to obtain an umbilical cord mesenchymal stem cell extract; the small molecular substance can be stored for a long time at normal temperature, and the repair capability to the skin barrier is obviously enhanced.
Description
Technical Field
The invention relates to the technical field of cosmetics, in particular to an umbilical cord mesenchymal stem cell extract, application and a detection method.
Background
With the improvement of living standard and the advance of science and technology, the basic functions of whitening, moisturizing and the like in the past cannot meet the requirements of people on cosmetics, and the anti-aging is a concern gradually. In the process of skin aging, active oxygen is an important cause of skin aging, and endogenous aging and exogenous aging act simultaneously. Endogenous aging is mainly characterized by gradual accumulation of oxidized and glycated proteins with the increase of age, so that collagen and elastin in the dermis are lost, the skin becomes thin, and keratinocytes are denatured. Extrinsic aging is caused by ultraviolet damage, environmental damage, inflammation, etc., resulting in deterioration and damage of epidermal tissues.
The mesenchymal stem cells are adult stem cells derived from early mesoderm, have high self-renewal capacity and multidirectional differentiation potential, are an important cell component in a hematopoietic microenvironment, can proliferate and differentiate to various tissues such as bone, cartilage, muscle, ligament, tendon, fat and stromal cells, have weak immunogenicity, and are a seed cell source for tissue engineering. Compared with mesenchymal stem cells from other sources, the human umbilical cord mesenchymal stem cells also have the advantages of convenient extraction, rich content, pure cells, lower immunogenicity, stronger original and differentiated cell capacity, no ethical limitation and the like, can provide sufficient cell sources for experiments and clinics, and have wide clinical application prospects. The research shows that the culture supernatant of the human umbilical cord mesenchymal stem cells contains various cytokines, such as vascular endothelial growth factor, hepatocyte growth factor, basic fibroblast growth factor, epidermal growth factor, granulocyte macrophage colony stimulating factor, transforming growth factor beta, nerve growth factor and the like. These factors play an important role in healing skin wounds, reconstruction of extracellular matrix, and stable production and distribution of collagen and elastin.
At present, the traditional essence in the market is mainly prepared by exogenous substances such as plant component extraction or fine chemical manufacturing process, and although the traditional essence can produce transient improvement effect on skin, the traditional essence does not fundamentally change the aging and repairing problems of the skin. The application number is CN201910177644.0, the invention name is a preparation method of a human umbilical cord mesenchymal stem cell preparation for conventional anti-aging treatment, and the preparation method comprises an umbilical cord tissue block acquisition step, a stem cell primary culture step, a stem cell subculture step, a stem cell cryopreservation step, a stem cell recovery and culture step and a human umbilical cord mesenchymal stem cell preparation step; the preparation method of the human umbilical cord mesenchymal stem cell preparation comprises the steps of adopting 5% glucose injection to perform resuspension subculture until Pe generation of the human umbilical cord mesenchymal stem cells. However, the mesenchymal stem cells themselves and the paracrine factors or exosomes secreted by the mesenchymal stem cells used in the prior art have poor skin barrier repair capability and cannot be stored at normal temperature, so that the effect of applying the mesenchymal stem cells to skin repair is greatly reduced.
Disclosure of Invention
In view of this, the present invention provides an extract of umbilical cord mesenchymal stem cells, so as to solve the technical problem in the prior art that the umbilical cord mesenchymal stem cell extract is easy to inactivate active ingredients when stored at normal temperature.
In order to solve the technical problems, the invention provides an extract of umbilical cord mesenchymal stem cells, which is extracted by the following steps: (1) separating umbilical cord mesenchymal stem cells from the umbilical cord tissue block or the umbilical cord blood; (2) directionally differentiating the umbilical cord mesenchymal stem cells obtained in the step (1) into skin stem cells; (3) collecting and cracking the skin stem cells obtained in the step (2), and enriching active small molecular substances through a molecular sieve to obtain the umbilical cord mesenchymal stem cell extract.
Preferably, it is extracted by the following steps: which is extracted by the following steps: (1) umbilical cord mesenchymal stem cells are separated from the umbilical cord tissue block; (2) directionally differentiating the umbilical cord mesenchymal stem cells obtained in the step (1) into skin stem cells; (3) normally culturing the skin stem cells obtained in the step (2) for 18-36 h, then culturing in a hypoxic environment with 5% of oxygen, 5% of carbon dioxide and 90% of nitrogen for 18-36 h, collecting and cracking the obtained skin stem cells, obtaining active small molecular substances through a molecular sieve with the pore diameter of 0.3-2.0 nm, and enriching the active small molecular substances to obtain the umbilical cord mesenchymal stem cell extract.
Preferably, in the step (1), the umbilical cord mesenchymal stem cells are obtained from umbilical cord tissue blocks or umbilical cord blood through a primary culture step, a subculture step of stem cells, a cryopreservation step of stem cells, a recovery and culture step of stem cells and a preparation step of a human umbilical cord mesenchymal stem cell preparation.
Preferably, in the step (1), the primary culture step of umbilical cord mesenchymal stem cells comprises the adherent inoculation of umbilical cord tissue blocks into a culture dish, the temperature is 37 ℃, and the volume fraction of CO is 5%2And (2) standing for 1-2 h in an incubator, adding complete culture solution into a culture dish for culture, replacing the complete culture solution every 3 days by half, after the culture solution and umbilical cord tissue blocks are subjected to standing culture for 14 days, removing the culture solution and umbilical cord tissue blocks, cleaning cells by using normal saline, adding 0.5wt% of Tryple-Express digestive juice, standing for 2-3 minutes at 37 ℃, adding the complete culture solution to stop digestion after the pseudopodium is completely retracted and the cells are rounded, and blowing to obtain the P1 generation human umbilical cord mesenchymal stem cell suspension.
Preferably, in step (2), the composition of the culture solution is: 100g/L fetal bovine serum, 5mg/L insulin-L, L-glutamine, 8 ug/L epidermal growth factor, 2 ug/L fibroblast growth factor, 0.88 ug/L nerve growth factor, 175 ug/L glucocorticoid, 0.1mg/L baicalein, and 35 ug/L delphinidin.
Preferably, in the step (2), the placental mesenchymal stem cells obtained in the step (1) are inoculated in a skin stem cell culture medium at 37 ℃ and 5% CO2And incubating for 15-25 h in the incubator, removing the culture medium, adding the culture medium into a fresh skin stem cell culture medium again, and carrying out subculture amplification when the skin stem cells are approximately 80-90% fused.
Preferably, the step (2) further comprises digesting the directionally differentiated skin stem cells with 0.1% pancreatic enzyme, and washing the cells with physiological salt at least once.
Preferably, in the step (3), the skin stem cells are cracked by ice bath ultrasonication, the ice bath ultrasonication condition is intermittent ultrasonication, the ultrasonication power is 300-350W, the work is 10s each time, the intermittent operation is 3s, and the circulation is performed for at least 20 times.
The application of the umbilical cord mesenchymal stem cell extract in preparing a beauty product.
A detection method of umbilical cord mesenchymal stem cell extract adopts reversed phase liquid chromatography-mass spectrometry.
Compared with the prior art, the invention has the beneficial technical effects that:
1. the method comprises the steps of directionally differentiating mesenchymal stem cells into skin stem cells, collecting the skin stem cells, collecting active substances in the skin stem cells by an ultrasonic crushing method, and enriching small molecular substances by a molecular sieve. The small molecular substance can be stored for more than 24 months at normal temperature, and the repair capability to the skin barrier is obviously enhanced. Compared with the prior art, the mesenchymal stem cells and the secreted paracrine factors or exosomes thereof are directly used, so that the capability of repairing skin barriers is poor, active substances in the mesenchymal stem cells are easy to inactivate at normal temperature, and the mesenchymal stem cells cannot be stored at normal temperature. Mesenchymal stem cells are directionally differentiated into skin stem cells, and then small molecular active substances of the mesenchymal stem cells are enriched, so that the mesenchymal stem cells can be stored for a long time at normal temperature, and the repair capability of the mesenchymal stem cells on the skin barrier is enhanced.
2. According to the invention, preferably, the umbilical cord mesenchymal stem cells are differentiated into the skin stem cells, the skin stem cells obtained in the step (3) are normally cultured for 18-36 h, then are cultured for 18-36 h in a hypoxia environment, then are normally treated to propagate the cells in a large area, and then are cultured in the hypoxia environment, so that the detection shows that the activity of the umbilical cord mesenchymal stem cell extract is greatly improved.
3. The prepared skin stem cell active substance applied to cream cosmetics can obviously improve skin aging, reduce the occurrence of adverse reactions and increase the water content and the oil content of the horny layer.
Drawings
FIG. 1 is a graph of cellular extracts versus the amount of collagen produced by fibroblasts in examples 1, 3-4;
FIG. 2 is a graph of the amount of collagen produced by fibroblasts from the cell extracts of examples 1, 5 to 10;
FIG. 3 is a graph of cellular extracts versus the amount of collagen produced by fibroblasts from examples 9, 11-14;
FIG. 4 is a graph of the amount of collagen produced by fibroblasts following 6 months of storage of the cell extracts of examples 9, 11-14;
FIG. 5 is a chromatogram of the base peak of a 200ng HeLa liquid mass analysis;
FIG. 6 is a plot of the parent ion error profile;
FIG. 7 is a chromatogram of the cell sample at the base peak for liquid chromatography;
FIG. 8 is a graph of the error distribution of the parent ion in a cell sample;
FIG. 9 shows the peptide truncation ratio distribution of the cell sample;
FIG. 10 is a BasePiak diagram of liquid chromatography of cell culture medium samples;
FIG. 11 is a graph showing the peptide truncation ratio distribution of a cell sample;
FIG. 12 is a graph showing the effect of using the umbilical cord mesenchymal stem cell extract of the present invention on patients suffering from red swelling allergy.
Detailed Description
The following examples are intended to illustrate the present invention in detail and should not be construed as limiting the scope of the present invention in any way. The instruments and devices referred to in the following examples are conventional instruments and devices unless otherwise specified; the industrial raw materials (reagents and raw materials are selected according to the situation) are all conventional industrial raw materials which are sold in the market if not specified; the processing and manufacturing methods (detection, test, preparation method and the like are selected according to the situation) are conventional methods unless otherwise specified.
Example 1: an umbilical cord mesenchymal stem cell extract, which is extracted by the following steps:
(1) umbilical cord mesenchymal stem cells are separated from the umbilical cord tissue block, an umbilical cord specimen meeting the collection standard is collected, and cell separation is carried out within 48 h. The umbilical cord preservation solution was discarded, washed 3 times with PBS, and the umbilical cord was transferred toAnd (3) intercepting small umbilical cord segments in a phi 10cm culture dish, wherein the small umbilical cord segments have good transparency, no damage and no edema area, and each segment is intercepted into 4 x 4 cm. The umbilical cord was dissected longitudinally with tissue scissors, the umbilical vein and umbilical artery were removed, and washed 4 times with PBS. Using special scissors for umbilical cord tissue to cut residual tissue of umbilical cord, namely Wharton's jelly into 1mm3Inoculating the tissue block into a new culture dish of phi 10cm, supplementing half of serum-free complete culture medium after 24 hours, and then discarding 3ml of half amount of culture medium for supplementing 4ml every 2 days;
(2) directionally differentiating the umbilical cord mesenchymal stem cells obtained in the step (1) into skin stem cells; in the step (2), the composition of the culture solution is as follows: insulin 20mg/L, L-glutamine 5mg/L, epidermal growth factor 8 μ g/L, fibroblast growth factor 2 μ g/L, nerve growth factor 0.88 μ g/L, glucocorticoid 175 μ g/L, baicalein 0.1mg/L and delphinidin 35 μ g/L;
(3) normally culturing the skin stem cells obtained in the step (2) for 24 hours, then culturing the skin stem cells in a hypoxic environment for 24 hours, cracking the skin stem cells by adopting ice bath ultrasonic crushing, wherein the ice bath ultrasonic crushing condition is intermittent ultrasound, the power of ultrasonic crushing is 320W, the time of working is 10s, the time of intermittent ultrasound is 3s, and the cycle is 40 times, collecting and cracking the obtained skin stem cells, and enriching active small molecular substances through a molecular sieve with the aperture of 1nm to obtain the umbilical cord mesenchymal stem cell extract. The low oxygen condition comprises 5% of oxygen, 5% of carbon dioxide and 90% of nitrogen.
Example 2: the difference from example 1 is in step (1):
(1) separating umbilical cord mesenchymal stem cells from umbilical cord blood:
extracting and collecting mononuclear cells in cord blood by using the separation liquid, and separating and extracting the mononuclear cells in the residual red blood cell component again; washing the mononuclear cells collected twice; coating nonspecific adherence-promoting protein and specific adherence-promoting protein at the bottom of a culture bottle; carrying out subculture when the fusion rate of the primary cells reaches 60%; and culturing the umbilical cord blood mesenchymal stem cells which are continuously subjected to passage, and performing passage after the cell fusion rate reaches 80%.
The influence of the examples 1 and 2 on the cell index shows that the umbilical cord mesenchymal stem cells obtained by the umbilical cord tissue blocks are more easily obtained and the operation is more convenient.
Example 3: the difference from example 1 is in step (3):
(3) normally culturing the skin stem cells obtained in the step (2) for 48h, collecting and cracking the skin stem cells to obtain the skin stem cells, and enriching active small molecular substances through a molecular sieve with the aperture of 1nm to obtain the umbilical cord mesenchymal stem cell extract.
Example 4: the difference from example 1 is in step (3):
(3) and (3) directly collecting and cracking the skin stem cells obtained in the step (2) in a low-oxygen environment for 48 hours to obtain the skin stem cells, and enriching active small molecular substances through a molecular sieve with the aperture of 1nm to obtain the umbilical cord mesenchymal stem cell extract.
The extracts of example 1, example 3 and example 4 were collected and added to fibroblasts for 48h of culture, the fibroblasts were lysed, the cell supernatants from each group were collected, 100uL of collagen separation and concentration reagent was added, and the supernatants were centrifuged and discarded after overnight at 4 ℃. The standard collagen was diluted to 0, 0.01, 0.05, 0.10, 0.20, 0.5, 1.00 mg/mL 1, respectively. mu.L of the srcolic dye was added to each tube and incubated for 30min, followed by washing with 1 × Acid-SaltWashreagent. 250uL of the alkali metal reagent was added to each tube and vortexed to mix. Transfer 200 μ L of sample to 96-well plate. Detecting with a microplate reader, adjusting the detection wavelength to 555nm, adjusting the absorbance to zero with water, measuring the absorbance of the blank reagent, the standard collagen and the detection sample blank reagent, repeating the test error by 10% for 3 times, and obtaining the collagen detection results of example 1, example 3 and example 4 as shown in figure 1.
As is clear from fig. 1, in examples 1, 5 and 6, the extracts obtained by culturing normally for 24 hours and then culturing in a hypoxic environment for 24 hours according to example 1 have an effect of promoting the production of collagen in fibroblast culture.
Example 5: the difference from example 1 is in step (3):
(3) normally culturing the skin stem cells obtained in the step (2) for 18h, then culturing in a low-oxygen environment for 24h, and enriching active small molecular substances through a molecular sieve with the aperture of 1nm to obtain the umbilical cord mesenchymal stem cell extract.
Example 6: the difference from example 1 is in step (3):
(3) normally culturing the skin stem cells obtained in the step (2) for 30h, then culturing in a low-oxygen environment for 24h, and enriching active small molecular substances through a molecular sieve with the aperture of 1nm to obtain the umbilical cord mesenchymal stem cell extract.
Example 7: the difference from example 1 is in step (3):
(3) normally culturing the skin stem cells obtained in the step (2) for 36h, then culturing in a low-oxygen environment for 24h, and enriching active small molecular substances through a molecular sieve with the aperture of 1nm to obtain the umbilical cord mesenchymal stem cell extract.
Example 8: the difference from example 1 is in step (3):
(3) normally culturing the skin stem cells obtained in the step (2) for 24h, then culturing in a low-oxygen environment for 18h, and enriching active small molecular substances through a molecular sieve with the aperture of 1nm to obtain the umbilical cord mesenchymal stem cell extract.
Example 9: the difference from example 1 is in step (3):
(3) normally culturing the skin stem cells obtained in the step (2) for 24h, then culturing in a low-oxygen environment for 30h, and enriching active small molecular substances through a molecular sieve with the aperture of 1nm to obtain the umbilical cord mesenchymal stem cell extract.
Example 10: the difference from example 1 is in step (3):
(3) normally culturing the skin stem cells obtained in the step (2) for 24h, then culturing in a low-oxygen environment for 36h, and enriching active small molecular substances through a molecular sieve with the aperture of 1nm to obtain the umbilical cord mesenchymal stem cell extract.
The extracts of example 1 and examples 5-10 were collected and added to fibroblasts for 48h of culture, the fibroblasts were lysed, the cell supernatants from each group were collected, 100uL of collagen was added to the separated and concentrated reagent, and the supernatant was centrifuged and discarded after overnight at 4 ℃. The specific operation is as described above. The effect of collagen production by fibroblasts from the extracts of examples 1 and 5 to 10 is shown in FIG. 2.
As shown in figure 2, the extract in example 9 has the highest content of collagen produced by fibroblasts, so the time parameters in example 9 are adopted, that is, the skin stem cells obtained in step (2) are normally cultured for 24 hours, then cultured in a hypoxic environment for 30 hours, and then passed through a molecular sieve with the pore diameter of 1nm, so that the umbilical cord mesenchymal stem cell extract is obtained by enriching active small molecular substances.
Example 11: the difference from example 9 is in step (3):
(3) normally culturing the skin stem cells obtained in the step (2) for 24h, then culturing in a hypoxia environment for 30h, and enriching active small molecular substances through a molecular sieve with the aperture of 0.3nm to obtain the umbilical cord mesenchymal stem cell extract.
Example 12: the difference from example 9 is in step (3):
(3) normally culturing the skin stem cells obtained in the step (2) for 24h, then culturing in a hypoxic environment for 30h, and enriching active small molecular substances through a molecular sieve with the aperture of 0.5nm to obtain the umbilical cord mesenchymal stem cell extract.
Example 13: the difference from example 9 is in step (3):
(3) normally culturing the skin stem cells obtained in the step (2) for 24h, then culturing in a hypoxic environment for 30h, and enriching active small molecular substances through a molecular sieve with the aperture of 1.5nm to obtain the umbilical cord mesenchymal stem cell extract.
Example 14: the difference from example 9 is in step (3):
(3) normally culturing the skin stem cells obtained in the step (2) for 24h, then culturing in a hypoxia environment for 30h, and enriching active small molecular substances through a molecular sieve with the aperture of 2.0nm to obtain the umbilical cord mesenchymal stem cell extract.
Comparative example 1: the difference from example 9 is in step (3):
(3) normally culturing the skin stem cells obtained in the step (2) for 24h, then culturing in a low-oxygen environment for 30h, and enriching the obtained active small molecular substances to obtain the umbilical cord mesenchymal stem cell extract without molecular sieve treatment.
The extracts of example 9, examples 11-14 and comparative example 1 were collected and added to fibroblasts within 1 day for culturing for 48h, the fibroblasts were lysed, the cell supernatants of each group were collected, 100uL of collagen was added to separate and concentrate the reagents, and the supernatants were centrifuged and discarded after overnight at 4 ℃. The specific operation is as described above. The effect of collagen production by fibroblasts from the extracts of example 9, examples 11 to 14 and comparative example 1 is shown in FIG. 3.
As is clear from FIG. 3, in each of examples 9, 13 and 14, the amount of collagen produced by fibroblasts was increased to 4.0mg/mL or more.
Meanwhile, the extracts of example 9, example 13-14 and comparative example 1 are stored at 37 ℃ for 6, 12, 24 and 36 months, the extracts of example 9, example 11-14 and comparative example 1 are collected and added into fibroblasts for culturing for 48 hours, the fibroblasts are cracked, the cell supernatants of each group are taken, 100uL of collagen separation and concentration reagent is added, and the supernatant is centrifuged and discarded after overnight at 4 ℃. The specific operation is as described above. The effect of collagen production by fibroblasts from the extracts of examples 9 and 11 to 14 is shown in FIG. 4.
As can be seen from fig. 4, examples 9 and 11 show that the collagen production of fibroblasts is most stable after 6, 12, 24 and 36 months at 37, and the content of the active substance contained therein is most stable.
The detection method of the umbilical cord mesenchymal stem cell extract comprises the following steps of:
1. the detection method comprises the following steps: protein extraction → protein treatment → C18 reverse phase Tip column desalination → reversed phase liquid chromatography-MS.
Protein extraction
(1) Cell sample: add 300. mu.L of 1% SDS solution containing 1% protease inhibitor to the EP tube and sonicate in ice for 2min (10 son, 10off, avoid overheating to lose protein); centrifuging at 21000 Xg 4 deg.C for 15min, and collecting supernatant; protein concentration was determined using BCA assay.
(2) Culture medium sample: adding 2mL of methanol into 1mL of culture medium, uniformly mixing by vortex, and standing for 5min on ice; centrifuging at 21000 Xg 4 deg.C for 15min, and removing supernatant; adding 300 μ L of 1% SDS solution, and vortexing to dissolve protein precipitate; protein concentration was determined using BCA assay.
Protein processing
(1) The two protein solutions were treated identically as follows: denaturing the solution at 95 ℃ for 10min, and cooling to room temperature; adding 3 μ L of 1M Dithiothreitol (DTT) into the centrifuge tube, and shaking at 56 deg.C for 1.5 h; after cooling to room temperature, 6. mu.L of 1M Iodoacetamide (IAA) was added and the reaction was carried out for 30min with exclusion of light.
(2) The enzymatic hydrolysis was carried out using the FASP (filter-aidedamplepreperation) enzymatic method: 200 μ L of 50mM Ammonium Bicarbonate (ABC) rinse the ultrafiltration membrane, load 50 μ g of the treated protein onto the ultrafiltration membrane, and centrifuge to remove the buffer solution; adding 200 mu L of 50mMABC solution, and fully removing SDS; adding 100 mu LpH8 buffer solution and 1 mu g Trypsin protease, and carrying out constant temperature oscillation reaction at 37 ℃ for 12 hours; the enzymatic hydrolysis was stopped by adding 5. mu.L of trifluoroacetic acid (TFA), the tubes were replaced, and the peptide mixture was collected by centrifugation.
Desalting with reversed phase Tip column
(1) And (3) activation: using 1.5mL ion tube (with sharp tool to punch a 0.3cm diameter hole) fixed Pep-Tip desalting column (only for processing 50u g samples), adding 200 u LBphase, 5000 x g (or 7000 rpm) centrifugation for 5 min;
(2) balancing: adding 200 μ LAPhase, and centrifuging at 5000 × g (or 7000 rpm) for 5 min;
(3) adsorbing a sample: adding the sample, and centrifuging for 10min at 3000 Xg (or 5000 rpm);
(4) desalting: 200 u LAPhase column washing, 5000 x g (or 7000 rpm) centrifugation for 5 min;
(5) and (3) elution: the sample was collected by changing the low adsorption centrifuge tube, centrifuged at 5000 Xg (or 7000 rpm) for 5min with 200. mu.LBphase, and the eluate was lyophilized.
(6) Dissolving: add 20. mu.L of LAPhase to the centrifuge tube and freeze-store at-20 ℃ until the sample is loaded.
Reversed phase liquid chromatography-mass spectrometry (RPLC-MS)
(1) Data acquisition software: xcalibur (Thermo, USA); information on the chromatographic column: single column mode, separation column: 75 μm × 200mm (C18, 1.9 μm particle size, 120A pore size); chromatography apparatus: easy-nano 1000; mass spectrometer: QOxactivePlus (Thermo, USA)
(2) Chromatographic conditions are as follows: column equilibrium volume: 5 mu L of the solution; sample loading amount: 3 mu L of the solution; loading volume: 8 μ L
Chromatographic separation time: 120 min; a: 0.1% aqueous formic acid; b: 80% acetonitrile, 0.1% formic acid
Flow rate: 200 nL/min; the gradients are shown in Table 1:
（3）
。
(4) mass spectrum conditions: MS scan range (m/z) 355-: high energy collision (HCD), fragmentation energy (NCE): 27; exclusion of ions at valency 1 and above 7, dynamic exclusion time 60s, data dependent acquisition mode (DDA), and one MS1 spectrum selected the 10 strongest parent ions for cascade scanning.
Data analysis
Raw data generated by mass spectrometry Raw files were qualitatively analyzed by ProteinDiscovery2.2.0 (Thermo), with ProteinDiscovery parameters as follows: the database is UniProtKB (2015 _04,42121), and the other parameters are set as follows: the mass tolerance of the parent ions is 10 ppm; fragment ion mass tolerance: 0.05 Da; protease: trypsin; a maximum of 2 leaky cleavage sites is allowed; fixing and modifying: carbamidomethyl (C); variable modification: oxidation (M) and acetyl (protein N-term).
The result of the detection
2.1 quality control data:
sample preparation: a chromatogram of a base peak of a 200ngHeLa liquid mass analysis; sample amount: the result of 200ng is shown in FIG. 5.
As can be seen from FIG. 5, the high-efficiency separation of peptide fragments is a precondition for ensuring the mass spectrometric detection, and in FIG. 5, the peak width of the extracted ion chromatogram is less than 0.5min, which indicates that the peptide fragments are well separated; the chromatographic peak is smooth, no obvious burr appears, which indicates that electrospray ionization (ESI) is stable, and high-efficiency ionization of the peptide fragment can be realized.
FIG. 6 shows that the mass spectrum mass axis deviation is less than 2ppm, which fully indicates that the mass spectrum state is normal; the number of peptide fragments identified from 20ngHeLa enzymolysis products is 9,505, the number of proteins identified is 2439, and the data also indicate that the LC-MS system is good in state and can be used for sample determination.
The list of protein identifications is illustrated below:
coverage: coverage rate; peptides: all peptide fragments; PSMs: the number of times of identification; UniquePeptides: the number of unique peptide segments of the proteome; AAs: the length of the protein sequence; MW: the molecular weight of the protein; pic.pi: isoelectric point of the protein. K-Q columns: quantification of each sample; R-X columns: a quantitative value for each sample; ScoreMascot: searching and scoring a protein library; PeptidesMascot: searching and scoring the peptide fragment library;
each column of the peptide fragment identification results is illustrated below:
sequence: a peptide fragment sequence; modifications: modifying the peptide segment; qvalityq-value: actual RDR (false positive rate), the smaller the value, the better; protein groups: appear in several ProteinGroups; proteins: occurs in several proteins; PSMs: the number of times of identification; masterprotein accesses: the best match protein number; misededclearages: a missed cut site; Theo.MH + [ Da ]: theoretical mass to charge ratio; J-P columns: quantification of the peptide stretch in each sample; quantitative values in Q-; IonsScoreMascot: scoring the parent ions; w: peptide fragments were present in each sample.
(2) Results of cell sample identification
Sample information: a human cell; sample loading amount: 200ng, the cell sample identification result is shown in figure 7, and the cell sample parent ion error distribution diagram is shown in figure 8; the peptide cleavage rate distribution of the cell sample is shown in FIG. 9.
Fig. 7 and 8 show that the LC-MS system can well realize the high-efficiency analysis of the peptide fragment after sample treatment and the mass spectrum state is stable.
FIG. 9 shows that the percentage of peptide fragments containing less than 2 cleavage sites reaches 99.5%, which fully indicates that the sample pretreatment method can completely and well perform sample pretreatment, i.e., ultrasonic extraction + FASP membrane enzymolysis. The number of proteins identified from a 200ng cell sample using this method was 3609.
(3) Cell culture Medium sample identification results
Sample information: a human cell; sample loading amount: 200 ng. The BasePaak diagram of the liquid mass analysis of the human-derived cell culture medium sample is shown in FIG. 10, and the peptide cleavage rate distribution of the cell sample is shown in FIG. 11.
FIGS. 10 and 11 show that the percentage of peptides containing less than 2 nick sites is 96%, and the base peak chromatogram shows that the peptides are fewer, indicating that the sample is mainly a high-abundance protein. Finally, only 7 proteins were identified from 200ng cell culture media samples.
Table 2 shows the result of detecting the extract of umbilical cord mesenchymal stem cells according to the present invention
A beauty cream containing umbilical cord mesenchymal stem cell extract comprises the following components and mass percent of the beauty cream, 10.5 percent of the mesenchymal stem cell extract prepared in example 13, 3.5 percent of lanonol, 7.5 percent of white beeswax, 2.3 percent of camellia extract, SOD5 percent, 2 percent of carnosine, 2 percent of chitosan oligosaccharide, 2.5 percent of xanthan gum, 3 percent of propolis and the balance of water.
Comparative example 2: the beauty cream comprises, by mass, 3.5% of lanolin alcohol, 7.5% of white beeswax, 2.3% of camellia extract, SOD 5%, 2% of carnosine, 2% of chitosan oligosaccharide, 2.5% of xanthan gum, 3% of propolis and the balance of water.
Comparative example 3: the subject did not use any skin care products.
The test method comprises the following steps: 150 female volunteers with aged skin were screened, aged 30-50 years, mean age 40 years, inclusion criteria: dry skin, reduced elasticity, appearance of wrinkles, pigmentation, dull skin tone, and other skin aging phenomena, with no other anti-aging products used throughout the test period.
150 female test volunteers were divided into 3 groups of 50 each, namely, the group of example 13 and the groups of comparative examples 2 to 3, wherein the control group 3 was a subject who did not use any skin care product, the group of example 13 and the groups of comparative examples 2 to 3 were subjects who used the beauty creams prepared in the example 13 and the comparative example 2 of the present invention, respectively, and after cleansing the face in the morning and evening, an appropriate amount of beauty cream was applied to the face, the whole test time of each group was 60 days, adverse reactions occurring during the test period were recorded, and the subjects were evaluated for effects, and part of the comparative test graphs are shown in fig. 12.
Skin aging evaluation: evaluation was performed by observing wrinkles in 5 test areas of the subject (glabellar lines, forehead wrinkles, crow's feet, nasolabial folds, perilabial wrinkles), and the criteria were evaluated: no wrinkles; mild (2-3 wrinkles, length <1.5 cm); moderate (2-6 shallow wrinkles, length <3 cm); severe (several major wrinkles up to 4cm in length with minor wrinkles).
And (3) safety evaluation: the occurrence of adverse reactions such as slight discomfort, erythema, dryness, pruritus and stabbing pain of a subject after using the product is mainly evaluated.
On the 100 th day, the water content and the oil content of the tested part of the tested subject are detected by a non-invasive instrument for statistical analysis. The test results are shown in tables 3-4.
TABLE 3 evaluation of the facial safety of the subjects with different cosmetics
TABLE 4 Water content and oil content of different cosmetics on the test part
As can be seen from tables 2 and 3, the beauty cream of the present invention can improve the dark yellow and red spots of the skin, and increase the water content and oil content of the horny layer of the skin.
While the present invention has been described in detail with reference to the drawings and examples, it will be understood by those skilled in the art that various changes in the specific parameters of the above examples may be made without departing from the spirit of the invention, and various specific examples are included in the scope of the present invention and will not be described in detail herein.
Claims (10)
1. An umbilical cord mesenchymal stem cell extract is characterized by being extracted by the following steps: (1) separating umbilical cord mesenchymal stem cells from the umbilical cord tissue block or the umbilical cord blood; (2) directionally differentiating the umbilical cord mesenchymal stem cells obtained in the step (1) into skin stem cells; (3) collecting and cracking the skin stem cells obtained in the step (2), and enriching active small molecular substances through a molecular sieve to obtain the umbilical cord mesenchymal stem cell extract.
2. The umbilical cord mesenchymal stem cell extract according to claim 1, which is extracted by the steps of: (1) umbilical cord mesenchymal stem cells are separated from the umbilical cord tissue block; (2) directionally differentiating the umbilical cord mesenchymal stem cells obtained in the step (1) into skin stem cells; (3) normally culturing the skin stem cells obtained in the step (2) for 18-36 h, then culturing in a hypoxic environment with 5% of oxygen, 5% of carbon dioxide and 90% of nitrogen for 18-36 h, collecting and cracking the obtained skin stem cells, obtaining active small molecular substances through a molecular sieve with the pore diameter of 0.3-2.0 nm, and enriching the active small molecular substances to obtain the umbilical cord mesenchymal stem cell extract.
3. The umbilical cord mesenchymal stem cell extract according to claim 1, wherein in the step (1), the umbilical cord mesenchymal stem cells are obtained from umbilical cord tissue mass or umbilical blood through a primary culture step, a subculture step of stem cells, a cryopreservation step of stem cells, a recovery and culture step of stem cells and a preparation step of a human umbilical cord mesenchymal stem cell preparation.
4. Umbilical cord mesenchymal stem cells according to claim 3A cell extract, wherein in step (1), the primary culture step of umbilical cord mesenchymal stem cells comprises adherent seeding of umbilical cord tissue blocks into a culture dish at 37 ℃ with 5% volume fraction of CO2And (2) standing for 1-2 h in an incubator, adding complete culture solution into a culture dish for culture, replacing the complete culture solution every 3 days by half, after the culture solution and umbilical cord tissue blocks are subjected to standing culture for 14 days, removing the culture solution and umbilical cord tissue blocks, cleaning cells by using normal saline, adding 0.5wt% of Tryple-Express digestive juice, standing for 2-3 minutes at 37 ℃, adding the complete culture solution to stop digestion after the pseudopodium is completely retracted and the cells are rounded, and blowing to obtain the P1 generation human umbilical cord mesenchymal stem cell suspension.
5. The umbilical cord mesenchymal stem cell extract of claim 3, wherein: in the step (2), the composition of the culture solution is as follows: insulin 20mg/L, L-glutamine 5mg/L, epidermal growth factor 8 μ g/L, fibroblast growth factor 2 μ g/L, nerve growth factor 0.88 μ g/L, glucocorticoid 175 μ g/L, baicalein 0.1mg/L and delphinidin 35 μ g/L.
6. The umbilical cord mesenchymal stem cell extract of claim 1, wherein: in the step (2), the placenta mesenchymal stem cells obtained in the step (1) are inoculated in a skin stem cell culture medium and are cultured at 37 ℃ and 5% CO2And incubating for 15-25 h in the incubator, removing the culture medium, adding the culture medium into a fresh skin stem cell culture medium again, and carrying out subculture amplification when the skin stem cells are approximately 80-90% fused.
7. The umbilical cord mesenchymal stem cell extract of claim 1, wherein: the step (2) also comprises digesting the directionally differentiated skin stem cells by using 0.1% pancreatic enzyme and washing the cells at least once by using physiological salt.
8. The umbilical cord mesenchymal stem cell extract of claim 1, wherein: in the step (3), the skin stem cells are cracked by adopting ice bath ultrasonic crushing, the ice bath ultrasonic crushing condition is intermittent ultrasonic, the ultrasonic crushing power is 300-350W, 10s of work is performed every time, the intermittent operation lasts for 3s, and the circulation is performed for at least 20 times.
9. Use of an extract of umbilical cord mesenchymal stem cells according to any one of claims 1-8, wherein: the umbilical cord mesenchymal stem cell extract is used for preparing a beauty product.
10. A detection method of an umbilical cord mesenchymal stem cell extract comprises the following steps: it is characterized by adopting a reversed phase liquid chromatography-mass spectrometry method.
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