CN114470002A

CN114470002A - Application of placenta stem cell freeze-dried powder in preparation of medicines and cosmetics

Info

Publication number: CN114470002A
Application number: CN202210272118.4A
Authority: CN
Inventors: 吕荣取; 张然
Original assignee: Beijing Yuetao Biotechnology Co ltd
Current assignee: Hebei Yihe Medical Laboratory Co ltd
Priority date: 2022-03-18
Filing date: 2022-03-18
Publication date: 2022-05-13
Anticipated expiration: 2042-03-18
Also published as: CN114470002B

Abstract

The invention relates to application of placenta stem cell freeze-dried powder in preparation of medicines and cosmetics. The protein with better anti-freezing activity is obtained by separating, identifying and preparing the adonis amurensis from the adonis amurensis, the protein can protect the activity of stem cells and improve the anti-oxidation activity when being used for preparing the freeze-dried powder of the stem cells, the prepared freeze-dried powder can be effectively used for preparing medicaments for repairing skin injuries and preparing cosmetics for resisting skin oxidation, and the protein has better effect and application value.

Description

Application of placenta stem cell freeze-dried powder in preparation of medicines and cosmetics

Technical Field

The invention relates to the field of biology, in particular to application of placenta stem cell freeze-dried powder in cosmetics and medicines.

Background

The placenta mesenchymal stem cells are multipotent stem cells, and can be differentiated into various mesenchymal cells such as bones, cartilages, muscles, fat and the like. In the normal tissue damage repair process of a body, the placenta mesenchymal stem cells are an important cell bank participating in tissue regeneration. Under the action of special signals caused by tissue injury, the placenta mesenchymal stem cells migrate to the injured part, gather and proliferate locally, and differentiate along different paths according to different injury signals. The placenta mesenchymal stem cells are easy to separate and amplify, have strong multiplication capacity in vitro, and can still maintain the multidirectional differentiation capacity even if the placenta mesenchymal stem cells are amplified by 1 hundred million times. Therefore, the placenta mesenchymal stem cell is a practical tissue repair seed cell.

The placenta mesenchymal stem cell is used as a good 'seed cell' source, and can repair and reconstruct various injured or diseased tissues and organs. Researches show that the injection of the placenta mesenchymal stem cells can inhibit myocardial inflammation, inhibit apoptosis of myocardial cells and stimulate vascular proliferation, and a certain curative effect on dilated cardiomyopathy can be observed, and the researches have great contribution to cytological treatment of heart failure. The liver cells of the human amniotic epithelial cells subjected to in-vitro directional differentiation can express at least 30 genes expressed by human adult livers, which shows that the liver cells derived from the human amniotic epithelial cells can effectively treat liver diseases. The function is observed to be gradually improved after the amniotic epithelial cells are transplanted into the damaged part of the spinal cord injury mouse model, the hindlimb capacity of the mouse is evaluated by a rat spinal cord injury scoring system, and finally the hindlimb capacity reaches 19 points which is only 2 points lower than that of a normal animal. In addition, the placenta mesenchymal stem cells have the functions of promoting wound healing, locally expanding and homing the wound and directionally differentiating into wound repairing cells and endothelial cells; can also be used as a carrier cell of VEGF to carry target genes to a target area to play a long-acting healing promoting function of VEGF. In addition, research shows that the multidrug resistance gene mdr1 retrovirus vector can effectively transfect the placenta mesenchymal stem cells, and the transfected exogenous gene not only can be fully expressed, but also can play a normal physiological function.

Based on the importance of the placental mesenchymal stem cells, preservation of the placental mesenchymal stem cells is becoming more important. CN108186682A discloses adding 10% BSA as excipient into the placenta stem cell culture stock solution, fully dissolving, filtering and sterilizing with a 0.45um sterile filter, and subpackaging into 2ml penicillin bottles, wherein each bottle contains 1ml BSA; after subpackaging, placing the penicillin bottles in an ultra-low temperature refrigerator at minus 80 ℃ for pre-freeze drying, taking out after pre-freezing for 12 hours, and then placing in a freeze dryer for freeze drying; and obtaining the freeze-dried powder of the placenta mesenchymal stem cells. The freeze-dried powder prepared by the preparation method effectively preserves various cell factor mixtures with biological activity in human placenta mesenchymal stem cells, the preservation period can be prolonged to 2 years, and the bottleneck that the preservation period of a cell factor solution is limited is effectively solved.

CN111658672A discloses a human placental stem cell lyophilized powder and a preparation method thereof, in particular to a preparation method of a human placental stem cell lyophilized powder, which comprises the following steps: s1, culturing human placenta mesenchymal stem cells by using a cell culture medium with a pH value of 5.6-6.8 and containing serum until 70-90% of the cells are fused, adding deionized water, a stabilizer and an excipient, mixing and dissolving until the mixture is transparent to obtain a mixed solution; s2, standing the mixed solution in an environment with a pH value of 5.6-6.8, and sterilizing at high temperature after standing; s3, cooling the mixed solution after high-temperature disinfection and sterilization to 2-5 ℃; s4 filtration was performed using precision filter paper. The lyophilized powder of human placental stem cells prepared by the method has the effects of comprehensively regulating aged organisms and recovering young organisms; it can greatly raise metabolism level of organism.

However, the effect of the freeze-dried powder prepared by the current placenta mesenchymal stem cell freeze-drying method is still to be further improved. CN112876545A provides a novel antifreeze protein with higher antifreeze activity, which has the functions of inhibiting ice crystal growth and recrystallization, has application potential in the aspects of structure protection, cell protection and the like, and provides a candidate for developing a cryoprotectant. The existing research also shows that part of antifreeze proteins also have antioxidant activity, so that the development of a specific antifreeze antioxidant protein for preparing freeze-dried powder aiming at placenta mesenchymal stem cells becomes more important.

Disclosure of Invention

The antifreeze protein with better antifreeze property is obtained by specific screening and preparation, and has better effect when being used for preparing the placenta mesenchymal stem cell freeze-dried powder.

In one aspect, the invention provides an antifreeze protein having a sequence as set forth in SEQ ID NO: 1 is shown.

The invention provides an antifreeze protein, which is a protein consisting of an amino acid sequence shown in SEQ ID No.1, or a protein with the same activity obtained by substituting, deleting or inserting one or more amino acids in the amino acid sequence shown in SEQ ID No. 1.

In particular, the antifreeze proteins can be produced by means of existing in vitro expression systems, for example E.coli expression systems.

In another aspect, the present invention provides a method for preparing a lyophilized powder of placental mesenchymal stem cells, the method comprising: adding serum-free antibiotic-free DMEM/F12 medium into the mesenchymal stem cells until the cell concentration is 10⁸Perml, centrifuged with isotonic PBS (1000r/min, 5min) and washed 3 times. Subpackaging the placenta mesenchymal stem cells into 1mL to 1.5mL centrifuge tubes, ensuring that each sample has enough cells, centrifuging for 1600r/min, centrifuging for 4min, removing supernatant, adding 30% of antifreeze protein (w/v) protective agent into the cells, cooling from room temperature to 80 ℃ below zero at the speed of 10 ℃/min, balancing for 10min, and freeze-drying at the freeze-drying temperature of 20 ℃ below zero to 35 ℃ under the vacuum degree of 50-200 Pa for 24-36 hours to obtain freeze-dried powder, namely the placenta stem cell freeze-dried powder.

Further, the invention provides application of the antifreeze protein in preparation of placenta mesenchymal stem cell freeze-dried powder.

Further, the invention provides application of the placenta mesenchymal stem cell freeze-dried powder in preparing a pharmaceutical composition for skin repair and skin oxidation resistance.

Further, the invention provides application of the placenta mesenchymal stem cell freeze-dried powder in preparation of cosmetics for skin repair and skin oxidation resistance.

Further, the freeze-drying and freeze-drying medium can also comprise mannitol, disaccharide or gelatin besides the antifreeze protein, wherein the disaccharide is selected from sucrose, trehalose and a mixture of sucrose and trehalose. In one embodiment, the lyophilization medium comprises about 1% mannitol, about 1% disaccharide selected from sucrose, trehalose and mixtures of sucrose and trehalose, or about 0.25% gelatin. In one embodiment. In one embodiment, the antifreeze protein is dissolved in a phosphate buffer, wherein the phosphate buffer has a concentration of about 10 mM. In one embodiment, the phosphate buffer is potassium phosphate buffer at a concentration of about 10mM and a pH of about 7.2. In one embodiment, the lyophilization medium comprises antifreeze protein in 10mM potassium phosphate buffer, ph7.2, along with mannitol, sucrose, and gelatin.

The compositions of the invention may be administered by any suitable means, for example orally, for example in the form of pills, tablets, capsules, granules or powders; under the tongue; a cheek portion; parenterally, e.g., by subcutaneous injection, intradermal injection, intranasal injection, intravenous injection, intramuscular injection, intraperitoneal injection, or intrasternal injection, or using infusion techniques (e.g., as a sterile injectable aqueous or nonaqueous solution or suspension); a nasal portion, e.g., by inhalation spray, aerosol, spray or nebulizer; topical application, such as creams, ointments, pastes, powders or gels; transdermal, such as in the form of a patch; transmucosal; or rectally, such as in the form of suppositories. The compositions of the present invention may also be administered in a form suitable for immediate release or extended release. Immediate release or extended release may be achieved by use of a suitable pharmaceutical composition, or, particularly in the case of extended release, by use of a device such as a subcutaneous implant or osmotic pump.

The freeze-dried stem cells provided by the invention have better viability and oxidation resistance

As used herein, "viability" refers to the number of viable bacteria or the percentage of viable to dead bacteria in a sample before freezing, after freezing but before freeze-drying, or after freeze-drying and storage for up to 3 years. Viability is typically provided in percent, meaning the percentage of bacteria in a sample that are viable after treatment (e.g., freezing or freeze-drying), compared to the number of viable bacteria prior to any such treatment. Alternatively, prior to any such treatment, the viability provided herein is the number of Colony Forming Units (CFUs) in the treated sample divided by the CFUs. Many methods for determining viability of a bacterial sample are known in the art, and any art-recognized technique or assay can be used. In some embodiments, viability after lyophilization is determined as CFU count/CFU count pre-lyophilization x 100% after reconstitution. The viability of the freeze-dried powder is close to 100%, and the freeze-dried powder has a good effect.

Advantageous effects

The protein with better anti-freezing activity is obtained by separating, identifying and preparing the adonis amurensis from the adonis amurensis, the protein can protect the activity of stem cells and improve the anti-oxidation activity when being used for preparing the freeze-dried powder of the stem cells, and the prepared freeze-dried powder can be effectively used for preparing medicaments for repairing skin injuries and cosmetics for resisting skin oxidation and has better effect and application value.

Drawings

FIG. 1 is a graph showing the results of thermal hysteresis activity

FIG. 2 is a graph showing the result of the identification of the antifreeze property

FIG. 3 functional verification diagram of placenta mesenchymal stem cell lyophilized powder

Detailed Description

The invention will be more readily understood by reference to the following examples, which are provided for purposes of illustration of the present invention and are not to be construed as limiting the scope of the invention in any way.

Unless defined otherwise or clear from context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be appreciated that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present technology.

Example 1 screening of antifreeze proteins

Adonis amurensis is first freeze-dried and then ground to pieces and stirred in 3 volumes of 10mmol/L PBS (pH8.0) for 3 h. Centrifuging the suspension at 3000r/min for 30 min; precipitating the supernatant by adopting 50-100% of saturated ammonium sulfate, centrifuging the salting-out solution at 3000r/min for 30min, dialyzing the precipitate for overnight against pure water, and freeze-drying to obtain the JZH anti-freeze crude protein. 100mg of the anti-freeze crude JZH protein was dissolved in 5mL of 10mmol/L PBS (pH8.0) and separated by a cation exchange column (2.6 cm. times.50 cm). The sample loading volume is 4mL, elution is carried out for 1h by using 10mmol/L PBS (pH8.0), then elution is carried out for 5h by using 0-1.5 mol/L NaCl (containing 10mmol/L PBS, pH8.0), the flow rate is 1.0mL/min, and the detection wavelength is 220 nm. The active fractions showing THA were collected, dialyzed against pure water, and lyophilized. 150mg of the lyophilized powder was reconstituted in 5mL of 10mmol/L PBS (pH8.0) and repurified with a DEAE column. Weighing 40g of DEAE-52, suspending in 100mL of distilled water, washing with distilled water, 0.5mol/L HCl and 0.5mol/L NaOH in turn, pouring out finely-divided particles, slowly adding DEAE-52 to a 0.8cm x 40cm column without bubbles, balancing with a balancing solution (5mmol/L Tris-HCl (pH 7.4),0.1mol/L KCl,0.1mol/L EDTA) or washing to A280 absorption value as a base level, subjecting the complex solution to column chromatography at 2mL, eluting with 25mmol/L Tris-HCl (pH 7.4),0.1mol/L EDTA and KCl gradient, eluting with 0.1-0.3 mol/L KCl gradient, eluting with 0.3mol/L KCl gradient to A280 absorption value as a base level, eluting with 0.3-0.6 mol/LKCl gradient, eluting with 0.6mol/L LKCl gradient, eluting with 1.0mol/L LKCl, collecting eluates, freeze drying, concentrating, adding propylene dextran S-300, purifying with 50mmol/LNH4HCO3 column, collecting active components showing THA peak, dialyzing with pure water, and lyophilizing. And (3) carrying out mass spectrometry (MALDI-TOF/TOF) identification on the component with the highest THA activity: the target protein was cut from the gel strip analyzed by Tricine-SDS-PAGE, processed and detected by MALDI-TOF/TOF tandem mass spectrometer, data acquisition was performed using auto-acquisition data and positive ion mode, primary and secondary mass spectral data were integrated, and data were analyzed comprehensively and protein identified using GPS 3.6(Applied Biosys-ters) and Mascot 2.3(Matrix Science). The sequence of the antifreeze protein is identified as SEQ ID NO: 1 is shown.

MGRQLGATDRYALKTSWYFLYCIEYMHSEYVTWEFYLSMKHNLITIGGYEYWVNGVERVTLSAVHCIVC EFYDYLVNSYIDKWQDDHIYKRCYIKLLCIIKLSRRACFQCECETPHYSDFIFDRVECINVPIINYDLR VWELIKCEYPQRFKFQQPDIACREFETIRLGQRQDWNCHAFWIFQEILNTVKKVCVDEYVIQHNVSLHG GYAIFHKWMCWAIDCQQFTGHTFKSNN。

The thermal hysteresis activity is an important index for judging the antifreeze activity of antifreeze protein. The Thermal Hysteresis Activity (THA) is determined with reference to methods of the prior art. Taking Tris-HC1 buffer solution as extracting solution, setting the extracting temperature to be 4 ℃, the extracting time to be 1.5h, and the material-liquid ratio to be 1: 3 (g: mL), the results of examining the influence of the extract solutions having different pH values on the thermal hysteresis activity of antifreeze protein are shown in FIG. 1.

As can be seen from FIG. 1, the thermal hysteresis activity of the antifreeze protein continuously increases with the increase of the pH value, and reaches the highest value (0.083 + -0.002) DEG C when the pH value of the extract is 8.0. When the pH value of the extract is more than 8.0, the thermal hysteresis activity of the extract is reduced, and strong alkali can damage the spatial structure of the antifreeze protein to influence the functional property of the antifreeze protein, so that the thermal hysteresis activity of the antifreeze protein is reduced.

Example 2 identification of anti-freezing Properties

Detecting activity of Escherichia coli added with antifreeze protein, collecting fresh activated Escherichia coli liquid, culturing until absorbance OD600 is about 1.0, diluting with sterile water 10⁴Double, split into sterile EP tubes, add purified SEQ ID NO: 1 and mixing the antifreeze protein with the bacterial liquid until the final concentration is 1%, 10%, 20% and 30% W/v respectively, simultaneously taking blank bacterial liquid and the bacterial liquid added with 20% glycerol as a control, placing all experimental groups at-10 ℃, freezing for 120h, absorbing 50uL of bacterial liquid, coating the bacterial liquid on an LB (LB) flat plate (each group is 3 times), and carrying out inverted culture at 37 ℃ for 24h, and then carrying out colony statistics. The results are shown in FIG. 2.

From the results shown in fig. 2, it can be seen that the survival rate of the bacteria added with the anti-freeze protein at a concentration of more than 10% is significantly higher than that of the control group of blank bacteria liquid and glycerin, and the relative survival rate of the bacteria is correspondingly higher as the concentration of the added protein is higher. And the more pronounced the antifreeze protection is with increasing protein concentration (P < 0.01).

Example 3 preparation of placental stem cells

Under aseptic condition, repeatedly washing human placenta tissue with PBS, removing placenta blood, cutting off residual umbilical cord tissue, and placing in aseptic square dish for use. Tissue mincing combined with an enzyme liquid digestion method: firstly, processing the placenta tissue by a tissue mincing and separating machine, and smashing the placenta to obtain placenta tissue fragments. ② the placenta tissue fragments are placed in a 200mL sterile centrifuge tube, and the placenta tissue fragments are digested by combined application of 0.25 percent trypsin and 0.1 percent collagenase type I. ③ digesting for 30min in a shaker at 37 ℃, removing tissues and reserving digestive juice, centrifuging for 5min at 1500 r/min, and collecting cell sediment obtained by centrifugation. And fourthly, suspending the cells collected after centrifugation in a-MEM culture medium containing 10 volume percent of fetal calf serum, and culturing in a CO2 culture box with the volume percent of 5 percent at 37 ℃. Fifthly, changing the liquid after 48h, removing the non-adherent cells, changing the liquid every three or four days for 1 time, digesting after the mesenchymal stem cells adhere to form a clone island to obtain the primary mesenchymal stem cells of the placenta source, and continuously subculturing or freezing in a liquid nitrogen tank.

Immunophenotyping: digesting the fused 3 rd generation placenta-derived mesenchymal stem cells by pancreatin, centrifuging and washing to prepare 2xl0⁵Respectively adding monoclonal antibody antibodies CD19, CD73, CD105, HLA-DR, CD11b, CD34, CD45 and CD90 into the tube cell suspension, incubating for 30min in a dark place, adding 1ml PBS, 1500r, centrifuging for 5min, discarding the supernatant, and repeating for 2 times; cells were resuspended in 500uL PBS and surface markers of cells were detected by flow cytometry. The results show that the obtained cells highly express CD 7399.92%, CD 9099.97% and CD 10599.34%, and do not express CD34, CD19, CD11b, CD45 and HLA-DR, which indicates that the prepared cells are mesenchymal stem cells.

Example 4 preparation and Activity detection of placental mesenchymal Stem cell lyophilized powder

The mesenchymal stem cells prepared in example 3 were added to serum-free, antibiotic-free DMEM/F12 medium to a cell concentration of 10⁸Perml, centrifuged with isotonic PBS (1000r/min, 5min) and washed 3 times.

Experimental grouping, wherein the base solution of the protective agent is DMEM, the protective agent is divided into the following groups, the first group is a control group, and the protective agent consists of 40% PVP (w/v) + 15% FBS (v/v) + 20% trehalose (w/v) + 10% glycerol (v/v); the second group is a blank control group; the third group is antifreeze protein 10% antifreeze protein (w/v), the fourth group is antifreeze protein 20% antifreeze protein (w/v), the third group is antifreeze protein 30% antifreeze protein (w/v). Subpackaging the placenta mesenchymal stem cells into 1mL to 1.5mL centrifuge tubes, ensuring that each sample has enough cells, centrifuging for 1600r/min, centrifuging for 4min, removing supernatant, adding each group of protective agent into the cells, carrying out low-temperature microscope observation, cooling from room temperature to-80 ℃ at the speed of 10 ℃/min, balancing for 10min, carrying out freeze-drying at the freeze-drying temperature of-35 ℃ and the vacuum degree of 100Pa for 36 hours, and obtaining freeze-dried powder, namely the placenta stem cells.

Dissolving lyophilized powder with PBS, and adjusting cell number to 5 × 10⁵and/mL, after trypan blue staining, randomly selecting 6 visual fields in each group under a common light microscope, counting the number of cells stained by trypan blue, and calculating the survival rate of the cells, namely [ (total number of cells-number of trypan blue stained cells)/the total number of counted cells X100%. The results are shown in table 1 below.

TABLE 1 Effect of different groups of lyophilized powder on cell survival

As can be seen from Table 1, the antifreeze protein of the invention has concentration dependence, and can improve the survival rate of resuscitation cells of the freeze-dried powder. The cell survival rate of the 30% antifreeze protein experimental group reaches 98.4 +/-2.7%, the control group has better survival effect than the control group, and the protective effect of the experimental group and the control group is obvious (P is less than 0.01) compared with the blank control group.

Example 5 functional verification of lyophilized powder of placental mesenchymal stem cells

After animals were anesthetized with ketamine (45mg/kg body mass) in the abdominal cavity, the back was prepared with sodium sulfide depilatory, the temperature of a thermostatic waterbath was adjusted to 75 ℃, a 50ml syringe with a diameter of 3cm and a base removed was placed on the back of the animals, and 20ml of water at 75 ℃ was kept for 12S to prepare a deep ii-degree scald model.

The prepared scald model was randomly divided into a stem cell transplantation group and a model control group, the stem cell transplantation group was immediately administered with DMEM/F12 medium to dissolve lyophilized powder (the control group prepared in example 4 and the 30% antifreeze protein experimental group, respectively) after scald, the model control group was treated with the same amount of medium, and the number of cells was adjusted to 1 × 10⁷Ml, 5 points are evenly selected on the scald part0.1ml of stem cells are locally injected into each point, a little supernatant (0.5m1) is dipped by a sterile cotton swab and evenly smeared on the scalded part, then sterile gauze is used for covering the wound surface, and the animal is kept warm. The control group was given an equal amount of physiological saline as the model group. Animals were housed individually in small cages. Feeding animals in a single cage, freely taking food and drinking water, observing wound healing conditions after 10 days, and calculating wound healing area proportion. The results are shown in FIG. 3.

From the results of fig. 3, it can be seen that, in terms of the healing rate, the wound healing ratio of the 30% antifreeze protein experimental group reaches (78.92 ± 4.2)%, the effect on the control group is good, and the difference between the experimental group and the model control group is significant (P < 0.05).

Experimental example 6 Oxidation resistance Effect experiment

Equal amounts of the lyophilized powder of example 4 were taken, incubated at 25 ℃ for 5 days, and a normal storage environment was simulated. Then, the cells were lysed with PBS, and the cells were disrupted to obtain cells.

Taking human epidermal keratinocyte HEK-A in logarithmic growth phase according to 1 × 10⁴cells/mL are inoculated into a 96-well plate with the concentration of 200 mu L per well, 10 mu L of cells are added after normal culture is carried out for 24H, and 1mM H is adopted after 5min₂O₂And performing oxidation damage treatment for 6 h. The MTT method measures the cell viability and the absorbance value A490. To pass through H₂O₂The number of cells subjected to oxidative damage treatment was 100%, and the cell survival% of each experimental sample was calculated. The results are shown in Table 2.

TABLE 2 Effect of the lyophilized powder of the present invention on Oxidation resistance

Group of	Cell viability%
		Control group	81.2±2.1*
Blank control group	51.3±1.9
		10% antifreeze protein test group	80.8±3.2*
20% antifreeze protein test group	92.3±2.8*
		30% antifreeze protein test group	98.5±2.4*

The results from table 2 show that there is a significant difference between the control and experimental and blank groups (P < 0.05). Particularly, the cell survival rate of the 30 percent antifreeze protein experimental group reaches (98.5 +/-2.4)%. The freeze-dried powder disclosed by the invention has a good antioxidant effect.

It should be understood that the above describes only some embodiments of the present invention and that various other changes and modifications may be affected therein by one of ordinary skill in the related art without departing from the scope or spirit of the invention.

Sequence listing

<110> Beijing Yue Pan Biotechnology Co., Ltd

<120> application of placenta stem cell freeze-dried powder in preparation of medicines and cosmetics

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 234

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Gly Arg Gln Leu Gly Ala Thr Asp Arg Tyr Ala Leu Lys Thr Ser

1 5 10 15

Trp Tyr Phe Leu Tyr Cys Ile Glu Tyr Met His Ser Glu Tyr Val Thr

20 25 30

Trp Glu Phe Tyr Leu Ser Met Lys His Asn Leu Ile Thr Ile Gly Gly

35 40 45

Tyr Glu Tyr Trp Val Asn Gly Val Glu Arg Val Thr Leu Ser Ala Val

50 55 60

His Cys Ile Val Cys Glu Phe Tyr Asp Tyr Leu Val Asn Ser Tyr Ile

65 70 75 80

Asp Lys Trp Gln Asp Asp His Ile Tyr Lys Arg Cys Tyr Ile Lys Leu

85 90 95

Leu Cys Ile Ile Lys Leu Ser Arg Arg Ala Cys Phe Gln Cys Glu Cys

100 105 110

Glu Thr Pro His Tyr Ser Asp Phe Ile Phe Asp Arg Val Glu Cys Ile

115 120 125

Asn Val Pro Ile Ile Asn Tyr Asp Leu Arg Val Trp Glu Leu Ile Lys

130 135 140

Cys Glu Tyr Pro Gln Arg Phe Lys Phe Gln Gln Pro Asp Ile Ala Cys

145 150 155 160

Arg Glu Phe Glu Thr Ile Arg Leu Gly Gln Arg Gln Asp Trp Asn Cys

165 170 175

His Ala Phe Trp Ile Phe Gln Glu Ile Leu Asn Thr Val Lys Lys Val

180 185 190

Cys Val Asp Glu Tyr Val Ile Gln His Asn Val Ser Leu His Gly Gly

195 200 205

Tyr Ala Ile Phe His Lys Trp Met Cys Trp Ala Ile Asp Cys Gln Gln

210 215 220

Phe Thr Gly His Thr Phe Lys Ser Asn Asn

225 230

Claims

1. Use of an antifreeze protein in the preparation of placenta mesenchymal stem cell lyophilized powder; wherein the sequence of the antifreeze protein is shown as SEQ ID NO: 1 is shown in the specification; the preparation method of the placenta mesenchymal stem cell freeze-dried powder comprises the steps of adding serum-free antibiotic-free DMEM/F12 culture medium into the placenta mesenchymal stem cells until the cell concentration is 10⁸Centrifuging with isotonic PBS solution for 1000r/min for 5min, and washing for 3 times; subpackaging the placenta mesenchymal stem cells into 1mL to 1.5mL centrifuge tubes, centrifuging for 1600r/min, centrifuging for 4min, removing supernatant, adding 30% antifreeze protein w/v protective agent into the cells, cooling to-80 ℃ from room temperature at 10 ℃/min, balancing for 10min, and freeze-drying for 24-36 hours at the freeze-drying temperature of-20 ℃ to-35 ℃ under the vacuum degree of 50-200 Pa to obtain freeze-dried powder, namely the placenta stem cell freeze-dried powder.

2. Use of the lyophilized powder of placental mesenchymal stem cells prepared according to the use of claim 1 in the preparation of a pharmaceutical composition for skin repair.

3. Use of the lyophilized powder of placental mesenchymal stem cells prepared according to the use of claim 1 in the preparation of cosmetics for skin antioxidation.

4. The use according to claim 2, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

5. Use according to claim 4, characterized in that the pharmaceutically acceptable carrier comprises an excipient.

6. The use according to claim 3, characterized in that moisturizing and whitening ingredients are further added to the cosmetic.