CN114470002B

CN114470002B - Application of placenta mesenchymal stem cell freeze-dried powder in preparation of medicine

Info

Publication number: CN114470002B
Application number: CN202210272118.4A
Authority: CN
Inventors: 吕荣取; 张然
Original assignee: Hebei Yihe Medical Laboratory Co ltd
Current assignee: Hebei Yihe Medical Laboratory Co ltd
Priority date: 2022-03-18
Filing date: 2022-03-18
Publication date: 2023-10-03
Anticipated expiration: 2042-03-18
Also published as: CN114470002A

Abstract

The invention relates to application of placenta mesenchymal stem cell freeze-dried powder in preparation of a medicament. The protein with better anti-freezing activity is prepared from the calendula through separation and identification, the activity of the placenta mesenchymal stem cells can be protected and the anti-oxidation activity can be improved when the protein is used for preparing the freeze-dried powder of the placenta mesenchymal stem cells, and the prepared freeze-dried powder can be effectively used for preparing medicaments for repairing skin injury and has better effects and application values.

Description

Application of placenta mesenchymal stem cell freeze-dried powder in preparation of medicine

Technical Field

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

Background

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

The placenta mesenchymal stem cells are used as a good source of seed cells, and can repair and reconstruct various tissues and organs of injury or lesions. The research finds that the placenta mesenchymal stem cells can inhibit myocardial inflammation, inhibit apoptosis of myocardial cells and stimulate vascular proliferation after being infused, and can be observed to have certain curative effects on dilated cardiomyopathy, and the research has great contribution to cytological treatment of heart failure. The liver cells of the in vitro directional differentiation of the human amniotic epithelial cells can express at least 30 genes expressed by human adult livers, which indicates that the liver cells of the human amniotic epithelial cells can effectively treat liver diseases. Gradual improvement of function was observed after implantation of amniotic epithelial cells into the injured site of the spinal cord injury mouse model, and the hindlimb capacity of the mice was evaluated by using a rat spinal cord injury scoring system, and finally 19 points were reached, which was only 2 points lower than that of normal animals. In addition, the placenta mesenchymal stem cells have the effect of promoting wound healing, and have the effects of locally expanding and homing to the wound and directionally differentiating to the wound repair cells-endothelial cells; can also be used as a carrier cell of VEGF, and can carry the target gene to the target area to exert the long-acting healing promoting function of VEGF. In addition, research shows that the multidrug resistance gene mdr1 retrovirus vector can effectively transfect placenta mesenchymal stem cells, and the transfected exogenous gene can not only be fully expressed, but also exert normal physiological functions.

Based on the importance of the placenta mesenchymal stem cells, the preservation of the placenta mesenchymal stem cells is particularly important at present. CN108186682a discloses that the stock solution of placenta stem cell culture is added with 10% bsa as excipient, after being fully dissolved, filtered and sterilized by a sterile filter of 0.45um, and split-packed into 2ml penicillin bottles, each bottle being 1ml; after sub-packaging, placing the penicillin bottles in an ultralow temperature refrigerator at-80 ℃ for pre-freeze drying, taking out after pre-freezing for 12 hours, and then placing the penicillin bottles in a freeze dryer for freeze drying; obtaining the placenta mesenchymal stem cell freeze-dried powder. The freeze-dried powder prepared by the preparation method effectively preserves various bioactive cytokine mixtures in human placenta mesenchymal stem cells, can prolong the preservation period to 2 years, and effectively solves the bottleneck of limited preservation period of cytokine solution.

CN111658672a discloses a human placenta stem cell freeze-dried powder and a preparation method thereof, in particular to a preparation method of human placenta stem cell freeze-dried powder, comprising the following steps: s1, culturing human placenta mesenchymal stem cells to 70-90% by using a cell culture medium with a pH value of 5.6-6.8 and containing serum, adding deionized water, a stabilizer and an excipient, mixing and dissolving until the mixture is transparent, and obtaining a mixed solution; s2, standing the mixed solution in an environment with the pH value of 5.6-6.8, and sterilizing by high-temperature disinfection after standing; s3, cooling the mixed solution after high-temperature sterilization to enable the temperature to be reduced to 2-5 ℃; s4, filtering by adopting precise filter paper. The human placenta stem cell freeze-dried powder prepared by the method has the effects of comprehensively regulating and recovering younger organisms; it can greatly raise metabolism level of human body.

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 new antifreeze protein with higher antifreeze activity, which has the functions of inhibiting ice crystal growth and recrystallization, has application potential in structural protection, cytoprotection and other aspects, and provides candidates for low-temperature protective agent development. The existing research also shows that part of antifreeze protein also has antioxidant activity, so that the development of a specific antifreeze antioxidant protein for preparing freeze-dried powder aiming at placenta mesenchymal stem cells becomes important.

Disclosure of Invention

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

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

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

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

In another aspect, the present invention provides a method for preparing placenta mesenchymal stem cell freeze-dried powder, the method comprising: serum-free addition of mesenchymal stem cellsAntibiotic-free DMEM/F12 medium to a cell concentration of 10 ⁸ Per ml, centrifuged (1000 r/min, 5 min) with isotonic PBS solution, washed 3 times. Subpackaging the placenta mesenchymal stem cells into centrifuge tubes of 1mL to 1.5mL to ensure that the cells in each sample are in sufficient quantity, 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 lyophilizing at a lyophilization temperature of-20 to 35 ℃ under a vacuum degree of 50-200 Pa for 24-36 hours to obtain lyophilized powder of the placenta stem cells.

Further, the invention provides application of the antifreeze protein in preparing freeze-dried powder for placenta mesenchymal stem cells.

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

Further, the freeze-dried lyophilization medium may include mannitol, disaccharides selected from sucrose, trehalose and mixtures of sucrose and trehalose or gelatin in addition to the antifreeze protein. In one embodiment, the lyophilization medium comprises about 1% mannitol, about 1% disaccharide, or about 0.25% gelatin, wherein the disaccharide is selected from sucrose, trehalose, and mixtures of sucrose and trehalose. 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 a 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, as well as mannitol, sucrose and gelatin.

The compositions of the invention may be administered by any suitable means, such as orally, for example in the form of pills, tablets, capsules, granules or powders; sublingual; cheek parts; parenteral, 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 non-aqueous solution or suspension); nose, for example by inhalation of a spray, aerosol, spray or nebulizer; topical application, such as cream, ointment, paste, powder or gel; transdermal, such as in the form of a patch; a transmucosal membrane; 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 or prolonged release may be achieved by the use of a suitable pharmaceutical composition or, particularly in the case of prolonged release, by the 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 bacteria 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 as a percentage, meaning the percentage of bacteria in a live sample after treatment (e.g., freezing or freeze drying), as compared to the number of any such live bacteria prior to treatment. Alternatively, the viability provided herein is the number of Colony Forming Units (CFUs) in the treated sample divided by the CFU prior to any such treatment. Many methods for determining viability of a bacterial sample are known in the art and any art recognized technique or assay may be used. In some embodiments, viability after lyophilization is determined as CFU count after reconstitution/CFU count pre-lyophilization by 100%. The freeze-dried powder has good effect, wherein the viability of the freeze-dried powder is close to 100%.

Advantageous effects

The protein with better anti-freezing activity is prepared from the calendula through separation and identification, and can protect the activity of stem cells and improve the antioxidant activity when being used for preparing the freeze-dried powder of the stem cells, thereby having better effect and application value.

Drawings

FIG. 1 shows the results of thermal hysteresis activity

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

FIG. 3 functional verification chart of placenta mesenchymal stem cell freeze-dried powder

Detailed Description

The invention will be more readily understood by reference to the following examples, which are provided to illustrate the invention and should not be construed to limit the scope of the invention in any way.

Unless defined otherwise or the context clearly indicates otherwise, 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 for antifreeze proteins

The calendula was first freeze-dried and then ground to break, and stirred in 10mmol/L PBS (pH 8.0) 3 times the volume for 3 hours. Centrifuging the suspension at 3000r/min for 30min, precipitating the supernatant by adopting ammonium sulfate with 50-100% saturation, centrifuging the salting-out solution at 3000r/min for 30min, dialyzing the precipitate against pure water overnight, and freeze-drying to obtain the JZH antifreeze crude protein. 100mg of JZH antifreeze crude protein was dissolved in 5mL of 10mmol/L PBS (pH 8.0), and the solution was separated by a cation exchange column (2.6 cm. Times.50 cm). The sample was applied in an amount of 4mL, eluted with 10mmol/L PBS (pH 8.0) for 1h, then eluted with 0 to 1.5mol/L NaCl (10 mmol/L PBS, pH 8.0) for 5h at a flow rate of 1.0mL/min, and the detection wavelength was 220nm. 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 (pH 8.0) and re-purified using DEAE column. 40g of DEAE-52 was weighed and suspended in 100mL of distilled water, washed successively with distilled water, 0.5mol/L of HCl and 0.5mol/L of NaOH, the finely crushed particles were decanted, DEAE-52 was slowly added to a 0.8 cm.times.40 cm column without bubbles, equilibrated or washed with an equilibration solution (5 mmol/L Tris-HCl (pH=7.4), 0.1mol/L KCl,0.1mol/L EDTA) to a level based on the A280 absorption value, 2mL of the above-mentioned complex solution was subjected to column chromatography, eluted with a gradient of 25mmol/L Tris-HCl (pH=7.4), 0.1mol/L EDTA and KCl, the steps were first eluted with a gradient of 0.1 to 0.3mol/L KCl, eluted with a gradient of 0.3mol/L KCl to a level based on the A280 absorption value, eluted with a gradient of 0.6mol/L KCl, each elution solution was collected, and dried, and the respective fraction was concentrated to obtain a fraction of the dried, and the fraction was subjected to freeze-drying, 50, and the fraction was concentrated to concentration, and dried to obtain the fraction. Mass spectrometry (MALDI-TOF/TOF) was performed to identify the highest THA active component: the target protein is cut from the gel strip analyzed by Tricine-SDS-PAGE, detected by MALDI-TOF/TOF tandem mass spectrometer after treatment, data acquisition is performed by adopting an automatic acquisition data and a positive ion mode, primary and secondary mass spectrum data are integrated, and the data are comprehensively analyzed and protein identified by using GPS 3.6 (Applied Biosys-ters) and Mascot 2.3 (Matrix Science). Identifying the sequence of the antifreeze protein as SEQ ID NO: 1.

MGRQLGATDRYALKTSWYFLYCIEYMHSEYVTWEFYLSMKHNLITIGGYEYWVNGVERVTLSAVHCIVCEFYDYLVNSYIDKWQDDHIYKRCYIKLLCIIKLSRRACFQCECETPHYSDFIFDRVECINVPIINYDLRVWELIKCEYPQRFKFQQPDIACREFETIRLGQRQDWNCHAFWIFQEILNTVKKVCVDEYVIQHNVSLHGGYAIFHKWMCWAIDCQQFTGHTFKSNN。

The heat stagnation activity is an important index for judging the antifreeze activity of the antifreeze protein. Thermal Hysteresis Activity (THA) was measured with reference to the methods of the prior art. Tris-HC1 buffer solution is used as extracting solution, the extracting temperature is set to be 4 ℃, the extracting time is 1.5h, and the feed-liquid ratio is 1:3 (g: mL), the effect of the extracts of different pH values on the heat-stagnation activity of the antifreeze protein was examined, and the results are shown in FIG. 1.

As shown in FIG. 1, the thermal hysteresis activity of antifreeze protein increases continuously with increasing pH, and the thermal hysteresis activity reaches the highest value when the pH of the extract is 8.0, which is (0.083.+ -. 0.002) °C. When the extracted pH value is more than 8.0, the thermal hysteresis activity is reduced, and the strong alkali can damage the space structure of the antifreeze protein so as to influence the functional property of the antifreeze protein, thereby reducing the thermal hysteresis activity.

Example 2 identification of antifreeze properties

Detecting activity of the escherichia coli added with antifreeze protein, taking fresh activated escherichia coli bacterial liquid, culturing until absorbance OD600 is about 1.0, and diluting with sterile water for 10 ⁴ Multiple split charging into sterilized EP tubes, adding purified SEQ ID NO:1 with final concentration of 1%, 10%, 20% and 30% w/v respectively, and simultaneously placing all experimental groups at-10deg.C and freezing for 12% with blank bacterial liquid and bacterial liquid containing 20% glycerol as controlAfter 0h, 50uL of the bacterial liquid was aspirated and plated on LB plates (3 replicates per group), and after incubation for 24h at 37℃with inversion, colony statistics were performed. The results are shown in FIG. 2.

As can be seen from the results of fig. 2, the bacterial survival rate of the antifreeze protein group added with the concentration of more than 10% is significantly higher than that of the blank bacterial liquid and the glycerol control group, and the higher the concentration of the added protein, the higher the bacterial relative survival rate. And the more pronounced the cryoprotection with increasing protein concentration (P < 0.01).

EXAMPLE 3 preparation of placental Stem cells

Repeatedly washing human placenta tissue with PBS under aseptic condition to remove placenta blood, cutting off residual umbilical cord tissue, and placing in aseptic square tray. The tissue mincing combined enzyme liquid digestion method comprises the following steps: (1) firstly, placenta tissues are treated through a tissue mincing separator, and placenta is smashed to finally obtain placenta tissue fragments. (2) Placenta tissue fragments were placed in a 200mL sterile centrifuge tube and digested with 0.25% trypsin and 0.1% collagenase type I in combination. (3) Digesting for 30min by shaking table at 37 ℃, removing tissues to obtain digestive juice, centrifuging for 5min by 1500r/min, and collecting cell sediment obtained by centrifugation. (4) The cells collected after centrifugation were resuspended in a-MEM medium containing 10% fetal bovine serum by volume and cultured in a 5% CO2 incubator at 37 ℃. (5) Culturing for 48h, changing liquid for half, removing non-adherent cells, changing liquid for 1 time every three or four days, and digesting to obtain primary mesenchymal stem cells of placenta source after the mesenchymal stem cells are adherent to form clone islands, and continuously subculturing or freezing in a liquid nitrogen tank.

Immunophenotype identification: digesting the fused 3 rd generation placenta source mesenchymal stem cells with pancreatin, centrifugally washing to prepare 2xl0 ⁵ Tube cell suspension, respectively adding monoclonal anti-human antibodies CD19, CD73, CD105, HLA-DR, CD11b, CD34, CD45 and CD90, incubating for 30min in dark place, adding 1mLPBS,1500r, centrifuging for 5min, discarding supernatant, and repeating for 2 times; cells were resuspended in 500uL PBS and surface markers of cells were detected with a flow cytometer. The results show that the obtained cells highly express CD 73.92%, CD 90.97%, CD 105.99%, CD34, CD19, CD11b, CD45 and HLA-DR, which indicates that the cells prepared are mesenchymal stem cells.

EXAMPLE 4 preparation and Activity detection of placenta 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 10 ⁸ Per ml, centrifuged (1000 r/min, 5 min) with isotonic PBS solution, washed 3 times.

The experiment groups are that the base solution of the protective agent is DMEM, the protective agent is divided into the following groups, the serial number of the first group is a control group, and the composition of the protective agent is 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 of serial numbers is antifreeze protein 10% antifreeze protein (w/v), the fourth group of serial numbers is antifreeze protein 20% antifreeze protein (w/v), and the third group of serial numbers is antifreeze protein 30% antifreeze protein (w/v). Subpackaging the placenta mesenchymal stem cells into centrifuge tubes of 1mL to 1.5mL to ensure that the cells in each sample are in sufficient quantity, centrifuging for 1600r/min, centrifuging for 4min, removing supernatant, adding each group of protective agents into the cells, performing low-temperature microscopic observation, cooling to-80 ℃ from room temperature at 10 ℃/min, balancing for 10min, performing freeze-drying at-35 ℃ under 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 quantity to 5×10 ⁵ After trypan blue staining per mL, 6 fields per group were randomly selected under a normal light microscope, the number of cells stained with trypan blue was counted, and the survival rate of cells was calculated = [ (total number of cells-trypan blue stained number of cells)/total number of counted cells X100%. The results are shown in Table 1 below.

TABLE 1 Effect of survival rates of different groups of lyophilized powder resuscitated cells

From Table 1, it can be seen that the antifreeze protein of the present invention has a concentration-dependent improvement in survival rate of resuscitated cells of the lyophilized powder. The cell survival rate of the 30% antifreeze protein experimental group reaches 98.4+/-2.7%, the experimental group has better survival effect than the control group, and the protection effect of the experimental group and the control group is obvious compared with the blank control group (P < 0.01).

Example 5 functional verification of placenta mesenchymal Stem cell lyophilized powder

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

Randomly dividing the prepared scald model into a stem cell transplantation group and a model control group, immediately after scalding, administering the stem cell transplantation group with DMEM/F12 culture medium-dissolved thawing dry powder (control group prepared in example 4 and 30% antifreeze protein experimental group respectively), treating the model control group with equal amount of culture medium, and adjusting the cell number to 1×10 ⁷ And (3) uniformly selecting 5 points at the scalded part, locally injecting 0.1ml of stem cells into each point, uniformly coating a small amount of supernatant (0.5 m 1) on the scalded part by using a sterile cotton swab, and then covering the wound surface by using sterile gauze, wherein the animal is kept warm. The control group was given an equal amount of physiological saline in the manner of the model group. Animals were housed individually in small cages. Animals are fed in a single cage, eat and drink water freely, the wound healing condition is observed after 10 days, and the wound healing area proportion is calculated. The results are shown in FIG. 3.

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

Experimental example 6 antioxidation Effect experiment

The same amount of each group of freeze-dried powder of the example 4 is taken, and the temperature is kept at 25 ℃ for 5 days, so that the normal storage environment is simulated. Then, the cells were lysed with PBS and disrupted to obtain cells.

Taking human epidermal keratinocyte HEK-A in logarithmic growth phase according to 1×10 ⁴ Inoculating 96-well plate with cell/mL concentration, culturing for 24 hr, adding 10 μL of cells, and culturing for 5minBy 1mM H ₂ O ₂ And (5) performing oxidative damage treatment for 6 hours. The MTT assay measures cell viability and absorbance value A490. To not pass through H ₂ O ₂ The number of cells subjected to the oxidative damage treatment was 100%, and the cell viability% of each experimental sample was calculated. The results are shown in Table 2.

TABLE 2 influence of the antioxidation of the lyophilized powder of the present invention

The results from table 2 show that there was a significant difference between the control and experimental groups and the blank group (P < 0.05). In particular, the cell survival rate of 30% antifreeze protein experimental group treated is up to (98.5+/-2.4)%. This shows that the freeze-dried powder of the invention has better antioxidation effect.

It should be understood that the foregoing is only a few embodiments of the present invention, and it should be noted that other modifications and improvements can be made by those skilled in the art without departing from the inventive concept of the present invention, which fall within the scope of the present invention.

Claims

1. An application of antifreeze protein in preparing freeze-dried powder of placenta mesenchymal stem cells; wherein the antifreeze protein has a sequence shown in SEQ ID NO:1 is shown in the specification; the preparation method of the placenta mesenchymal stem cell freeze-dried powder comprises the following steps: adding serum-free and antibiotic-free DMEM/F12 culture medium into placenta mesenchymal stem cells to reach cell concentration of 10 ⁸ Centrifuging with isotonic PBS solution for 1000r/min, centrifuging for 5min, and washing for 3 times; subpackaging the placenta mesenchymal stem cells into a centrifuge tube with the volume of 1mL to 1.5mL, centrifuging for 1600r/min, centrifuging for 4min, removing supernatant, adding DMEM containing 30% antifreeze protein into the cells, cooling the cells from room temperature to-80 ℃ at the mass-volume ratio of 10 ℃/min, balancing for 10min, and lyophilizing at the lyophilization temperature of-20 to 35 ℃ under the vacuum degree of 50-200 Pa for 24-36 hours to obtain lyophilized powder, namely the placenta mesenchymal stem cell lyophilized powder.

2. Placental mesenchymal stem cellsThe application of the freeze-dried powder in preparing the pharmaceutical composition for skin repair is provided, wherein the preparation method of the freeze-dried powder of the placenta mesenchymal stem cells is as follows: adding serum-free and antibiotic-free DMEM/F12 culture medium into placenta mesenchymal stem cells to reach cell concentration of 10 ⁸ Centrifuging with isotonic PBS solution for 1000r/min, centrifuging for 5min, and washing for 3 times; subpackaging the placenta mesenchymal stem cells into a centrifuge tube with the volume of 1mL to 1.5mL, centrifuging for 1600r/min, centrifuging for 4min, removing supernatant, adding DMEM containing 30% antifreeze protein into the cells, wherein the mass volume ratio of 30% is that the temperature is reduced 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, wherein the obtained freeze-dried powder is the placenta mesenchymal stem cell freeze-dried powder, and the antifreeze protein has the sequence shown in SEQ ID NO: 1.

3. Use according to claim 2, characterized in that: the pharmaceutical composition also comprises a pharmaceutically acceptable carrier.

4. A use according to claim 3, wherein: the pharmaceutically acceptable carrier includes an excipient.