CN111793108B - Application of cryopreservation solution containing peptide compounds in cryopreservation of organs and tissues - Google Patents

Abstract

The invention discloses the application of a cryopreservation solution containing a peptide compound in the cryopreservation of organs and/or tissues. The peptide compound is composed of an ice-philic amino acid and a hydrophilic group, has good ability to inhibit the growth of ice crystals and modify the morphology of ice crystals in aqueous solution, has no thermal hysteresis, has good biocompatibility, and is an ideal ice control material. . The cryopreservation solution containing the above-mentioned peptide compounds contains the above-mentioned peptide compounds, polyols, water-soluble sugars and other components, and can be used for cryopreservation of ovarian tissues or organs to maintain a complete follicle structure. The cryopreservation solution has simple components and stable performance.

Description

Application of cryopreservation solution containing peptide compounds in cryopreservation of organs and tissues

This application claims the priority of the patent application No. 201910281986.7 submitted to the State Intellectual Property Office of China on April 9, 2019, and the invention title is "a peptide compound and a cryopreservation solution containing the compound". The entire contents of this prior application are incorporated herein by reference.

technical field

The invention belongs to the technical field of biomedical materials, in particular to the application of a cryopreservation solution containing a peptide compound in the cryopreservation of organs and tissues.

Background technique

Cryopreservation refers to the preservation of biological materials under ultra-low temperature, which slows down or stops cell metabolism and division, and can continue to develop once the normal physiological temperature is restored. Since its inception, this technology has become one of the indispensable research methods in the field of natural sciences and has been widely used. In recent years, with the increase of life pressure, human fertility has been declining year by year, and fertility preservation has attracted more and more attention. The cryopreservation of human germ cells (sperm, oocytes) and gonad tissue has become important means of fertility. In addition, as the world's population ages, the need for cryopreservation of donated human-derived cells, tissues or organs for use in regenerative medicine and organ transplantation is rapidly increasing. Therefore, how to efficiently cryopreserve precious cells, tissues and organ resources for emergencies has become an urgent scientific and technical problem to be solved.

The most commonly used cryopreservation method is vitrification. Although the vitrification technology can make the liquid inside and outside the cell into a vitrified state during the rapid freezing process, the damage during the freezing process is avoided. However, during the rewarming process, the existing cryopreservation reagents cannot effectively control the growth of ice crystals, thereby damaging the cells. The high concentration (≥15%) of toxic organic solvents used in the current vitrification method, such as: DMSO, leads to the toxic and side effects of cryopreservation reagents on cells, and seriously affects the survival rate of cryopreserved objects after resuscitation and even (offspring) safety. sexuality and functional expression. To sum up, the currently used cryopreservation reagents do not have the ability to effectively control the growth of ice crystals during the rewarming process, and at the same time have the problem of poor reagent safety.

SUMMARY OF THE INVENTION

In order to improve the above-mentioned defects of the prior art, the present invention provides a peptide compound, a method for synthesizing the same, a cryopreservation solution containing the compound, and an application thereof.

The present invention is achieved through the following technical solutions:

A peptide compound composed of ice-friendly amino acids, such as: threonine (L-Thr), glutamine (L-Gln), aspartic acid (L-Asn), etc. and other hydrophilic amino acids or glucose The other hydrophilic amino acids can be selected from arginine, proline, alanine and the like.

According to the present invention, the peptide compound is a polypeptide compound formed by two or more amino acid units, such as: 2-8 amino acid units, specifically 2, 3, 4, 5, or 6 amino acid units ; The polypeptide compound is composed of two or more different amino acids.

As an exemplary technical solution, the molar ratio of the ice-philic amino acid to other hydrophilic amino acids in the peptide compound is (0.1-3):1, preferably (0.5-2):1.

According to the present invention, the arrangement of ice-philic amino acids and other hydrophilic amino acids in the peptide compound is not particularly limited, and amino acid linking groups or chemical bonds known in the art can be used for connection. For example, ice-philic amino acids and hydrophilic amino acids can be A single inter-order arrangement can also be connected with multiple ice-friendly amino acids or multiple hydrophilic amino acids to form ice-friendly amino acid fragments or hydrophilic amino acid fragments, and then connected with hydrophilic amino acids (or fragments) and ice-friendly amino acids (or fragments) respectively. .

According to the present invention, the peptide compounds are L-Thr-L-Arg(TR), L-Thr-L-Pro(TP), L-Arg-L-Thr(RT), L-Pro-L-Thr (PT), L-Thr-L-Arg-L-Thr(TRT), L-Thr-L-Pro-L-Thr(TPT), L-Ala-L-Ala-L-Thr(AAT), L - At least one of Thr-L-Cys-L-Thr (TCT).

According to the present invention, the peptide compound is a molecule composed of glucolactone or other saccharides and ice-philic amino acids through chemical bonding, for example:

GDL-L-Thr, GDL-L-Gln, GDL-L-Asn, GDL-L-Phe, GDL-L-Tyr, -GDL-L-Val, GDL-L-Ser.

According to the present invention, the peptide compound has any structure represented by formula (1)-formula (8):

The preparation method of the above-mentioned peptide compounds can be synthesized by using a polypeptide synthesis method known in the art, for example, a solid-phase synthesis method.

The preparation method according to the present invention comprises the following steps: resin swelling, covalent attachment of an amino-protected amino acid to the swollen resin, deprotection, condensation reaction of adding another amino-protected amino acid, deprotection, cleavage, and purification.

According to the preparation method of the present invention, the glycopeptide derivatives can be prepared by the known methods of the present invention, for example, the glycopeptide derivatives can be prepared by reacting glucolactone or other saccharides with amino acids in an organic solvent, or using The glycopeptide derivatives are prepared by solid phase synthesis. In one embodiment, glucose lactone (GDL) is dissolved in an organic solvent, and amino acids and a basic catalyst are added to the organic solvent, and the reaction is carried out to synthesize glycopeptide derivatives.

According to the preparation method of the present invention, the organic solvent can be selected from methanol, ethanol and the like. According to the preparation method of the present invention, the GDL-L-Thr is prepared by the following synthetic route:

The present invention also provides the application of the above-mentioned peptide compound for controlling the growth of ice crystals in an aqueous solution, and the application of the above-mentioned peptide compound for preparing a cell or tissue cryopreservation solution.

In one embodiment, the glycopeptide derivative is prepared by solid phase synthesis, comprising: swelling of the resin, covalently attaching an amino-protected amino acid to the swollen resin, deprotection, addition of a carbohydrate (eg, glucose) ester) condensation reaction, cleavage, purification. The synthesis method of GDL-L-Val and GDL-L-Ser refers to the synthesis method of GDL-L-Thr.

The present invention also provides the peptide compound represented by formula (9):

Wherein, R is selected from substituted or unsubstituted alkyl, and the substituent can be selected from -OH, -NH ₂ , -COOH, -CONH ₂ etc., for example, R is substituted or unsubstituted C _1-6 alkyl , preferably R is -CH ₃ , -CH ₂ CH ₃ , -CH ₂ CH ₂ COOH; n is an integer greater than or equal to 1 and less than or equal to 1000, for example, an integer in the range of 1-100. In some embodiments of the invention, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10.

As an embodiment of the present invention, the compound represented by the formula (9) has any of the following structures:

According to the present invention, the compound shown in the formula (9) is prepared by the following synthetic route:

The present invention also provides the use of the compound of the above formula (9) for controlling the growth of ice crystals in an aqueous solution, and the use of the compound of the formula (9) for preparing a cell or tissue cryopreservation solution.

According to the application of the present invention, the peptide compound can be used in combination with other ice control materials, and the ice control materials are selected from at least one of PVA, amino acids, polyamino acids, and the like.

The present invention also provides a cryopreservation solution containing the above-mentioned peptide compound.

The cryopreservation solution according to the present invention contains 0.1-50 g of the peptide compound per 100 mL.

The cryopreservation solution according to the present invention further contains one or more of PVA, amino acids, polyamino acids, DMSO, and polyols.

The cryopreservation solution according to the present invention may further contain serum.

The cryopreservation solution according to the present invention, per 100 mL, contains 0.1-50 g of the peptide compound, 0-6.0 g of PVA, 0-9.0 g of polyamino acid, 0-15 mL of DMSO, and 5-45 mL of polyol , 0.1-1.0mol L ^-1 of water-soluble sugar, 0-30mL of serum, and the balance is buffer.

As an embodiment of the present invention, the cryopreservation solution, per 100 mL, contains 0.1-50 g of the peptide compound, 0.1-15 mL of DMSO, 5.0-45 mL of polyol, and 0.1-1.0 mol L ^-1 water-soluble Sexual sugar, 0-30mL of serum, does not contain PVA and polyamino acids, and the balance is buffer.

As an embodiment of the present invention, the cryopreservation solution contains, per 100 mL, 0.1-50 g of the peptide compound, 0.1-6.0 g of PVA, 0-9.0 g of polyamino acid, 0-15 mL of DMSO, 5.0 -45mL of polyol, 0.1-1.0mol L ^-1 of water-soluble sugar, 0-30mL of serum, the balance is buffer.

As an embodiment of the present invention, the cryopreservation solution contains, per 100 mL, 0.1-50 g of the peptide compound, 0.1-6.0 g of PVA, 0.1-9.0 g of polyamino acid, 0.1-7.5 mL of DMSO, 5.0-45mL of polyol, 0.1-1.0mol L ^-1 of water-soluble sugar, and the balance is buffer.

In a specific embodiment, the cryopreservation solution contains 0.1-10 mL of DMSO, eg, 0.1-7.5 mL of DMSO.

In a specific embodiment, the cryopreservation solution contains 0.1-6.0 g of PVA, such as 0.1-4.0 g of PVA.

In a specific embodiment, the cryopreservation solution contains 5-30 mL of polyol, eg, 8.0-25 mL, 20 mL, 15 mL.

In a specific embodiment, the cryopreservation solution contains 0.1-0.8 mol L ^-1 of water-soluble sugar, such as 0.2-0.6 mol L- ¹ of sucrose.

In a specific embodiment, the cryopreservation solution contains 5.0-30 mL of serum, such as 5.0-20 mL of fetal bovine serum; in another specific embodiment, the cryopreservation solution does not contain fetal bovine serum.

According to the present invention, the PVA is selected from one or more combinations of isotactic PVA, syndiotactic PVA and atactic PVA, for example, the syndiotacticity of the PVA is 15%-60%, specifically for example 50 %-60%, 50%-55%.

According to the present invention, the PVA may be selected from PVA with a molecular weight of 10-500 kDa, eg, a molecular weight of 10-30 kDa, 30-50 kDa, 80-90 kDa, 200-500 kDa.

According to the present invention, the polyamino acid can be selected from a homopolymer of at least one of lysine, arginine, proline, threonine, histidine, etc. or a copolymer of two or more amino acids.

According to the present invention, the polyhydric alcohol can be a polyhydric alcohol with 2-5 carbon atoms, preferably a dihydric alcohol with 2-3 carbon atoms, and/or a trihydric alcohol, such as ethylene glycol, propylene glycol, and glycerin Any of; preferably ethylene glycol.

According to the present invention, the water-soluble sugar can be at least one of non-reducing disaccharides, water-soluble polysaccharides, and sugar anhydrides, such as selected from sucrose, trehalose, water-soluble cellulose (such as hydroxypropyl methylcellulose) etc.), polysucrose; preferably sucrose, hydroxypropyl methylcellulose. The water-soluble sugar can act to protect cell membranes and prevent cell sedimentation.

According to the present invention, the buffer may be selected from at least one of DPBS or hepes-buffered HTF buffer, or other cell culture medium buffers.

According to the present invention, the serum can be selected from human serum albumin or its analogs, such as sodium dodecyl sulfonate (SDS), for human-derived cryopreservation objects; fetal bovine serum can be selected for non-human-derived cryopreservation objects or bovine serum albumin.

The preparation method of the above cryopreservation solution includes dissolving the peptide compound in a buffer solution, adjusting the pH after cooling to room temperature, dissolving other components in another buffer solution, mixing after cooling, adjusting the pH, and using the buffer solution. Make up to a predetermined volume and optionally add serum at the time of use.

The preparation method according to the present invention comprises the steps:

(1) dissolving the peptide compound in a part of the buffer solution, and adjusting the pH after cooling to room temperature to obtain solution 1;

(2) dissolving the water-soluble sugar in a part of the buffer solution, adding other components after the water-soluble sugar is completely dissolved to obtain solution 2;

(3) Optionally, dissolve PVA and/or polyamino acid in another part of the buffer solution, and adjust the pH after cooling to room temperature to obtain solution 3;

(4) After the solution 1, the solution 2, and optionally the solution 3 are cooled to room temperature, they are mixed, the pH is adjusted, and the volume is adjusted to a predetermined volume with a buffer to obtain the cryopreservation solution.

According to the preparation method of the present invention, in the step (3), the PVA is heated and dissolved in a warm bath; for example, the temperature of the water bath is 60-85°C, preferably 80°C. In the step (3), the dissolving includes a stirring step.

According to the preparation method of the present invention, in the step (2), the dissolution is ultrasonic-assisted dissolution.

In the present invention, hydrophilic means that it can form non-covalent interactions with water molecules, such as hydrogen bonds, van der Waals interactions, electrostatic interactions, hydrophobic interactions or π-π interactions with water;

Iceophilicity refers to the ability to form non-covalent interactions with ice, such as the ability to form hydrogen bonds with ice, van der Waals interactions, electrostatic interactions, hydrophobic interactions, or π-π interactions.

In the present invention, when using the cryopreservation solution, a freezing balance solution known in the art can be selected. Preferably, the DMSO content in the cryopreservation solution is 0, and the cryopreservation solution contains 7.5 polyols per 100 mL. -15mL, serum 10-20mL, buffer balance; preferably, when the serum content in the cryopreservation solution is also 0, the cryo-equilibrium solution contains PVA 1.0-5.0g per 100mL, polyol 7.5-15mL , the buffer balance. The components in the frozen equilibration solution have the same meaning as in the cryopreservation solution.

The present invention also provides the application of the above-mentioned cryopreservation solution or cryopreservation solution or cryopreservation reagent for cryopreservation of cells, tissues or organs; for example, for oocytes, sperm or stem cells, ovarian tissues, embryos or ovarian organs Store frozen.

The present invention provides the application of the cryopreservation solution in the cryopreservation of organs and/or tissues.

The present invention provides the application of the freezing balance solution in the cryopreservation of organs and/or tissues.

The present invention provides the application of the cryopreservation reagent in the cryopreservation of organs and/or tissues.

Further, the application of the cryopreservation solution of the present invention includes: balancing the organ and/or tissue in the cryopreservation solution, then placing the organ and/or tissue in the cryopreservation solution, and then placing the organ and/or tissue in the cryopreservation solution. On frozen slides, cryopreserved in liquid nitrogen.

In one embodiment, the organ and/or tissue is an ovarian tissue or an ovarian organ, eg, a section of ovarian tissue or an intact ovarian tissue.

beneficial effect

The inventors of the present invention found that in the process of controlling the growth of ice crystals in the mixed phase of ice and water, the material needs to have a good interaction with both ice and water. Therefore, according to the ice-philic-hydrophilic ice-controlling mechanism, an ice-philic amino acid such as thru Peptide compounds in which amino acids are combined with other hydrophilic amino acids or carbohydrates. The synthesized peptide compounds have good ice control effect, can modify the morphology of ice crystals, have no thermal hysteresis, and can maintain excellent performance when used in cryopreservation solutions. cell viability.

Description of drawings

Figure 1: Electron micrographs of GDL-L-Thr's activity in inhibiting ice crystal growth and statistics of ice crystal size.

Figure 2: The effect of GDL-L-Thr on the modification of ice crystal morphology in pure water.

Figure 3: Electron micrograph of the ice crystal growth inhibition activity of the TR short-chain peptide prepared in Example 2 and a statistical graph of the ice crystal size.

Figure 4: The effect of modifying the morphology of ice crystals with the TR short-chain peptide prepared in Example 2 in pure water.

Figure 5: The effect of Example 7 peptides R-COOH, R-CH ₃ and R-CH ₂ CH ₃ on ice crystal growth inhibition activity.

Figure 6: Modification of ice crystal morphology by peptoids (A) R-COOH, (B) R-CH ₃ and (C) R-CH ₂ CH ₃ in pure water.

Figure 7: Photographs of sections of fresh unfrozen ovarian organoids from 3 day old mice.

Figure 8: Sectional photos of intact ovarian organs cryopreserved in the cryopreservation solution of Comparative Example 4 after thawing.

Figure 9: Sectional photos of frozen intact ovarian organs using the cryopreservation solution of Example 6 after thawing.

Figure 10: Photographs of sections of fresh unfrozen ovarian tissue from sexually mature mice.

FIG. 11 : Photographs of the cryopreserved ovarian tissue sections in the cryopreservation solution of Comparative Example 5 after thawing.

Figure 12: Photographs after thawing of cryopreserved ovarian tissue sections using the cryopreservation solution of Example 7.

Detailed ways

The preparation method of the present invention will be described in further detail below with reference to specific examples. It should be understood that the following examples are only for illustrating and explaining the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the intended protection scope of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified; the reagents, materials, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1 Synthesis of compound of formula (1)

(1) Put 2-chlorotrityl chloride resin (2-Chlorotrityl Chloride Resin) into a reaction tube, add DCM (20 mL g ⁻¹ ), and shake for 30 minutes. Sand core suction filtration to remove the solvent, add three times molar excess of Fmoc-L-Pro-OH, then add 8 times molar excess of DIEA, and finally add DMF to dissolve, and shake for 30 minutes. Methanol cap for 30 minutes.

(2) Remove the solvent DMF, add 20% piperidine/DMF solution (10 mL g ^-1 ), remove the solvent after 5 minutes, then add 20% piperidine/DMF solution (10 mL g ^-1 ), remove the piperidine after 15 minutes solution. Take a small amount of resin, wash it with ethanol three times, add ninhydrin reagent, heat at 105-110 °C for 5 minutes, and turn dark blue as a positive reaction.

(3) The product obtained by the above reaction was washed with DMF (15 mL g ^-1 , twice), methanol (15 mL g ^-1 , twice) and DMF (15 mL g ^-1 , twice) in sequence, and then added to the reaction tube. Dissolve Fmoc-L-Thr(tBu)-OH with as little DMF as possible; a two-fold excess, a two-fold excess of HBTU. Immediately thereafter, an 8-fold excess of DIEA was added, and the reaction was carried out for 30 minutes.

(4) After removing the solution, take a small amount of resin, wash with ethanol three times, add ninhydrin reagent, heat at 105-110°C for 5 minutes, colorless is negative reaction, that is, the reaction is complete.

(5) The product obtained by the above reaction was washed with DMF (15 mL g ^-1 , twice), methanol (15 mL g ^-1 , twice) and DMF (15 mL g ^-1 , twice) successively, and then the solvent was removed, and 20% Piperidine/DMF solution (10mL g ^-1 ), remove the solvent after 5 minutes, then add 20% piperidine/DMF solution (10mL g ^-1 ), remove the piperidine solution after 15 minutes, take a small amount of resin, wash with ethanol , add ninhydrin reagent, heat at 105 ~ 110 ℃ for 5 minutes, turn dark blue as a positive reaction.

(6) The product obtained from the above reaction was washed with DMF (15 mL g -1 , twice), methanol (15 mL g ^-1 , twice) and DCM (15 mL g ^-1 , twice) successively, and the resin was drained.

(7) The product was cut for 90 minutes using cutting solution (15 mL g ⁻¹ , TFA:water:EDT:Tis=95:1:2:2, V/V). The cleavage solution was blown dry with nitrogen, and then lyophilized to obtain crude polypeptide.

(8) Purify the polypeptide by HPLC and transfer salt or desalt, HPLC: tR=6.1mins (purification column model: Kromasil100-5C18, 4.6mm*250mm; gradient eluent: 0.1% TFA acetonitrile solution and 0.1% TFA aqueous solution, 0mins -1:99, 20mins-1:9). The purified solution was lyophilized to obtain the finished product L-Thr-L-Pro (referred to as TP). The yield is about 80%. Mass spectrometry identified 217.3 as [M+H]+.

Example 2 Synthesis of compound of formula (2)

(1) Put 2-chlorotrityl chloride resin into a reaction tube, add DCM (20 mL g ⁻¹ ), and shake for 30 minutes. Sand core suction filtration to remove the solvent, add three times molar excess of Fmoc-L-Thr(tBu)-OH, then add 8 times molar excess of DIEA, finally add DMF to dissolve, and shake for 30 minutes. Methanol cap for 30 minutes.

(2) Remove the solvent DMF, add 20% piperidine/DMF solution (10 mL g ^-1 ), remove the solvent after 5 minutes, then add 20% piperidine/DMF solution (10 mL g ^-1 ), remove the piperidine after 15 minutes solution. Take a small amount of resin, wash it with ethanol three times, add ninhydrin reagent, heat at 105-110 °C for 5 minutes, and turn dark blue as a positive reaction.

(3) The product obtained by the above reaction was washed with DMF (15 mL g ^-1 , twice), methanol (15 mL g ^-1 , twice) and DMF (15 mL g ^-1 , twice) in sequence, and then added to the reaction tube. Dissolve Fmoc-Arg(Pbf)-OH with as little DMF as possible; a two-fold excess and a two-fold excess of HBTU. Immediately thereafter, an 8-fold excess of DIEA was added, and the reaction was carried out for 30 minutes.

(4) After removing the solution, take a small amount of resin, wash with ethanol three times, add ninhydrin reagent, heat at 105-110°C for 5 minutes, colorless is negative reaction, that is, the reaction is complete.

(5) The product obtained by the above reaction was washed with DMF (15 mL g ^-1 , twice), methanol (15 mL g ^-1 , twice) and DMF (15 mL g ^-1 , twice) successively, and then the solvent was removed, and 20% Piperidine/DMF solution (10mL g ^-1 ), remove the solvent after 5 minutes, then add 20% piperidine/DMF solution (10mL g ^-1 ), remove the piperidine solution after 15 minutes, take a small amount of resin, wash with ethanol , add ninhydrin reagent, heat at 105 ~ 110 ℃ for 5 minutes, turn dark blue as a positive reaction.

(6) The product obtained from the above reaction was washed with DMF (15 mL g ^-1 , twice), methanol (15 mL g ^-1 , twice) and DCM (15 mL g ^-1 , twice) successively, and the resin was drained.

(7) The product was cut for 90 minutes using cutting solution (15 mL g ⁻¹ , TFA:water:EDT:Tis=95:1:2:2, V/V). The cleavage solution was blown dry with nitrogen, and then lyophilized to obtain crude polypeptide.

(8) Purify the polypeptide by HPLC and transfer salt or desalt, HPLC: tR=4.8mins (purification column model: Kromasil100-5C18, 4.6mm*250mm; gradient eluent: 0.1% TFA acetonitrile solution and 0.1% TFA aqueous solution, 0mins -1:99, 20mins-1:4). The purified solution was lyophilized to obtain the finished product L-Thr-L-Arg(TR). The yield is about 80%. Mass spectrometry identified 276.2 as [M+H]+.

Example 3 Synthesis of compound of formula (3)

(1) Put 2-chlorotrityl chloride resin into a reaction tube, add DCM (20 mL g ⁻¹ ), and shake for 30 minutes. Sand core suction filtration to remove the solvent, add three times molar excess of Fmoc-L-Thr(tBu)-OH, then add 8 times molar excess of DIEA, finally add DMF to dissolve, and shake for 30 minutes. Methanol cap for 30 minutes.

(2) Remove the solvent DMF, add 20% piperidine/DMF solution (10 mL g ^-1 ), remove the solvent after 5 minutes, then add 20% piperidine/DMF solution (10 mL g ^-1 ), remove the piperidine after 15 minutes solution. Take a small amount of resin, wash it with ethanol three times, add ninhydrin reagent, heat at 105-110 °C for 5 minutes, and turn dark blue as a positive reaction.

(3) The product obtained by the above reaction was washed with DMF (15 mL g ^-1 , twice), methanol (15 mL g ^-1 , twice) and DMF (15 mL g ^-1 , twice) in sequence, and then added to the reaction tube. Dissolve Fmoc-Arg(Pbf)-OH with as little DMF as possible; a two-fold excess and a two-fold excess of HBTU. Immediately thereafter, an 8-fold excess of DIEA was added, and the reaction was carried out for 30 minutes.

(4) After removing the solution, take a small amount of resin, wash with ethanol three times, add ninhydrin reagent, heat at 105-110°C for 5 minutes, colorless is negative reaction, that is, the reaction is complete.

(5) The product obtained by the above reaction was washed with DMF (15 mL g ^-1 , twice), methanol (15 mL g ^-1 , twice) and DMF (15 mL g ^-1 , twice) successively, and then the solvent was removed, and 20% Piperidine/DMF solution (10mL g ^-1 ), remove the solvent after 5 minutes, then add 20% piperidine/DMF solution (10mL g ^-1 ), remove the piperidine solution after 15 minutes, take a small amount of resin, wash with ethanol , add ninhydrin reagent, heat at 105 ~ 110 ℃ for 5 minutes, turn dark blue as a positive reaction.

(6) The product obtained from the above reaction was washed with DMF (15 mL g ^-1 , twice), methanol (15 mL g ^-1 , twice) and DMF (15 mL g ^-1 , twice) successively, and the resin was drained.

(7) Repeat operations (3) to (5) to link the amino acid Fmoc-L-Thr(tBu)-OH. The product obtained by the reaction was washed sequentially with DMF (15 mL g ^-1 , twice), methanol (15 mL g ^-1 , twice) and DCM (15 mL g ^-1 , twice) and the resin was drained.

(8) The product was cut for 90 minutes using cutting solution (15 mL g ⁻¹ , TFA:water:EDT:Tis=95:1:2:2, V/V). The cleavage solution was blown dry with nitrogen, and then lyophilized to obtain crude polypeptide.

(9) Purify the polypeptide by HPLC and transfer salt or desalt, HPLC: tR=3.9mins (purification column model: Kromasil100-5C18, 4.6mm*250mm; gradient eluent: 0.1% TFA acetonitrile solution and 0.1% TFA aqueous solution, 0mins -1:99, 20mins-1:4). The purified solution was lyophilized to obtain the finished product L-Thr-L-Arg-L-Thr (TRT). The yield is about 75%. Mass spectrometry identified 377.4 as [M+H]+.

Example 4 Synthesis of compound of formula (4)

(1) Put 2-chlorotrityl chloride resin into a reaction tube, add DCM (20 mL g ⁻¹ ), and shake for 30 minutes. Sand core suction filtration to remove the solvent, add three times molar excess of Fmoc-L-Thr(tBu)-OH, then add 8 times molar excess of DIEA, finally add DMF to dissolve, and shake for 30 minutes. Methanol cap for 30 minutes.

(2) Remove the solvent DMF, add 20% piperidine/DMF solution (10 mL g ^-1 ), remove the solvent after 5 minutes, then add 20% piperidine/DMF solution (10 mL g ^-1 ), remove the piperidine after 15 minutes solution. Take a small amount of resin, wash it with ethanol three times, add ninhydrin reagent, heat at 105-110 °C for 5 minutes, and turn dark blue as a positive reaction.

(3) The product obtained by the above reaction was washed with DMF (15 mL g ^-1 , twice), methanol (15 mL g ^-1 , twice) and DMF (15 mL g ^-1 , twice) in sequence, and then added to the reaction tube. Dissolve Fmoc-L-Pro-OH with as little DMF as possible; a two-fold excess and a two-fold excess of HBTU. Immediately thereafter, an 8-fold excess of DIEA was added, and the reaction was carried out for 30 minutes.

(4) After removing the solution, take a small amount of resin, wash with ethanol three times, add ninhydrin reagent, heat at 105-110°C for 5 minutes, colorless is negative reaction, that is, the reaction is complete.

(5) The product obtained by the above reaction was washed with DMF (15 mL g ^-1 , twice), methanol (15 mL g ^-1 , twice) and DMF (15 mL g ^-1 , twice) successively, and then the solvent was removed, and 20% Piperidine/DMF solution (10mL g ^-1 ), remove the solvent after 5 minutes, then add 20% piperidine/DMF solution (10mL g ^-1 ), remove the piperidine solution after 15 minutes, take a small amount of resin, wash with ethanol , add ninhydrin reagent, heat at 105 ~ 110 ℃ for 5 minutes, turn dark blue as a positive reaction.

(6) The product obtained from the above reaction was washed with DMF (15 mL g ^-1 , twice), methanol (15 mL g ^-1 , twice) and DMF (15 mL g ^-1 , twice) successively, and the resin was drained.

(7) Repeat operations (3) to (5) to link the amino acid Fmoc-L-Thr(tBu)-OH. The product obtained by the reaction was washed sequentially with DMF (15 mL g ^-1 , twice), methanol (15 mL g ^-1 , twice) and DCM (15 mL g ^-1 , twice) and the resin was drained.

(8) Use cutting solution (15 mL g-1, TFA:water:EDT:Tis=95:1:2:2, V/V) to cut the product for 90 minutes. The cleavage solution was blown dry with nitrogen, and then lyophilized to obtain crude polypeptide.

(9) Purify the polypeptide by HPLC and transfer salt or desalt, HPLC: tR=7.6mins (purification column model: Kromasil100-5C18, 4.6mm*250mm; gradient eluent: 0.1% TFA acetonitrile solution and 0.1% TFA aqueous solution, 0mins -1:99, 20mins-2:8). The purified solution was lyophilized to obtain the finished product L-Thr-L-Pro-L-Thr (TPT). The yield is about 70%. Mass spectrometry identified 318.3 as [M+H]+

Example 5 Synthesis of compound of formula (5)

(1) Put 2-chlorotrityl chloride resin into a reaction tube, add DCM (20 mL g ⁻¹ ), and shake for 30 minutes. Sand core suction filtration to remove the solvent, add three times molar excess of Fmoc-L-Thr(tBu)-OH, then add 8 times molar excess of DIEA, finally add DMF to dissolve, and shake for 30 minutes. Methanol cap for 30 minutes.

(2) Remove the solvent DMF, add 20% piperidine/DMF solution (10 mL g ^-1 ), remove the solvent after 5 minutes, then add 20% piperidine/DMF solution (10 mL g ^-1 ), remove the piperidine after 15 minutes solution. Take a small amount of resin, wash it with ethanol three times, add ninhydrin reagent, heat at 105-110 °C for 5 minutes, and turn dark blue as a positive reaction.

(3) The product obtained by the above reaction was washed with DMF (15 mL g ^-1 , twice), methanol (15 mL g ^-1 , twice) and DMF (15 mL g ^-1 , twice) in sequence, and then added to the reaction tube. Dissolve Fmoc-L-Ala-OH with as little DMF as possible; two-fold excess and two-fold excess of HBTU. Immediately thereafter, an 8-fold excess of DIEA was added, and the reaction was carried out for 30 minutes.

(4) After removing the solution, take a small amount of resin, wash with ethanol three times, add ninhydrin reagent, heat at 105-110°C for 5 minutes, colorless is negative reaction, that is, the reaction is complete.

(5) The product obtained by the above reaction was washed with DMF (15 mL g ^-1 , twice), methanol (15 mL g ^-1 , twice) and DMF (15 mL g ^-1 , twice) successively, and then the solvent was removed, and 20% Piperidine/DMF solution (10mL g ^-1 ), remove the solvent after 5 minutes, then add 20% piperidine/DMF solution (10mL g ^-1 ), remove the piperidine solution after 15 minutes, take a small amount of resin, wash with ethanol , add ninhydrin reagent, heat at 105 ~ 110 ℃ for 5 minutes, turn dark blue as a positive reaction.

(6) The product obtained from the above reaction was washed with DMF (15 mL g ^-1 , twice), methanol (15 mL g ^-1 , twice) and DMF (15 mL g ^-1 , twice) successively, and the resin was drained.

(7) Repeat operations (3) to (5) to link the amino acid Fmoc-L-Ala-OH. The product obtained by the reaction was washed with DMF (15 mL g ^-1 , twice), methanol (15 mL g ^-1 , twice) and DCM (15 mL g ^-1 , twice) successively, and the resin was drained.

(8) The product was cut for 90 minutes using cutting solution (15 mL g ⁻¹ , TFA:water:EDT:Tis=95:1:2:2, V/V). The cleavage solution was blown dry with nitrogen, and then lyophilized to obtain crude polypeptide.

(9) Purify the polypeptide by HPLC and transfer salt or desalting, HPLC: tR=7.9mins (purification column model: Kromasil100-5C18, 4.6mm*250mm; gradient eluent: 0.1% TFA acetonitrile solution, acetonitrile solution and 0.1% TFA Aqueous solution in water, 0mins-1:99, 20mins-1:9). The purified solution was lyophilized to obtain the finished product L-Ala-L-Ala-L-Thr (AAT). The yield is about 70%. Mass spectrometry identified 260.1 as [M-8H]+.

Example 6 Synthesis of compound of formula (6)

(1) 1.0 g (0.56 mol) of glucolactone (GDL) and 0.56 mol of L-Thr were prepared by solid-phase synthesis method.

(2) HPLC purification, HPLC: tR=3.4mins (purification column model: Kromasil100-5C18SHIMADZUIntertsil ODS-SP (4.6mm*250mm*5μm); gradient eluent: 0.1% TFA acetonitrile solution and 0.1% TFA aqueous solution, 0.01- 20mins-1:99, 20-30mins-21:79, 30-40mins-100:0, 40-50mins-1:99), the yield was about 50%, and mass spectrometry identified 296.099 as [M-H]+.

The GDL-L-Thr prepared by solid-phase synthesis has higher purity, which is more conducive to product separation. The experimental results show that GDL-L-Thr prepared by solid-phase synthesis has higher purity and maintains a good ability to inhibit ice crystal growth (Figure 1). .

Example 7 Synthesis of compound of formula (9)

(1) Pour the DCM solution of Dichlorodimethylsilane into the peptide synthesis tube, let it stand for 30 minutes, and then dry the synthesis tube for use.

(2) Take 100 mg of resin into a synthesis tube, add 2 mL of DMF, pass nitrogen, and swell the resin for 10 minutes and then filter with suction.

(3) Add 1 mL of 4-methylpiperidine/DMF solution for deprotection, remove after 5 minutes, add 1 mL of 4-methylpiperidine/DMF solution, remove after 15 minutes, bubbling, and suction filtration.

(4) DMF rinse 5 times, bubbling, suction filtration.

(5) 2M, 0.5 mL of bromoacetic acid/DMF solution, and N,N-diisopropylcarbodiimide/DMF solution were sequentially added, bubbled for 20 minutes, suction filtered, and rinsed with DMF three times.

(6) Add 1 M, 1 mL of primary amine/DMF solution, bubble for 30 minutes, rinse with DMF, rinse with dichloromethane (x3).

(7) Repeat steps (5), (6) until the desired molecular weight.

(8) Add 4 mL of lysis solution, shake well, blow dry with nitrogen, and finally freeze-dry and purify to obtain the final product.

(9) A peptoid in which R is -CH ₃ , -CH ₂ CH ₃ and -CH ₂ CH ₂ COOH. Mass spectrometry identified 444.6 as [M+H] + where R was -CH ₃ , 528.8 was [M+H] ⁺ where R was -CH ₂ CH ₃ , 792.1 was [M ⁺ H] where R was -CH ₂ CH ₂ COOH ⁺ .

The ice crystal recrystallization inhibition (IRI) activity adopts the "sputter freezing method", the sample is dissolved and dispersed in DPBS solution, and 10-30 μL of the above solution is added dropwise to the surface of a clean silicon wafer pre-cooled at -60 °C at a height of more than 1.0 m. , using a hot and cold stage to heat up to -6 °C at a rate of 20 °C/min, and annealed at this temperature for 30 mins, using a polarizing microscope and a high-speed camera to observe and record the size of the ice crystals, and the hot and cold stage was sealed. Each sample was repeated at least three times, and the size of the ice crystals was counted using Nano Measurer 1.2. The error of the statistical results was the standard deviation.

Ice crystal morphology (DIS) observation and thermal hysteresis (TH) measurement were performed using a nanoliter osmometer. First, the capillary was melted with an alcohol lamp external flame, and the ultra-fine pore capillary was produced by stretching at the same time. connected. The immersion lens oil with higher viscosity is injected into the micro-aperture disc, and the aqueous solution of the dissolved material is injected into the micro-pore using a micro-injector. By controlling the temperature, the droplets are rapidly frozen, and the temperature is slowly increased to obtain single crystal ice, and the temperature is slowly lowered with an accuracy of 0.01 °C, and the ice crystal morphology and TH test are observed using a microscope equipped with a high-speed camera.

The IRI activity test was performed on 20 μL of the DPBS solution of TR prepared in the above Example 2 using the "sputter freezing method". The maximum ice crystal size (%) measured relative to DPBS is shown in Figure 3 . The maximum ice crystal size of TR bound by chemical bonding is significantly smaller than that of arginine in DPBS and threonine in DPBS at the same concentration.

Take the deionized aqueous solution of TR prepared in the above Example 2, and observe the morphology of ice crystals with nanoliters. shown in Figure 4. And no thermal hysteresis was measured.

The IRI activity test was performed on 20 μL of the DPBS solution of GDL-L-Thr prepared in the above Example 6 using the "sputter freezing method". The maximum ice crystal size (%) measured relative to DPBS is shown in Figure 1 . The maximum ice crystal size of GDL-L-Thr bound by chemical bonds is significantly smaller than the maximum ice crystal size of GDL in DPBS solution and L-Thr in DPBS solution at the same concentration, and smaller than the maximum ice crystal size of DPBS solution mixed with GDL and L-Thr at the same concentration size.

Take the deionized aqueous solution of GDL-L-Thr prepared in Example 6 above, and observe the morphology of ice crystals using nanoliters. -0.4～0.01℃), as shown in Figure 2. And no thermal hysteresis was measured.

The IRI activity test was performed on 20 μL of the DPBS solution of the compound prepared in Example 7 above using the “sputter freezing method”. The maximum ice crystal size (%) measured relative to DPBS is shown in FIG. 5 .

Taking the deionized aqueous solution of the three peptoids prepared in the above Example 7, and using nanoliters to observe the morphology of ice crystals, it is found that the peptoids whose _R is _-CH3 and _-CH2CH3 have a relatively obvious effect of modifying the morphology of ice crystals. R is -CH ₂ CH ₂ COOH without the effect of modifying the ice crystal morphology (supercooling degree -0.1 ℃, -0.4 ~ 0.01 ℃), the obtained morphology is shown in Figure 6, and the three peptoids have no heat detected lag.

The above results show that the prepared peptide compounds have the activity of inhibiting the growth of ice crystals, the effect of modifying the morphology of ice crystals, and there is no thermal hysteresis, which can control the growth of ice crystals, and can be used for cryopreservation.

Example 8 Oocyte and embryo cryopreservation experiments

(1) The TR synthesized in Example 2 and the TPT synthesized in Example 4 were prepared as a cryopreservation solution as follows:

Cryopreservation solution 1: the total volume is 100mL, 28g TR is ultrasonically dissolved in 25mL DPBS, and the pH is adjusted to 7.0 to obtain solution 1; 0.05mol sucrose is ultrasonically dissolved in 25mL DPBS, and 10mL of ethylene glycol, 7.5mL of DMSO is solution 2. After solution 1 and solution 2 return to room temperature, mix the two solutions, adjust the pH, and make up to 80% of the total volume with DPBS. Finally, add 20mL of serum before use.

Cryopreservation solution 2: the total volume is 100 mL, 28 g TPT is dissolved in 25 mL of DPBS by ultrasonic, and the pH is adjusted to 7.0, which is solution 1; 0.05 mol of sucrose is ultrasonically dissolved in 25 mL of DPBS, and 10 mL of ethylene glycol and 7.5 mL of DMSO are added in sequence after the sucrose is completely dissolved. , is solution 2. After solution 1 and solution 2 return to room temperature, mix the two solutions, adjust the pH, and make up to 80% of the total volume with DPBS. Finally, add 20 mL of serum before use.

Comparative Example 1:

Cryopreservation solution a: each 1 mL contains 15% (v/v) DMSO, 15% (v/v) ethylene glycol, 20% (v/v) serum, 0.5M sucrose, and the balance is DPBS.

(2) Balance solution and thawing solution:

Freezing Balance Solution A: Each 1mL contains 7.5% (v/v) DMSO, 7.5% (v/v) ethylene glycol, 20% (v/v) serum, and the balance is DPBS;

Thawing solution B: Thawing solution I (containing 1.0 mol L ^-1 sucrose, 20% serum, and the balance being DPBS); Thawing solution II (containing 0.5 mol L ^-1 sucrose, 20% serum, and the balance being DPBS); Thawing solution III (containing 0.25mol L ^-1 sucrose, 20% serum, the balance is DPBS); Thawing solution IV (20% serum, the balance is DPBS).

(3) Preserve mouse oocytes with the above cryopreservation solution and the formula of the comparative example

The cryopreservation solutions 1 and 2 prepared in the above steps, and the cryopreservation solution and the freezing balance solution of Comparative Example 1 were used to cryopreserve the oocytes of mice. Specifically, the oocyte cryopreservation method used in the present invention is that the oocyte is first placed in the cryopreservation solution for equilibration for 5 minutes, then placed in the prepared cryopreservation solution for 50 seconds, and the oocyte that has been equilibrated in the cryopreservation solution is placed in the cryopreservation solution for 50 seconds. Place it on a frozen carrier, then quickly put it into liquid nitrogen (-196°C), and seal the carrier for further storage; when thawing, place the frozen oocytes in Thawing Solution I at 37°C to equilibrate for 5 minutes , and then equilibrated in the thawing solution II-IV for 3 minutes; the thawed oocytes were cultured for 2 hours and the number of viable cells was observed. After thawing, oocytes were cultured for 2 hours to calculate oocyte viability (Table 1).

(4) Preserve mouse embryos with the above cryopreservation solution and the formula of the comparative example

The cryopreservation solutions 1 and 2 prepared in the above steps, and the cryopreservation solution and the freezing balance solution of Comparative Example 1 were used to cryopreserve the embryos of mice. The embryo cryopreservation method used in the present invention is specifically that the embryos are first placed in the cryopreservation solution for equilibration for 5 minutes, then placed in the prepared cryopreservation solution for 50 seconds, and the embryos that have been equilibrated in the cryopreservation solution are placed on the freezing carrier rod , and then quickly put it into liquid nitrogen (-196°C), and keep the carrier after sealing; when thawing, place the frozen embryos in thawing solution I at 37 °C for 5 minutes, and then in thawing solution II- Equilibrate for 3 minutes in IV; culture the thawed embryos for 2 hours and observe the number of viable cells. After thawing, embryos were cultured for 2 hours to calculate viability (Table 2).

The survival rate in the examples is the average of the survival rate of 3-12 repeated experiments.

Table 1 Survival rate of cryopreservation of mouse oocytes

Numbering balance fluid refrigerant Thaw solution Total number of frozen eggs Survival after 2 hours Comparative Example 1 A cryopreservation a B 146 95% Application example 1 A cryopreservation solution 1 B 93 96.2% Application example 2 A cryopreservation solution 2 B 48 90%

Table 2 The survival rate of cryopreservation of mouse embryos

Numbering balance fluid refrigerant Thaw solution total number of embryos Survival after 2 hours Comparative Example 2 A cryopreservation a B 38 94.3% Application example 3 A cryopreservation solution 1 B 41 95.9%

The data in Table 1 and Table 2 show that the polypeptide of the present invention is used for cryopreservation of oocytes and embryos, and only adding a small amount of DMSO (7.5%) can reach the oocytes and Embryos survival rate, and the data of Application Example 1 and Application Example 3 show that TR polypeptides have more excellent effects for cryopreservation of oocytes and embryos.

Example 9: Stem Cell Cryopreservation

Cryopreservation solution 1: the total volume is 100mL, 28g TR is ultrasonically dissolved in 25mL DPBS, and the pH is adjusted to 7.0 to obtain solution 1; 0.05mol sucrose is ultrasonically dissolved in 25mL DPBS, and 10mL of ethylene glycol, 7.5mL of DMSO is solution 2. After solution 1 and solution 2 return to room temperature, mix the two solutions, adjust the pH, and make up to 80% of the total volume with DPBS. Finally, add 20mL of serum before use.

Cryopreservation solution 3: the total volume is 100mL, 28g TR is ultrasonically dissolved in 25mL DPBS, and the pH is adjusted to 7.0, which is solution 1; Solution 2. After solution 1 and solution 2 return to room temperature, mix the two solutions, adjust the pH, and make up to 80% of the total volume with DPBS. Finally, add 20 mL of serum before use.

Comparative Example 2:

Cryopreservation solution b: 10% (v/v) DMSO, 15% (v/v) serum in each 1 mL, and the balance is medium a-MEM (USA, Invitrogen, C12571500BT).

The cryopreservation of human umbilical cord mesenchymal stem cells was carried out respectively according to the scheme in Table 3 using the above cryopreservation solution. The cryopreservation method of human umbilical cord stem cells is specifically the microdrop method: the human umbilical cord mesenchymal stem cells on the culture dish are digested with 25% trypsin for 2 minutes, and then placed in an equal volume of culture medium (10% FBS+a-MEM medium). ), gently pipetting until all the stem cells fall off, add a 1.5ml centrifuge tube, centrifuge at 1000 rmp for 5 minutes, discard the supernatant, separate the cells from the medium, add 10uL of freezing solution to the bottom of the centrifuge tube, and gently pipette to disperse the stem cell mass. The freezing solution with the stem cells was placed on a freezing slide and placed in liquid nitrogen (-196 degrees Celsius) for cryopreservation. When thawing, place the frozen carrier with cells and freezing solution directly into a-MEM medium at 37°C for thawing. After thawing, trypan blue staining was used to check the viability, and the number of cells was counted using the instrument JIMBIO-FIL, viability=number of viable cells/total number of cells (see Table 3).

Table 3 Cryopreservation survival rate of human umbilical cord mesenchymal stem cells

Numbering cryopreservation solution cryopreservation method survival rate Application example 4 cryopreservation solution 1 droplet method 87.8% Application example 5 cryopreservation solution 3 droplet method 75.1% Comparative Example 3 cryopreservation solution b droplet method 76.6%

As can be seen from the results in Table 3, when the cryopreservation solution of the present invention does not add DMSO or only adds a small amount of DMSO (7.5%), it can achieve a level of cell survival comparable to the cryopreservation solution added with 10% DMSO in the prior art It can greatly reduce the dosage of DMSO, reduce the damage and toxicity of DMSO to cells, and can greatly improve the passage stability and cell viability of stem cells after freezing.

Example 10: Cryopreservation of Ovarian Organs and Ovarian Tissue

Cryopreservation solution 1: the total volume is 100mL, 28g TR is ultrasonically dissolved in 25mL DPBS, and the pH is adjusted to 7.0 to obtain solution 1; 0.05mol sucrose is ultrasonically dissolved in 25mL DPBS, and 10mL of ethylene glycol, 7.5mL of DMSO is solution 2. After solution 1 and solution 2 return to room temperature, mix the two solutions, adjust the pH, and make up to 80% of the total volume with DPBS. Finally, add 20mL of serum before use.

Cryopreservation solution a: each 1 mL contains 15% (v/v) DMSO, 15% (v/v) ethylene glycol, 20% (v/v) serum, 0.5M sucrose, and the balance is DPBS.

Freezing Balance Solution A: Each 1mL contains 7.5% (v/v) DMSO, 7.5% (v/v) ethylene glycol, 20% (v/v) serum, and the balance is DPBS;

Thawing solution B: Thawing solution I (containing 1.0 mol L ^-1 sucrose, 20% serum, and the balance being DPBS); Thawing solution II (containing 0.5 mol L ^-1 sucrose, 20% serum, and the balance being DPBS); Thawing solution III (containing 0.25mol L ^-1 sucrose, 20% serum, the balance is DPBS); Thawing solution IV (20% serum, the balance is DPBS).

The above-mentioned cryopreservation solution and the cryopreservation solution of the comparative example and the cryopreservation solution were respectively cryopreserved according to the schemes in Table 4 and Table 5 to cryopreserve intact ovarian organs of mice and ovarian tissue sections of sexually mature mice within 3 days of birth.

The whole ovarian organ or ovarian tissue section was first placed in the frozen equilibration solution for 25 minutes at room temperature, and then placed in the prepared cryopreservation solution for 15 minutes. stored in nitrogen. After thawing, intact ovarian organs or ovarian tissue slices were placed in culture medium (10% FBS+a-MEM), placed in a 37°C, 5% CO ₂ incubator for 2 hours, and then fixed with 4% paraformaldehyde and paraffin. Embedding and HE staining were used to observe the morphology. The results are shown in Figures 7-12. Figure 7 is a photo of a fresh unfrozen ovarian organ section, and Figure 10 is a photo of a fresh and unfrozen ovarian tissue section.

Table 4 Ovarian Organ Cryopreservation Scheme

Numbering balance fluid cryopreservation solution Thaw solution form Application example 6 A cryopreservation solution 1 B Figure 9 Comparative Example 4 A cryopreservation a B Figure 8

Table 5 Ovarian tissue cryopreservation scheme

According to Figures 7-9, compared with Comparative Example 4 and fresh unfrozen ovarian organs using no peptide bionic ice-controlling material, the follicle structure is relatively complete in the section photos of the cryopreserved ovarian organs using Application Example 6 after thawing , the interstitial structure is relatively complete, the cytoplasm of the cells is homogeneous, and the cytoplasm is relatively more pale, and the nuclei are relatively shrunken and hyperchromatic. Relatively more staining, nuclei shrinkage, hyperchromatic relatively less. It can be seen that the application example 6 group has better cryopreservation effect on ovarian organs.

According to Figures 10-12, compared with the fresh unfrozen adult mouse ovarian tissue in the scheme of Application Example 7 and Comparative Example 5, the structures of growth follicles and antral follicles are relatively complete, and it can be seen that the cryopreservation solution of the present invention is used for cryopreservation Ovarian tissue also had better results than existing techniques.

It can be seen that the cryopreservation solution prepared with the peptide bionic ice-controlling material as the main component of the present invention can be simultaneously applied to cryopreservation of oocytes, embryos, stem cells, reproductive organs and tissues, and can achieve a good cell survival rate and biological activity.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (34)

1. The application of cryopreservation liquid containing peptide compounds in organ and/or tissue cryopreservation comprises 0.1-50g of peptide compounds in terms of volume of cryopreservation liquid per 100mL, wherein the peptide compounds are obtained by reacting hydrophilic amino acid and hydrophilic amino acid or Glucolactone (GDL); the ice-philic amino acid refers to an amino acid which can form non-covalent interaction with ice, the hydrophilic amino acid refers to an amino acid which can form non-covalent interaction with water molecules, the ice-philic amino acid is selected from at least one of threonine (L-Thr), glutamine (L-Gln) and aspartic acid (L-Asn), and the hydrophilic amino acid is selected from at least one of arginine, proline and alanine;

the peptide compound is a polypeptide compound with 2-8 amino acid units, and the polypeptide compound contains more than two amino acid units.

2. The use according to claim 1, wherein said polypeptide compound is at least one of L-Thr-L-Arg (TR), L-Thr-L-Pro (TP), L-Arg-L-Thr (RT), L-Pro-L-Thr (PT), L-Thr-L-Arg-L-Thr (TRT), L-Thr-L-Pro-L-Thr (TPT), L-Ala-L-Ala-L-Thr (AAT), L-Thr-L-Cys-L-Thr (TCT).

3. The use according to claim 1, wherein the peptidic compound has any one of the structures of formula (1) to formula (8):

formula (1)

Formula (2)

Formula (3)

Formula (4)

Formula (5)

Formula (6)

Formula (7)

Formula (8).

4. The use according to claim 1, said peptidic compound having a structure according to formula (9):

the compound of the formula (9),

wherein R is selected from substituted or unsubstituted C_1-6And an alkyl group, wherein the substituent can be selected from-OH and-COOH, and n is an integer of more than or equal to 1 and less than or equal to 8.

5. The use of claim 4, R is-CH₃、-CH₂CH₃、-CH₂CH₂COOH。

6. The use according to claim 4, wherein the compound of formula (9) has a structure represented by any one of the following:

。

7. use according to any one of claims 1 to 6, said peptidic compound being used in combination with other ice-controlling materials selected from at least one of PVA, amino acids, polyamino acids.

8. The use according to any one of claims 1 to 6, wherein the cryopreservation solution comprises serum.

9. The use according to any one of claims 1 to 6, wherein the cryopreservation solution comprises, per 100mL, 0.1 to 50g of the peptide compound, 0 to 6.0g of PVA, 0 to 9.0g of the polyamino acid, 0 to 15mL of DMSO, 5 to 45mL of the polyol, 0.1 to 1.0mol L of the polyol^-10-30mL of serum, and the balance of buffer solution.

10. The use according to claim 9, wherein the cryopreservation solution contains 0.1-15mL of DMSO.

11. The use according to claim 10, wherein the cryopreservation solution contains 0.1-10mL of DMSO.

12. The use according to claim 9, wherein the cryopreservation solution contains 0.1 to 6.0g of PVA.

13. The use according to claim 12, wherein the cryopreservation solution contains 0.1 to 4.0g of PVA.

14. The use according to claim 9, wherein the cryopreservation solution contains 5.0-30mL of serum.

15. The use according to claim 14, wherein the cryopreservation solution contains 5.0-20mL of serum.

16. Use according to claim 9, wherein the PVA is selected from one or a combination of two or more of isotactic PVA, syndiotactic PVA and atactic PVA.

17. Use according to claim 16, the PVA having a syndiotacticity of from 15% to 60%.

18. Use according to claim 17, the PVA having a syndiotacticity of from 50% to 60%.

19. Use according to claim 18, the PVA having a syndiotacticity of from 50% to 55%.

20. The use according to claim 9, wherein the polyamino acid is selected from a homopolymer of at least one of lysine, arginine, proline, threonine, histidine or a copolymer of two or more amino acids.

21. Use according to claim 9, wherein the polyol is a polyol having 2 to 5 carbon atoms.

22. Use according to claim 9, wherein the polyol is any one of ethylene glycol, propylene glycol and glycerol.

23. The use according to claim 9, wherein the water-soluble saccharide is at least one of a non-reducing disaccharide, a water-soluble polysaccharide, and a sugar anhydride.

24. The use according to claim 23, wherein the water-soluble sugar is selected from sucrose, trehalose, polysucrose.

25. The use of claim 23, wherein the water-soluble sugar is hydroxypropylmethylcellulose.

26. The use according to claim 9, wherein the buffer is selected from at least one of DPBS or hepes-buffered HTF buffer or other cell culture medium buffer.

27. The use according to any one of claims 1 to 6, wherein the cryopreservation solution is prepared by the following preparation method: dissolving the peptide compound in a buffer solution, cooling to room temperature, adjusting pH, dissolving other components in another buffer solution, cooling, mixing, adjusting pH, and diluting to a predetermined volume with the buffer solution.

28. Use according to claim 27, wherein serum is added at the time of use.

29. The use according to claim 27, wherein the preparation method of the cryopreservation liquid comprises the following steps:

(1) dissolving a peptide compound in a part of buffer solution, cooling to room temperature, and adjusting the pH value to obtain a solution 1;

(2) dissolving water-soluble sugar in a part of buffer solution, and adding other components after the water-soluble sugar is completely dissolved to prepare a solution 2;

(3) and (3) mixing the solution 1 and the solution 2 after cooling to room temperature, adjusting the pH value, and fixing the volume to a preset volume by using a buffer solution to obtain the frozen preservation solution.

30. The use of claim 29, said step (3) further comprising, before said step of: dissolving PVA and/or polyamino acid in the other part of buffer solution, cooling to room temperature, and adjusting pH to obtain solution 3.

31. The use of claim 30, said step (3) comprising the steps of: and (3) cooling the solution 1, the solution 2 and the solution 3 to room temperature, mixing, adjusting the pH value, and fixing the volume to a preset volume by using a buffer solution to obtain the frozen preservation solution.

32. The use according to any one of claims 1-6, comprising: balancing the organ and/or tissue in the freezing balancing liquid, then putting the organ and/or tissue into the freezing preservation liquid, then putting the organ and/or tissue on a freezing slide and freezing and preserving by liquid nitrogen.

33. The use according to any one of claims 1 to 6, wherein the organ and/or tissue is ovarian tissue or an ovarian organ.

34. The use of claim 32, wherein the organ and/or tissue is ovarian tissue sections or whole ovarian tissue.

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