CN110074097B - Cryopreservation resuscitation solution system for preserving ovarian activity - Google Patents
Cryopreservation resuscitation solution system for preserving ovarian activity Download PDFInfo
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- CN110074097B CN110074097B CN201910478788.XA CN201910478788A CN110074097B CN 110074097 B CN110074097 B CN 110074097B CN 201910478788 A CN201910478788 A CN 201910478788A CN 110074097 B CN110074097 B CN 110074097B
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
- A01N1/126—Physiologically active agents, e.g. antioxidants or nutrients
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
- A01N1/125—Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
本发明的一种保存卵巢活性的冻存复苏溶液体系，是以浓度为10～100g/L的甘油明胶复合物水溶液作为基础溶液，甘油明胶复合物中甘油：明胶的质量比为10∶1，基础溶液中添加香蜂草苷、生长因子、棕榈酰三肽‑1、肝素泊洛沙姆和维生素C，将卵巢置于该溶液体系中进行冻存，可以提高冻存卵巢复苏后的生物学活性。A cryopreservation and resuscitation solution system for preserving ovarian activity of the present invention uses an aqueous solution of a glycerol-gelatin complex with a concentration of 10-100 g/L as a basic solution, and the mass ratio of glycerol:gelatin in the glycerol-gelatin complex is 10:1, Adding melanoside, growth factor, palmitoyl tripeptide-1, heparin poloxamer and vitamin C to the basic solution, and placing the ovaries in the solution system for cryopreservation can improve the biology of cryopreserved ovaries after resuscitation active.
Description
Technical Field
The invention relates to a method for recovering the activity of a cryopreserved ovary, in particular to a cryopreserved preservation solution of the ovary and a using method thereof.
Background
The ovary is the reproductive organ of the female animal. The ovarian microenvironment refers to the developmental maturity of the ovary, whether the histiocyte structure of the ovary is complete, the number of primordial follicles contained in the ovary, whether the primordial follicles can develop to maturity and ovulate, the thickness of the ovarian serosal layer and the mature ova can be smoothly discharged, the inflammation and adhesion conditions of the pelvic cavity, the functions of ovarian hormone secreting cells, the level and proportion of sex hormones in vivo, the functions and regulation conditions of peripheral nerves, the functions of the microvasculature and the blood supply (supplying oxygen and nutrient substances and excluding cell metabolites), and the like, and the factors jointly form the ovarian microenvironment. Whether the discharged follicle is mature or not is influenced by the ovarian microenvironment, the discharged follicle quality is improved by conditioning the ovarian microenvironment, and the conception probability is improved.
Ovarian tissue cryopreservation is one of the most potential and most interesting techniques for ensuring female fertility. During cryopreservation, maintenance of the ovaries is the most difficult bottleneck to overcome in the microenvironment. The ovarian cryopreservation liquid is reported to be a cryoprotectant such as dimethyl sulfoxide (DMSO), propylene glycol, sucrose, trehalose, glycerol, animal serum and the like, but the cryoprotectant has undesirable effects and has cytotoxicity (such as DMSO) or potential safety problems (such as animal serum). Therefore, the research on a cryoprotectant which is non-toxic and has high cell survival rate becomes a problem to be solved urgently in the field of ovarian freezing. Has no toxicity and cell preservation activity, and has become a hotspot in the field of cryoprotectants in recent years.
The cryoprotectant plays an important role in avoiding freezing damage of human ovarian tissues in a freezing process, but the cryoprotectant also has tissue cytotoxicity, in a freezing pre-balancing process, the ovarian tissues are placed in the cryoprotectant for a long time (30-90 minutes), and due to the overlong balancing time, the cryoprotectant can cause the ovarian tissues to be damaged by the cytotoxicity, so that the development capacity of oocytes is reduced.
Although embryo in vitro production by cryopreservation of ovaries has been successful, the number and quality of thawed oocytes are greatly reduced, resulting in great waste of oocytes and low oocyte maturation rate and embryo development ability.
Although various cryopreservation solutions have been developed, the preservation time and the resuscitation activity are not ideal, and a preservation solution system capable of maintaining the biological activity of the ovary for a long time is urgently needed to be developed.
To meet the needs of clinical treatment, the ideal cryopreservation resuscitation solution system for preserving ovarian activity needs to meet the following requirements: (1) the microenvironment of the ovary is maintained; (2) the preparation has high component safety, convenient preparation, long preservation time by matching with a freezing preservation mode; (3) is easy to be cleaned and removed, does not produce side effect on the organism and does not influence the normal function and the biological activity of the ovary; (4) the activity of the frozen ovary is recovered quickly, and the quantity and the quality of the thawed oocyte are ensured.
At present, no report of ovary cryopreservation solution system meeting all the requirements is found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art (namely the existing cryopreservation protection solution can not keep the biological activity of the ovary after long-term cryopreservation and influence the quantity and quality of oocytes), provides a cryopreservation resuscitation solution system for preserving the ovarian activity, provides the maximum microenvironment guarantee for keeping the biological activity of the ovary after long-term cryopreservation, simultaneously meets various requirements of the ideal cryopreservation resuscitation solution system for preserving the ovarian activity, and lays a foundation for realizing the safety, effectiveness, convenience and economy of clinical treatment.
The cryopreservation resuscitation solution system for preserving ovarian activity is characterized in that a glycerol-gelatin compound water solution with the concentration of 10-100 g/L is used as a base solution, the mass ratio of glycerol to gelatin in the glycerol-gelatin compound is 10: 1, melicitrin, a growth factor, palmitoyl tripeptide-1, heparin poloxamer and vitamin C are added into the base solution, the concentration of the melicitrin in the base solution is 0.001-0.5 g/L, the concentration of the growth factor is 0.001-0.05 g/L, the concentration of the palmitoyl tripeptide-1 is 0.001-0.5 g/L, the concentration of the poloxamer is 10-60 g/L, and the concentration of the vitamin C is 0.001-0.5 g/L.
The solution system takes a glycerol-gelatin compound aqueous solution with the concentration of 20-50 g/L as a basic solution, wherein the concentration of melissoside in the basic solution is 0.005-0.01 g/L, the concentration of growth factor is 0.005-0.01 g/L, the concentration of palmitoyl tripeptide-1 is 0.01-0.2 g/L, the concentration of heparin poloxamer is 15-30 g/L, and the concentration of vitamin C is 0.05-0.2 g/L.
The above base solution may further contain a polyol comprising: ethylene glycol, propylene glycol, polyethylene glycol, mannitol, sorbitol, maltitol, xylitol, lactitol.
Sugar may be further added to the base solution, including: sucrose, lactose, galactose, ribose, deoxyribose, maltose, glucose, fructose, trehalose, sorbose, xylose, raffinose, mannose, starch, dextrin.
The growth factor comprises: ovarian granulosa cytokine, vascular endothelial growth factor, fibroblast growth factor, neurotrophic factor, transforming growth factor.
The growth factor is preferably basic fibroblast growth factor.
The above freezing process is a freezing process at a temperature of 0 ℃ or lower.
The preparation method of the cryopreservation resuscitation solution system for preserving ovarian activity comprises the following steps:
(1) putting gelatin into a container, adding water 2 times of the mass of the gelatin for soaking, fully swelling, then putting into a water bath at 100 ℃ for heating to dissolve, adding glycerol after fully dissolving, uniformly stirring, and cooling to 15 ℃ to form a glycerol-gelatin compound aqueous solution;
(2) dissolving heparin poloxamer in 4 times of water, adding melissoside, growth factor and palmitoyl tripeptide-1, stirring, adding water solution of glycerogelatin complex, mixing, adding vitamin C, and mixing to obtain the final product.
The cryopreservation resuscitation solution system for preserving the ovarian activity can be further added with osmotic pressure regulator, electrolyte, antioxidant and bacteriostatic agent commonly used in pharmacy.
The cryopreservation resuscitation solution system for preserving the ovarian activity is preserved in a dark environment at the temperature of 0-4 ℃.
Compared with the existing ovary preservation solution, the cryopreservation resuscitation solution system for preserving the ovarian activity has the following advantages: no reagent with potential safety risk such as DMSO, animal serum and the like is used, and toxic and side reactions caused by reagent residues are avoided; the maintenance of the microenvironment of the ovary is facilitated; the preparation has high component safety, convenient preparation, long preservation time by matching with a freezing preservation mode; fourthly, the ovary cleaning agent is easy to clean and remove, does not produce side effect on the organism and does not influence the normal function and the biological activity of the ovary; the activity of the frozen ovary is recovered quickly, and the quantity and the quality of the thawed oocyte are ensured.
Detailed Description
Hereinafter, specific embodiments of the present invention will be described in detail. It should be noted that technical features or combinations of technical features described in the following embodiments should not be considered in isolation, and they may be combined with each other to achieve better technical effects.
EXAMPLE 1 preparation of a cryopreservation Resuscitation solution System for preserving ovarian Activity
According to the component proportion of the table 1, a cryopreservation resuscitation solution system for preserving the ovarian activity is prepared.
The preparation method of the cryopreservation resuscitation solution system for preserving the ovarian activity comprises the following steps:
(1) weighing a specified amount of gelatin, placing the gelatin in a proper container, adding water 2 times the mass of the gelatin for soaking, fully swelling, placing the gelatin in a water bath at 100 ℃ for heating to dissolve, adding glycerol after fully dissolving, uniformly stirring, and cooling to 15 ℃ to form a glycerol-gelatin compound aqueous solution;
(2) dissolving heparin poloxamer in 4 times of water, adding melissoside, growth factor and palmitoyl tripeptide-1, stirring, adding water solution of glycerogelatin complex, mixing, adding vitamin C, and mixing to obtain the final product.
Each experimental group is configured according to components and proportion within the protection scope of the claims of the application, and each control group is a sample with a certain component missing, replaced or the concentration of the component exceeding the protection scope of the claims of the application.
TABLE 1 compositions of experimental and control groups of cryopreserved resuscitation solution systems for preserving ovarian activity
Note: "/" indicates that the item component is not present, and "+" indicates that the item component (column name component) is replaced by a component in the table.
Example 2 evaluation of Freeze Resuscitation Activity of sheep ovaries
The solutions of the experimental group and the control group prepared in example 1 were subjected to the following freezing and thawing processes, and then the thawed sheep ovarian activity was measured for evaluation of the effect of the application of the freezing and thawing solution system for preserving ovarian activity.
Freezing and storing: respectively putting the separated sheep ovaries into 50mL freezing bags filled with solutions for promoting the activity recovery of the frozen ovaries, freezing to-90 ℃ in a programmed manner, keeping the temperature for 5min, and then putting the freezing bags filled with the ovaries into liquid nitrogen for storage.
And (3) resuscitation: after one week the samples of each group were taken out of the liquid nitrogen and placed in a 37 ℃ water bath to melt to an ice-water mixture, shaking constantly until completely melted.
And (3) activity determination: according to the literature [ Fundamentals of biotechnology in productive media.reprod Biomed online.2004, 9 (6): 680 ion 691 and wheel sheet over cryoprediction: evaluation of a slow freezing protocol with dimethylsulphoxide. J Assist Reprod Gene.2011, 28 (1): 7-14. the application effect of the cryopreservation resuscitation solution for preserving ovarian activity is evaluated according to indexes such as ovarian tissue morphology, primordial follicle count and apoptotic cell staining. The normal ovary is used as a control (each index is full), each group is scored, the higher the effect is, the higher the score is, the full score is 10. The evaluation results of the application effect of the cryopreservation resuscitation solution system for preserving ovarian activity of the experimental group and the control group on the sheep ovaries are shown in table 2.
TABLE 2 evaluation results of sheep ovary application effects of experimental group and control group
The observation result of tissue morphology shows that the oocytes in the sheep ovaries of the experimental group are spherical, contain complete and round cell nucleuses, and are surrounded by uniformly distributed monolayer granular cells. While the follicles in the sheep ovaries of the control group are shown to be cell nucleus solid and contracted, the basement membrane is incomplete, and the arrangement of the peripheral granular cells is disordered.
Primordial follicle count results indicated that the number of primordial follicles in the ovine ovaries of the experimental group was high, with a primordial follicle ratio of normal morphology higher than 90% approaching the 94% ratio of the normal group. The control group had a small number of primary follicles in the sheep ovary, and the ratio of the primary follicles in the normal form was less than 80%, which is obviously different from the experimental group.
The staining result of the apoptotic cells further shows that the apoptosis rate of the sheep ovarian cortex part of the experimental group is obviously lower than that of the control group, which indicates that the component combination is favorable for maintaining the ovarian microenvironment.
From the overall score, the results were better in groups 2 and 8, followed by 7, 6 and 1. The control group was significantly inferior to the experimental group, with 4 groups having the worst effect, followed by 9, 10, 12, 17, and 18 groups, and the remaining control groups had close results.
The above detailed description is specific to possible embodiments of the invention, and the embodiments are not intended to limit the scope of the invention, and all equivalent implementations or modifications that do not depart from the scope of the invention should be construed as being included within the scope of the invention. In addition, various modifications, additions and substitutions in other forms and details may occur to those skilled in the art within the scope and spirit of the invention as disclosed in the claims. It is understood that various modifications, additions, substitutions and the like can be made without departing from the spirit of the invention as disclosed in the accompanying claims.
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| CN100999722A (en) * | 2007-01-08 | 2007-07-18 | 安徽省立医院 | In-vitro cultivating matural process of immatural ovocyte in ovarium organized block |
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