CN117004557A - Application of 4-phenylbutyric acid or derivative thereof in preparation of product for promoting in-vitro embryo development - Google Patents
Application of 4-phenylbutyric acid or derivative thereof in preparation of product for promoting in-vitro embryo development Download PDFInfo
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- CN117004557A CN117004557A CN202310861287.6A CN202310861287A CN117004557A CN 117004557 A CN117004557 A CN 117004557A CN 202310861287 A CN202310861287 A CN 202310861287A CN 117004557 A CN117004557 A CN 117004557A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0604—Whole embryos; Culture medium therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
Abstract
The invention discloses application of 4-phenylbutyric acid or a derivative thereof in preparing a product for promoting in-vitro embryo development. The component can improve the blastula formation rate of embryo during embryo development before implantation, and promote embryo proliferation to increase total cell number of embryo; the composition can also increase endoplasmic reticulum abundance of early embryo and reduce endoplasmic reticulum stress level, thereby protecting endoplasmic reticulum; the composition can also reduce the level of apoptosis related gene CAS3 in early embryo, inhibit excessive apoptosis of embryo, thereby improving in vitro embryo culture quality and development ability, and facilitating in vitro embryo culture.
Description
Technical Field
The invention relates to the technical field related to embryo engineering technology, in particular to application of 4-phenylbutyric acid or derivatives thereof in preparing products for promoting in-vitro embryo development.
Background
The in vitro embryo production technology is the basis of embryo engineering technology, and the process involvesIn vitro maturation of oocytes, sperm capacitation and in vitro fertilization, and in vitro culture of embryos. In vitro embryo culture means placing the embryo in a special container at 38deg.C (artificial mixed gas: 5% CO is sometimes added) 2 、5％O 2 、90％N 2 Or 5% CO 2 95% air), allowing the embryo to develop continuously in vitro. The in vitro culture of embryo has strict requirements on culture solution, sterility and other conditions, and the method is commonly used for identifying embryo survival rate and carrying out in vitro embryo research. In the in vitro development of mammalian embryos, smooth development to blastocysts is a very critical step. The blastocysts have tightly packed trophoblasts and loosely packed inner cell clusters, and only the best quality embryos develop into the final blastocysts. Judging whether blastula is good or bad according to the quantity and quality of embryo cells. Blastocysts are usually formed on days 5-7 after fertilization of the ovum and are also the final stage of embryo in vitro culture.
In vitro embryo production is a technique for creating new life by manipulating an ovum, sperm, or fertilized egg developing embryo in vitro and in vivo. Due to the difference of in-vitro and in-vivo operations and the increase of female fertility age, the technology faces serious challenges, mainly including maternal factors, embryo disorders, insufficient nutritional factors, fertilization failure and the like, so that problems of bad birth or health of offspring and the like are caused. In a natural state, the embryo is implanted into a mother body in a blastula form, so that a high embryo implantation rate can be obtained. However, compared to the microenvironment of in vivo dynamic equilibrium, in vitro culture embryos are affected by physical and chemical factors such as temperature, humidity, oxygen, carbon dioxide and amino acids, so that the embryo development quality produced in vitro is low, the blastocyst rate is lower than that of in vivo embryos, and the problem of low pregnancy rate after embryo implantation is caused. To address these problems, some prior art techniques add melatonin during in vitro embryo culture. However, melatonin has a large first pass effect, has a low oral bioavailability, and requires a large dose to act, whereas the administration of melatonin in large amounts can disrupt the endocrine system and thus has a limitation [1].
Therefore, there is an urgent need to find new compounds that improve the composition of in vitro embryo culture media to increase the developmental capacity and quality of in vitro embryos.
[1] Liu Zheyu, sun Zheng, research on melatonin metabolic patterns [ J ]. Life sciences, 2017,29 (2): 209-214.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides the application of 4-phenylbutyric acid or the derivative thereof in preparing the product for promoting the development of the in-vitro embryo, and the substance can effectively improve the culture quality and the development capacity of the in-vitro embryo and is beneficial to the culture of the in-vitro embryo.
According to one aspect of the invention, there is provided the use of 4-phenylbutyric acid or a derivative thereof for the preparation of a product for promoting in vitro embryonic development.
According to a preferred embodiment of the invention, there is at least the following advantageous effect: the component can improve the blastula formation rate of embryo during embryo development before implantation, and promote embryo proliferation to increase total cell number of embryo; the composition can also increase endoplasmic reticulum abundance of early embryo and reduce endoplasmic reticulum stress level, thereby protecting endoplasmic reticulum; the composition can also reduce the level of apoptosis related gene CAS3 in early embryo, inhibit excessive apoptosis of embryo, thereby improving in vitro embryo culture quality and development ability, and facilitating in vitro embryo culture.
In some embodiments of the invention, the chemical formula of 4-phenylbutyric acid is as follows:
4-phenylbutyric acid is a chemical substance that can be used in pharmaceutical applications. In recent years, the FDA approved drug 4-phenylbutyric acid (4-PBA) has been increasingly used as an in vitro and in vitro nonspecific chemical partner. Studies have shown that 4-PBA reduces endoplasmic reticulum stress in thalassemia-treated melanoma cell lines, and that 4-PBA treatment significantly reduces liver injury and apoptosis, and can also inhibit hypoxia-induced lipolysis in rat adipose tissue and lipid accumulation in the liver by modulating endoplasmic reticulum stress. However, it is not known whether 4-PBA has an accelerating effect on blastocyst stage embryo development.
In some embodiments of the invention, the promoting in vitro embryo development comprises at least one of improving embryo quality or improving embryo developmental capacity.
In some embodiments of the invention, the increasing embryo developmental capacity, if any, includes decreasing in vitro embryo endoplasmic reticulum stress-related gene GRP78 or decreasing in vitro apoptosis-related gene CAS3 levels.
In some preferred embodiments of the invention, the product comprises at least one of a kit, a medium, or a medicament.
In some more preferred embodiments of the invention, the promoting in vitro embryo development comprises at least one of increasing embryo blastocyst rate, promoting embryo proliferation or increasing endoplasmic reticulum abundance of early embryo, decreasing endoplasmic reticulum stress level, decreasing apoptosis level in early embryo. 4-phenylbutyric acid or derivatives thereof can improve embryo blastula rate, promote embryo proliferation or increase endoplasmic reticulum abundance of early embryo, reduce endoplasmic reticulum stress level, reduce apoptosis related gene CAS3 level in early embryo, thereby effectively improving embryo quality and development ability in vitro.
In some more preferred embodiments of the invention, the quality and viability of in vitro embryos are assessed by detecting/calculating the total cell number of the in vitro embryo. An increase in total cell number in the embryo represents an increase in embryo division levels, thereby reflecting the proliferation capacity of the embryo in vitro.
In some more preferred embodiments of the invention, the quality and developmental capacity of an in vitro embryo is assessed by detecting/calculating the endoplasmic reticulum abundance of the in vitro embryo. The endoplasmic reticulum connects various structures in the cell into a whole and has the function of bearing the transport of substances in the cell, and the main function of the endoplasmic reticulum is to participate in the synthesis, processing, packaging and transport of proteins and lipids. Endoplasmic reticulum plays a central role in the synthesis of macromolecules. All macromolecular substances, including proteins, lipids, and polysaccharide complexes, which are transported to the golgi apparatus, plasma membrane, lysosome, or extracellular space in the future are synthesized in the presence of the endoplasmic reticulum. In certain cell types, the distribution of the endoplasmic reticulum also varies with the degree of differentiation and the functional state of the cell. For example, the rough endoplasmic reticulum in the pancreatic exocrine cells of highly differentiated synthetic secreted proteins is very developed; immature or undifferentiated cells, such as various stem cells, embryonic cells, etc., have a rough endoplasmic reticulum which is less developed than its corresponding mature cells.
In some preferred embodiments of the invention, the quality and developmental capacity of an in vitro embryo is assessed by detecting/calculating an endoplasmic reticulum stress-related marker (GRP 78) of the in vitro embryo, wherein the degree of stress of the in vitro embryo can be assessed by detecting/calculating the relative fluorescence intensity of the in vitro embryo endoplasmic reticulum stress-related marker (GRP 78). GRP78, an intrinsic component in the endoplasmic reticulum, is a binding protein that is uniquely localized on the endoplasmic reticulum and is capable of recognizing the endoplasmic reticulum protein signal sequence, and is widely found in various organisms. There are a number of soluble chaperones and foldases within the lumen of the endoplasmic reticulum of GRP78, with GRP78 being the first chaperone to be discovered. It is a stress protein belonging to a family of heat shock proteins 70 (heat shock protein, hsp70). GRP78 is involved in both regulation of calcium homeostasis and regulation of unfolded protein responses. The relative fluorescence intensity of the GRP78 in the endoplasmic reticulum can be detected using an appropriate fluorescence detection device to evaluate the degree of stress of the embryo by calculating the level of fluorescence intensity.
In some preferred embodiments of the invention, the quality and developmental capacity of in vitro embryos are assessed by detecting/calculating apoptosis-related genes (Caspase-3, CAS 3) of in vitro embryos. Apoptosis is an important process for maintaining cell homeostasis, and excessive apoptosis can disrupt homeostasis of the cell's internal environment, thereby compromising in vitro embryonic development. CAS3 is a member of the cysteine-aspartic protease (caspase) family, and activation of CAS3 causes apoptosis. The relative fluorescence intensity of CAS3 in the embryo cytoplasm can be detected using an appropriate fluorescence detection device, so that the apoptosis level of the embryo can be evaluated by calculating the level of fluorescence intensity.
In some preferred embodiments of the invention, the promoting in vitro embryo development comprises increasing in vitro embryo blastogenesis rate and in vitro embryo total cell count, increasing in vitro embryo endoplasmic reticulum abundance, decreasing in vitro embryo endoplasmic reticulum stress level, and decreasing in vitro embryo apoptosis level.
In some preferred embodiments of the invention, the increase in vitro embryo developmental capacity is an increase in vitro embryo blastogenesis rate. Preferably, administration of the product of the invention results in an increase in vitro embryo blastocyst formation of 21.65% relative to the absence of administration of the product of the invention, as demonstrated by the in vitro embryo blastocyst formation test.
In some preferred embodiments of the invention, the enhancing the in vitro embryo developmental capacity is promoting embryo proliferation, increasing the total number of cells in the in vitro embryo. Preferably, administration of the product of the invention results in an increase in total in vitro embryo cell count of 22.73% relative to the absence of administration of the product of the invention, as demonstrated by the in total in vitro embryo cell count test.
In some preferred embodiments of the invention, the increasing in vitro embryo developmental capacity is increasing in vitro embryo endoplasmic reticulum abundance. Preferably, administration of the product of the invention results in an increase in vitro embryo endoplasmic reticulum abundance of 16.33% relative to the absence of administration of the product of the invention, as demonstrated by the in vitro embryo endoplasmic reticulum abundance test.
In some preferred embodiments of the invention, the increase in vitro embryo developmental capacity is a decrease in vitro embryo endoplasmic reticulum stress related genes (glucose regulatory protein 78, GRP 78). Preferably, administration of the product of the invention results in a 8.31% reduction in the level of the in vitro embryo endoplasmic reticulum stress-related marker (GRP 78) relative to the absence of administration of the product of the invention, as demonstrated by the in vitro embryo endoplasmic reticulum stress-related gene (GRP 78) fluorescence intensity test.
In other preferred embodiments of the invention, administration of the product of the invention results in a 0.92-fold decrease in fluorescence intensity of the endoplasmic reticulum stress-associated gene (GRP 78).
In some preferred embodiments of the invention, the increase in vitro embryo developmental capacity is a decrease in apoptosis levels of in vitro embryos. Preferably, administration of the product of the invention reduces in vitro embryo CAS3 levels by 9.1% relative to the in vitro embryo apoptosis-related gene CAS3 levels tested without the product of the invention.
According to a further aspect of the present invention there is provided the use of 4-phenylbutyric acid or a derivative thereof in the preparation of a product for increasing the rate of embryo blastogenesis in vitro.
According to a further aspect of the present invention there is provided the use of 4-phenylbutyric acid or a derivative thereof in the preparation of a product for increasing the total cell number of an outer embryo. Promote the proliferation of in vitro embryo and increase the total cell number of in vitro embryo.
According to a further aspect of the present invention there is provided the use of 4-phenylbutyric acid or a derivative thereof in the preparation of a product for increasing the abundance of the endoplasmic reticulum of an embryo in vitro.
According to a further aspect of the present invention there is provided the use of 4-phenylbutyric acid or a derivative thereof for the preparation of a product for reducing in vitro embryo endoplasmic reticulum stress.
According to a further aspect of the present invention there is provided the use of 4-phenylbutyric acid or a derivative thereof in the preparation of a product associated with reduced apoptosis in vitro.
In some preferred embodiments of the invention, the product comprises at least one of a kit, a medium, or a medicament.
In the context of a value for improving the developmental capacity effect of an embryo in vitro, the value of "improvement" used is calculated by: [ (measured values for the product of the invention) - (measured values for the product of the invention) v ] (measured values for the product of the invention; the value of "decrease" used is calculated by: [ (measured without application of the product according to the invention) - (measured with application of the product according to the invention) ]/(measured without application of the product according to the invention).
According to a further aspect of the invention, there is also provided a product for promoting in vitro embryo development, the starting material of which comprises 4-phenylbutyric acid or a derivative thereof.
In some embodiments of the invention, the product comprises at least one of a drug, a medium, or a kit.
In some embodiments of the invention, the embryo comprises a mammalian in vitro embryo. Preferably, the mammal includes, but is not limited to, humans, domesticated animals, farm animals, zoo animals, sports animals, pet animals (e.g., dogs, cats, guinea pigs, rabbits, rats, mice, horses, camels, bison, cows), primates (e.g., apes, monkeys, gorillas, and chimpanzees), canines (e.g., dogs and wolves), felines (e.g., cats, lions, and tigers), equines (e.g., horses, donkeys, and zebras), eating animals (e.g., cows, pigs, and sheep), ungulates (e.g., deer and giraffes), and rodents (e.g., mice, rats, hamsters, and guinea pigs).
In some embodiments of the invention, the mammal comprises a pig.
In some embodiments of the invention, the mammalian in vitro embryo comprises a mammalian in vitro parthenogenesis activated embryo, a mammalian in vitro fertilization embryo. Preferably, the mammalian in vitro embryo comprises a parthenogenetic activated oocyte, a fertilized egg cell, and a 2-cell, 4-cell, 8-cell, 16-cell, morula and blastocyst developed from the parthenogenetic activated oocyte and/or fertilized egg cell.
In some embodiments of the invention, the medicament further comprises a pharmaceutically acceptable excipient. Preferably, the auxiliary materials comprise pharmaceutically acceptable diluents, excipients, carriers, binders, lubricants, suspending agents, coating agents and solubilizers. The drug may be in a variety of forms, such as liquid, semi-solid, and solid dosage forms. In other embodiments of the invention, the pharmaceutical forms include liquid solutions (e.g., injectable and infusible solutions), sprays, dispersions, or suspensions. In some embodiments of the invention, the drug is a liquid solution.
Preferably, the culture medium further comprises basal medium, solvent, antibiotics, nutritional functional factors that facilitate in vitro embryonic cell development.
Preferably, the basal medium comprises TCM-199 medium, PZM-5 medium, CR1-aa medium, ham' S F medium, NCSU-37 medium, NCSU-23 medium.
Preferably, the inorganic salt includes sodium chloride, potassium dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, sodium bicarbonate.
Preferably, the antibiotics include gentamicin, penicillin, streptomycin, and the like.
Preferably, the nutritional functional factors that facilitate in vitro embryonic cell development include hormones, energy substrates, amino acids, growth factors.
In some embodiments of the invention, the hormone comprises Follicle Stimulating Hormone (FSH), luteinizing Hormone (LH), human chorionic gonadotrophin (hCG), gestagen serum gonadotrophin (PMSG), estradiol (E 2 )。
In some embodiments of the invention, the energy substrate comprises glucose, pyruvate, lactate.
In some embodiments of the invention, the amino acid comprises an essential amino acid, an optional amino acid. According to some embodiments of the invention, the amino acid comprises L-cysteine, L-isoleucine, L-leucine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-arginine hydrochloride, L-cystine dihydrochloride, L-histidine hydrochloride monohydrate, L-lysine hydrochloride.
In some embodiments of the invention, the growth factor comprises Epidermal Growth Factor (EGF), insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF), nerve Growth Factor (NGF).
According to a further aspect of the invention there is also provided the use of 4-phenylbutyric acid or a derivative thereof for the promotion of embryo development in vitro.
According to a further aspect of the invention, there is also provided the use of 4-phenylbutyric acid or a derivative thereof for the promotion of the quality of in vitro embryo development.
According to a further aspect of the invention there is also provided the use of 4-phenylbutyric acid or a derivative thereof for the promotion of in vitro embryo development.
According to a further aspect of the invention, there is also provided the use of 4-phenylbutyric acid or a derivative thereof for increasing the blastocyst rate of an embryo in vitro.
According to a further aspect of the invention, there is also provided the use of 4-phenylbutyric acid or a derivative thereof for increasing the total cell number of an outer embryo.
According to a further aspect of the invention, there is also provided the use of 4-phenylbutyric acid or a derivative thereof for increasing the abundance of the endoplasmic reticulum of an embryo in vitro.
According to a further aspect of the invention, there is also provided the use of 4-phenylbutyric acid or a derivative thereof for decreasing in vitro embryo endoplasmic reticulum stress.
According to a further aspect of the invention there is also provided the use of 4-phenylbutyric acid or a derivative thereof for reducing apoptosis in an embryo in vitro.
Preferably, the embryo comprises a mammalian in vitro embryo. Preferably, the in vitro embryo comprises a mammalian in vitro parthenogenesis activated embryo. More preferably, the in vitro embryo comprises a pig in vitro parthenogenesis activated embryo. More preferably, the in vitro embryo comprises a pig in vitro parthenogenesis activated oocyte.
According to yet another aspect of the present invention, there is also presented a method for improving the ability of an embryo to develop in vitro, comprising the steps of: 4-phenylbutyric acid or a derivative thereof is administered to an in vitro embryo.
The method may be used for non-therapeutic purposes. In particular, the method can be used for scientific research, such as in vitro cell culture research, in vitro embryo culture research, pre-implantation embryo development mechanism research, and the like.
According to a further aspect of the present invention there is also presented a method for promoting porcine embryo development in vitro comprising the steps of: 4-phenylbutyric acid or a derivative thereof is administered to an in vitro embryo.
In some embodiments of the invention, the method comprises the steps of:
s1, applying 4-phenylbutyric acid or a derivative thereof to an in vitro embryo;
s2, culturing the in-vitro embryo until the blastula stage.
Preferably, 4-phenylbutyric acid or a derivative thereof may be administered in the form of a kit, a medium or a pharmaceutical product.
Preferably, 4-phenylbutyric acid or a derivative thereof in S1 may be applied at any point during the in vitro embryo development to the blastocyst stage, including after parthenogenesis of the oocyte, after fertilization of the sperm egg, and at any point during the development to the 2-cell, 4-cell, 8-cell, 16-cell, morula, blastocyst stage.
Preferably, the 4-phenylbutyric acid or derivative thereof in S1 may be administered once, but more preferably a plurality of times. In some embodiments of the invention, 4-phenylbutyric acid or a derivative thereof may be administered from once daily to once weekly. Specifically, 4-phenylbutyric acid or a derivative thereof may be administered once daily, once every two days, once every three days, once every four days, once every five days, once every six days, once weekly.
Preferably, the effective amount of 4-phenylbutyric acid or derivative thereof in S1 is 1 to 100. Mu. Mol/L, more preferably 60 to 100. Mu. Mol/L, most preferably 75. Mu. Mol/L. The term "effective amount" as used in the present specification and in the claims means an amount of a compound sufficient to provide the desired effect, relative to the mass of 4-phenylbutyric acid applied and the volume of the liquid environment (e.g., medium) applied, to which the compound is administered, as compared to the case where the compound is not administered; the expected effect comprises obviously improving the formation rate of embryo blastula in vitro, and/or obviously promoting the proliferation of embryo in vitro so as to increase the total cell number of embryo in vitro, improve the abundance of endoplasmic reticulum of embryo in vitro, obviously reduce the stress level of endoplasmic reticulum of embryo in vitro, and/or obviously reduce the apoptosis level of embryo in vitro. Significantly increasing the blastocyst formation of an in vitro embryo means that the blastocyst formation of an in vitro embryo is increased by at least 21.65% compared to the situation without administration of the compound. Significantly promoting embryo proliferation in vitro means that the total number of cells in vitro is increased by at least 22.73% compared to the case without the compound. Significantly increasing the in vitro embryo endoplasmic reticulum abundance means that the in vitro embryo endoplasmic reticulum abundance is increased by at least 16.33% compared to the case without administration of the compound. Significantly reducing the endoplasmic reticulum stress capacity of an in vitro embryo means that the fluorescence intensity of the in vitro embryo endoplasmic reticulum stress-related gene (GRP 78) is reduced by at least 8.31% compared to the case without administration of the compound. Significantly reducing the level of apoptosis in an in vitro embryo means that the fluorescence intensity of the apoptosis-related gene (CAS 3) in vitro is reduced by at least 8.31% compared to the case without administration of the compound.
Preferably, the cultivation temperature in S2 is 36-40℃and the cultivation time is 40-50 hours.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a graph showing the effect of in vitro embryo blastogenesis in the culture media of experimental group (4-PBA) and control group (Con) according to example 2 of the present invention (A) and a graph showing the statistical result of the formation rate (B); data are expressed as mean ± standard deviation; n=4 independent parallel experiments; the significance difference is expressed as: * P <0.05.
FIG. 2 is a graph (A) showing quantitative detection results of the functions of the endoplasmic reticulum of in vitro embryos in culture medium of experimental group (4-PBA) and control group (Con) and a graph (B) showing statistical results of the abundance of the endoplasmic reticulum of the in vitro embryos according to the embodiment 3 of the invention; data are expressed as mean ± standard deviation; n=3 independent parallel experiments; the significance difference is expressed as: * P <0.001.
FIG. 3 is a graph (A) and a graph (B) of the results of in vitro embryo total cell count detection in culture medium of experimental group (4-PBA) and control group (Con) according to example 4 of the present invention; data are expressed as mean ± standard deviation; n=3 independent parallel experiments; the significance difference is expressed as: * P <0.05.
FIG. 4 is a graph (A) showing the results of in vitro embryo endoplasmic reticulum stress function detection in the culture medium of experimental group (4-PBA) and control group (Con) and a graph (B) showing the statistical results of endoplasmic reticulum stress marker GRP78 relative to fluorescence intensity according to the embodiment 4 of the present invention; data are expressed as mean ± standard deviation; n=3 independent parallel experiments; the significance difference is expressed as: * P <0.05.
FIG. 5 is a graph (A) showing the results of in vitro apoptosis level detection in culture medium of experimental group (4-PBA) and control group (Con) and a graph (B) showing the quantitative statistical results of apoptosis-related gene CAS3 of embryo according to example 5 of the present invention; data are expressed as mean ± standard deviation; n=3 independent parallel experiments; the significance difference is expressed as: * P <0.05.
Data statistics: the data in the above figures were all statistically analyzed using SPSS software and student t-test was used to compare the data between the two groups.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention. The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available. Unless otherwise indicated, the same parameter is the same in each embodiment. The following examples are illustrative only and are not to be construed as limiting the invention.
In the description of the present invention, the descriptions of the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The term "room temperature" as used herein means a temperature of 25.+ -. 5 ℃ and in the examples 25 ℃.
Example 1
This example is the use of 4-phenylbutyric acid in the preparation of a product for promoting in vitro embryonic development. The method specifically comprises the following steps: 4-phenylbutyric acid was added to the porcine embryo in vitro medium in an effective amount of 75. Mu. Mol/L (. Mu.M).
The method for using the culture medium to promote the development of porcine oocyte embryos in vitro comprises the following steps:
1. collection of pig oocytes:
ovaries from prepubertal gilts were collected from the slaughterhouse, placed in 0.9% physiological saline at 37 ℃ and transported to the laboratory. The cumulus-oocyte complexes (COCs) and follicular fluid in 3-8mm diameter follicles in the ovaries were aspirated with a syringe, placed in 15ml shaking tubes, and allowed to stand in a water bath at 38 ℃ for 15min until COCs precipitated to the bottom of the tubes, and the supernatant was aspirated. The precipitate was resuspended in an appropriate amount of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer (SIGMA, H6147) containing 0.1% (w/v) polyvinyl alcohol (PVA, SIGMA, P8136) and 0.05g/L gentamicin (SIGMA, E003632), and the supernatant was aspirated after 15min, and the procedure was repeated three times. Transferring the COCs precipitate to a culture dish, adding a proper amount of HEPES buffer solution, and selecting COCs with uniform cytoplasm of oocytes and three or more layers of dense cumulus cells around the oocytes under a split microscope by using a glass pipette.
2. In vitro maturation of porcine oocytes:
washing the selected COCs three times by using a maturation medium IVM; the maturation medium IVM was: to TCM-199 (GIBCO, 11150-059) were added 0.1g/L sodium pyruvate (SGMA, P4562), 0.6mmol/L L-cysteine (SIGMA, C7477), 10ng/mL epidermal growth factor (SIGMA, E4127), 10% porcine follicular fluid, 10IU/mL luteinizing hormone (Ningbo second hormone works), 10IU/mL follicle stimulating hormone (Ningbo second hormone works). 500. Mu.L of maturation medium IVM was added to a 4-well dish, the medium was covered with 500. Mu.L of mineral oil, and then the washed three times of COC were placed in the maturation medium IVM. The 4-well plates were placed at 38.5℃with 5% CO 2 Culturing in a humidity saturated incubator for about 45h (44-46 h, and maturing oocytes after culturing to achieve MII stage).
3. Parthenogenesis activation of early porcine embryos:
and transferring COCs reaching the MII stage into 0.1% hyaluronidase by a pipette, repeatedly blowing for about 30 times, and removing cumulus cells around the oocyte to obtain the naked oocyte. The naked oocyte was transferred to a 297mmol/L mannitol (pH 7.2, SIGMA, M9546) solution containing 0.1mmol/L CaCl 2 (SIGMA，C7902)、0.05mmol/L MgSO 4 (SIGMA, M1880), 0.01% (w/v) PVA, 0.5mmol/L HEPES. The parthenogenetic activation of the naked oocyte was carried out by a total of two direct current pulses with an electric field strength of 1.2kV/cm of 60. Mu.S each time.
4. Applying 4-phenylbutyrate to pig in vitro parthenogenetic embryos and in vitro embryo culture:
1) The parthenoestrus activated early embryo was cultured in porcine embryo in vitro medium (PZM-5 medium containing 7.5mg/mL cytochalasin B) for 3-5h. Wherein, the PZM-5 culture medium consists of the following components: 108mmol/L (mM) sodium chloride (sigma, S5886), 10mM potassium chloride (sigma, P5405), 0.35mM potassium dihydrogen phosphate (sigma, P5655), 0.4mM magnesium sulfate (sigma, M2643), 25mM sodium bicarbonate (sigma, S5761), 0.2mM sodium pyruvate (sigma, P5280), 2.0mM calcium lactate (sigma, C8356), 2.0mM glutathione (sigma, G8540), 5.0mM hypotaurine (sigma, H1384), 20mL/L essential amino acids (sigma, B6766), 10mL/L non-essential amino acids (sigma, M7145), 25mg/mL gentamycin (sigma, B6766), 4mg/mL bovine serum albumin (sigma, A8806), and adding ultrapure water to a certain volume, and adjusting the pH to 7.2-7.4.
2) Removing the culture medium, applying pig embryo in vitro culture medium (PZM-5 culture medium) containing 4-phenylbutyric acid to parthenoelectrically activated early embryo, and setting PZM-5 culture medium containing 75 μmol/L (μM) 4-phenylbutyric acid in experimental group; and 1 control group, PZM-5 medium containing no 4-phenylbutyrate was applied to parthenogenetically activated early embryos.
3) Early embryo was placed at 38.5℃with 5% CO 2 Culturing in a humidity saturated incubator for 3-7 days without changing the culture medium.
After the culture is completed, the embryo development capability and quality of the experimental group culture are found to be obviously superior to those of the control group.
Example 2
The embodiment is the application of 4-phenylbutyric acid in preparing the product for improving the embryo blastocyst rate in vitro. The method specifically comprises the following steps: 4-phenylbutyric acid was added to the porcine embryo in vitro medium (PZM-5 medium) in an effective amount of 75. Mu. Mol/L (. Mu.M).
The medium was used for pig in vitro embryo culture by the same procedure as in example 1, and the quality and development ability of in vitro embryos were evaluated by detecting and calculating the blastocyst rate in the culture medium of the experimental group and the control group at day 7, and the results are shown in fig. 1. Wherein, blastula ratio= (number of blastula formed/number of all early parthenogenetic embryos cultured) ×100%.
As can be seen from FIG. 1, the addition of 75. Mu. Mol/L (. Mu.M) of 4-phenylbutyric acid to the in vitro culture medium (PZM-5 medium) of porcine embryos significantly increased the blastocyst formation rate of early parthenogenetic embryos of pigs relative to the control group, which had a blastocyst formation rate of 40.305%; the formation rate of blastula added with 4-phenylbutyrate was 49.03%, which is significantly higher than that of the control group (P < 0.05).
The addition of 4-phenylbutyric acid at an effective concentration (75. Mu.M) to the medium helps promote smooth development of parthenogenesis-activated early embryos to blastula. The activation of oocyte to develop smoothly to blastocyst is the key of in vitro embryo culture, and only early embryo with good development capacity and development quality can develop to blastocyst, and then embryo implantation rate can be improved by using blastocyst for in vivo transplantation. Therefore, the 4-phenylbutyric acid improves the development capacity and the development quality of the embryo at the early stage before implantation, lays a good foundation for embryo implantation, and simultaneously shows that the 4-phenylbutyric acid has good application prospect in preparing the product for improving the formation rate of embryo blastula in vitro.
Example 3
The embodiment is the application of 4-phenylbutyric acid in preparing a product for improving the abundance of an embryo endoplasmic reticulum in vitro. The method specifically comprises the following steps: 4-phenylbutyric acid was added to the porcine embryo in vitro medium (PZM-5 medium) in an effective amount of 75. Mu. Mol/L (. Mu.M).
The medium was used in pig in vitro embryo culture by the same procedure as in example 1, and quality and development ability of in vitro embryos were evaluated by detecting endoplasmic reticulum abundance of 4-cells of early parthenogenetic embryos of pigs. Specifically, an endoplasmic reticulum Red fluorescent probe (ER-Tracker Red) is adopted to detect the endoplasmic reticulum abundance of the 4-cell of the pig early parthenogenetic embryo:
early embryos of experimental group (PZM-5 medium supplemented with 4-phenylbutyrate) and control group (PZM-5 medium without 4-phenylbutyrate) were cultured in vitro to 4-cell stage of early parthenogenetic embryos of pigs, the 4-cell embryos were washed 5 times with PBS buffer, and placed at 38deg.C in 5% CO in medium containing 1. Mu. Mol/L (μM) endoplasmic reticulum Red fluorescent probe (ER-Tracker Red) 2 Is incubated for 1h in an incubator. Images were taken using a fluorescence microscope (Nikon, tokyo, japan). Images were analyzed using Image J software and endoplasmic reticulum abundance was calculated as red fluorescence intensity. The fluorescence intensity of the control group was set to 1, and the relative fluorescence intensity of the treatment group with respect to the control group was calculated.
As shown in fig. 2, endoplasmic reticulum abundance was measured by endoplasmic reticulum staining of 4-cell stage embryos, and endoplasmic reticulum abundance of 4-phenylbutyrate treated group (75 μm) was significantly higher than that of control group (P < 0.001), specifically 1.16-fold higher than that of control group by 16.33%; the main function of the endoplasmic reticulum is to participate in the synthesis, processing, packaging and transport of proteins and lipids. The distribution of the endoplasmic reticulum also varies depending on the degree of cell differentiation and the functional state. Such as the well-differentiated synthetic secretin, the rough endoplasmic reticulum is well developed in pancreatic exocrine cells; immature or undifferentiated cells, such as various stem cells, embryonic cells, etc., have a rough endoplasmic reticulum which is less developed than its corresponding mature cells. Therefore, the results show that 75 mu M of 4-phenylbutyric acid can effectively improve the abundance of endoplasmic reticulum and the function of endoplasmic reticulum, thereby improving the development quality and development capacity of early embryo. Meanwhile, the 4-phenylbutyric acid has good application prospect in preparing the product for improving the abundance of the endoplasmic reticulum of the embryo in vitro.
Example 4
This example is the use of 4-phenylbutyric acid in the preparation of a product for reducing the ability of the endoplasmic reticulum of an embryo to stress in vitro. The method specifically comprises the following steps: 4-phenylbutyric acid was added to the porcine embryo in vitro medium (PZM-5 medium) in an effective amount of 75. Mu. Mol/L (. Mu.M).
The medium was used in pig in vitro embryo culture by the same procedure as in example 1, and quality and development ability of in vitro embryos were evaluated by detecting endoplasmic reticulum abundance of early parthenogenetic embryos of pigs. Specifically, total cell number of pig in vitro parthenogenetic embryo and fluorescence intensity of GRP78 were detected using Anti-GRP78 antibody (Abcam, ab 21685):
early embryos of experimental group (PZM-5 medium supplemented with 4-phenylbutyrate) and control group (PZM-5 medium without 4-phenylbutyrate) were cultured in vitro to day 7, swine parthenogenetic activated in vitro embryos were fixed with 3.7% paraformaldehyde for 30min, permeabilized with 0.3% Triton X-100 for 30min, and then blocked with 3% BSA for 1h at 37 ℃. After 5 washes with PBS-PVA, porcine in vitro embryos were incubated with rabbit anti-GRP78 primary antibody additive solution overnight at 4℃in the refrigerator protected from light. The next day, the sample was taken out, washed 5 times with PBS-PVA, and then put into goat anti-rabbit secondary antibody (Abcam, ab 150077) and incubated for 1h at normal temperature in a dark place. After 1h, the porcine in vitro embryo was incubated with 10. Mu.g/mL DNA fluorescent dye Hoechst 33342 for 8min at room temperature in the absence of light for labeling the nuclei, washed 5 times with PBS-PVA. Images were taken using a fluorescence microscope (Nikon, tokyo, japan). Images were analyzed using Image J software, and the fluorescence intensity and total cell count of GRP78 of porcine in vitro embryos were counted, and the lower the fluorescence, the lower the endoplasmic reticulum stress was indicated.
The results are shown in figures 3 and 4, wherein figure 3 shows that the total number of embryo cells is significantly higher in the 4-phenylbutyrate treated group (75 μm) than in the control group (P < 0.05), wherein the control group is 41.83, the treated group is 51.34, and the treated group is 22.73% higher than the control group; FIG. 4 shows that GRP78 relative fluorescence intensity was significantly lower in the 4-phenylbutyrate treated group (75. Mu.M) than in the control group (P < 0.05), where the control group was set to 1 and the treated group was 0.9169, and the treated group was reduced by 8.31% relative to the control group. The results show that the 4-phenylbutyric acid can effectively reduce the endoplasmic reticulum stress function of the pig parthenogenesis in-vitro embryo, thereby improving the development quality and the development capacity of the in-vitro embryo. Meanwhile, the 4-phenylbutyric acid has good application prospect in preparing products for improving the proliferation capacity of in-vitro embryos.
Example 5
This example is the use of 4-phenylbutyric acid in the preparation of a product for reducing the level of apoptosis in an embryo in vitro. The method specifically comprises the following steps: 4-phenylbutyric acid was added to the porcine embryo in vitro medium (PZM-5 medium) in an effective amount of 75. Mu. Mol/L (. Mu.M).
The medium was used in pig in vitro embryo culture by the same procedure as in example 1, and the quality and development ability of in vitro embryos were evaluated by detecting the fluorescence intensity of the apoptosis-related gene CAS3 of early parthenogenetic embryos of pigs. Specifically, pig in vitro parthenogenetic embryo apoptosis-related gene CAS3 fluorescence intensity was detected using Anti-CAS3 antibody (Sigma-Aldrich, cat# C8487-100 ul):
early embryos of experimental group (PZM-5 medium supplemented with 4-phenylbutyrate) and control group (PZM-5 medium without 4-phenylbutyrate) were cultured in vitro to day 7, swine parthenogenetic activated in vitro embryos were fixed with 3.7% paraformaldehyde for 30min, permeabilized with 0.3% Triton X-100 for 30min, and then blocked with 3% BSA for 1h at 37 ℃. After washing 5 times with PBS-PVA, the pig in vitro embryos were incubated with rabbit anti-CAS3 primary antibody additive solution at 4℃overnight in the absence of light. The next day, the sample was taken out, washed 5 times with PBS-PVA, and then put into goat anti-rabbit secondary antibody (Abcam, ab 150077) and incubated for 1h at normal temperature in a dark place. After 1h, the porcine in vitro embryo was incubated with 10. Mu.g/mL DNA fluorescent dye Hoechst 33342 for 8min at room temperature in the absence of light for labeling the nuclei, washed 5 times with PBS-PVA. Images were taken using a fluorescence microscope (Nikon, tokyo, japan). The Image was analyzed by Image J software, and the fluorescence intensity and total cell number of apoptosis-related gene CAS3 of porcine in vitro embryos were counted, and the lower the fluorescence, the lower the apoptosis level of the embryos was indicated.
The results are shown in FIG. 5, which shows that the relative fluorescence intensity of apoptosis-related gene CAS3 was significantly lower in the 4-phenylbutyrate-treated group (75. Mu.M) than in the control group (P < 0.05), wherein the control group was set to 1, the treated group was set to 0.909, and the treated group was reduced by 9.1% relative to the control group. The results show that the 4-phenylbutyric acid can effectively reduce apoptosis level of pig parthenogenesis activated in-vitro embryos, thereby improving development quality and development capacity of the in-vitro embryos.
Example 6
The embodiment provides an in vitro embryo culture medium which comprises 75 mu M of 4-phenylbutyric acid, a basal culture medium, inorganic salts, solvents, antibiotics and nutritional functional factors which are beneficial to in vitro embryo development.
The substitution of 4-phenylbutyric acid in the above examples with its derivatives also has the effect of promoting in vitro embryo development.
In conclusion, the invention proves that the 4-phenylbutyric acid can improve the blastula formation rate of in vitro embryo culture, promote the proliferation of in vitro embryos, increase the total cell number of the embryos, protect the endoplasmic reticulum function of the in vitro embryos, reduce the endoplasmic reticulum stress level of the in vitro embryos, and reduce the apoptosis level of the embryos, thereby effectively improving the quality and the development capability of the in vitro embryos. 4-phenylbutyric acid has good market application prospect in preparing products for improving the quality and the development ability of in-vitro embryos.
The invention discloses the application of 4-phenylbutyric acid or a derivative thereof in preparing products for improving in-vitro embryo development for the first time, and the application range of the 4-phenylbutyric acid or the derivative thereof is widened because the 4-phenylbutyric acid or the derivative thereof is not reported to be related to the in-vitro embryo development capability. 4-phenylbutyric acid or derivatives thereof can promote embryo proliferation or increase endoplasmic reticulum abundance of early embryo, reduce endoplasmic reticulum stress level, reduce apoptosis related gene CAS3 level in early embryo by improving blastula rate, thereby effectively improving development quality and development ability of in vitro embryo. 4-phenylbutyric acid or derivatives thereof have no toxic or side effect on mammalian cells, wide sources and easy acquisition, and the addition of 4-phenylbutyric acid or derivatives thereof into a culture medium has simple operation, easy control and remarkable effect. The 4-phenylbutyric acid or the derivative thereof can be used for preparing a pharmaceutical composition, a culture medium or a kit for promoting the development of in vitro embryos, and has good industrial application prospect.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.
Claims (10)
- Use of 4-phenylbutyric acid or a derivative thereof for the preparation of a product for promoting in vitro embryonic development.
- 2. The use according to claim 1, characterized in that: the promoting in vitro embryo development includes at least one of improving embryo quality or improving embryo developmental capacity.
- 3. The use according to claim 2, characterized in that: if included, the enhancing embryonic development capacity includes decreasing in vitro embryo endoplasmic reticulum stress-related gene GRP78 or decreasing in vitro apoptosis-related gene CAS3 levels.
- 4. A use according to any one of claims 1 to 3, characterized in that: the product is a kit, a culture medium or a medicament.
- Use of 4-phenylbutyric acid or a derivative thereof in the preparation of a product, characterized in that: the product has at least one of the following functions:(1) Improving embryo blastula rate;(2) Increasing the total cell number of the outer embryo;(3) Increasing endoplasmic reticulum abundance of early embryo;(4) Reducing the stress level of the endoplasmic reticulum;(5) Reduce the apoptosis level in early embryo.
- 6. A product for promoting embryo development in vitro, characterized in that: the starting material of the product comprises 4-phenylbutyric acid or a derivative thereof.
- 7. The product of claim 6, wherein: the product is a kit, a culture medium or a medicament.
- 8. A method for improving the developmental capacity of an embryo in vitro, comprising: the method comprises the following steps: 4-phenylbutyric acid or a derivative thereof is administered to an in vitro embryo.
- 9. A method for promoting porcine embryo development in vitro, which is characterized in that: the method comprises the following steps: 4-phenylbutyric acid or a derivative thereof is administered to an in vitro embryo.
- 10. The method according to claim 9, wherein: the method comprises the following steps:s1, applying 4-phenylbutyric acid or a derivative thereof to an in vitro embryo;s2, culturing the in-vitro embryo until the blastula stage.
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