CN111019885A - Pig endometrium-derived exosome and extraction method and application thereof - Google Patents
Pig endometrium-derived exosome and extraction method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a porcine endometrium-derived exosome, and an extraction method and application thereof. The invention firstly separates to obtain endometrial tissue, and then cuts the endometrial tissue into tissue blocks; centrifuging for 3-10 min at the temperature of 20-30 ℃ and the weight of 80-100 g after washing, and removing supernatant; then placing the tissue blocks in Hanks' Balanced Salt Solution for incubation, and collecting a pig endometrium culture Solution; and finally, extracting the exosome by an ultracentrifugation method to obtain the exosome derived from the porcine endometrium. Compared with the prior art, the method provided by the invention is simpler and more convenient, saves the sample collection cost, reduces the labor loss, saves the sampling time and increases the sampling efficiency. The shape of the extracted exosome is more obvious under an electron microscope, the background is clean and free of impurities, and the exosome can be used as an additive for promoting the in-vitro culture efficiency of pig embryos.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a porcine endometrium-derived exosome, and an extraction method and application thereof.
Background
The endometrium is a complex dynamic tissue that undergoes periodic remodeling and differentiation throughout the female reproductive life. Successful conception requires the cooperation of a high quality embryo and an endometrium with good receptivity, and the success of implantation of the embryo into the endometrium determines the result of the pregnancy. During embryo implantation, cross-talk between the endometrial epithelium and the blastocyst, especially trophoblasts, is a prerequisite for successful implantation. During this crosstalk, various molecular and functional changes occur to promote synchronization between the embryo and the endometrium and uterine cavity microenvironment. In particular, the trophectoderm is modulated to achieve attachment of the endometrium. Therefore, intercellular communication between the trophectoderm and the endometrial epithelium is critical for the establishment of embryo implantation and pregnancy.
The exosome is a double-layer membrane vesicle with the diameter of 30-150 nm, can carry substances such as protein, miRNA, mRNA, lncRNA and circRNA, can be selectively fused with target cells, transports the carried genetic substances to the target cells to play a role, and remotely plays a role in intercellular signal transmission, so that the exosome is considered to be a novel intercellular signal communication mode. Research shows that some proteins contained in exosomes help exosomes stimulate immune cells to eliminate diseased cells (Wujinen, Dingwar. exosomes biological function and application research progress. animal medicine progress. 2016; 37(12): 90-4.); exosomes can carry RNAs from secretory cells to target cells and function within the target cells, and mRNA can be converted to a functional protein after RNAs reach recipient cells through exosomes; miRNA can degrade mRNA or inhibit the translation process of mRNA, so that the expression of protein is regulated and controlled (Kudzuvine, extraction and identification of cow uterine fluid exosome in estrus and influence thereof on in vitro fertilization embryo development [ Master ]: northwest university of agriculture and forestry science and technology; 2017.); thakur (Thakur BK, Zhang H, Becker A, Matei I, Huang Y, Costa-Silva B, et al. double-stranded DNA exosomes: a novel biomarker in cancer detection. cell research. 2014; 24(6):766-9.) et al, when studying exosomes derived from pancreatic cancer, also found that they contained stable double-stranded DNA structures and suggested that they might be potential biomarkers for cancer diagnosis. Studies by Testa et al (Testa JS, Apcher GS, Comber JD, Eisenlohr LC. isomer-driven anti-gene transfer for MHC class II presented with efficacy by the receptor binding activity of antibiotic in. journal of immunology (baseball: 1950). 2010; 185(11):6608-16.) indicate that exosomes can carry some functional antigenic peptide complexes to mediate antiviral immunity and thereby reduce host susceptibility to infection. Animal experiments show that the clinical symptoms of collagen-induced arthritic mice are reduced and delayed hypersensitivity reactions in the mice are inhibited (Kim SH, Bianco NR, Shufesky WJ, Morelli AE, Robbins PD. effective treatment of inflammatory diseases with surfaces derived from dendritic cells modified to express IL-4.Journal of immunology (Md: 1950). 2007; 179(4): 2242-9.).
It was found that exosomes are present in the preparations of uterine fluid/mucus and endometrial epithelial cells. The pregnant uterus not only contains exosomes derived from a mother body, but also contains exosomes derived from an embryo, in maternal-fetal exchange, substances carried by the exosomes derived from the mother body and the exosomes derived from the embryo are exchanged, and the exosomes can be orderly expressed in time and space in the whole pregnancy process. Therefore, in the process of embryo implantation, the maternal uterine cavity fluid exosome can mutually transmit information (including RNAs, protein and lipid molecules) between endometrial cells and embryonic cells, thereby playing the function of maternal-fetal conversation and having important influence on the success rate of embryo implantation.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention mainly aims to provide an extraction method of exosomes derived from pig endometrium.
The invention also aims to provide the porcine endometrium-derived exosome extracted by the extraction method.
The invention further aims to provide application of the porcine endometrium-derived exosome.
The purpose of the invention is realized by the following technical scheme:
a method for extracting exosomes from pig endometrium comprises the following steps:
(1) taking out a uterus after a pig is slaughtered, fastening two sides of a uterine horn to be taken to ensure that the uterine cavity is sealed, cutting the uterine horn along a longitudinal axis after cleaning, and exposing the uterine cavity; separating endometrial tissue from the myometrium and shearing the endometrial tissue into tissue pieces;
(2) washing the tissue blocks cut up in the step (1) by using sterile DPBS containing 1% (v/v) of isotatic-antimycotic, 0.002mg/ml of amphotericin B and 0.05mg/ml of gentamicin to remove impurities, centrifuging for 3-10 min at the temperature of 20-30 ℃ and under the condition of 80-100 g, and removing supernatant;
(3) placing the tissue block centrifuged and supernatant removed in step (2) in Hanks' blannced Salt Solution at 37 ℃ and 5% CO2Incubating for 20-28 h under the condition of (1), and collecting a pig endometrium culture solution;
(4) extracting exosomes from the porcine endometrium culture solution collected in the step (3) by an ultracentrifugation method to obtain exosomes from the porcine endometrium;
the size of the tissue block in the step (1) is preferably 1-5 mm3Size;
the specific operation of the washing in the step (1) is preferably:
cleaning 2-3 times by using alcohol with the volume fraction of 75%, then washing 2-3 times by using physiological saline containing 1% (v/v) of acetylic-antimycotic, 0.002mg/ml of amphotericin B and 0.05mg/ml of gentamicin, and finally washing 1-2 times by using sterile DPBS containing 1% (v/v) of acetylic-antimycotic, 0.002mg/ml of amphotericin B and 0.05mg/ml of gentamicin; through the cleaning, the purposes of sterilizing, disinfecting and cleaning the uterus, removing impurities and preventing pollution are achieved;
the centrifugation condition in the step (2) is preferably 25 ℃ and centrifugation for 5min under the condition of 90 g;
the incubation time in the step (3) is preferably 24 h;
the specific operation of the ultracentrifugation method described in step (4) is preferably:
① centrifuging the collected endometrium culture solution at 4 deg.C and 2000g for 20min, and collecting supernatant;
② centrifuging the supernatant fluid collected in step ① at 4 deg.C under 10000g for 30min, and collecting the supernatant fluid;
③ centrifuging the supernatant obtained in step ② at 4 deg.C and 120000g for 2h, discarding the supernatant, and collecting the precipitate;
④ resuspending the precipitate collected in step ③ with DPBS, centrifuging at 4 deg.C and 120000g for 2h, and discarding the supernatant to obtain total exosomes derived from endometrium;
an exosome derived from porcine endometrium is extracted by the extraction method;
the application of the pig endometrium-derived exosome in preparing an embryo in-vitro culture additive;
the embryo in vitro culture additive is preferably a product for promoting the proliferation of embryo trophoblast cells;
the application preferably comprises the following steps:
inoculating the embryo trophoblast cells into a culture medium containing the pig endometrium-derived exosomes to culture for 24-48 h;
the concentration of the pig endometrium-derived exosome in the culture medium is preferably more than 0 mug/mL and not more than 50 mug/mL;
compared with the prior art, the invention has the following advantages and effects:
(1) the invention provides an extraction method of exosomes from pig endometrium, compared with the existing method, the method provided by the invention is simpler and more convenient, and the existing method is optimized and innovated; and the cost of sample collection is saved, the manpower loss is reduced, the sampling time is saved, and the sampling efficiency is improved.
(2) Compared with an enzymatic hydrolysis method, the method selects Hanks' Balanced Salt Solution to process the cut tissue blocks, the shape of the extracted exosomes under an electron microscope is more obvious, the background is clean and free of impurities, and meanwhile, a thought is provided for extracting the exosomes from other tissues.
(2) The invention discovers for the first time that the exosome derived from the pig endometrium can promote the proliferation of the embryo trophoblast cells and can be used as an additive for promoting the in-vitro culture efficiency of pig embryos.
Drawings
FIG. 1 is a transmission electron micrograph of exosomes extracted from minced endometrial tissue pieces treated by enzymatic method (A) and Hanks' Balanced Salt Solution (B).
FIG. 2 is a graph of particle size analysis of the exosomes extracted in the present invention.
FIG. 3 shows the detection results of Western blot expressed by proteins CD63 and CD9 extracted from exosomes according to the present invention.
FIG. 4 is a graph showing the results of uterine-derived exosomes affecting proliferation of porcine embryonic trophoblast cells, with significant differences indicated by different lettering, showing that endometrial-derived exosomes promote proliferation of embryonic trophoblast cells.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Hanks' Balanced Salt Solution was purchased from Gibco, and the other components were all commercially available.
Example 1
(1) Preparation of endometrial tissue blocks
① taking out uterus after slaughtering pig, fastening on two sides of uterine horn to ensure uterine cavity sealing, cleaning with 75% alcohol for 3 times, washing with physiological saline containing 1% (v/v) acetylic-antimycotic, 0.002mg/ml amphotericin B and 0.05mg/ml gentamicin for 3 times, cutting, putting into ice box, and taking back to laboratory;
② uterus is brought back to the laboratory and washed 1 time with sterile DPBS containing 1% (v/v) of an antigenic-antimycotic, 0.002mg/ml amphotericin B and 0.05mg/ml gentamicin, and the uterine horn is incised along the longitudinal axis to expose the uterine cavity;
③ separating endometrium tissue from myometrium, and cutting the endometrium tissue into 1-5 mm pieces with scissors3A size tissue mass;
(2) collecting the tissue blocks cut in the step (1) into a 50ml centrifuge tube, slowly adding sterile DPBS containing 1% (v/v) of antimitotic-antimycotic, 0.002mg/ml of amphotericin B and 0.05mg/ml of gentamicin into the centrifuge tube to wash the tissue blocks cut in the centrifuge tube to remove impurities, and then centrifuging the tissue blocks at 25 ℃ and 90g for 5 min;
(3) discarding the supernatant, adding the minced tissue pieces to a cell culture plate containing Hanks' Balanced Salt Solution, and placing the cell culture plate at 37 ℃ and 5% CO2Incubating for 24h in the incubator, and collecting an endometrial culture solution;
(4) centrifuging the collected endometrium culture solution at 4 deg.C and 2000g for 20min, and collecting supernatant;
(5) transferring the supernatant collected in the step (4) into a new centrifugal tube, centrifuging for 30min under the centrifugation condition of 10000g at 4 ℃, and collecting the supernatant;
(6) transferring the supernatant collected in the step (5) into a super centrifuge (Beckmann Optima XPN-100Ultracentrifuge as an Ultracentrifuge), placing the super centrifuge in a vertical rotor VTi50, centrifuging for 2h under the centrifugation condition of 4 ℃ and 120000g, removing the supernatant, and collecting the precipitate;
(7) and (4) resuspending the precipitate collected in the step (6) by using DPBS, centrifuging for 2h at 4 ℃ under 120000g, and discarding the supernatant, wherein the obtained precipitate is the total exosomes derived from endometrium.
Example 2
(1) Preparation of endometrial tissue blocks
① taking out uterus after slaughtering pig, fastening on two sides of uterine horn to ensure uterine cavity sealing, cleaning with 75% alcohol for 2 times, washing with 1% (v/v) alcoholic-antimycotic, 0.002mg/ml amphotericin B and 0.05mg/ml gentamicin saline for 3 times, cutting, putting into ice box, and taking back to laboratory;
② uterus is brought back to the laboratory and washed 1 time with sterile DPBS containing 1% (v/v) of an antigenic-antimycotic, 0.002mg/ml amphotericin B and 0.05mg/ml gentamicin, and the uterine horn is incised along the longitudinal axis to expose the uterine cavity;
③ separating endometrium tissue from myometrium, and cutting the endometrium tissue into 1-5 mm pieces with scissors3A size tissue mass;
(2) collecting the tissue blocks cut in the step (1) into a 50ml centrifuge tube, slowly adding sterile DPBS containing 1% (v/v) of antimitotic-antimycotic, 0.002mg/ml of amphotericin B and 0.05mg/ml of gentamicin into the centrifuge tube to wash the tissue blocks cut in the centrifuge tube to remove impurities, and then centrifuging the tissue blocks at the temperature of 20 ℃ and the speed of 100g for 3 min;
(3) discarding the supernatant, adding the minced tissue pieces to a cell culture plate containing Hanks' Balanced Salt Solution, and placing the cell culture plate at 37 ℃ and 5% CO2Incubating for 28h in the incubator, and collecting an endometrial culture solution;
(4) centrifuging the collected endometrium culture solution at 4 deg.C and 2000g for 20min, and collecting supernatant;
(5) transferring the supernatant collected in the step (4) into a new centrifugal tube, centrifuging for 30min under the centrifugation condition of 10000g at 4 ℃, and collecting the supernatant;
(6) transferring the supernatant collected in the step (5) into a super centrifuge (Beckmann Optima XPN-100Ultracentrifuge as an Ultracentrifuge), placing the super centrifuge in a vertical rotor VTi50, centrifuging for 2h under the centrifugation condition of 4 ℃ and 120000g, removing the supernatant, and collecting the precipitate;
(7) and (4) resuspending the precipitate collected in the step (6) by using DPBS, centrifuging for 2h at 4 ℃ under 120000g, and discarding the supernatant, wherein the obtained precipitate is the total exosomes derived from endometrium.
Example 3
(1) Preparation of endometrial tissue blocks
① taking out uterus after slaughtering pig, fastening on two sides of uterine horn to ensure uterine cavity sealing, cleaning with 75% alcohol for 3 times, washing with 1% (v/v) alcoholic-antimycotic, 0.002mg/ml amphotericin B and 0.05mg/ml gentamicin saline for 2 times, cutting, putting into ice box, and taking back to laboratory;
② uterus is brought back to the laboratory and washed 2 times with sterile DPBS containing 1% (v/v) of an antigenic-antimycotic, 0.002mg/ml amphotericin B and 0.05mg/ml gentamicin, and the uterine horn is incised along the longitudinal axis to expose the uterine cavity;
③ separating endometrium tissue from myometrium, and cutting the endometrium tissue into 1-5 mm pieces with scissors3A size tissue mass;
(2) collecting the tissue blocks cut in the step (1) into a 50ml centrifuge tube, slowly adding sterile DPBS containing 1% (v/v) of antimitotic-antimycotic, 0.002mg/ml of amphotericin B and 0.05mg/ml of gentamicin into the centrifuge tube to wash the tissue blocks cut in the centrifuge tube to remove impurities, and then centrifuging the tissue blocks at 30 ℃ and 80g for 10 min;
(3) discarding the supernatant, adding the minced tissue pieces to a cell culture plate containing Hanks' Balanced Salt Solution, and placing the cell culture plate at 37 ℃ and 5% CO2Incubating for 20h in the incubator, and collecting an endometrial culture solution;
(4) centrifuging the collected endometrium culture solution at 4 deg.C and 2000g for 20min, and collecting supernatant;
(5) transferring the supernatant collected in the step (4) into a new centrifugal tube, centrifuging for 30min under the centrifugation condition of 10000g at 4 ℃, and collecting the supernatant;
(6) transferring the supernatant collected in the step (5) into a super centrifuge (Beckmann Optima XPN-100Ultracentrifuge as an Ultracentrifuge), placing the super centrifuge in a vertical rotor VTi50, centrifuging for 2h under the centrifugation condition of 4 ℃ and 120000g, removing the supernatant, and collecting the precipitate;
(7) and (4) resuspending the precipitate collected in the step (6) by using DPBS, centrifuging for 2h at 4 ℃ under 120000g, and discarding the supernatant, wherein the obtained precipitate is the total exosomes derived from endometrium.
Comparative examples
(1) Preparation of endometrial tissue blocks
① taking out uterus after slaughtering pig, fastening on two sides of uterine horn to ensure uterine cavity sealing, cleaning with 75% alcohol for 3 times, washing with physiological saline containing 1% (v/v) acetylic-antimycotic, 0.002mg/ml amphotericin B and 0.05mg/ml gentamicin for 3 times, cutting, putting into ice box, and taking back to laboratory;
② uterus is brought back to the laboratory and washed 1 time with sterile DPBS containing 1% (v/v) of an antigenic-antimycotic, 0.002mg/ml amphotericin B and 0.05mg/ml gentamicin, and the uterine horn is incised along the longitudinal axis to expose the uterine cavity;
③ separating endometrium tissue from myometrium, and cutting the endometrium tissue into 1-5 mm pieces with scissors3A size tissue mass;
(2) collecting the tissue blocks cut in the step (1) into a 50ml centrifuge tube, slowly adding sterile DPBS containing 1% (v/v) of antimitotic-antimycotic, 0.002mg/ml of amphotericin B and 0.05mg/ml of gentamicin into the centrifuge tube to wash the tissue blocks cut in the centrifuge tube to remove impurities, and then centrifuging the tissue blocks at 25 ℃ and 90g for 5 min;
(3) discarding the supernatant, adding the minced tissue pieces to a cell culture plate containing collagenase at a concentration of 0.1mg/ml, and placing the cell culture plate at 37 deg.C and 5% CO2Then DMEM F12 is added with 20% FBS culture medium to stop enzyme digestion, and an endometrial culture solution is collected;
(4) centrifuging the collected endometrium culture solution at 4 deg.C and 2000g for 20min, and collecting supernatant;
(5) transferring the supernatant collected in the step (4) into a new centrifugal tube, centrifuging for 30min under the centrifugation condition of 10000g at 4 ℃, and collecting the supernatant;
(6) transferring the supernatant collected in the step (5) into a super centrifuge (Beckmann Optima XPN-100Ultracentrifuge as an Ultracentrifuge), placing the super centrifuge in a vertical rotor VTi50, centrifuging for 2h under the centrifugation condition of 4 ℃ and 120000g, removing the supernatant, and collecting the precipitate;
(7) and (4) resuspending the precipitate collected in the step (6) by using DPBS, centrifuging for 2h at 4 ℃ under 120000g, and discarding the supernatant, wherein the obtained precipitate is the total exosomes derived from endometrium.
Effect example 1
(1) Fig. 1 is a transmission electron micrograph of the exosomes prepared in example 1 and comparative example respectively, and it can be seen from the transmission electron micrograph that the exosomes prepared in example 1 have more obvious shapes under the electron micrograph, clean background and no impurities compared with the enzymolysis method.
(2) FIG. 2 is a particle size analysis diagram of the exosomes prepared in example 1, and it can be seen from the diagram that most of the detected particle sizes in the sample are distributed between 30-150 nm, which conforms to the diameter range of the exosomes, and the detected sample is the exosomes.
(3) FIG. 3 is a graph showing the results of detection of Western blot expressed by the proteins CD63 and CD9 in exosomes prepared in example 1, from which it can be seen that bands of exosome marker proteins CD63 and CD9 were detected, indicating that exosome surface marker proteins CD63 and CD9 are present in porcine endometrial tissue (wherein, the detection method: Western blot; antibodies: CD63 antibody (abcam) and CD9 (production) antibodies).
Effect example 2
(1) In reference (Corps AN, Brigstock D R, Littlewood C J, et al. receptors for growth factor and insulin growth factor-I on preimplantation growth factor of the pig [ J ]. Development,1990,110(1): 221-:
① slaughtering pig 12 days of gestation, taking out uterus, placing on ice, and taking back to laboratory;
② Each uterine horn was rinsed with DMEM to obtain a blastocyst and to separate the trophoblast from the embryo, yolk sac and allantoic membrane;
③ cutting the trophoblast with scissors, and gently sucking the cut tissue repeatedly through a syringe needle to prepare a cell suspension;
④ centrifuging the cell suspension at 4 deg.C and 100g for 10min, repeating for 2-3 times, and suspending in DMEM medium containing 5% FBS and 0.1U/ml insulin at 37 deg.C and 5% CO2Incubating for 2 days in the incubator;
(2) the embryonic trophoblast cells incubated in step (1) were seeded into 100. mu.L of complete medium in 96-well plates and treated with 0. mu.g/mL, 5. mu.g/mL, 20. mu.g/mL and 50. mu.g/mL exosomes/well in 96-well plates for 24h to 48 h.
(3) After 24h and 48h, 10. mu.L of CCK 8/well were added, incubated at 38.5 ℃ for a further 2h, and the absorbance measured at 450nm with a microplate reader.
The results are shown in fig. 4 and show that endometrium-derived exosomes promote proliferation of embryonic trophoblast cells (significant differences are indicated by different lettering).
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for extracting exosomes from pig endometrium is characterized by comprising the following steps:
(1) taking out a uterus after a pig is slaughtered, fastening two sides of a uterine horn to be taken to ensure that the uterine cavity is sealed, cutting the uterine horn along a longitudinal axis after cleaning, and exposing the uterine cavity; separating endometrial tissue from the myometrium and shearing the endometrial tissue into tissue pieces;
(2) washing the tissue blocks cut up in the step (1) by using sterile DPBS containing 1% (v/v) of isotatic-antimycotic, 0.002mg/ml of amphotericin B and 0.05mg/ml of gentamicin to remove impurities, centrifuging for 3-10 min at the temperature of 20-30 ℃ and under the condition of 80-100 g, and removing supernatant;
(3) placing the tissue block centrifuged and supernatant removed in step (2) in Hanks' blannced Salt Solution at 37 ℃ and 5% CO2Incubating for 20-28 h under the condition, and collecting pigsEndometrial culture fluid;
(4) and (4) extracting exosomes from the porcine endometrium culture solution collected in the step (3) by an ultracentrifugation method to obtain exosomes from the porcine endometrium.
2. The method for extracting an exosome derived from a porcine endometrium according to claim 1, characterized in that:
the tissue block in the step (1) has a size of 1-5 mm3Size.
3. The method for extracting an exosome derived from a porcine endometrium according to claim 1, characterized in that:
the specific operation of the cleaning in the step (1) is as follows:
the method comprises the steps of firstly cleaning 2-3 times by using 75% alcohol by volume fraction, then washing 2-3 times by using physiological saline containing 1% (v/v) of acetylic-antimycotic, 0.002mg/ml of amphotericin B and 0.05mg/ml of gentamicin, and finally washing 1-2 times by using sterile DPBS containing 1% (v/v) of acetylic-antimycotic, 0.002mg/ml of amphotericin B and 0.05mg/ml of gentamicin.
4. The method for extracting an exosome derived from a porcine endometrium according to claim 1, characterized in that:
and (3) centrifuging for 5min under the condition of 90g at the temperature of 25 ℃.
5. The method for extracting an exosome derived from a porcine endometrium according to claim 1, characterized in that:
the incubation time in step (3) was 24 h.
6. The method for extracting an exosome derived from a porcine endometrium according to claim 1, characterized in that:
the ultracentrifugation method in the step (4) comprises the following specific operations:
① centrifuging the collected endometrium culture solution at 4 deg.C and 2000g for 20min, and collecting supernatant;
② centrifuging the supernatant fluid collected in step ① at 4 deg.C under 10000g for 30min, and collecting the supernatant fluid;
③ centrifuging the supernatant obtained in step ② at 4 deg.C and 120000g for 2h, discarding the supernatant, and collecting the precipitate;
④ the pellet collected in step ③ was resuspended in DPBS, centrifuged again at 120000g at 4 ℃ for 2h and the supernatant discarded, the pellet obtained was total exosomes from endometrium.
7. An exosome derived from a porcine endometrium, characterized by being extracted by the extraction method according to any one of claims 1 to 6.
8. Use of the porcine endometrium-derived exosome of claim 7 for preparing an embryo in vitro culture additive.
9. Use of porcine endometrium-derived exosomes according to claim 8 in the preparation of embryo in vitro culture additive, characterized in that:
the embryo in-vitro culture additive is a product for promoting the proliferation of embryo trophoblast cells.
10. Use of porcine endometrium-derived exosomes according to claim 8 or 9 for preparing embryo in vitro culture additive, characterized by comprising the steps of:
inoculating the embryo trophoblast cells into a culture medium containing the porcine endometrium-derived exosome according to claim 7 and culturing for 24-48 h.
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| CN114323863B (en) * | 2021-12-31 | 2023-12-22 | 吉林大学 | Preparation method and application of exosome array capture probe |
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