CN111019885B - Exosome of pig endometrium source and extraction method and application thereof - Google Patents
Exosome of pig endometrium source and extraction method and application thereof Download PDFInfo
- Publication number
- CN111019885B CN111019885B CN201911112690.9A CN201911112690A CN111019885B CN 111019885 B CN111019885 B CN 111019885B CN 201911112690 A CN201911112690 A CN 201911112690A CN 111019885 B CN111019885 B CN 111019885B
- Authority
- CN
- China
- Prior art keywords
- endometrium
- exosomes
- supernatant
- pig
- centrifuging
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
-
- 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
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention belongs to the technical field of biology, and particularly relates to an exosome of pig endometrium sources, and an extraction method and application thereof. Separating to obtain endometrial tissue, and then shearing to form tissue blocks; centrifuging for 3-10 min at the temperature of 20-30 ℃ and the condition of 80-100 g after flushing, and discarding supernatant; then placing the tissue blocks in Hanks' Balanced Salt Solution for incubation, and collecting pig endometrium culture solution; finally, extracting exosomes by adopting an ultracentrifugation method to obtain the exosomes from the pig endometrium. Compared with the prior art, the method provided by the invention is simpler and more convenient, saves the cost of sample collection, reduces the loss of manpower, saves the sampling time and increases the sampling efficiency. The extracted exosomes have more obvious exosome shape under an electron microscope, have clean background and no impurities, and 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 an exosome of pig endometrium sources, and an extraction method and application thereof.
Background
Endometrium is a complex dynamic tissue that undergoes periodic remodeling and differentiation throughout the reproductive life of women. Successful conception requires that a good quality embryo co-operate with a well-tolerated endometrial, and whether an embryo can be successfully implanted into the endometrial determines the outcome of the pregnancy. Cross-talk between the endometrial epithelium and blasts, particularly trophoblasts, is a prerequisite for successful implantation during embryo implantation. During this crosstalk, various molecular and functional changes occur to promote synchronicity between the embryo and the endometrial and uterine cavity microenvironment. In particular, the trophectoderm is modulated to achieve endometrial attachment. Thus, intercellular communication between the trophectoderm and the endometrial epithelium is critical for embryo implantation and establishment of pregnancy.
The exosomes are bilayer membrane vesicles with the diameter of 30-150 nm, can carry proteins, miRNA, mRNA, lncRNA, circRNA and other substances, can be selectively fused with target cells, transport the carried genetic materials into the target cells to play a role, and remotely play a role in intercellular signal transmission, so that the exosomes are considered to be a novel intercellular signal communication mode. Studies have shown that some proteins contained in exosomes help exosomes stimulate immune cells to destroy diseased cells (Wu Jinen, ding Juntao. Development of exosome biological function and application, animal medicine development 2016;37 (12): 90-4.); exosomes can carry RNAs from the secretory cells to the target cells and function within the target cells, and after the RNAs reach the recipient cells through the exosomes, the mRNA can be converted to a functional protein; miRNA can degrade mRNA or inhibit the translation process of mRNA so as to regulate and control the expression of protein (Ge Hui. Extraction and identification of exosomes of estrus cow uterine fluid and influence on development of in vitro fertilized embryo [ Shuoshi ]: 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 in exosomes: a novel biomarker in cancer detection cell research 2014;24 (6): 766-9.) et al also found stable double-stranded DNA structures in the exosomes of pancreatic cancer origin and indicated that they were biomarkers for potential cancer diagnosis. Studies by Testa et al (Testa JS, apcher GS, comber JD, eisenlohr LC. Exosome-driven antigen transfer for MHC class II presentation facilitated by the receptor binding activity of influenza hemagglutin. Journal of immunology (Baltimore, md: 1950) 2010;185 (11): 6608-16.) have shown that exosomes can carry some antigen-peptide complexes with functionality to mediate anti-viral immunity, thereby reducing the susceptibility of the host to infection. The exosomes of the dendritic cells expressing IL-4 were genetically modified, and it was found through animal experiments that the clinical symptoms of collagen-induced arthritis mice were reduced, and that delayed hypersensitivity reactions in the mice were also inhibited (Kim SH, bianco NR, shufesky WJ, morelli AE, robbins PD. Effective treatment of inflammatory disease models with exosomes derived from dendritic cells genetically modified to express IL-4.Journal of immunology (Baltimore, md: 1950). 2007 (4): 2242-9.).
Exosomes were found to be present in preparations of uterine fluid/mucus and endometrial epithelial cells. The pregnant uterus contains not only the exosomes derived from the mother, but also the exosomes derived from the embryo, and in maternal-fetal communication, substances carried by the exosomes derived from the mother and the exosomes derived from the embryo are exchanged, and the exosomes are expressed orderly in time and space in the whole gestation process. Therefore, in the embryo implantation process, the maternal uterine cavity fluid exosome can mutually transmit information (including RNAs, proteins and lipid molecules) between endometrial cells and embryo cells, so that the function of maternal-fetal dialogue is exerted, and the embryo implantation success rate is greatly influenced.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the primary aim of the invention is to provide a method for extracting exosomes derived from pig endometrium.
Another object of the present invention is to provide an exosome derived from pig endometrium extracted by the above extraction method.
It is a further object of the present invention to provide the use of exosomes of endometrial origin in pigs as described above.
The aim of the invention is achieved by the following technical scheme:
a method for extracting exosomes derived from porcine endometrium, comprising the steps of:
(1) Taking out the uterus after slaughtering pigs, fastening the two sides of the uterine horn to be taken to ensure the sealing of the uterine cavity, and cutting the uterine horn along a longitudinal axis after cleaning to expose the uterine cavity; separating endometrial tissue from the myometrium and shearing endometrial tissue into tissue pieces;
(2) Flushing the chopped tissue blocks in the step (1) with sterile DPBS containing 1% (v/v) of anti-ntimycin, 0.002mg/ml of amphotericin B and 0.05mg/ml of gentamicin to remove impurities, and centrifuging for 3-10 min at 20-30 ℃ and 80-100 g, and discarding the supernatant;
(3) Placing the tissue mass after centrifugation of the supernatant of step (2) in Hanks' Balanced Salt Solution at 37℃with 5% CO 2 Incubating for 20-28 h under the condition of (1) and collecting the pig endometrium culture solution;
(4) Extracting exosomes from the pig endometrium culture solution collected in the step (3) by adopting an ultracentrifugation method to obtain exosomes from pig endometrium sources;
the size of the tissue mass in step (1) is preferably 1 to 5mm 3 Size of the material;
the specific operation of the washing described in step (1) is preferably:
firstly, washing with 75% alcohol for 2-3 times, then washing with physiological saline containing 1% (v/v) of antimiotic-antimycotic, 0.002mg/ml of amphotericin B and 0.05mg/ml of gentamicin for 2-3 times, and finally washing with sterile DPBS containing 1% (v/v) of antimiotic-antimycotic, 0.002mg/ml of amphotericin B and 0.05mg/ml of gentamicin for 1-2 times; through the cleaning, the purposes of sterilizing and cleaning uterus, removing impurities and preventing pollution are achieved;
the centrifugation conditions in step (2) are preferably 25℃and 90g for 5min;
the incubation time in step (3) is preferably 24 hours;
the specific operation of the ultracentrifugation method described in step (4) is preferably:
(1) centrifuging the collected endometrium culture solution at 4deg.C under 2000g centrifugation condition for 20min, and collecting supernatant;
(2) centrifuging the supernatant collected in the step (1) at 4 ℃ and 10000g for 30min, and collecting the supernatant;
(3) centrifuging the supernatant collected in the step (2) at 4 ℃ under the centrifugation condition of 120000g for 2 hours, discarding the supernatant, and collecting the precipitate;
(4) re-suspending the precipitate collected in the step (3) by using DPBS, centrifuging again at 4 ℃ under the centrifugation condition of 120000g for 2 hours, and discarding the supernatant to obtain the precipitate which is the total exosomes derived from endometrium;
an exosome derived from pig endometrium is extracted by the above extraction method;
the application of the exosome of the pig endometrium source in preparing embryo in vitro culture additive;
the embryo in-vitro culture additive is preferably a product for promoting proliferation of embryo trophoblast cells;
the application preferably comprises the following steps:
inoculating the embryo trophoblast cells into a culture medium containing the exosomes derived from the pig endometrium for culturing for 24-48 hours;
the concentration of exosomes derived from pig endometrium 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) 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 loss of manpower is reduced, the sampling time is saved, and the sampling efficiency is increased.
(2) Compared with the enzymolysis method, the method has the advantages that Hanks' Balanced Salt Solution is selected for treating the sheared tissue blocks, the shape of the extracted exosomes is more obvious under an electron microscope, the background is clean and no impurities exist, and the method also provides a thought for extracting the exosomes from other tissues.
(2) The invention discovers for the first time that the exosome from pig endometrium can promote proliferation of embryo trophoblast cells, and can be used as an additive for promoting the in vitro culture efficiency of pig embryo.
Drawings
FIG. 1 is a transmission electron microscope image of exosomes extracted by enzymatic hydrolysis (A) with Hanks' Balanced Salt Solution (B) treatment of sheared pieces of endometrial tissue.
FIG. 2 is a graph showing the particle size analysis of the extracted exosomes of the present invention.
FIG. 3 shows the detection results of Western blot of CD63 and CD9 expression of proteins extracted from exosomes according to the present invention.
Fig. 4 is a graph of the results of uterine-derived exosomes affecting porcine embryonic trophoblast cell proliferation, with distinct letter superscripts indicating 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 embodiments of the present invention are 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 the uterus after slaughtering pigs, fastening on two sides of the uterine horn to be taken out to ensure the sealing of the uterine cavity, firstly cleaning 3 times by using alcohol with the volume fraction of 75%, then flushing 3 times by using physiological saline containing 1% (v/v) anti-antimycotic, 0.002mg/ml amphotericin B and 0.05mg/ml gentamicin, and immediately taking the mixture back to a laboratory after cutting;
(2) after the uterus is brought back to the laboratory, the uterine horn is incised along the longitudinal axis to expose the uterine cavity by 1 time of rinsing with sterile DPBS containing 1% (v/v) of anti-ntimycin, 0.002mg/ml of amphotericin B and 0.05mg/ml of gentamicin;
(3) separating the endometrium tissue from the myometrium, and shearing the endometrium tissue into 1-5 mm pieces by scissors 3 A tissue block of size;
(2) Collecting the sheared tissue blocks in the step (1) into a 50ml centrifuge tube, slowly adding sterile DPBS containing 1% (v/v) anti-ntimycin, 0.002mg/ml amphotericin B and 0.05mg/ml gentamicin into the centrifuge tube, flushing the sheared tissue blocks in the centrifuge tube to remove impurities, and centrifuging at 25 ℃ and 90g for 5min;
(3) Removing supernatant, adding the sheared tissue mass into a cell culture plate containing Hanks' Balanced Salt Solution, and placing the cell culture plate at 37 ℃ and 5% CO 2 Incubating for 24 hours in an incubator of (2) and collecting endometrial culture solution;
(4) Centrifuging the collected endometrium culture solution at 4deg.C under 2000g centrifugation condition for 20min, and collecting supernatant;
(5) Transferring the supernatant collected in the step (4) into a new centrifuge 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-separation tube (the super-separation tube is Beckmann Optima XPN-100 Ultrafuge), placing the super-separation tube in a vertical rotor VTi50, centrifuging for 2 hours under the centrifugation condition of 120000g at 4 ℃, discarding the supernatant, and collecting the precipitate;
(7) Resuspension the sediment collected in step (6) with DPBS, centrifuging again at 4deg.C under 120000g for 2h, discarding supernatant, and obtaining sediment which is total exosomes derived from endometrium.
Example 2
(1) Preparation of endometrial tissue blocks
(1) Taking out the uterus after slaughtering pigs, fastening on two sides of the uterine horn to be taken out to ensure the sealing of the uterine cavity, firstly cleaning the uterine cavity for 2 times by using alcohol with the volume fraction of 75%, then flushing the uterine cavity for 3 times by using physiological saline containing 1% (v/v) anti-antimycotic, 0.002mg/ml amphotericin B and 0.05mg/ml gentamicin, and immediately taking the uterine cavity back to a laboratory after cutting off the uterine cavity;
(2) after the uterus is brought back to the laboratory, the uterine horn is incised along the longitudinal axis to expose the uterine cavity by 1 time of rinsing with sterile DPBS containing 1% (v/v) of anti-ntimycin, 0.002mg/ml of amphotericin B and 0.05mg/ml of gentamicin;
(3) separating the endometrium tissue from the myometrium, and shearing the endometrium tissue into 1-5 mm pieces by scissors 3 A tissue block of size;
(2) Collecting the sheared tissue blocks in the step (1) into a 50ml centrifuge tube, slowly adding sterile DPBS containing 1% (v/v) anti-ntimycin, 0.002mg/ml amphotericin B and 0.05mg/ml gentamicin into the centrifuge tube, flushing the sheared tissue blocks in the centrifuge tube to remove impurities, and centrifuging at 20 ℃ for 3min under the condition of 100 g;
(3) Removing supernatant, adding the sheared tissue mass into a cell culture plate containing Hanks' Balanced Salt Solution, and placing the cell culture plate at 37 ℃ and 5% CO 2 Incubating for 28 hours in an incubator, and collecting endometrial culture fluid;
(4) Centrifuging the collected endometrium culture solution at 4deg.C under 2000g centrifugation condition for 20min, and collecting supernatant;
(5) Transferring the supernatant collected in the step (4) into a new centrifuge 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-separation tube (the super-separation tube is Beckmann Optima XPN-100 Ultrafuge), placing the super-separation tube in a vertical rotor VTi50, centrifuging for 2 hours under the centrifugation condition of 120000g at 4 ℃, discarding the supernatant, and collecting the precipitate;
(7) Resuspension the sediment collected in step (6) with DPBS, centrifuging again at 4deg.C under 120000g for 2h, discarding supernatant, and obtaining sediment which is total exosomes derived from endometrium.
Example 3
(1) Preparation of endometrial tissue blocks
(1) Taking out the uterus after slaughtering pigs, fastening on two sides of the uterine horn to be taken out to ensure the sealing of the uterine cavity, firstly cleaning 3 times by using alcohol with the volume fraction of 75%, then flushing 2 times by using physiological saline containing 1% (v/v) anti-antimycotic, 0.002mg/ml amphotericin B and 0.05mg/ml gentamicin, and immediately taking the mixture back to a laboratory after cutting;
(2) after the uterus is brought back to the laboratory, the uterine horn is incised along the longitudinal axis and the uterine cavity is exposed by flushing 2 times with sterile DPBS containing 1% (v/v) of anti-ntimycin, 0.002mg/ml amphotericin B and 0.05mg/ml gentamicin;
(3) separating the endometrium tissue from the myometrium, and shearing the endometrium tissue into 1-5 mm pieces by scissors 3 A tissue block of size;
(2) Collecting the sheared tissue blocks in the step (1) into a 50ml centrifuge tube, slowly adding sterile DPBS containing 1% (v/v) anti-ntimycin, 0.002mg/ml amphotericin B and 0.05mg/ml gentamicin into the centrifuge tube, flushing the sheared tissue blocks in the centrifuge tube to remove impurities, and centrifuging at 30 ℃ and 80g for 10min;
(3) Removing supernatant, adding the sheared tissue mass into a cell culture plate containing Hanks' Balanced Salt Solution, and placing the cell culture plate at 37 ℃ and 5% CO 2 Incubating for 20 hours in an incubator of (2) and collecting endometrial culture solution;
(4) Centrifuging the collected endometrium culture solution at 4deg.C under 2000g centrifugation condition for 20min, and collecting supernatant;
(5) Transferring the supernatant collected in the step (4) into a new centrifuge 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-separation tube (the super-separation tube is Beckmann Optima XPN-100 Ultrafuge), placing the super-separation tube in a vertical rotor VTi50, centrifuging for 2 hours under the centrifugation condition of 120000g at 4 ℃, discarding the supernatant, and collecting the precipitate;
(7) Resuspension the sediment collected in step (6) with DPBS, centrifuging again at 4deg.C under 120000g for 2h, discarding supernatant, and obtaining sediment which is total exosomes derived from endometrium.
Comparative examples
(1) Preparation of endometrial tissue blocks
(1) Taking out the uterus after slaughtering pigs, fastening on two sides of the uterine horn to be taken out to ensure the sealing of the uterine cavity, firstly cleaning 3 times by using alcohol with the volume fraction of 75%, then flushing 3 times by using physiological saline containing 1% (v/v) anti-antimycotic, 0.002mg/ml amphotericin B and 0.05mg/ml gentamicin, and immediately taking the mixture back to a laboratory after cutting;
(2) after the uterus is brought back to the laboratory, the uterine horn is incised along the longitudinal axis to expose the uterine cavity by 1 time of rinsing with sterile DPBS containing 1% (v/v) of anti-ntimycin, 0.002mg/ml of amphotericin B and 0.05mg/ml of gentamicin;
(3) separating the endometrium tissue from the myometrium, and shearing the endometrium tissue into 1-5 mm pieces by scissors 3 A tissue block of size;
(2) Collecting the sheared tissue blocks in the step (1) into a 50ml centrifuge tube, slowly adding sterile DPBS containing 1% (v/v) anti-ntimycin, 0.002mg/ml amphotericin B and 0.05mg/ml gentamicin into the centrifuge tube, flushing the sheared tissue blocks in the centrifuge tube to remove impurities, and centrifuging at 25 ℃ and 90g for 5min;
(3) Removing supernatant, adding the sheared tissue mass into cell culture plate containing collagenase solution with concentration of 0.1mg/ml, placing the cell culture plate at 37deg.C and 5% CO 2 After 2.5h incubation in incubator (2), DMEM F12 was added with 20% fbs medium to stop enzyme digestion, and endometrial culture medium was collected;
(4) Centrifuging the collected endometrium culture solution at 4deg.C under 2000g centrifugation condition for 20min, and collecting supernatant;
(5) Transferring the supernatant collected in the step (4) into a new centrifuge 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-separation tube (the super-separation tube is Beckmann Optima XPN-100 Ultrafuge), placing the super-separation tube in a vertical rotor VTi50, centrifuging for 2 hours under the centrifugation condition of 120000g at 4 ℃, discarding the supernatant, and collecting the precipitate;
(7) Resuspension the sediment collected in step (6) with DPBS, centrifuging again at 4deg.C under 120000g for 2h, discarding supernatant, and obtaining sediment which is total exosomes derived from endometrium.
Effect example 1
(1) Fig. 1 is a transmission electron microscope image of the exosomes prepared in example 1 and comparative example, respectively, and it can be seen from the image that the exosomes prepared in example 1 have more obvious exosomes shape under electron microscope, clean background and no impurity compared with the enzymolysis method.
(2) FIG. 2 is a graph showing the particle size analysis of the exosomes prepared in example 1, and it can be seen from the graph that most of the detected particle sizes in the samples are distributed between 30 and 150nm, and conform to the range of exosomes diameter, indicating that the detected samples are exosomes.
(3) FIG. 3 is a graph showing the results of Western blot of the expression of 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 marker proteins CD63 and CD9 are present in porcine endometrial tissues (wherein, detection method: western blot; antibodies: CD63 antibody (abcam) and CD9 (bio) antibody).
Effect example 2
(1) Reference (Corps AN, brigstock D R, littlewood C J, et al Receptors for epidermal growth factor and insulin growth factor-I on preimplantaion trophoderm of the pig [ J ]. Development,1990,110 (1): 221-227.) the following methods are used to isolate porcine embryonic trophoblast cells:
(1) taking out uterus after slaughtering pigs 12 days gestation, and putting the pigs on ice to be brought back to a laboratory;
(2) each uterine horn was rinsed with DMEM to obtain blastocysts and trophoblasts were isolated from embryos, yolk sac and allantois;
(3) cutting the trophoblast with scissors, and repeatedly gently sucking the cut tissue through a syringe needle to prepare a cell suspension;
(4) centrifuging the above cell suspension at 4deg.C and 100g for 10min, repeating for 2-3 times, and re-suspending in DMEM medium, adding 5% FBS and 0.1U/ml insulin, placing at 37deg.C and 5% CO 2 Incubating for 2 days in an incubator;
(2) The embryonic trophoblast cells after the incubation of step (1) were seeded into 100. Mu.L of 96-well plates in complete medium and 96-well plates were treated with 0. Mu.g/mL, 5. Mu.g/mL, 20. Mu.g/mL and 50. Mu.g/mL exosomes/well for 24h to 48h.
(3) After 24h and 48h, 10. Mu.L of CCK 8/well was added and incubated for a further 2h at 38.5℃and absorbance was measured at 450nm using a microplate reader.
The results are shown in fig. 4, which shows that endometrial-derived exosomes promote proliferation of embryonic trophoblast cells (the different letter superscripts indicate significant differences).
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (4)
1. A method for extracting exosomes derived from porcine endometrium, which is characterized by comprising the following steps:
(1) Taking out the uterus after slaughtering pigs, fastening the two sides of the uterine horn to be taken to ensure the sealing of the uterine cavity, and cutting the uterine horn along a longitudinal axis after cleaning to expose the uterine cavity; separating endometrial tissue from the myometrium and shearing endometrial tissue into tissue pieces;
(2) Flushing the sheared tissue mass in step (1) with sterile DPBS containing 1% (v/v) anti-antimycotic, 0.002mg/ml amphotericin B and 0.05mg/ml gentamicin to remove impurities, then centrifuging at 25deg.C for 5min at 90g, and discarding supernatant;
(3) Placing the tissue mass after centrifugation of the supernatant of step (2) in Hanks' Balanced Salt Solution at 37℃with 5% CO 2 Is incubated for 24 hours under the condition of (2) and the pig endometrium is collectedA culture solution;
(4) Extracting exosomes from the pig endometrium culture solution collected in the step (3) by adopting an ultracentrifugation method to obtain exosomes from pig endometrium sources;
the size of the tissue block in the step (1) is 1-5 mm 3 Size of the material;
the specific operation of the ultracentrifugation method described in step (4) is:
(1) centrifuging the collected endometrium culture solution at 4deg.C under 2000g centrifugation condition for 20min, and collecting supernatant;
(2) centrifuging the supernatant collected in the step (1) at 4 ℃ and 10000g for 30min, and collecting the supernatant;
(3) centrifuging the supernatant collected in the step (2) at 4 ℃ under the centrifugation condition of 120000g for 2 hours, discarding the supernatant, and collecting the precipitate;
(4) resuspension the precipitate collected in step (3) with DPBS, centrifuging again at 4deg.C under 120000g for 2h, and discarding supernatant to obtain precipitate as total exosomes derived from endometrium.
2. The method for extracting pig endometrium-derived exosomes according to claim 1, wherein:
the specific operation of the cleaning in the step (1) is as follows:
the method comprises the steps of firstly washing 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 antimycotic-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 antimycotic-antimycotic, 0.002mg/ml of amphotericin B and 0.05mg/ml of gentamicin.
3. The application of the pig endometrium-derived exosomes prepared by the extraction method of any one of claims 1-2 in preparing embryo in vitro culture additives, characterized in that:
the in vitro embryo culture additive is a product for promoting proliferation of embryo trophoblast cells.
4. Use according to claim 3, characterized by comprising the steps of:
inoculating the embryo trophoblast cells into a culture medium containing the pig endometrium-derived exosomes prepared by the extraction method according to any one of claims 1-2, and culturing for 24-48 h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911112690.9A CN111019885B (en) | 2019-11-14 | 2019-11-14 | Exosome of pig endometrium source and extraction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911112690.9A CN111019885B (en) | 2019-11-14 | 2019-11-14 | Exosome of pig endometrium source and extraction method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111019885A CN111019885A (en) | 2020-04-17 |
| CN111019885B true CN111019885B (en) | 2023-05-05 |
Family
ID=70201648
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911112690.9A Active CN111019885B (en) | 2019-11-14 | 2019-11-14 | Exosome of pig endometrium source and extraction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111019885B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113234665A (en) * | 2021-05-13 | 2021-08-10 | 华南农业大学 | Primary endometrial cell line for PEDV replication and separation method thereof |
| CN114323863B (en) * | 2021-12-31 | 2023-12-22 | 吉林大学 | Preparation method and application of exosome array capture probe |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016033695A1 (en) * | 2014-09-05 | 2016-03-10 | Exerkine Corporation | Exosome isolation |
| CN107523536A (en) * | 2017-10-20 | 2017-12-29 | 上海市同济医院 | A kind of extracting method of tissue-derived excretion body and application |
| CN109512469A (en) * | 2018-12-19 | 2019-03-26 | 华南农业大学 | A method of uterine cavity liquid excretion body is extracted from pig living body |
| JP2019528085A (en) * | 2016-07-26 | 2019-10-10 | 広州海力特生物科技有限公司 | Separation and extraction method of exosomes by stacked centrifugal filtration |
-
2019
- 2019-11-14 CN CN201911112690.9A patent/CN111019885B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016033695A1 (en) * | 2014-09-05 | 2016-03-10 | Exerkine Corporation | Exosome isolation |
| JP2017526388A (en) * | 2014-09-05 | 2017-09-14 | エクサーカイン コーポレイションＥｘｅｒｋｉｎｅ Ｃｏｒｐｏｒａｔｉｏｎ | Isolation of exosomes |
| JP2019528085A (en) * | 2016-07-26 | 2019-10-10 | 広州海力特生物科技有限公司 | Separation and extraction method of exosomes by stacked centrifugal filtration |
| CN107523536A (en) * | 2017-10-20 | 2017-12-29 | 上海市同济医院 | A kind of extracting method of tissue-derived excretion body and application |
| CN109512469A (en) * | 2018-12-19 | 2019-03-26 | 华南农业大学 | A method of uterine cavity liquid excretion body is extracted from pig living body |
Non-Patent Citations (6)
| Title |
|---|
| Review: Embryo- and endometrium-derived exosomes and their potential role in assisted reproductive treatmentseliquid biopsies for endometrial receptivity;Hayden Homer et al;《Placenta》;20181231;第89-94页 * |
| 发情期牛子宫液外泌体的提取与鉴定及其对体外受精胚胎发育的影响;葛卉;《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑》;20180215(第02期);摘要 * |
| 奶牛子宫内膜细胞体外培养及应用研究概况;邝晓娇等;《动物医学进展》;20141220(第12期);第148-152页 * |
| 子宫内膜细胞外泌体的研究进展;杨红等;《国际妇产科学杂志》;20181015(第05期);第574-578页 * |
| 小鼠子宫内膜原代上皮细胞及间质细胞的优化分离和培养;夏宇红等;《现代妇产科进展》;20130730(第07期);第549-552页 * |
| 猪子宫内膜基质细胞和腺上皮细胞的分离培养;赵诚悦等;《吉林农业大学学报》;20130603;第35卷(第04期);第471-474页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111019885A (en) | 2020-04-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2017047799A1 (en) | Culture method for differentiating primordial germ cells into functionally mature oocytes | |
| Kautz et al. | Expression of genes involved in the embryo–maternal interaction in the early-pregnant canine uterus | |
| CN106350479A (en) | Composition and method for energy transfer of autologous germline mitochondria | |
| Matoba et al. | Optimizing production of in vivo-matured oocytes from superstimulated Holstein cows for in vitro production of embryos using X-sorted sperm | |
| CN111019885B (en) | Exosome of pig endometrium source and extraction method and application thereof | |
| Lee et al. | Principal protocols for the processing of cultured meat | |
| Yong et al. | Production of a transgenic piglet by a sperm injection technique in which no chemical or physical treatments were used for oocytes or sperm | |
| CN108060117B (en) | Method for improving pig cloned embryo development efficiency | |
| CN110577928A (en) | Oocyte in-vitro maturation culture solution and application thereof | |
| CN111893088A (en) | Method for in vitro isolated culture of Mongolian horse testis spermatogonium stem cells | |
| JP5234535B2 (en) | Additive for in vitro maturation culture of mammalian immature ovum and method for producing mature ovum using the same | |
| KR101763343B1 (en) | Method for Producing Cloned Canidae Using Nuclear Transfer of Somatic Cells or Stem Cells | |
| KR100767289B1 (en) | Embryonic stem cell culture method | |
| Hanin et al. | Mammary adipocyte flow cytometry as a tool to study mammary gland biology | |
| Lange-Consiglio et al. | Endometrial and oviduct extra-cellular vescicles for in vitro equine sperm hyperactivation and oocyte fertilization | |
| Ham et al. | Clonal growth of cells taken directly from adult rabbits | |
| He et al. | Immunosuppressive Factor MNSFβ Regulates Cytokine Secretion by Mouse Lymphocytes and Is Involved in Interactions between the Mouse Embryo and Endometrial Cells In Vitro | |
| CN108913653B (en) | Method for improving nuclear transplantation efficiency of pigs | |
| Madhukaran et al. | Protocol to dissociate epithelia from non-pregnant and pregnant mouse cervical tissue for single-cell RNA-sequencing | |
| AU2017201404B2 (en) | Compositions And Methods For Autologous Germline Mitochondrial Energy Transfer | |
| Fufana-Duran | Studies for the Improvement of in vitro culture systems of oocytes and embryos in water buffalo | |
| Cunha et al. | Transplantation and tissue recombination techniques to study mammary gland biology | |
| CN114134107A (en) | Artificial ovary participated by mesenchymal stem cells and preparation method and application thereof | |
| CN114369567A (en) | Method for establishing bovine expanded pluripotent embryonic stem cells and culture solution | |
| CN111925981A (en) | Method for separating chicken male and female primordial germ cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |