TWI753404B - Oviductal stem cell culture promotes oocyte maturation and embryo development in vitro, biopreparation comprining them the method of preparation and uses thereof. - Google Patents

Abstract

The oocytes in vitro maturation (IVM) rate and in vitro culture (IVC) development rate could be enhanced by the addition of serum, electron carrier, animo acids and anti-oxidants, but development rate still lower than in vivo. Also, the matured oocytes would suffer from polyspermy easily.

In IVM, porcine oocytes were matured under PFTSCs conditioned M199 and BFTSCs conditioned M199. In IVC, matured procine oocytes were cultured under PFTSCs conditioned PZM and BFTSCs conditioned PZM.

PFTSCs and BFTSCs promoted the porcine oocytes maturation rate, and had a trend to improve the blastocysts rate which had the potential to the clinic medical applications.

Description

Preparation, preparation method and use of tubal stem cells for promoting oocyte maturation and embryo development in vitro

The invention relates to the use of a fallopian tube stem cell for preparing a preparation for promoting the in vitro maturation and embryo development of oocytes, and the use of preparing a preparation for promoting the in vitro maturation and embryo development of oocytes, and the use of establishing a treatment experimental platform system; in particular, it relates to a pig and cattle The use of fallopian tube stem cells to prepare preparations for promoting oocyte maturation and embryo development in vitro, and the use of preparations for promoting oocyte maturation and embryo development in vitro, and the use of establishing a therapeutic experimental platform system.

Fallopian tube (FT) has secretory cells that can provide the functions required for egg maturation and development. Previous studies have confirmed that fallopian tube fluid can enhance egg maturation, and after co-culture with fallopian tube cells, in addition to enhancing egg maturation and development, It can also improve the hardening ability of the zona pellucida and reduce the problem of polyspermia.

In addition, previous studies have pointed out that stem cells have a paracrine effect, which can promote cell growth and repair. Fallopian tubes are medical wastes in slaughterhouses. cells, FTSCs) have the potential of pluripotency and also have excellent proliferation and differentiation ability, and the growth factors secreted by them can promote the repair of reproductive tract damage and egg development.

The US20160237402A1 patent mentions that this technology provides a method for the directed differentiation of pluripotent cells, female germline stem cells or oocytes into oocytes, granulosa cells and/or granulosa precursor cells, ie "synthetic granulosa cells". Synthetic granulosa cells can be used in methods of growth and maturation of follicles or follicle-like structures and immature oocytes. Additionally, synthetic granulosa cells can be used in methods of increasing ovarian-derived hormones and growth factors in a subject in need thereof. The technology provides a method for differentiating pluripotent cells into granule cells and/or granule precursor cells, the method comprising: culturing the pluripotent cells under culture conditions in which the pluripotent cells are differentiated into granules. Cells and/or granule precursor cells, wherein the culture conditions are in the absence of mouse embryonic fibroblast (MEF) and leukemia inhibitory factor (LIF) and in the presence of a GSK inhibitor.

The I322183 patent relates to a method for producing non-human mammalian chimeric embryos using non-human 1-cell stage embryos from zona pellucida removed to morula in a microcentrifuge tube ( Eppendorf vial) with fresh or freshly thawed cells in a mixed culture medium (coculture) to obtain non-human mammalian chimeric embryos; the aforementioned chimeric embryos are transferred to non-human embryonic recipient animals through embryo transfer (embryo transfer), After the end of pregnancy, non-human chimeric animals with gonadal genetic ability and animals formed from non-human embryonic stem cells (embryonic stem, ES, cell-derived animals) can be obtained. The patent relates to a method for producing mouse chimeric embryos, the steps of which include: obtaining mouse cells; obtaining mouse 1-cell stage embryos from zona pellucida removed to morulas; The aforementioned mouse cells and the aforementioned zona pellucida-removed mouse 1-cell stage embryos to morulas were co-cultured in a microcentrifuge tube (Eppendorf vial) for a period of time to obtain small cells. Mouse "embryo-cell" aggregates; and continued culture of the aforementioned mouse "embryo-cells" Aggregates to obtain mouse chimeric embryos.

The US20190002919A1 patent and the EP3495470A1 patent refer to a method of improving the fertilization capacity of a female mammal comprising administering one or more bioenergetic agents effective to enhance the oocyte or oocyte stem cell (OSC) of the female mammal. female mammalian subjects, thereby enhancing the fertility of the female mammalian subjects. The method of producing oocytes includes culturing stem cells in the presence of, but not limited to, oocyte stem cells, embryonic stem cells, skin stem cells, pancreatic stem cells and induced pluripotent stem cells (iPS cells). Bioenergetic agents or functional derivatives thereof are prepared into oocytes under conditions sufficient to differentiate stem cells. The cultivation method is through the administration of one or more bioenergetic agents and thus still has technical differences. The main difference of the present invention is that the differentiation of stem cells is verified and the separation and culture method is gradually established, which has the effect of enhancing the maturation of oocytes and assisting the development of eggs.

The problem to be solved by the present invention is as follows:

Although serum, electron carriers, amino acids or antioxidants can be used in the prior art to improve the maturation rate of in vitro maturation (IVM) and the development rate of in vitro culture (IVC) in oocytes, due to the The effect is not good, the development rate of in vitro culture is 8.0%, and the development rate of in vivo culture is 20.6%. The development rate of in vitro culture is still lower than that of in vivo culture (8.0% vs 20.6%), and in fact, there is still the disadvantage of polyspermia. .

Follicular fluid (FF) is a fluid that fills the follicular cavity and surrounds the egg in the ovarian follicle, which can be used for modified intracytoplasmic sperm injection.

Follicle-stimulating hormone (Follicle-stimulating hormone) is a hormone secreted and synthesized by the pituitary gland. It is a glycosylated protein hormone that regulates development, sexual maturation, and reproductive reproductive-related physiology.

Human chorionic gonadotropin (Human chorionic gonadotropin) is a glycoprotein hormone, which is secreted by the trophoblast cells of the placenta.

M199 (Sigma, M4530), used in cell culture, was originally developed for complete medium formulations. These media have broad applicability, especially for untransformed cells.

Porcine oocyte culture has the following in vitro culture basal medium, such as PZM series (PZM-3/PZM-4/PZM-5) medium and TCM-199. It is used for the metabolic and nutritional needs of pig oocyte in vitro development.

M199 was used as oocyte maturation medium, and PZM-3 was used as porcine zygote medium-3, PZM-3. PFTSCs are porcine fallopian tube stem cells, and BFTSCs are bovine fallopian tube stem cells.

The invention discloses a preparation method of porcine fallopian tube stem cells, which comprises the following steps: culturing porcine oocytes in IVM in M199 culture medium treated with porcine fallopian tube stem cells (PFTSCs), culturing in IVC in PZM-treated PFTSCs 3 culture medium.

Sources of stem cells are, for example, cord blood stem cells, embryonic stem cells, skin cells, blood cells, peripheral blood stem cells, and the like.

In 2006, the International Society for Cell Therapy (ISCT) standardized the definition of mesenchymal stem cells: adherent growth, expression of specific cell surface antigens (markers), such as CD44 and CD105, etc., in vitro (in vitro) with adipocytes. , osteoblast, chondrocyte differentiation capacity.

Among embryonic stem cells, OCT-4 is often used as a marker of stem cells, OCT-4 (Octamer-binding transcription factor 4): OCT-4 is involved in the control of cell self-renewal and stem cell differentiation.

Cluster of differentiation (CD) antigens: CD antigens are cell surface proteins that can be divided into different types, such as receptors. Since different cells have different CD antigens, antibodies are used to identify CD antigens on the cell surface for cell analysis.

The invention discloses a preparation method of porcine fallopian tube stem cells, which comprises the following steps: maturing and culturing porcine oocytes in vitro in M199 culture medium treated with porcine oviduct stem cells, and culturing them in vitro in PZM-treated porcine oviduct stem cells. 3 culture medium.

As described above, the pig fallopian tube stem cells express CD44 at a high level and CD105 at a moderate level.

As mentioned above, wherein the porcine fallopian tube stem cells have the potential to induce differentiation into scleroblasts, adipocytes and chondrocytes.

The present invention discloses a porcine fallopian tube stem cell preparation, which is prepared by the above-mentioned preparation method isolated from porcine fallopian tube stem cells, and the preparation comprises a composition prepared by the preparation method isolated from porcine fallopian tube stem cells.

The invention discloses a preparation method of porcine fallopian tube stem cells isolated from the porcine fallopian tube stem cells, which are used for preparing preparations for promoting oocyte maturation and embryo development in vitro and for establishing a treatment experimental platform system.

The invention discloses a preparation method of oviduct stem cells isolated from cattle, which comprises the following steps: maturing and culturing porcine oocytes in vitro; In the M199 medium, the PZM-3 medium that was matured in vitro by bovine fallopian tube stem cells.

As mentioned above, among the bovine fallopian tube stem cells, it highly expresses CD44.

As mentioned above, the bovine fallopian tube stem cells have the potential to induce differentiation into scleroblasts, adipocytes and chondrocytes.

The present invention discloses a porcine fallopian tube stem cell preparation, which is prepared by the above-mentioned preparation method isolated from cattle fallopian tube stem cells. The preparation comprises a composition prepared by the preparation method isolated from cattle fallopian tube stem cells.

The invention discloses a preparation method of fallopian tube stem cells isolated from cattle, the use of the cattle fallopian tube stem cells for preparing preparations for promoting oocyte maturation and embryo development in vitro and the use of establishing a treatment experimental platform system.

The effect of the present invention is mainly manifested in:

1. After adding the conditioned medium of porcine tubal stem cells and bovine tubal stem cells, the oocyte maturation rate was significantly improved (P<0.05). When added to IVC, the conditioned medium of the two cells significantly reduced the blastocyst development rate (P<0.05). <0.05).

2. Experiments have shown that bovine fallopian tube stem cells have the potential of regenerating stem cells and the ability of mesoderm-related cells to differentiate.

3. Experiments show that porcine fallopian tube stem cells and cattle fallopian tube stem cells can improve oocyte maturation.

4. Porcine fallopian tube stem cells and bovine fallopian tube stem cells, which can be used to prepare preparations for promoting oocyte maturation in vitro, embryo development, and to establish a therapeutic experimental platform system.

S101~S102: Steps of porcine fallopian tube stem cells to promote oocyte maturation in vitro and embryo development

S201~S202: cattle fallopian tube stem cells promote oocyte maturation in vitro and embryo development steps

Figure 1-1 shows the steps of promoting oocyte maturation and embryo development in vitro by porcine fallopian tube stem cells of the present invention;

Figure 1-2 shows the steps of promoting oocyte maturation and embryo development in vitro by cattle fallopian tube stem cells of the present invention;

Figure 2 is a cell culture state diagram of porcine fallopian tube stem cells and cattle fallopian tube stem cells of the present invention;

Fig. 3, Fig. 4 are RT-PCR analysis diagrams of the present invention;

Figure 5 and Figure 6 are flow cytometer analysis diagrams of the present invention;

Fig. 7 and Fig. 8 are cell diagrams of the present invention in osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation.

Experimental example:

material method

Animal source:

Taiwanese cattle (Bos indicus) and pig fallopian tubes were collected and rinsed immediately from the slaughterhouse with saline containing 1% penicillin/streptomycin (P/S, Gibco ^™ , 15140122, euApproved, south American), and within 2 hours the These tissues are brought to the cell lab.

Fallopian tube stem cell isolation experiment:

The fallopian tubes were washed twice with saline containing ¹ % penicillin/streptomycin, finely cut to a size of 0.1–0.5 mm, and placed in a serum supplemented with 10% fetal bovine serum (FBS, Gibco ^™ 10270106 and 1% penicillin/ Streptomycin-based α-MEM cell culture medium (Sigma M0894) was placed in a 1.5 ml microcentrifuge tube (Eppendorf, or called microcentrifuge tube) at 37° C. Subsequently, the fine tissue was placed in a 10 cm culture dish (TPP 93100), and then placed in Cultures were performed in cell culture conditions (Thermo Scientific ^™ ) at 38.5°C and 5% CO ₂ .

Flow cytometry analysis:

PFTSCs and BFTSCs of stem cells cultured to passage 5 (THE PASSAGE 5, P5) were added with 0.25% trypsin-EDTA (Gibco ^™ , 25200072) for 3 minutes, followed by centrifugation at 270G for 5 minutes. Subsequent addition of 100 μl Dulbecco's phosphate buffered saline (DPBS) (Gibco ^™ , 21600010) to suspend the cells, anti-porcine CD4 (Bio-rad, MCA1749GA), anti-porcine CD44 (anti-bovine) CD44) (Thermo Fisher Scientific, MA1-19277), anti-bovine CD44 (Bio-rad, MCA1653F), anti-bovine CD44 (Bio-rad, MCA2433F), anti-porcine CD105 (anti-porcine CD105) ( Thermo Fisher Scientific, MA5-11854) and anti-bovine CD105 (Thermo Fisher Scientific, MA5-11854), anti-mouse IgG1-PE (PE anti-mouse IgG1 Antibody) (eBioscience ^™ , 12-4015- 82) Add the appropriate concentration and place on ice for 30 minutes. Then, 800 [mu]l of DPBS was added and the antibody was removed by centrifugation. Finally, cells were suspended in 500 [mu]l DPBS, and the surface antigens were analyzed by flow cytometry and the background value of the secondary antibody was added.

RT-PCR analysis:

PFTSCs and BFTSCs cultured to passage 5 (THE PASSAGE 5, P5) were suspended in 0.25% trypsinized cell digest and washed twice with DPBS. RNA extraction and cDNA production were continued following the instructions of Qiagen RNeasy Micro Kit (74004). In the PCR experiment, referring to Mierni et al., Hu et al. and Gao et al., respectively, using porcine and bovine primers, GAPDH, Oct 4, CD29 and CD34 were analyzed, and analyzed at 95°C-45 seconds, 55°C-30 seconds and 30 PCR cycles were performed under the conditions of 72°C-50 seconds, please refer to Table 1 for the primer sequences of pigs and the primer sequences of cattle.

Table 1 Swine primer sequences and cattle primer sequences

Tri-lineage differentiation:

Osteogenic differentiation: For osteogenic differentiation, 1x10 ⁴ P5 PFTSCs and BFTSCs were cultured in 6 well dishes in differentiation medium containing 10% FBS. α-MEM, 10 mM glycerol phosphate (Sigma 17766), 50 μM ascorbic acid (Sigma, 33034) and 0.1 μM dexamethasone (Sigma, 31381) were maintained for 21 days.

Adipogenic differentiation: For adipogenic differentiation, 1x10 ⁵ P5 PFTSCs and BFTSCs were cultured in 6 well dishes (TPP 92006) in differentiation medium consisting of 10% FBS in α- MEM, 0.5 mM isobutyl-methylxanthine (Sigma 15879), 100 μM indomethacin (USpharmacopeia, 1341001), 10 μg/mL beef insulin (USpharmacopeia, 1342208) and 1 μM dexamethasone were maintained for 21 days.

Chondrogenic differentiation: For chondrogenic differentiation, 5x10 ⁵ P5 PFTSCs and BFTSCs were cultured in a 15 ml centrifuge tube in differentiation medium consisting of α-MEM with 1% FBS only, 50 μM ascorbic acid, 10 ng/mL Transforming growth factor-β2 (Sigma, T5300) and 6.25 μg/mL beef insulin were maintained for 21 days.

Differentiation verification: In 21 days, the differentiation medium was changed every 3 days by using Oil red O (Sigma, O0625), Alizarin red S (Sigma, A5533) and toluidine blue O (Toluidine blue O) (Sigma, T3260), the cells induced to differentiate were stained with the above reagents for confirmation.

Condition maturation medium production:

When the saturation of P5 cells in a 10 cm culture dish reached 80–90%, the medium was completely removed by washing with DPBS. Then, 3 ml of oocyte maturation medium (M199, Sigma, M4530) containing 10% FBS, 10 μL of follicle-stimulating hormone (FSH, Sigma, F-2293), 20 μL of human Human chorionic gonadotropin (hCG, Biovision, 4778-1000), 1% antibiotic-antimycotic (ABAM, Gibco, 15240-062) and 10% porcine follicular fluid ( porcine follicular fluid, PFF) or porcine zygote medium-3 (PZM-3) was added to each culture dish, and the cells were cultured for 3 hours. The prepared conditioned medium was filtered at 0.22 μm and placed in 4 Store at ℃.

Pig oocyte in vitro maturation (IVM) and in vitro culture (IVC) testing:

The obtained slaughterhouse pig ovaries were kept in 1% penicillin/streptomycin saline at 37°C, and after transport to the cell laboratory, the follicles were removed after washing 3 times with 1% penicillin/streptomycin saline within 1 hour. The solution was aspirated into a 15mL centrifuge tube with an 18G needle and a 10~20mL syringe, and incubated in a water bath at 37°C for 5 minutes to pellet the oocytes. The cumulus-oocyte complexes (COCs) were selected by pipetting the pelleted liquid into a 10 cm culture dish.

1. IVM test: The cumulus-oocyte complexes were washed twice with DPBS and then transferred to (1) normal maturation M199 medium (C), (2) PFTSCs-treated M199 maturation medium (porcine fallopian). tube stem cell treated M199 medium) (P), (3) PFTSCs-treated M199 maturation medium (bovine fallopian tube stem cell treated M199 medium) (B), 20-30 cumulus-oocyte complexes/100 μL, with Mineral oil was overlaid and incubated for 44 hours. After 44 hours, the oocyte complexes were treated with 0.1% hyaluronidase to remove cumulus cells and count the mature oocytes that released the first oocytes from each treatment. first polar body. Finally, mature oocytes were activated by treatment with calcium ionophore A23187 (Calcium Ionophore, A23187) for 5 minutes and 6-dimethylaminopurine (6-DMAP) for 4 hours and cultured under PZM-3 for 7 days .

2. IVC test: Cumulus-oocyte complexes were washed twice through DPBS and transferred to maturation medium for 44 hours. Mature and activated cumulus-free oocytes in (1) normal PZM-3 (normal PZM medium) (C), (2) PFTSCs-treated PZM-3 (porcine fallopian tube stem cell treated PZM medium) (P ), (3) BFTSCs-treated PZM-3 (bovine fallopian tube stem cell treated PZM medium) (B) for 7 days.

3. Different concentrations of IVC: Diluted porcine fertilized egg culture medium PZM-3 (conditioned PZM-3) with general PZM-3. Combine mature and activated oocytes with (1) PZM-3 (normal PZM medium) (C), (2) PZM-3 with 25% PFTSCs (25% PFTSCs treated PZM medium) (P25), (3) PZM-3 with 50% PFTSCs (50% PFTSCs treated PZM medium) ( P50), (4) PZM-3 with 25% BFTSCs (25% BFTSCs treated PZM medium) (B25), (5) PZM-3 with 50% BFTSCs (50% BFTSCs treated PZM medium) (B50) were cultured for 7 days ( Replace every 2 days). Record embryo stage results for each test on day 2, day 4 and day 7.

Statistical Analysis:

The data were analyzed and statistically analyzed by SAS 9.4 (Duncan method), and the eggs were cultured in the test conditioned medium. mother cells to test whether their maturation rate is increased.

Experimental results

Characterization, Cellular Expression, and Cell Differentiation of PFTSC and BFTSC:

When the finely divided tissue of the oviduct was cultured for 3 days, referring to Fig. 2, long stick-like cells were successfully isolated (Fig. 2A), which showed proliferative potential (Fig. 2B).

Referring to Figure 3, CD29 is expressed, but Oct4 and CD34 are not expressed.

Please refer to Figure 2, Short stick-like cells from bovine showed strong proliferative potential (Figure 2C and Figure 2D), please refer to Figure 4, and expressed CD29 and OCT4 mRNA, which is similar to MSCs related to pluripotency.

Referring to Figure 5, the expression of stem cell (MSCs) markers CD44 and CD105 were 76% and 8.0%, respectively, and the expression of T lymphocyte-related CD4 was 0.4%.

Referring to Figure 6, BFTSCs expressed 76.7% CD44, 7.6% CD105 and 3.7% CD4 in the surface antigen assay.

Please refer to FIG. 7A and FIG. 8A , after culturing PFTSCs and BFTSCs in osteogenic differentiation medium, calcium deposits gradually formed, and red complexes were shown by Alizarin Red S staining reagent.

Referring to Figures 7B and 8B, in adipocyte differentiation, both cell types showed the potential to differentiate into oil droplets (oil generation), which was confirmed by Oil Red O staining reagent.

Please refer to Fig. 7C and Fig. 8C, in the chondrocyte differentiation, cell sediments formed by PFTSCs and BFTSCs can be observed on the 3rd day of culture, and on the 21st day, the differentiated cartilage was embedded in the tissue and sliced through Toluidine blue O staining reagent showed evidence of matrix and intercellular space.

Pig oocyte maturation and development

Please refer to Table 2 below. After culturing porcine cumulus-oocyte complexes with conditioned maturation medium from PFTSCs and BFTSCs treatment, the maturation rate was higher than that in normal maturation medium, but There were no differences between groups in blastocyst rate.

Table 2. Effects of PFTSCs and BFTSCs-treated medium on in vitro maturation (IVM) of porcine oocytes

C: In vitro culture - in normal M199 maturation medium (IVM with normal maturation M199 medium).

P: In vitro culture-PFTSCs-treated M199 maturation medium (IVM with porcine fallopian tube stem cell treated M199 medium).

B: In vitro culture-BFTSCs-treated M199 maturation medium (IVM with bovine fallopian tube stem cell treated M199 medium).

The experiment was repeated 5 times.

^a , ^b in Table 2 represent significant differences between C, P and B groups.

Please refer to Table 3 below, in the in vitro culture test, compared with the general PZM-3 group, the morula rate of PZM-3 treated with PFTSCs was lower than that of the general PZM-3 group, and the morula rate from PFTSCs treatment was lower than that of the general PZM-3 group. and BFTSCs-treated PZM-3 had a decreased blastocyst rate.

Table 3. Effects of PFTSCs and BFTSCs-treated media on in vitro culture (IVC) porcine oocytes

C: In vitro culture in normal PZM-3 medium (IVC with normal PZM medium).

P: In vitro culture in PFTSCs-treated PZM-3 medium (IVC with porcine fallopian tube stem cell treated PZM medium).

B: In vitro culture in BFTSCs-treated PZM-3 medium (IVC with bovine fallopian tube stem cell treated PZM medium).

The experiment was repeated 6 times.

^a , ^b , ^ab in Table 3 represent significant differences between C, P and B groups.

Please refer to Table 4 below, when the porcine fertilized egg medium PZM-3 (conditioned PZM-3) was diluted with general PZM-3, the morula rate of the P50 group decreased on the 4th day of culture, while the P25 group and The morula rate and blastocyst rate of the P50 group were lower than those of the C group on the last day. in addition, There was no difference between the B25 group and the B50 group compared with the C group for the duration of 7 days.

Table 4. Effects of different concentrations of PFTSCs and BFTSCs treated with conditioned medium on IVC

C: In vitro culture - in normal PZM-3 medium (IVC with normal PZM medium).

P25: In vitro culture in 25% PFTSCs treated PZM-3 medium (IVC with 25% PFTSCs treated PZM medium).

P50: In vitro culture in 50% PFTSCs treated PZM-3 medium (IVC with 50% PFTSCs treated PZM medium).

B25: In vitro culture in 25% BFTSCs treated PZM-3 medium (IVC with 25% BFTSCs treated PZM medium).

B50: In vitro culture in 50% BFTSCs treated PZM-3 medium (IVC with 50% BFTSCs treated PZM medium).

The experiment was repeated 5 times.

^a , ^b , ^ab in Table 4 represent significant differences between C, P25, P50, B25 and B25.

It is proved from the above that the IVC of different concentrations of the present invention: wherein the porcine fertilized egg medium PZM-3 diluted with general PZM-3, preferably the PZM-3 medium treated with 25% PFTSCs in vitro, can still significantly reduce cysts. embryo development rate.

According to our experimental results, PFTSCs and BFTSCs can increase the maturation rate of porcine oocytes during IVM and contribute to the maturation of oocytes.

The present invention merely presents the preferred embodiments or examples of the technical means used to solve the problem, and is not intended to limit the scope of the patent application of the present invention. Equivalent changes and modifications are all covered by the patent of the present invention.

S101~S102: Steps of porcine fallopian tube stem cells to promote oocyte maturation in vitro and embryo development

S201~S202: cattle fallopian tube stem cells promote oocyte maturation in vitro and embryo development steps

Claims (10)

A method for preparing stem cells isolated from pig fallopian tubes, comprising the following steps: collecting and washing pig fallopian tubes; separating pig fallopian tube stem cells from the pig fallopian tubes and performing first culture to obtain pig fallopian tube stem cells after the first culture; preparing conditioned medium, and using The conditioned medium continues the second culture of the porcine fallopian tube stem cells after the first culture to obtain the isolated porcine fallopian tube stem cells; wherein, the conditioned medium is M199 medium or PZM-3 medium; the porcine oocytes are matured and cultured in vitro In M199 culture medium treated with porcine fallopian tube stem cells, it was cultured in vitro in PZM-3 culture medium treated with porcine fallopian tube stem cells. A method for preparing stem cells isolated from porcine fallopian tube according to claim 1, wherein the isolated porcine fallopian tube stem cells have a CD44 expression of 76-76.7% and a CD105 expression of 7.6-8.0%. A method for preparing stem cells isolated from porcine fallopian tube as claimed in claim 1, wherein the stem cells isolated from porcine fallopian tube have the potential to induce differentiation into osteocytes, adipocytes and chondrocytes. A porcine fallopian tube stem cell preparation, which is prepared from the porcine fallopian tube stem cells isolated as described in claim 1. A porcine fallopian tube stem cell isolated as claimed in claim 1 is used for preparing preparations for promoting oocyte maturation and embryo development in vitro and for establishing a therapeutic experimental platform system. A method for preparing oviduct stem cells isolated from cattle, which comprises the following steps: collecting and washing the oviducts of cattle; separating porcine oviduct stem cells from the oviducts of cattle and firstly culturing them to obtain the first cultured oviduct stem cells. Bovine fallopian tube stem cell; prepare conditioned medium, and continue the second culture of the first cultured bovine fallopian tube stem cell with the conditioned medium to obtain the isolated cattle fallopian tube stem cell; wherein, the conditioned medium is M199 medium or PZM-3 medium ; Pig oocytes were matured in vitro in M199 culture medium treated with bovine oviduct stem cells, and matured in vitro in PZM-3 culture medium treated with bovine oviduct stem cells. A method for preparing fallopian tube stem cells isolated from cattle as described in claim 6, wherein the CD44 expression of the isolated cattle fallopian tube stem cells is 76-76.7%. As claimed in claim 6, a method for preparing stem cells isolated from bovine fallopian tube, the stem cells isolated from bovine fallopian tube have the potential to induce differentiation to produce osteocytes, adipocytes and chondrocytes. A bovine fallopian tube stem cell preparation, which is prepared from the bovine fallopian tube stem cell isolated as described in claim 6. A fallopian tube stem cell isolated from cattle according to claim 6, used for preparing preparations for promoting oocyte maturation and embryo development in vitro and for establishing a therapeutic experimental platform system.

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