Sheep ovarian granular cell separation, culture and identification method

Abstract

The invention discloses a sheep ovarian granular cell separation, culture and identification method, and belongs to the technical field of animal cell culture. Ovarian granular cells are obtained through special treatment modes of collection, disinfection, cutting and the like. The method has the advantages of being simple, convenient, labor-saving, low in pollution rate, high in follicular fluid obtaining rate and the like, and a foundation is laid for research on development of ovarian granular cells, oocytes and follicles.

Description

technical field

[0001] The invention belongs to the technical field of animal cell culture, and in particular relates to a method for separating, cultivating and identifying sheep ovary granulosa cells. Background technique

[0002] The development of sheep follicles is a very complex and orderly process that combines endocrine regulation and external regulation. Granulosa cells and oocytes are the main components of follicles. all play an important role. Understanding the development process and regulation mechanism of sheep follicles can effectively improve the ovulation rate of sheep and further improve the reproductive efficiency of sheep.

[0003] Granulosa cells (GCs), as the main functional cells of the mammalian ovary, play a vital role in the development of follicles. There are follicle stimulating hormone (FSH) and luteinizing hormone (LH) receptors on the surface of ovarian granulosa cells, which can promote the proliferation of granulosa cells and the secretion...

Examples

Embodiment 1

[0045] (1) Kill the adult sheep by neck bleeding, collect the ovaries, wash and disinfect them with alcohol, and place them in preheated normal saline (containing 1% double antibody, 100 U / mL penicillin and 100 μg / mL streptomycin) (37 ℃) to the sterile room within 2h-4h.

[0046] (2) Soak the ovary collected in step (1) in 75% alcohol for 10 seconds, wash it with 37°C normal saline three times, remove excess fat and mesentery on the ovary, wash it with 37°C normal saline three times, and transpose 10mL DMEM / F12 complete medium prepared in a clean bench [DMEM / F12 (45mL) + FBS (5mL) + double antibody (500μL, the double antibody includes 100U / mL penicillin and 100μg / mL streptomycin) + 50 μg / mL sodium pyruvate (1 μL)], cut 3-7 mm follicles with a blade (eg figure 1 shown), release follicular fluid, and operate under the liquid surface throughout the process.

[0047] (3) Place the cell culture medium suspension in a 15mL centrifuge tube, centrifuge at 1500r / min for 10min, discar...

Embodiment 2

[0050](1) Kill the adult sheep by neck bleeding, collect the ovaries, wash and disinfect them with alcohol, and place them in preheated normal saline (containing 1% double antibody, 100 U / mL penicillin and 100 μg / mL streptomycin) (37 ℃) within 4 hours to transfer to a sterile room.

[0051] (2) Soak the ovary collected in step (1) in 75% alcohol for 10 seconds, wash it with 37°C normal saline three times, remove excess fat and mesentery on the ovary, wash it with 37°C normal saline for 5 times, and transpose 10mL DMEM / F12 complete medium prepared in a clean bench [DMEM / F12 (45mL) + FBS (5mL) + double antibody (500μL, the double antibody includes 100U / mL penicillin and 100μg / mL streptomycin) + 50 μg / mL sodium pyruvate (1 μL)], cut 3-7 mm follicles with a blade, and release follicular fluid.

[0052] (3) Place the cell culture medium suspension in a 15mL centrifuge tube, centrifuge at 1500r / min for 10min, discard the supernatant; wash the pellet with PBS, centrifuge at 1500r / mi...

Embodiment 3

[0055] (1) The adult multiparous sheep were sacrificed by neck bleeding, and the ovaries were collected and placed in preheated normal saline (37°C) (containing 1% double-antibody, 100U / mL penicillin and 100μg / mL streptomycin) within 4h Transfer to a sterile room.

[0056] (2) Soak the ovary collected in step (1) in 75% alcohol for 10 seconds, wash it with 37°C normal saline three times, remove excess fat and mesentery on the ovary, wash it with 37°C normal saline for 5 times, and transpose 10mL DMEM / F12 complete medium prepared in a clean bench [DMEM / F12 (45mL) + FBS (5mL) + double antibody (500μL, the double antibody includes 100U / mL penicillin and 100μg / mL streptomycin) + 50 μg / mL sodium pyruvate (1 μL)], cut 3-7 mm follicles with a blade, and release follicular fluid.

[0057] (3) Place the cell culture medium suspension in a 15mL centrifuge tube, centrifuge at 1500r / min for 10min, discard the supernatant; wash the pellet with PBS, centrifuge at 1500r / min for 10min, disca...

technical field

[0001] The invention belongs to the technical field of animal cell culture, and in particular relates to a method for separating, cultivating and identifying sheep ovary granulosa cells. Background technique

[0002] The development of sheep follicles is a very complex and orderly process that combines endocrine regulation and external regulation. Granulosa cells and oocytes are the main components of follicles. all play an important role. Understanding the development process and regulation mechanism of sheep follicles can effectively improve the ovulation rate of sheep and further improve the reproductive efficiency of sheep.

[0003] Granulosa cells (GCs), as the main functional cells of the mammalian ovary, play a vital role in the development of follicles. There are follicle stimulating hormone (FSH) and luteinizing hormone (LH) receptors on the surface of ovarian granulosa cells, which can promote the proliferation of granulosa cells and the secretion...

Examples

Embodiment 1

[0045] (1) Kill the adult sheep by neck bleeding, collect the ovaries, wash and disinfect them with alcohol, and place them in preheated normal saline (containing 1% double antibody, 100 U / mL penicillin and 100 μg / mL streptomycin) (37 ℃) to the sterile room within 2h-4h.

[0046] (2) Soak the ovary collected in step (1) in 75% alcohol for 10 seconds, wash it with 37°C normal saline three times, remove excess fat and mesentery on the ovary, wash it with 37°C normal saline three times, and transpose 10mL DMEM / F12 complete medium prepared in a clean bench [DMEM / F12 (45mL) + FBS (5mL) + double antibody (500μL, the double antibody includes 100U / mL penicillin and 100μg / mL streptomycin) + 50 μg / mL sodium pyruvate (1 μL)], cut 3-7 mm follicles with a blade (eg figure 1 shown), release follicular fluid, and operate under the liquid surface throughout the process.

[0047] (3) Place the cell culture medium suspension in a 15mL centrifuge tube, centrifuge at 1500r / min for 10min, discar...

Embodiment 2

[0050](1) Kill the adult sheep by neck bleeding, collect the ovaries, wash and disinfect them with alcohol, and place them in preheated normal saline (containing 1% double antibody, 100 U / mL penicillin and 100 μg / mL streptomycin) (37 ℃) within 4 hours to transfer to a sterile room.

[0051] (2) Soak the ovary collected in step (1) in 75% alcohol for 10 seconds, wash it with 37°C normal saline three times, remove excess fat and mesentery on the ovary, wash it with 37°C normal saline for 5 times, and transpose 10mL DMEM / F12 complete medium prepared in a clean bench [DMEM / F12 (45mL) + FBS (5mL) + double antibody (500μL, the double antibody includes 100U / mL penicillin and 100μg / mL streptomycin) + 50 μg / mL sodium pyruvate (1 μL)], cut 3-7 mm follicles with a blade, and release follicular fluid.

[0052] (3) Place the cell culture medium suspension in a 15mL centrifuge tube, centrifuge at 1500r / min for 10min, discard the supernatant; wash the pellet with PBS, centrifuge at 1500r / mi...

Embodiment 3

[0055] (1) The adult multiparous sheep were sacrificed by neck bleeding, and the ovaries were collected and placed in preheated normal saline (37°C) (containing 1% double-antibody, 100U / mL penicillin and 100μg / mL streptomycin) within 4h Transfer to a sterile room.

[0056] (2) Soak the ovary collected in step (1) in 75% alcohol for 10 seconds, wash it with 37°C normal saline three times, remove excess fat and mesentery on the ovary, wash it with 37°C normal saline for 5 times, and transpose 10mL DMEM / F12 complete medium prepared in a clean bench [DMEM / F12 (45mL) + FBS (5mL) + double antibody (500μL, the double antibody includes 100U / mL penicillin and 100μg / mL streptomycin) + 50 μg / mL sodium pyruvate (1 μL)], cut 3-7 mm follicles with a blade, and release follicular fluid.

[0057] (3) Place the cell culture medium suspension in a 15mL centrifuge tube, centrifuge at 1500r / min for 10min, discard the supernatant; wash the pellet with PBS, centrifuge at 1500r / min for 10min, disca...

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