Extraction and culture method of mouse ovarian granular cells

A technology of granulosa cells and culture methods, which is applied in the field of extraction and cultivation of mouse ovary granulosa cells, can solve the problems of few mouse ovary granulosa cells and unstable effects, and achieve the effect of simple ingredients, easy operation and short time-consuming

Pending Publication Date: 2021-10-01
JIANGSU PROVINCE INST OF TRADITIONAL CHINESE MEDICINE
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Problems solved by technology

At present, there are few studies on the in vitro isolation of mouse ovary granulosa cells in vitro, and the effect is unstabl

Method used

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  • Extraction and culture method of mouse ovarian granular cells
  • Extraction and culture method of mouse ovarian granular cells

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Embodiment Construction

[0024] The present invention selects 3-week-old, 10-15g immature female mice, and separates ovarian granulosa cells.

[0025] The specific operation is as follows:

[0026] Primary Granulosa Cell Extraction:

[0027] 1. Take 3-week-old immature female mice, inject PMSG 8IU intraperitoneally, and kill the mice by neck dislocation 46-48 hours later.

[0028] 2. Fix the mice, soak them in 75% alcohol for 10 seconds to disinfect, remove the ovaries quickly under aseptic conditions, wash away the blood with phosphate buffer saline (PBS), put the clean ovaries in a mixture of 2% Penicillin + 2% mycoplasma scavenger PBS (preheated at 37 ℃) in a 35mm culture dish, quickly remove the capsule and tissue around the ovary under a microscope, and transfer the stripped ovary to a new one. Wash 3 times in PBS buffer;

[0029] 3. Transfer the ovary to a 35mm Petri dish containing the operating solution, and incubate at 37°C for 10-15min;

[0030] 4. Gently puncture the follicle with a 1mL...

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Abstract

The invention belongs to a cell biology technology, and particularly relates to an extraction and culture method of mouse ovarian granular cells. According to the technical scheme, the method comprises the following specific steps: (1) injecting pregnant mare serum gonadotropin PMSG into the abdominal cavity of a female mouse; (2) killing the mouse by using a cervical vertebra dislocation method, separating the ovary of the mouse, cleaning with PBS (Phosphate Buffer Solution), and removing a fat envelope around the mouse under a microscope; (3) puncturing follicles in the mixed operation solution by using a syringe needle to release granular cells and oocytes, filtering by using a screen, centrifuging the filtrate, and discarding the supernatant; and (4) mixing the complete culture medium, resuspending the cells, precipitating and inoculating, wherein the operation liquid includes DME/F-12:1(1X), 3% of fetal calf serum, 2% of mycillin mixed liquid and 2% of mycoplasma scavenger. Furthermore, the mouse is an immature female mouse which is 3 weeks old and 10-13g in weight, the injection amount of the PMSG is 5-10IU, the injection method is intraperitoneal injection, the injection amount of the PMSG is preferably 8IU, and the time for killing the mouse after cervical vertebra dislocation is 46-48h after injection. The mechanical method provided by the invention is simple and convenient to operate and short in time consumption, and the obtained ovarian granular cells are high in yield.

Description

technical field [0001] This research belongs to cell biology technology, and specifically relates to a method for extracting and culturing mouse ovarian granulosa cells. Background technique [0002] Follicles are mainly composed of oocytes, granulosa cells and theca cells. Granulosa cells are a kind of somatic cells in the ovary. They develop earlier in the follicle than other cells. When their shape and number reach a certain level, the oocytes are Start to develop, so granulosa cells are the key factor in initiating follicular development. At the same time, granulosa cells express various gonadotropin receptors to further regulate the growth and maturation of follicles, and are also the initiators of follicular atresia. Therefore, granulosa cells can be used as a good cell model for studying reproductive endocrine changes, physiological function regulation and evaluating pharmacology in vitro in women and female animals. At present, there are few studies on the in vitro...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0682C12N2509/00
Inventor 陈玥陈思赵伟博卢燕王佩娟叶宇齐
Owner JIANGSU PROVINCE INST OF TRADITIONAL CHINESE MEDICINE
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