Mouse ovarian granulosa cell separation, culture and in-vitro injury model construction method

A granulosa cell and cell culture technology, applied in the field of cell culture, can solve the problems of poor understanding of the maintenance of ovarian granulosa cells after passage, apoptosis of ovarian granulosa cells, etc., to improve the yield and avoid the effects of influence.

Pending Publication Date: 2021-01-15
NANCHANG UNIV
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AI Technical Summary

Problems solved by technology

[0005] So far, the extraction method of ovarian granulosa cells and the maintenance of performance after passage are not well unders

Method used

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  • Mouse ovarian granulosa cell separation, culture and in-vitro injury model construction method
  • Mouse ovarian granulosa cell separation, culture and in-vitro injury model construction method
  • Mouse ovarian granulosa cell separation, culture and in-vitro injury model construction method

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Embodiment 1

[0057] The PMSG hormone was injected into 3-4 week-old mice by intraperitoneal injection, and 48 hours later, the abdominal cavity of the mice was opened to expose bilateral ovary tissues. Remove the mouse ovarian tissue with sterile surgical scissors, wash the ovarian tissue with PBS, and remove the adipose tissue and connective tissue around the ovarian tissue. The ovarian granulosa cells were released by puncturing the follicles with a 1 mL syringe needle under a stereomicroscope. After puncturing the follicles, the ovarian tissue was digested in 0.25% trypsin for 30 minutes, and then the digestion was terminated. The two were combined into a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 minutes. After removing the supernatant, resuspend the cells with the complete medium of ovarian granulosa cells, and then inoculate them in 12-well or 6-well plates at 37°C, 5% CO 2 cultured in an incubator. After 48 hours, the original medium was discarded, and fresh complete ...

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Abstract

The invention discloses a mouse ovarian granulosa cell extraction and in-vitro chemotherapy injury model construction method. The method mainly comprises the following steps of 1) injecting PMSG to promote ovulation of a young mouse with age of 3-4 weeks; 2) performing mouse ovary separation and follicle breakage: dissecting the abdomen of the mouse subjected to ovulation induction for 48 hours, taking out ovaries on two sides, stripping fat tissues and connective tissues around the ovaries, puncturing follicles by using a 1 mL syringe needle under a microscope to release ovarian granulosa cells, sieving the cells with a 200-mesh sieve, and then putting the cells into a 15 mL centrifuge tube for later use; 3) digesting the ovaries with 0.25% pancreatin: putting ovary tissues after folliclebreakage into the 0.25% pancreatin, digesting theovary tissues in a water bath kettle of 37 DEG C for 30 minutes, sieving the ovary tissues with the 200-mesh sieve, putting the ovary tissues into the15 mL centrifuge tube, and centrifuging the ovary tissues to obtain a large amount of ovarian granulosa cells with higher purity; and 4) constructing an ovarian granulosa cell in-vitro chemotherapy injury model: treating the ovarian granulosa cells with cyclophosphamide (CTX) with the concentration of 5 mg/mL for 24 hours to obtain the ovarian granulosa cell chemotherapy injury model.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a method for separating and culturing mouse ovarian granulosa cells and constructing an in vitro injury model. Background technique [0002] The ovary is an important reproductive organ for women, and it is also a very important endocrine organ. In addition to the function of ovulation, the ovary also has the ability to secrete hormones, which is very important for women to maintain the balance of hormone levels in the body. Ovarian granulosa cells are very important functional cells in the ovary. The proliferation and differentiation of ovarian granulosa cells will directly affect the development, growth, atresia and secretion of hormones of follicles, thereby affecting the function of the ovary. [0003] Premature ovarian failure is an ovarian functional disease that seriously affects women's physical and mental health. It mainly refers to the irreversible damage to ovarian functio...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0682C12N2500/84C12N2509/00C12N2509/10
Inventor 柳全文辛洪波顾皓铖李婧嫄
Owner NANCHANG UNIV
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