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Bovine theca cell in vitro culture modifier and application thereof

A technology of follicular theca and cell body, applied in the field of in vitro regulator of bovine follicular theca cells, achieving good application value

Active Publication Date: 2017-08-11
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

To date, there are no reports of the effects of melatonin on bovine theca cells

Method used

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  • Bovine theca cell in vitro culture modifier and application thereof
  • Bovine theca cell in vitro culture modifier and application thereof
  • Bovine theca cell in vitro culture modifier and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Effect of Melatonin on Steroid Synthesis in Bovine Large Follicular Theca Cells

[0036] 1. Cell Culture

[0037] The ovaries of non-pregnant cows were collected from a large slaughterhouse in the outer suburbs of Beijing. After collection, they were stored in 0.9% saline containing 1% penicillin and streptomycin, and arrived at the laboratory within 1 hour of transportation on ice. Theca cells are isolated from large follicles with a diameter of 8-22 mm, and the follicular blood vessels are required to be clear and the follicular fluid is transparent. After extracting the follicular fluid from the large follicle, cut it longitudinally with a scalpel, and scrape off the residual granulosa cells by blunt separation. The membranous cells were isolated by microdissection and digested on a shaker at 37°C for 1 hour. Undigested membranous cells were filtered through a 149 μm filter. The membranous cells were centrifuged at 50g at 4°C for 7 minutes, and the cultu...

Embodiment 2

[0046] Example 2 Effect of melatonin on CYP11A1, CYP17A1, CASP3 and StAR mRNA expression

[0047] 1. Cell Culture

[0048] The membranous cells obtained by cell culture in Example 1 were cultured for 24 hours in a medium containing 10% FBS, and three treatments were used: control, IGF1 (30ng / ml) and melatonin (2.3 μg / ml)+ IGF1 (30ng / ml), all treatments included 30ng / ml LH. After culturing for 24 hours, the medium was aspirated, and the cells were lysed for RNA extraction. The experiment was designed with 3 different repetitions. Each replicate of theca cells originates from 5-7 large follicles on 4-6 bovine ovaries.

[0049] 2. RNA extraction and RT-PCR quantitative determination of expression

[0050] After the cultivation, the culture medium of each well was aspirated; 0.5 ml of TRNzol reagent (Tiangen Biochemical Technology Co., Ltd., Beijing) was added to the well, and RNA was extracted. Each treatment included 2 RNA samples. RNA samples were measured at 260 nm absor...

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Abstract

The invention provides a bovine theca cell in vitro culture modifier. A bovine theca cell in vitro culture solution is taken as a substrate, and melatonin is contained in the substrate. When the in vitro culture solution where the melatonin is added to culture bovine theca cells, the melatonin can inhibit synthesis of bovine theca cell progesterone, the melatonin inhibits StAR and CASP3 expression induced by LH+IGF1, but has no influence on CYP11A1 and CYP17A1 expression. However, the melatonin can promote bovine theca cell proliferation, the melatonin serves as an endocrine regulating factor of a bovine ovary function, effects of LH and IGF1 are inhibited by down-regulation of synthesis of steroid hormones, and membrane cell differentiation is delayed. The melatonin has good application values by being applied to preparation for promoting bovine theca cell proliferation or inhibiting steroidogenesis.

Description

technical field [0001] The invention relates to the technical field of livestock gamete embryo engineering, in particular to an in vitro regulator of bovine theca cells and an application thereof. Background technique [0002] As one of the three main cells in the ovary, theca cells play a role in various functions of the ovary, such as steroid synthesis, follicular development and follicular atresia. Theca cells originate from fibroblast-like stromal cells in the ovary, which can secrete androgen, interact with follicle-stimulating hormone (FSH), and promote the proliferation of granulosa cells and the development of follicles. [0003] Melatonin, N-acetyl-5-methoxytryptamine (Melatonine, MT), is mainly secreted by the pineal gland, and its biological functions are diverse, including regulation of biological cycle rhythm, sleep function, reproduction and anti-oxidative stress functions, etc. . Studies have shown that in mammals, melatonin affects sexual development and re...

Claims

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Application Information

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IPC IPC(8): C12N5/075
CPCC12N5/0609C12N2501/105C12N2501/30C12N2501/31
Inventor 冯涛刘彦白佳桦许晓玲宋玉清肖霖力肖银霞
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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