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Protective agent for improving ovarian function based on excessive autophagy injury and application of protective agent

A technology for ovarian function and protective agent, which is applied in the field of drug screening for early-onset ovarian insufficiency, and can solve the problem of no in vitro model and the like

Pending Publication Date: 2021-09-07
THE FIRST AFFILIATED HOSPITAL OF ANHUI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] So far, there is no good in vitro model that can provide theoretical basis and technical support for screening drugs for clinical treatment of POI

Method used

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  • Protective agent for improving ovarian function based on excessive autophagy injury and application of protective agent
  • Protective agent for improving ovarian function based on excessive autophagy injury and application of protective agent
  • Protective agent for improving ovarian function based on excessive autophagy injury and application of protective agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Melatonin (MT) inhibits excessive autophagy in hamster ovary epithelial cells CHO

[0031] 1) LC3-EGFP was transfected into CHO cells, and the LC3-CHO cell line stably expressing autophagy was selected by sorting flow;

[0032] The CHO cell line of ovarian epithelial cells was purchased from the cell bank of the Type Culture Collection, Chinese Academy of Sciences, trypsinized CHO cells, and seeded in 6-well plates (3×10 5 LC3-EGFP and control N1-EGFP were transfected into hamster ovary epithelial cells CHO after 24 hours of culture, using the transfection reagent lipofectamine3000; CHO cells were sorted out; in G418 complete medium containing 600ug / ml (including Dubucco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS), 2mM glutamine (L-glutamine) , 100 units / ml penicillin (penicillin), 0.1 mg / L streptomycin (streptomycin)), and screened out the LC3-CHO cell line stably expressing autophagy.

[0033] 2) Western Blot was used to detect the effe...

Embodiment 2

[0038] Example 2: Detecting the reduction of mitochondrial function in ovarian CHO cells after melatonin improves excessive autophagy injury;

[0039] 1) Using MitroTracker red staining to detect 10 -9 M Melatonin (MT) ameliorated the reduction of mitochondrial expression levels in CHO cells overexpressing autophagy, such as figure 2 shown.

[0040] 5μM MitoTracker Red dye was added to the cell culture medium of each group of CHO-N1, CHO-LC3 and MT+CHO-LC3, and incubated at 37°C for 30 minutes. Cells in each group were washed three times with PBS, and then the fluorescence intensity was detected by laser confocal microscope (LSM 800, Zeiss). The parameters used to obtain images remain the same. Compared with the control group (CHO-N1), the mean fluorescence intensity of MitroTracker red staining (representing the expression of mitochondria) in the CHO-LC3 group was significantly reduced, and the mitochondrial expression in the melatonin-treated group (MT+CHO-LC3) was signi...

Embodiment 3

[0051] Example 3: Detecting the increase of mitochondrial oxidative stress level in ovarian CHO cells after melatonin improves excessive autophagy injury;

[0052] 1) Using MitoSox staining to detect the effect of overexpression of autophagy on the oxidative stress level of CHO cells, such as Image 6 shown;

[0053] 5 µM MitoSox dye was added to the cell culture medium of each group of CHO-N1, CHO-LC3 and MT+CHO-LC3, and incubated at 37°C for 10 minutes. Cells in each group were washed three times with PBS, and then the fluorescence intensity was detected by laser confocal microscope (LSM 800, Zeiss). The parameters used to obtain images remain the same. Compared with the control group (CHO-N1), the mean fluorescence intensity of MitoSOX staining (representing the level of mitochondrial oxidative stress) in the CHO-LC3 group was significantly increased, and the mitochondrial oxidative stress in the melatonin-treated group (MT+CHO-LC3) was significantly increased. Levels we...

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Abstract

The invention discloses a protective agent for improving ovarian function based on excessive autophagy injury, develops a melatonin-containing agent for preventing or improving ovarian function reduction by taking autophagy as a target spot, and discloses application of the protective agent. The invention not only can improve the reduction of the mitochondrial function of the CHO cells, the increase of the oxidative stress level of the mitochondrial and the cell cycle arrest caused by the excessive autophagy injury, but also can improve the reduction of the ovarian reserve function of the CHO cells caused by the excessive autophagy injury.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of a protective agent for improving ovarian function with autophagy as a target in drug screening for premature ovarian insufficiency (POI). Background technique [0002] Reproductive health is a major issue in the contemporary world and has received extensive attention from the international community. As a country with a large population, the infertility rate of women of childbearing age in my country is as high as 15%. Premature ovarian insufficiency (POI) is one of the main causes of infertility in women of reproductive age. POI refers to the premature loss of ovarian function in women before the age of 40 due to various reasons. In recent years, the proportion of POI in women with infertility has been increasing year by year. [0003] At present, the etiology and pathogenesis of POI are still unclear. In the process of exploring the etiology and pathogenesis o...

Claims

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Application Information

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IPC IPC(8): A61K31/4045A61P15/08
CPCA61K31/4045A61P15/08
Inventor 刘雅静曹云霞谢庆娥纪冬梅梁丹杜馨王梦瑶魏兆莲周平
Owner THE FIRST AFFILIATED HOSPITAL OF ANHUI MEDICAL UNIV
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