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Method for testing influence of midazolam on development of testicular interstitial cells cultured in vitro

A technology of Leydig cells and midazolam, which is applied in the field of biochemical experiments, can solve the problems of unclear long-term effects on the development of Leydig cells

Active Publication Date: 2022-03-15
THE SECOND HOSPITAL AFFILIATED TO WENZHOU MEDICAL COLLEGE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the potential long-term effects of repeated use of the drug during adolescence on the development of Leydig cells are unknown;

Method used

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  • Method for testing influence of midazolam on development of testicular interstitial cells cultured in vitro
  • Method for testing influence of midazolam on development of testicular interstitial cells cultured in vitro
  • Method for testing influence of midazolam on development of testicular interstitial cells cultured in vitro

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Embodiment Construction

[0060] Midazolam of the present invention is to the embodiment of the test method of the Leydig cell development influence of in vitro culture: comprise the following steps,

[0061] Step 1, select adult male SD rats of 2-4 months as the experimental subjects, dissolve EDS in the mixture of DMSO:PBS=1:3, and give SD rats intraperitoneal injection at a dose of 80mg / kg, after After four days of EDS treatment, SD rats were sacrificed;

[0062]Step 2, quickly take out the SD rat testis and place it in ice-cold Hanks' balanced salt solution, remove the testicular capsule, place the parenchymal tissue in DMEM / F12 medium containing 1 mg / ml collagenase IV, 34°C water bath shaker, shake 90 times / min, digest the tissue for 30 minutes; after the digested tissue precipitates at room temperature for 1 minute, filter the supernatant with a nylon mesh with a pore size of 70 μm; use magnetic bead sorting (MACS) for CD90+ cells For forward sorting, the obtained cell pellets were suspended in ...

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Abstract

The invention discloses a method for testing influence of midazolam on development of testicular interstitial cells cultured in vitro. Comprising the following steps: culturing rats, separating CD90 positive cells, culturing testicular mesenchymal stem cells (SLC), detecting the influence of midazolam on proliferation and differentiation, amplifying the SLCs and detecting the cell activity, carrying out DAPI dyeing and counting, analyzing cells by a flow cytometer, analyzing testosterone (T) and progesterone (P4), carrying out mRNA and protein expression detection through QPCR (Quantitative Polymerase Chain Reaction) and WB (White Brown) experiments, and determining significant differences and drawing a chart through SNK (Sodium Nitrogen Kinase) detection. The steps are simple, and the influence of midazolam medicine on proliferation and differentiation of the testicular mesenchymal stem cells can be effectively verified.

Description

technical field [0001] The invention relates to the technical field of biochemical experiments. Background technique [0002] Midazolam is a neurological drug with multiple functions, including sedative, hypnotic, anxiety-reducing, anterograde amnesia, brain-mediated muscle relaxation, and anticonvulsant activity. Because the drug is often used long-term in children and adolescents, the possible effects of the drug on other physiological systems also need to be studied. Male infertility rates have been rising in recent decades, but the reasons behind it remain unclear. A recent study found that men's average sperm count has declined by an average of 1.4 percent per year over the past 50 years, with an overall decline of 52.4 percent between 1973 and 2011. Leydig cells (LCs) are the major source of testosterone (T) in male mammals, including male humans. Androgens are essential for the maintenance of male reproduction (including spermatogenesis) and normal male physiologic...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12Q1/6851C12Q1/02G01N33/68
CPCC12N5/0683C12Q1/6851G01N33/5044G01N33/68C12N2503/02C12N2509/00C12Q2600/158C12Q2531/113
Inventor 陈浩林季敏鹏赵星仪温馨陈丹
Owner THE SECOND HOSPITAL AFFILIATED TO WENZHOU MEDICAL COLLEGE
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