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Transgenic cell assay for combined endocrine disrupting effects of glucocorticoids

A technology of transgenic cells and glucocorticoids, which is applied in the determination/inspection of cells and microorganisms modified by the introduction of foreign genetic material, and the screening of compounds. It can solve the problems of long detection time, high cost, and inability to detect, and achieve sensitivity High, low cost, cost reduction effect

Active Publication Date: 2020-12-18
INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, there are in vitro bioassay methods based on cell proliferation, and in vivo biological methods through animal experiments such as organ weight gain and growth and development, but there are some bottlenecks and deficiencies in the above methods, mainly reflected in: (1) glucocorticoid Glucocorticoid EDCs are ubiquitous in the environment and food, hospital discharge, use of plasticizers and packaging materials, agricultural input residues, environmental heavy metal background, etc., a large number of unknown or known glucocorticoid EDCs prototypes and their metabolites may be Missed or impossible to detect, the existing methods such as mass spectrometry and ELISA are unbalanced between high-throughput, sensitivity, time and cost. At present, such investigation and monitoring have not been carried out in large quantities, mainly because of high cost and long detection time , so it is very important to develop new detection methods with higher throughput, lower cost, good sensitivity and reasonable detection time; (2) Substances that induce glucocorticoid effects exist widely, and there are many known and unknown chemicals and For its structural analogues, the urgency of developing a more broad-spectrum, non-targeting, and overall screening method for glucocorticoid EDCs is particularly prominent

Method used

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  • Transgenic cell assay for combined endocrine disrupting effects of glucocorticoids
  • Transgenic cell assay for combined endocrine disrupting effects of glucocorticoids
  • Transgenic cell assay for combined endocrine disrupting effects of glucocorticoids

Examples

Experimental program
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Effect test

Embodiment 1

[0057] A method for screening transgenic cell lines with combined effects of glucocorticoid endocrine disruption

[0058] 1) Extraction and sequencing of plasmids

[0059] Plasmid pTet-GFP-GR[ was produced by pTet-splice (such as figure 1 shown) and GFP-GR whole gene recombination], pTet-tTAK (such as figure 2 shown) and pSV2-neo (as shown image 3 shown) were transformed into Escherichia coli Trans5α competent cells by heat shock method, spread on LB (Amp) plates for culture, cultured at 37°C overnight, and single clone strains were picked respectively. The obtained monoclonal strain was transferred to 30 mL LB (Amp) liquid medium for overnight culture, and the plasmid was extracted using the endotoxin-free plasmid mini-extraction kit.

[0060] The plasmid concentration is 147-508ng / μL (OD 260 / 280 Between 1.82-1.85), 20 μL samples were taken respectively and sent to Sangon for sequencing, and the sequencing results were all correct, such as Figure 10 shown, the rest ar...

Embodiment 2

[0074] Determination method of transgenic cell line for combined effect of glucocorticoid endocrine disruption (taking dexamethasone as an example)

[0075] 1) Dosing time and dosing concentration tests on the screened Hela-GFP-GR stable strain cells.

[0076] In order to eliminate the influence factors in the medium and serum as much as possible, the CD-FBS was treated with phenol red-free MEM medium and activated carbon. Hela-GFP-GR removed tet and cultured for 1d, digested and counted, according to 10 4 Each well was inserted into a black 96-well plate, and after culturing overnight, Dexa 10 -6 mol / L, 10 -7 mol / L, 10 -9 mol / L, 10 -11 mol / L and 10 -13 mol / L, a blank control (DMSO) was set, at least 3 parallels were performed for each concentration, and the cells were fixed and stained with Hochest33342 (Ex / Em 350 / 361nm) after 0.5h, 1h and 4h after administration, respectively.

[0077] The result is as Figure 7 Shown: The results of high-content acquisition images sh...

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Abstract

The invention discloses a transgenic cell strain of a glucocorticoid endocrine disrupting joint effect. A Hela cell is selected for transfecting glucocorticoid receptors with a green fluorescent protein tag; and through antibiotics screening, a stable GFP-GR transfection Hela cell strain is obtained. The invention also discloses an assay method of the transgenic cell strain. The assay method comprises the following steps of Step 1, building a transgenic cell strain of the glucocorticoid endocrine disrupting joint effect according to an action mechanism of glucocorticoids and glucocorticoid receptors; and Step 2, through administration on the transgenic cell strain, the motion rate of the cell strain from a cytoplasmic region to a nuclear region is quantitatively detected. The method has the advantages that a tested cell carrier can be used through infinite amplification without additional cost loss; through a high throughput screening technology, the synchronous detection of a plurality of samples can be realized; and the characteristics of low cost, ultrahigh flux and high sensitivity are realized. The method can be used for detecting glucocorticoid EDCs such as dairy products, sewage and urine of raised animals at a living body level.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a transgenic cell assay method for combined effects of glucocorticoid endocrine interference. Background technique [0002] Endocrine disrupting chemicals (EDCs), also known as environmental hormones (Environmental Hormone), are substances present in food and the environment that can interfere with the endocrine system of humans or animals and cause abnormal effects. EDCs can affect many endocrine systems such as reproduction, mammary gland development, neuroendocrine, thyroid metabolism, cancer, obesity, and cardiovascular system by interfering with the synthesis, secretion, transportation, and metabolism of natural hormones in animals. It can also cause endocrine disorders. Endocrine effects involve estrogen, androgen, glucocorticoid and mineralocorticoid effects, etc. Glucocorticoids are a kind of adrenocortical hormones, which exert their functions through glucocort...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/65C12Q1/02C12R1/91
CPCC07K14/721C12N15/65C12N15/85G01N33/5029G01N2500/10
Inventor 李耘刘畅钱永忠邱静任亚林李金娟
Owner INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS