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Application of FRT cell strain in preparation of preparation or kit for screening CaCC regulator

A cell line and regulator technology, applied in the field of biomedicine, can solve the problems of cumbersome operation, long detection period, and the inability of fluorescent proteins to penetrate cell membranes.

Pending Publication Date: 2021-01-29
JINLIN MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ca 2+ The disadvantage of the activated fluorescent protein detection method is low sensitivity, fluorescent protein cannot penetrate the cell membrane, and requires microinjection and other methods to load cells, which requires high technical requirements
The disadvantages of the fluorescent indicator method are cumbersome operation, high reagent consumption, long detection cycle and poor repeatability.
The disadvantage of patch clamp technology is that it requires specific instruments and equipment, requires high technical requirements for operators, and is expensive.

Method used

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  • Application of FRT cell strain in preparation of preparation or kit for screening CaCC regulator
  • Application of FRT cell strain in preparation of preparation or kit for screening CaCC regulator
  • Application of FRT cell strain in preparation of preparation or kit for screening CaCC regulator

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1 Construction of co-expression ANO1 and YFP-H148Q / I152L cell model

[0087] 1. Construction of cell lines co-expressing ANO1 and YFP-H148Q / I152L: According to the Lipofectamine 3000 instructions, the ANO1 plasmid was transiently transfected into FRT cells, screened with zeocin antibiotics, and observed with an inverted fluorescence microscope two weeks later. The cells with green fluorescence were limitedly diluted, and the cell lines of the positive clones obtained were expanded and cultured. The cells still expressing ANO1 after two passages were FRT cell lines stably expressing ANO1. According to the instructions of Lipofectamine 3000, the YFP-H148Q / I152 plasmid was transfected into the FRT cells that had stably expressed ANO1, screened with G418 antibiotics, observed with an inverted fluorescence microscope two weeks later, and the cells with green fluorescence in the cytoplasm were picked for limited After dilution, the cell lines of the obtained positive ...

Embodiment 2

[0089] Embodiment 2 electrophysiological recording

[0090] Whole-cell (whole-cell) patch-clamp technique recording was carried out at room temperature, and FRT cells transfected with ANO1 and YFP-H148Q / I152L in good condition were inoculated on glass slides, placed under an inverted fluorescent microscope on the second day, and externally detected with electrodes. Liquid perfusion, the resistance of the electrode after entering the water is about 4 ~ 6MΩ, after the negative pressure is applied to form a GΩ high-impedance seal, the negative pressure is quickly applied to rupture the cell membrane at the tip of the electrode, forming a whole-cell recording mode. The initial clamping voltage was 0mV. After recording for 10ms, a stepwise voltage stimulus was given. The voltage was from -80mV to +80mV. Each increase of 20mV was recorded for 800ms. After 800ms, the voltage became 0mV, and the recording was continued for 100ms. Electrode liquid: 140mmol / L NMDG-Cl, 2mmol / L MgCl 2 , ...

Embodiment 3

[0091] Example 3 Fluorescence Quenching Kinetics Experiments to Identify the Validity of Cell Models

[0092] The FRT cells stably co-transfected with ANO1-YFP-H148Q / I152L were inoculated into 96-well plates with black walls and transparent bottom, and cultured for 18 hours. Divide the cells into two groups: the experimental group and the control group. The cells of the two groups were washed 3 times with PBS buffer containing calcium and magnesium ions, 50 μL of PBS buffer containing calcium and magnesium was added, 120 μL of sodium iodide PBS buffer containing ionomycin (CaCC activator) was added to the experimental group, and T16Ainh was added to the control group. -A01 (CaCC-specific inhibitor) was incubated for 10 min, and then 120 μL of PBS buffer containing ionomycin sodium iodide was added, and the dynamic change of relative fluorescence intensity was detected with a Fluo star multifunctional microplate reader. The specific settings are as follows: the emission wavele...

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Abstract

The invention relates to the field of biological medicine, in particular to application of an FRT cell strain in preparation of a preparation or a kit for screening a CaCC regulator.] The ANO1 is observed to be expressed on a cell membrane under an inverted fluorescence microscope, and YFP-H148Q / I152L is expressed in cytoplasm; a model has the physiological characteristics of a classic calcium-activated chloride ion channel; an FRT cell model for co-expression of ANO1 and YFP-H148Q / I152L is successfully constructed; the model can be used for screening a CaCC regulator, and a fluorescence change slope value and the concentration of the CaCC regulator form a dose-dependent relationship; and the fluorescence change slope value can reflect the concentration of Ca<2+> in the cytoplasm, and themodel can sensitively detect the concentration of the Ca<2+> in the cytoplasm. The cell model can efficiently and sensitively detect the concentration of a second messenger Ca<2+> in the cytoplasm, and a simple, convenient and rapid method is provided for research of Ca<2+> signal-related targets.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the application of FRT cell lines in preparing preparations or kits for screening CaCC regulators. Background technique [0002] The second messenger is one of the starting components of intracellular signal transduction, mainly including: calcium ion (Ca2+), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), inositol triphosphate (IP3), Diacylglycerol (DAG), etc. Ca 2+ As a ubiquitous second messenger in the cytoplasm, it can regulate a variety of important physiological processes in the cell, such as participating in the synthesis and release of neurotransmitters, regulating the maturation and fertilization of germ cells, and regulating the activities of various enzymes in the body. intracytoplasmic Ca 2+ It is also involved in the occurrence of diseases such as hypertension, coronary atherosclerotic heart disease, and Alzheimer's disease. Therefore, th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02C12N5/10G01N21/64
CPCG01N33/5044G01N21/6428C12N5/0617C12N5/0625C07K14/47G01N2500/10C12N2510/00
Inventor 郝峰郑锴朱靓陈爽杨娟洪啟元解宇浩张嘉琪
Owner JINLIN MEDICAL COLLEGE
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