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Cell autophagy monitoring probe, preparation method therefor and use of cell autophagy monitoring probe

A cell and autophagy technology, applied in the fields of biochemical equipment and methods, chemical instruments and methods, measuring devices, etc., can solve the problems of autophagy monitoring is not special, cannot monitor the autophagy state of living cells, and is expensive. , to achieve the effect of simple structure, simple operation and low cytotoxicity

Active Publication Date: 2017-04-26
ANHUI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These traditional monitoring methods have their limitations, such as scanning electron microscopy and Western blot, they cannot monitor the autophagy state in living cells, and at the same time, these methods are very complicated to operate and expensive, and the monitoring of autophagy not a special image

Method used

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  • Cell autophagy monitoring probe, preparation method therefor and use of cell autophagy monitoring probe
  • Cell autophagy monitoring probe, preparation method therefor and use of cell autophagy monitoring probe
  • Cell autophagy monitoring probe, preparation method therefor and use of cell autophagy monitoring probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: the synthesis of fluorescent probe molecule Lyso-OSC

[0034]7-((4-methoxyphenyl)ethylene)-3-carboxylic acid-coumarin (1g, 3mmol), N,N-diisopropylethylamine (1.2g, 9mmol), 1-hydroxy Benzotriazole (0.65g, 4.5mmol), EDC·HCl (0.65g, 3.3mmol) and 2-morphine-1-ethylamine (1.2g, 9mmol) were added to the Shrek bottle, under anhydrous and oxygen-free conditions Add 30mL of N,N-dimethylformamide, stir and react at room temperature for 24h; after the reaction is completed, add 50mL of dichloromethane to the reaction solution to dissolve the reactant, and then extract with 30mL of water (3 times) to obtain an organic phase. Through column chromatography on 200-300 mesh silica gel (the eluent is composed of ethyl acetate and petroleum ether mixed at a volume ratio of 1:1), 0.35 g of the target product was obtained with a yield of 40%.

[0035] 1 H NMR (400MHz, CDCl 3 )δ9.13(s,1H),8.86(s,1H),7.63(d,J=8.0Hz,1H),7.53(s,1H),7.50(s,1H),7.49(s,1H), 7.46(s,1H),6.95(d,J=8...

Embodiment 2

[0037] Example 2: Two-photon test of fluorescent probe molecule Lyso-OSCC

[0038] Using two-photon measurement technology, test the two-photon absorption cross section of fluorescent probe molecules, from Figure 8 It can be seen that the maximum effective absorption cross section of the fluorescent probe molecule is 93GM, and the two-photon excitation wavelength is 760nm. The two-photon absorption of fluorescent probe molecules was verified. Under the two-photon excitation wavelength of 760nm, the voltage was gradually adjusted from 200w to 800w. Two-photon excitation is possible.

Embodiment 3

[0039] Example 3: Cytotoxicity Test

[0040] The MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide) experiment is based on the reported literature, and the cytotoxicity test is done. Add 0, 10, 20, and 30 μM fluorescent probe molecules to the same batch of MCF-7 cells, and the conditions are 37 ° C, 5% CO 2 Incubate in a cell incubator for 24 hours, according to the formula of cell viability: cell viability%=OD 570 (sample) / OD 570 (control group)×100, the cell survival rate can be calculated ( Figure 5 ). From Figure 5 We can see that when the concentration is 5 μM, the cell survival rate is about 98%, and when the probe concentration reaches 15 μM, the cell survival rate is still about 82%, which shows that the fluorescent probe molecule of the present invention has basically no toxic effect on cells , so it can be used for cell detection and monitoring of polarity changes in lysosomes.

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Abstract

The invention discloses a cell autophagy monitoring probe, a preparation method therefor and use of the cell autophagy monitoring probe. The structure of the cell autophagy monitoring probe is represented by a formula shown in the description. Cellular co-localization experiments confirm that the cell autophagy monitoring probe can be specifically located to a lysosome; and shown by two-photon fluorescence microscopic imaging experiments, the cell autophagy monitoring probe disclosed by the invention has relatively good infiltration to MCF-7, Hela cells and the like, can be used for detecting changes of polarity of the lysosome and is suitable for being located to the lysosome in cells and detecting the change condition of the polarity of the lysosome. The cell autophagy monitoring probe disclosed by the invention can be used for monitoring an autophagy process of the cells through instantly detecting the change of internal polarity of the lysosome.

Description

[0001] 1. Technical field [0002] The invention relates to a two-photon fluorescent probe, in particular to a cell autophagy monitoring probe and its preparation method and application. [0003] 2. Background technology [0004] Autophagy is when cells are stimulated by nutrient deficiency, oxidative stress, high temperature, injury, etc., and a free bilayer membrane wraps the cell fluid or damaged organelles to form autophagosomes, which combine with lysosomes to form autophagy Lysosome, the process of degrading the content into amino acids, nucleotides, and free fatty acids to maintain the cell's own homeostasis benefits. Autophagy can be divided into 4 parts, including: induction, formation of autophagosomes, membrane fusion of autophagosomes and lysosomes, and degradation of autolysosomes. As a very important metabolic pathway in organisms, autophagy regulates the continuous renewal of peroxisomes, mitochondria and endoplasmic reticulum, and can also remove damaged organe...

Claims

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Application Information

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IPC IPC(8): C07D311/16C09K11/06C12Q1/02G01N21/64
CPCC07D311/16C09K11/06C09K2211/1033C09K2211/1088G01N21/6428G01N21/6486G01N33/502G01N33/5076
Inventor 孟祥明李龙春江嘉诚郭蒙蒙朱满洲
Owner ANHUI UNIVERSITY
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