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Fluorochrome for cytolysosome positioning and preparation method and application thereof

A technology of lysosome localization and fluorescent dyes, applied in biochemical equipment and methods, azo dyes, organic dyes, etc., can solve the problems of quantum dot surface property fluorescence quenching, limit the application of quantum dots, etc., and achieve anti-photobleaching Excellent effect, good anti-photobleaching effect, wide application prospect

Inactive Publication Date: 2016-02-10
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Another type of lysosome staining method is to use quantum dot fluorescence imaging [LCao, XWang, et al. Biological toxicity, and changes in the surface properties of quantum dots may lead to fluorescence quenching, which also limits the application of quantum dots in fluorescence imaging

Method used

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  • Fluorochrome for cytolysosome positioning and preparation method and application thereof
  • Fluorochrome for cytolysosome positioning and preparation method and application thereof
  • Fluorochrome for cytolysosome positioning and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Preparation of 1,2-bis(4-methyl-phenyl)-1,2-diphenylethene

[0048] Add 4-methylbenzophenone (10.80g, 30mmol), Zn powder (9.75g, 150mmol) into 650mL anhydrous tetrahydrofuran, add titanium tetrachloride (8.3mL, 75mmol) dropwise under ice-cooling, add 1.6mL pyridine, then heated to reflux. The reaction process was monitored by thin-layer chromatography. After the reaction of the raw materials was completed (about 8 hours), it was cooled to room temperature, and the solvent was distilled off. Then it was extracted with dichloromethane, and the organic phase was washed with saturated brine and water, respectively. Separate the organic phase, add anhydrous sodium sulfate to dry, distill off the solvent, separate and purify by column chromatography (developing solvent is petroleum ether: dichloromethane=5:1, R f =0.8) to obtain 1,2-bis(4-methyl-phenyl)-1,2-diphenylethylene as a light yellow solid, with a yield of 9.80 g and a yield of 90.6%.

[0049] Preparation of 1,2-bi...

Embodiment 2

[0057] Actual Staining of Live Cells with Lysosome-Targeting Fluorescent Dyes

[0058] Dissolve the fluorescent dye in DMSO to obtain a probe solution (0.001mol / L), filter and sterilize it with a 0.22 μm filter membrane, and add it to the high-glucose DMEM / F12 medium containing 15% FBS and 1% double antibody Shake well to obtain the working solution for cell staining. After co-cultivating the adherent cells with the working solution for a certain period of time, the staining was observed under a confocal laser microscope. figure 2 and image 3 It shows that the cytotoxicity of the dye is very small, and the survival rate of L929 cells and MCF-7 cells is above 90% after cultured for 48 hours within the concentration of 10 μmol / L. Proved by co-staining experiments (experimental results see Figure 4 ), the dye has highly lysosome-targeting properties and can quickly and accurately stain vital lysosomes. Figure 5 and Figure 6 It shows that the dye has excellent anti-photo...

Embodiment 3

[0060] Lysosome-targeted Fluorescent Dye Tracking Observation of Lysosome Morphology During Apoptosis

[0061] Adhered L929 cells in logarithmic growth phase were taken and cultured by adding medium containing 10 μmol / L dye. Add 5-fluorouracil to the culture medium of some cells, so that the concentration of 5-fluorouracil in the cell culture system is 300 μg / mL; add H to the culture medium of some cells 2 o 2 , so that H in the cell culture system 2 o 2 The concentration is 100 μg / mL; cis-DDP is added to the medium of some cells to make the concentration in the cell culture system 100 μg / mL; the effect of three drugs is used to induce apoptosis of L929 cells. The morphology of cell lysosomes was observed at the 3h, 12h, and 24h time points of drug action, respectively. The results showed that when L929 cells undergo apoptosis, the volume of intracellular lysosomes increases, and the proportion of lysosomes in the cytoplasm increases. The observation of the morphological ...

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Abstract

The invention discloses fluorochrome for cytolysosome positioning and a preparation method and application thereof. The preparation method of the fluorochrome compound includes the steps that a benzophenone derivative containing methyl substituents is reacted under the catalysis of Zn / TiC14, an obtained product and N-bromosuccinimide are subjected to a bromination reaction, then a product is reacted with morpholine, and the tetraphenyl ethylene derivative stain containing morpholine groups is obtained. The preparation method is easy to implement, raw materials are easy to get, and reaction conditions are mild. The obtained fluorochrome contains tetraphenyl ethylene fluorescence groups capable of gathering the activity of inducing fluorescence emission and can be used for performing specific staining on all kinds of cytolysosome. The fluorochrome has the advantages of being small in cytotoxicity, stable in cell metabolism resistance, good in photobleaching resisting effect and the like and hardly affects normal physiological activities of cells, and a new method is provided for observing the morphologic changes of cytolysosome for a long time. The fluorochrome can be widely applied to monitoring the morphology of cytolysosome, observing the apoptosis process and the like.

Description

technical field [0001] The invention relates to a fluorescent dye for cell lysosome localization, a preparation method and application thereof, and belongs to the technical field of biological analysis and detection. Background technique [0002] Lysosome is an important organelle in cells, which plays important functions such as intracellular material catabolism, immune recognition, and immune defense. At the same time, lysosome also plays an important role in the process of cell apoptosis. Therefore, the location, tracking and morphological observation of lysosomes in cells are of great significance to understand the life process of the whole cell. [0003] The imaging of lysosomes usually adopts the method of fluorescent targeted imaging [HZhu, JFan, et al. Chem. Commun. 2012, 48, 11766]. Currently commercialized lysosome-targeting fluorescent dyes include RedDND, LysoTrackerGreenDND-26, Lyso-TrackerBlue, etc. These fluorescent dyes are lysosome-targeted and can local...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06C09B57/00C07D295/023C12Q1/02G01N21/64
Inventor 刘又年臧启光王立强邓留李娟
Owner CENT SOUTH UNIV
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