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Method for preparing and marking bcl-2 (b-cell lymphoma-2) protein of oral squamous cell carcinoma cells through imine acridine fluorescent probe

A technology of bcl-2 and fluorescent probes, applied in the direction of fluorescence/phosphorescence, chemical instruments and methods, luminescent materials, etc., can solve the problems of short fluorescence lifetime, poor photostability, and limited applications, and achieve simple synthesis routes and excellent reaction conditions Gentle, easy post-processing results

Inactive Publication Date: 2019-02-19
FUJIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the shortcomings of existing fluorescent materials, such as poor photostability and short fluorescence lifetime, their application as markers in biomolecules, cells, and in vivo bioimaging research is limited.

Method used

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  • Method for preparing and marking bcl-2 (b-cell lymphoma-2) protein of oral squamous cell carcinoma cells through imine acridine fluorescent probe
  • Method for preparing and marking bcl-2 (b-cell lymphoma-2) protein of oral squamous cell carcinoma cells through imine acridine fluorescent probe
  • Method for preparing and marking bcl-2 (b-cell lymphoma-2) protein of oral squamous cell carcinoma cells through imine acridine fluorescent probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Synthesis of small molecule fluorescent probes for protein labeling

[0030] (1) Synthesis of intermediate compound I:

[0031]

[0032] Add 3 mmol of acridone into a two-necked flask equipped with a condenser tube and a stirring bar, and fill with N 2 , add 6ml SOCl in nitrogen atmosphere 2 , reflux at 80 °C for 3 h, after the reaction is completed, cool to room temperature, slowly drop the reaction solution into ice water while stirring, mix ice, concentrated ammonia water and chloroform (mass ratio: 1:1:1) solution was added to the above liquid, the chloroform layer was separated, dried (anhydrous magnesium sulfate), and filtered to obtain a light yellow liquid, which was purified by column chromatography with ethyl acetate: petroleum ether volume ratio = 1:3 to obtain white needle crystals , an intermediate compound I with strong fluorescence, with a yield of 86%.

[0033]

[0034] Dissolve the intermediate compound I (9-chloroacridine) in phenol...

Embodiment 2

[0038] Example 2: Fluorescence changes after fluorescent probes and bcl-2 protein labeling of oral squamous cell carcinoma cells

[0039] The Tris-HCl solution was prepared to be 0.05 mol / LPH=7.4 (with a concentration of 0.1 mol / L NaCl solution). Add 1 ml of Tris-HCl solution to 4 ml of EP tube, the concentration of 1 ml of Example 1 is 1 × 10 -5 mol / L solution of fluorescent compound (fluorescent probe III) and 1 ml of ultrapure water were prepared as a control solution, and the added concentration was 1×10 -5 The fluorescence intensity after mol / L bcl-2 protein was compared. Using a 2mm quartz cuvette, after repeated measurements, the excitation wavelength of 260 nm is the best, the sensitivity of the instrument is adjusted to 1, the wavelength range is between 285 and 500 nm, the excitation and emission slits are 5 nm, and the voltage is 500 V. The scanning speed was 1200 nm / min, and the change of the fluorescence intensity of the compound (fluorescent probe III) was me...

Embodiment 3

[0041] Embodiment 3: the kinetics of fluorescent probe and bcl-2 protein reaction

[0042]The Tris-HCl solution was prepared to be 0.05 mol / LPH=7.4 (with a concentration of 0.1 mol / L NaCl). Add 1 ml Tris-HCl solution to 4 ml of EP tube, the concentration prepared in 1 ml of Example 1 is 1×10 -5 mol / L solution of fluorescent compound (fluorescent probe III) and 1 ml concentration of 1×10 -5 mol / L bcl-2 protein. Placed in a 37°C incubator for incubation. get figure 2 , the results showed that the fluorescence intensity changed most obviously when the time reached 120 min, and the fluorescence intensity did not change much when the time was extended, so the best time for the fluorescent substance (fluorescent probe III) to bind to the protein was 2 h.

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Abstract

The invention discloses a method for preparing and marking bcl-2 protein of oral squamous cell carcinoma cells through an imine acridine fluorescent probe. Imine acridine is applied to performing fluorescence marking on the bcl-2 protein of the oral squamous cell carcinoma cells and compared on photostability with Fluorescein insothiocyanate (FITC) marks. The applied imine acridine fluorescent probe technically has the advantages that the imine acridine fluorescent probe can reach a maximum emission wavelength of 470 nm under a fluorescence microscope, viewed under a fluorescence inversion microscope, the imine acridine fluorescent probe can clearly mark the bcl-2 protein in tongue cancer ca8113 cells; the method for preparing and marking the bcl-2 protein of the oral squamous cell carcinoma cells through the imine acridine fluorescent probe is high in sensitivity, simple rapid and accurate and is superior to traditional FITC marking methods.

Description

technical field [0001] The invention relates to the field of luminescent material detection, in particular to a method for preparing an imine acridine fluorescent probe and a method for using the probe to mark bcl-2 protein in oral squamous cell carcinoma cells. Background technique [0002] Oral cancer is one of the common malignant tumors in the head and neck, and its occurrence and development are caused by multiple factors. Studies have shown that cell apoptosis plays an important role in the occurrence and development of oral cancer. Bcl-2 protein is an apoptosis-related protein that has been studied more in this field, and bcl-2 regulatory related proteins are abnormally expressed in oral squamous cell carcinoma. For a long time, organic fluorescence has been used to label biological macromolecules to study the interaction between various proteins and the effect on the function of cells. However, due to the shortcomings of the existing fluorescent materials, such as ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D401/12C09K11/06G01N21/64
CPCC07D401/12C09K11/06C09K2211/1029G01N21/6486
Inventor 柯方骆兵卢梅秀吴斌苏慧敏林媚陈艳
Owner FUJIAN MEDICAL UNIV
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