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Method for screening DOX (doxorubicin)-nephrotoxicity-resistant active substances through three-color fluorescence labeling

A fluorescent labeling and anti-doxorubicin technology, which is applied in the direction of fluorescence/phosphorescence, material excitation analysis, etc., can solve the problems of single information, not conducive to the comprehensive evaluation of the effect of compound cells, and missing screening, so as to reduce the dosage, screening and evaluation The results are comprehensive and rigorous, and the effect of eliminating experimental errors

Inactive Publication Date: 2014-08-06
ZHEJIANG UNIV
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Problems solved by technology

[0007] The disadvantage of this method is that when single-color fluorescent labeling is used in drug screening research, the information obtained is single, which is not conducive to a comprehensive evaluation of the effect of compounds on cells, and there is a phenomenon of missing screening.
[0008] However, there are no relevant reports introducing a high-content screening method for simultaneously labeling kidney cells with multiple fluorescent dyes and determining the degree of renal cell damage and the protective effect of drugs through cell fluorescence imaging

Method used

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  • Method for screening DOX (doxorubicin)-nephrotoxicity-resistant active substances through three-color fluorescence labeling
  • Method for screening DOX (doxorubicin)-nephrotoxicity-resistant active substances through three-color fluorescence labeling
  • Method for screening DOX (doxorubicin)-nephrotoxicity-resistant active substances through three-color fluorescence labeling

Examples

Experimental program
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Effect test

Embodiment 1 3

[0056] Example 1 Three-color fluorescent probe labeling kidney cells to evaluate the damage effect of doxorubicin hydrochloride

[0057] HK-2 cells were labeled with three fluorescent probes FDA, MTR, and Hoechst33342, and the damage degree of doxorubicin hydrochloride to HK-2 cells was reflected by the change of fluorescence intensity.

[0058] HK-2 cells were inoculated in 96-well plates at 5000 cells / well, and incubated in a cell culture incubator for 24 hours to allow the cells to adhere to the wall; the old culture solution was sucked off, and 90 μL of culture solution (high-glucose DMEM basal medium) was added to each well. Then add 10 μL of culture solution containing 7.8125, 15.625, 31.25, 62.5, 125, 250, 500 and 1000 μmol / L doxorubicin hydrochloride in sequence; a solvent control group (containing 0.1% DMSO) was also set up in the experiment, and each group had 3 replicate wells, and incubated 24h; Aspirate the old culture medium, add 100μL high-sugar DMEM basal mediu...

Embodiment 2

[0062] Example 2 Screening Active Components with Anti-Adriamycin Hydrochloride Damaged Kidney Cells from Safflower Extract

[0063] Weigh 200g of safflower medicinal material, heat and reflux extraction with 50% ethanol twice, each time for 1h, combine the extracts, filter, concentrate and dry to obtain the A01 component. Fraction A01 was separated by macroporous resin, eluted with water, 20% ethanol, 40% ethanol, and 95% ethanol in sequence, and the eluents were collected and concentrated respectively to obtain B01, B02, B03, and B04 fractions. B02, B03 and B04 components were further separated by preparative liquid chromatography, and C01-C20, D01-D20 and E01-E20 components were prepared respectively. 53 safflower fractions (D15, D17, D19, D20, E05, E07, E11, E13, E16, E17, E19, E20 fractions were prepared too little to meet the screening requirements) were evaluated for renal cytoprotective activity. The specific process is:

[0064] An appropriate amount of components w...

Embodiment 3

[0074] Example 3 Study on the activity of four active ingredients in safflower against doxorubicin hydrochloride-injured kidney cells

[0075] Select four active ingredients from Table 1: isoquercetin, rutin, kaempferol-3-O-rutinoside and hydroxysafflower yellow A, and dilute them into 5 concentrations (100, 50, 25, 12.5 and 6.25 μmol / L), using the method disclosed in the present invention (Example 2) to evaluate the protective effect of each compound on HK-2 cells damaged by doxorubicin hydrochloride, the evaluation results are shown in figure 2 , image 3 , Figure 4 , Figure 5 .

[0076] The experimental results show that the four compounds all have a certain degree of protective effect on HK-2 cells damaged by doxorubicin hydrochloride. Among them, the protective effect of hydroxysafflor yellow A is relatively weak, the protective effect of kaempferol-3-O-rutinoside and rutin is stronger, and the protective effect of isoquercetin is the strongest. Moreover, isoquerc...

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Abstract

The invention discloses a method for screening DOX (doxorubicin)-nephrotoxicity-resistant active substances through three-color fluorescence labeling. The method comprises the following steps: kidney cells are taken and added into to-be-screened substances for pre-protection; DOX is added for damage after the pre-protection is finished; a cell membrane integrity fluorescent probe, a mitochondrial membrane potential fluorescent probe and a cell nucleus fluorescent probe which have different excitation and emission wavelengths are added simultaneously into the damaged kidney cells for fluorescence labeling; fluorescence images of the cells are collected and analyzed after dying is finished, and the protection rates of the to-be-screened substances for cell membranes, mitochondria and cell nucleuses are calculated; and the active substances whose protection rates for the cell membranes, mitochondria and cell nucleuses are all higher than 15% are screened out from the to-be-screened substances. Three fluorescent probes are adopted to label the kidney cells simultaneously, the protection effects of the to-be-screened substances for the kidney cells are surveyed from different action mechanisms, the screening method has the characteristics of fastness, economy, high throughput and high content, and the screening result is more comprehensive and precise.

Description

technical field [0001] The invention belongs to the field of drug screening and evaluation methods, and in particular relates to a method for screening anti-adriamycin nephrotoxic active substances with three-color fluorescent labels. Background technique [0002] Chronic kidney disease is a common complex disease, with an incidence rate of 10.8% among adults in my country, and has become one of the major diseases affecting public health in my country. Renal tubular epithelial cell injury is an important factor for the continuous progression of chronic kidney disease. Therefore, screening for substances that have a protective effect on renal tubular epithelial cells is of great significance for the discovery of therapeutic drugs for chronic kidney disease. [0003] The causes of renal tubular epithelial cell injury are diverse, and the side effects of drugs are one of the inducing factors. For example, doxorubicin (DOX) is a broad-spectrum anti-tumor drug commonly used in c...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 程翼宇赵筱萍王毅
Owner ZHEJIANG UNIV
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