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Application of FasL protein serving as biomarker to qualitative Aspirin sensitivity detection of colorectal cancer stem cell

A qualitative detection, aspirin technology, applied in the biological field, can solve problems such as lack of, difficult to predict curative effect, lack of pertinence in patient medication, etc., to achieve rapid and sensitive results

Inactive Publication Date: 2015-09-30
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although Aspirin has a strong tumor inhibitory effect on colorectal cancer, there are differences in the therapeutic effect of Aspirin on different malignant tumors or different types of the same tumor.
At present, the main problem facing Aspirin in the clinical adjuvant treatment of colorectal cancer is the lack of reference indicators to guide individualized drug use, the lack of targeted drug use for patients, and its curative effect is difficult to predict
In addition, with the increasing clinical application of Aspirin, the emergence of drug resistance and side effects is an important reason to limit its clinical application. How to eliminate tumor stem cells while minimizing its side effects is also a problem to be solved in the clinical application of Aspirin

Method used

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  • Application of FasL protein serving as biomarker to qualitative Aspirin sensitivity detection of colorectal cancer stem cell
  • Application of FasL protein serving as biomarker to qualitative Aspirin sensitivity detection of colorectal cancer stem cell
  • Application of FasL protein serving as biomarker to qualitative Aspirin sensitivity detection of colorectal cancer stem cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0090] (1) Cell proliferation experiment

[0091] 1) TC cells of HT29 cell line and P1 cell line (1×10 4 / mM) were inoculated into three low-adhesion 96-well plates with the same amount, and five test groups were set up: ① blank group, ② Aspirin (5mM) group, ③ control antibody (IgG) group, ④ FasL neutralizing antibody Nok -1 (1μg / mL) and Aspirin (5mM) group, ⑤ FasL neutralizing antibody Nok-1 (2μg / mL) and Aspirin (5mM) group, each group was set up with 6 duplicate holes, and the 96-well plate was placed in a carbon dioxide incubator Culture overnight;

[0092] 2) Take out the 96-well plate, add 20 μl of MTS to each well, shake the culture plate gently and put it in the cell incubator; after 4 hours, put the culture plate in the 680 microplate reader to detect the absorbance value at 490nm; for the test results, see Figure 2A and 2B .

[0093] Such as Figure 2A and 2B As shown, in HT29 cells and P1 cells, FasL neutralizing antibody Nok-1 can reverse the killing of Aspir...

Embodiment 3

[0114] (1) Western Blotting western blotting experiment

[0115] TC cells and AC cells of the HT29 cell line and the P1 cell line (both at a concentration of 1×10 6 / mL) After being treated with different concentrations of Aspirin for 48 hours, the cells were digested with ACCUTASE enzyme, and the total protein was extracted:

[0116] 1) Collect the cell pellet by centrifugation, and wash the cells with PBS;

[0117] 2) The cell lysate M-PER was pre-cooled at 4°C, and 100×protease inhibitor mixture was added before use, and mixed evenly;

[0118] 3) Add 50 μl of lysate per 1×106 cells, pipette repeatedly and place on ice for 30 minutes to fully lyse;

[0119] 4) Centrifuge at 13000rpm for 1 minute, collect the supernatant into a new 1.5ml eppendorf tube, and put it on ice for later use.

[0120] SDS-PAGE discontinuous electrophoresis gel configuration is as follows:

[0121]

[0122] 1) Prepare the separating gel, mix it well, pour it into the rubber plate, and slowly a...

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Abstract

The invention discloses application of FasL protein serving as a biomarker to qualitative Aspirin sensitivity detection of a colorectal cancer stem cell, and further discloses application of mRNA coding the FasL protein and serving as a biomarker to qualitative Aspirin sensitivity detection of the colorectal cancer stem cell, as well as application of acetylated H3K9 protein serving as a biomarker to qualitative Aspirin sensitivity detection of the colorectal cancer stem cell. According to the invention, Aspirin can selectively remove tumor stem cells of colorectal cancer, and can obviously raise the FasL protein expression level in a tumor stem cell of colorectal cancer, so that the FasL protein is Aspirin response protein, the Aspirin sensitivity of the tumor stem cell can be evaluated accurately and fast by detecting the expression level of an FasL gene or the FasL protein in the tumor stem cell. Therefore, Aspirin effectiveness can be evaluated directly from genetic transcription and protein expression / modification level.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of human FasL protein as a biomarker in qualitatively detecting the sensitivity of colorectal cancer stem cells to aspirin. Background technique [0002] Aspirin (aspirin), chemical name acetylsalicylic acid, is a small molecular compound with simple structure synthesized by German chemist Felix Hoffman in the early 20th century. Aspirin is the most used non-steroidal anti-inflammatory drug in the world with the longest use time (more than 100 years of use history), and will not produce drug dependence. Epidemiological and clinical trials have shown that in addition to being used for antipyretic and anti-platelet aggregation, Aspirin can also prevent and treat various tumors including colorectal cancer (CRC), esophageal cancer, and lung cancer. Among them, the tumor inhibitory effect on colorectal cancer is particularly prominent, and it is a hot spot that continues ...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/6812
Inventor 黄建陈志刚孔冬
Owner ZHEJIANG UNIV
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