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Method for verifying eltrombopag mediated anti-angiogenesis function of macrophage

An anti-angiogenesis and macrophage technology, applied in the field of verification of the anti-angiogenesis effect of macrophages mediated by Eltrombopag, can solve problems such as no related reports

Inactive Publication Date: 2020-01-07
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that ELB is cytotoxic to breast cancer cells, but there is no relevant report on the application of ELB in the field of anti-angiogenic therapeutic drugs

Method used

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  • Method for verifying eltrombopag mediated anti-angiogenesis function of macrophage
  • Method for verifying eltrombopag mediated anti-angiogenesis function of macrophage
  • Method for verifying eltrombopag mediated anti-angiogenesis function of macrophage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: MTT assay detects the effect of ELB on the proliferation of Raw264.7 cells

[0064] Take the Raw264.7 cells in the logarithmic growth phase, discard the old medium, wash with 3mL PBS, and discard the supernatant. Add 3mL PBS to each dish and pipette the cells repeatedly to make them fall off into a single cell suspension. Transfer the cell suspension into a 15mL centrifuge tube and centrifuge at 1000rpm for 5min.

[0065] Discard the supernatant, add an appropriate amount of complete culture medium, mix well, take a small amount of cell suspension for dilution, absorb 60 μL of the diluted liquid and add it to a cell counting plate for cell counting. Adjust the density of the cell suspension, inoculate 6,000 cells per well in a 96-well plate, add 100 μL per well, and add a circle of PBS to the outermost circle of the 96-well plate. Place the 96-well plate with cells in a 37°C, 5% CO2 cell incubator and culture overnight until it adheres to the wall. (The bl...

Embodiment 2

[0069] Example 2: Detection of mRNA expression of angiogenesis-related factors in Raw264.7 cells by q-PCR

[0070] Take the Raw264.7 cells in the logarithmic growth phase, discard the old medium, wash with 3mL PBS, and discard the supernatant. Add 3mL PBS to each dish and pipette the cells repeatedly to make them fall off into a single cell suspension. Transfer the cell suspension into a 15mL centrifuge tube. Centrifuge at 1000rpm for 5min.

[0071] Discard the supernatant, add an appropriate amount of PBS, mix well, take a small amount of cell suspension for dilution, absorb 60 μL of the diluted liquid and add it to a cell counting plate for cell counting. Adjust the cell suspension density to 1.5 x 10 6 Cells were seeded in 60 mm cell culture dishes, 2 mL per dish. Place the culture dish in a cell culture incubator for 3 h to make it adhere to the wall.

[0072] After 3 hours, the cell culture dishes were taken out, the supernatant was discarded, and 2 mL of RPMI medium...

Embodiment 3

[0074] Embodiment 3: ELISA detects the release of macrophage VEGF protein

[0075] Take the Raw264.7 cells in the logarithmic growth phase, discard the old medium, wash with 3mL PBS, and discard the supernatant. Add 3mL PBS to each dish and pipette the cells repeatedly to make them fall off into a single cell suspension. Transfer the cell suspension into a 15mL centrifuge tube. Centrifuge at 1000rpm for 5min.

[0076] Discard the supernatant, add an appropriate amount of RPMI 1640 and mix well, take a small amount of cell suspension for dilution, pipette 60 μL of the diluted liquid and add it to a cell counting plate for cell counting. Adjust the density of the cell suspension, inoculate every 12×106 cells in a 100mm cell culture dish, then replenish to 6mL with RPMI 1640, and put the culture dish into a 37°C, 5% CO2 incubator to balance for 12h.

[0077] After 12 hours, the culture dish was taken out, the supernatant was discarded, 4 mL of RPMI 1640 medium was added to eac...

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Abstract

The invention provides a method for verifying the Eltrombopag (ELB) mediated anti-angiogenesis function of macrophage. Through MTT assay, q-PCR and ELISA (enzyme linked immunosorbent assay), influenceof the ELB upon macrophage proliferation, expression of mRNA (messenger ribonucleic acid) of related angiogenesis factors (VEGF-A, bFGF, MMP-9) and VEGF (vascular endothelial growth factor) proteinscan be detected; and furthermore, through MTT experiments, cell wound scratch assay and Matrigel pipe formation experiments, influence of the ELB upon endothelial cell proliferation, migration and tube cavity formation through macrophage can be observed. The method verifies that the ELB is capable of inhibiting proliferation of the macrophage, inhibiting mRNA of the expression of related angiogenesis factors and inhibiting release of the VEGF proteins, and meanwhile, finds that the ELB acts on supernate of the macrophage and is capable of remarkably inhibiting proliferation, migration and tubule formation of the endothelial cells, and thus the macrophage can be mediated by the ELB and thus has the anti-angiogenesis function.

Description

technical field [0001] The invention relates to the field of drug testing, in particular to a method for verifying the anti-angiogenesis effect of macrophages mediated by Eltrombopag. Background technique [0002] Breast cancer is a common malignant tumor in women. Its incidence rate is increasing year by year, and it presents a younger trend, seriously endangering women's health. Currently, drug therapy (chemotherapy) is still the main treatment for breast cancer, but chemotherapy drugs can reduce the patient's platelet count during treatment. Therefore, finding a drug that inhibits breast cancer growth without affecting platelet levels is critical. Studies have found that tumor growth and metastasis depend on new blood vessels to provide oxygen and nutrients, and inhibiting tumor angiogenesis is an effective strategy for treating tumors. At the same time, in the tumor microenvironment, a large number of macrophages can release a variety of pro-angiogenic factors, thereby...

Claims

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Application Information

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IPC IPC(8): C12Q1/02
CPCG01N33/5055C12N2503/02G01N2500/10
Inventor 杨喜燕张建革杨柳青栾朋伟崔乾飞周静
Owner ZHENGZHOU UNIV
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