阅读说明：本技术 一种艾曲波帕介导巨噬细胞抗血管生成作用验证方法 (Method for verifying anti-angiogenesis effect of eltrombopag-mediated macrophages ) 是由 杨喜燕 张建革 杨柳青 栾朋伟 崔乾飞 周静 于 2019-10-11 设计创作，主要内容包括：本发明提供一种艾曲波帕(Eltrombopag,ELB)介导巨噬细胞抗血管生成作用验证方法,通过MTT实验、q-PCR和ELISA检测ELB对巨噬细胞增殖、血管生成相关因子(VEGF-A、bFGF、MMP-9)mRNA的表达及VEGF蛋白的影响；再通过MTT实验、细胞划痕实验、Matrigel基质胶成管实验,观察ELB通过巨噬细胞对内皮细胞增殖、迁移和管腔形成的影响。本发明证实了ELB能抑制巨噬细胞的增殖,抑制血管生成相关因子mRNA的表达及VEGF蛋白的释放,同时发现ELB作用于巨噬细胞的上清能显著抑制内皮细胞的增殖、迁移和小管形成,说明ELB可以介导巨噬细胞而具有抗血管生成作用。(The invention provides a verification method for the anti-angiogenesis effect of Eltrombopag (ELB) mediated macrophages, which detects the influence of ELB on macrophage proliferation, the expression of angiogenesis related factors (VEGF-A, bFGF and MMP-9) mRNA and VEGF protein through MTT experiment, q-PCR and ELISA; and then the influence of the ELB on the proliferation and migration of endothelial cells and the formation of a lumen through macrophages is observed through an MTT experiment, a cell scratch experiment and a Matrigel tube forming experiment. The invention proves that ELB can inhibit the proliferation of macrophages, inhibit the expression of mRNA (messenger ribonucleic acid) related to angiogenesis and release of VEGF (vascular endothelial growth factor) protein, and simultaneously finds that ELB acts on the supernatant of the macrophages and can obviously inhibit the proliferation, migration and tubule formation of endothelial cells, thereby indicating that the ELB can mediate the macrophages to have the effect of resisting angiogenesis.)

1. A method for verifying the anti-angiogenesis effect of Eltrombopag (ELB) mediated macrophages is characterized by comprising the following steps:

preparing reagents at least comprising cell culture solution, cell cryopreservation solution, ELB mother solution and MTT solution; performing cell culture on a macrophage Raw264.7, wherein the cell culture comprises cell recovery, cell passage, cell cryopreservation and cell counting;

the MTT method is used for detecting the influence of ELB mother liquor with different concentrations on the proliferation of the macrophage Raw264.7 cells, and comprises the following steps:

taking macrophage Raw264.7 in logarithmic phase, discarding the old culture medium, washing once with 3mL PBS, and discarding the supernatant; adding 3mL of PBS into each dish, repeatedly blowing and beating macrophage Raw264.7 to enable the macrophage to fall off into single cell suspension, and transferring the cell suspension into a 15mL centrifuge tube; centrifuging at 1000rpm for 5 min; discarding the supernatant, adding a proper amount of complete culture solution, mixing uniformly, taking a small amount of cell suspension for dilution, sucking 60 mu L of diluted liquid, and adding the liquid into a cell counting plate for cell counting; adjusting the density of cell suspension, inoculating 6000 cells per hole into a 96-hole plate, wherein each hole is 100 mu L, and a circle of PBS is added at the outermost circle of the 96-hole plate;

putting the 96-well plate with the well-paved cells into a cell culture box with the temperature of 37 ℃ and the CO2 of 5 percent for culture overnight until the cells adhere to the wall; discarding the supernatant, adding 200 μ L of cell culture solution containing drugs with different concentrations into each well, and setting 6 multiple wells for each group of concentration;

incubating in a cell culture box at 37 deg.C and 5% CO2 for 48 h; after 48 hours, adding 20 mu L of MTT solution into each hole, and continuously incubating for 4 hours; discarding the supernatant, adding 150 μ L DMSO into each well, and shaking on a shaker for 10min to completely dissolve the bluish purple crystals; measuring absorbance value at 570nm wavelength by using an enzyme-labeling instrument;

calculating the survival rate of cells according to the following formula, drawing survival curves under different concentrations, and calculating the IC after the ELB acts on macrophage Raw264.748h₅₀A value; cell viability (%) × (experimental OD-blank OD/control OD-blank OD) × 100%.

2. The method for verifying the anti-angiogenesis effect of the ELB-mediated macrophage as claimed in claim 1, wherein the q-PCR method is used for detecting the mRNA expression of the angiogenesis-related factor Raw264.7 of the macrophage, and comprises a cell administration experiment, the extraction of RNA in a cell, the measurement of RNA concentration, the reverse transcription of the RNA and a q-PCR amplification reaction; the cell administration experiment comprises:

taking macrophage Raw264.7 in logarithmic phase, discarding the old culture medium, washing once with 3mL PBS, and discarding the supernatant; adding 3mL of PBS into each dish to repeatedly blow and beat the macrophages, so that the macrophages fall off to form single cell suspension; transferring the cell suspension into a 15mL centrifuge tube; centrifuging at 1000rpm for 5 min;

discarding the supernatant, adding a proper amount of PBS, mixing uniformly, taking a small amount of cell suspension for dilution, sucking 60 mu L of diluted liquid, and adding the liquid into a cell counting plate for cell counting; cell suspension density was adjusted to 1.5X 10⁶The cells were seeded in 60mm cell culture dishes, 2mL each; placing the culture dish in a cell culture box for culturing for 3 hours to make the culture dish adhere to the wall;

taking out cell culture dishes after 3h, discarding the supernatant, adding 2mL of RPMI culture medium containing drugs with different concentrations into each dish, wherein the drug concentrations are 5 mu M and 10 mu M respectively, and placing the culture dishes in an incubator for incubation for 24 h;

the culture dish was removed, the supernatant was discarded, 500. mu. LTrizol reagent was added to each dish, cells were repeatedly blown on ice to completely lyse them, the lysates were transferred to 1.5mL EP tubes, numbered, and quickly stored in a-80 ℃ freezer overnight for subsequent experiments.

3. The method for verifying the anti-angiogenesis effect of the ELB-mediated macrophage according to claim 1 or 2, wherein the effect of ELB mother liquor on the release of VEGF protein of the macrophage Raw264.7 cells is analyzed, and the method comprises the following steps:

taking macrophage Raw264.7 in logarithmic phase, discarding the old culture medium, washing once with 3mL PBS, and discarding the supernatant; adding 3mL of PBS into each dish, and repeatedly blowing and beating the cells to ensure that the cells fall off to form single cell suspension; transferring the cell suspension into a 15mL centrifuge tube, and centrifuging at 1000rpm for 5 min;

discarding the supernatant, adding an appropriate amount of RPMI 1640, mixing uniformly, taking a small amount of cell suspension for dilution, sucking 60 mu L of diluted liquid, and adding the liquid into a cell counting plate for cell counting; cell suspension density was adjusted to 12X 10⁶Inoculating each cell in a cell culture dish with the diameter of 100mm, supplementing the cell culture dish to 6mL by RPMI 1640, and balancing the culture dish in a 5% CO2 incubator at the temperature of 37 ℃ for 12 hours;

taking out the culture dish after 12h, discarding the supernatant, adding 4mL of RPMI 1640 culture medium into each dish of the control group, adding 4mL of RPMI 1640 culture medium containing 10 mu M ELB into each dish of the experimental group, and culturing for 24h in an incubator;

taking out the culture dish after 24h, collecting the supernatant, placing the supernatant in a 15mL centrifuge tube, centrifuging the supernatant at 1000rpm for 3min, sucking the supernatant into a 5mL centrifuge tube, and writing a mark; the content of VEGF protein was detected by ELISA.

4. The method for verifying the anti-angiogenesis effect of ELB-mediated macrophages according to claim 3, wherein the influence of ELB stock solution on endothelial cell proliferation through the macrophages Raw264.7 is analyzed, and the method comprises the steps of preparing macrophage supernatant and performing proliferation experiments on endothelial cells, wherein the preparation of macrophage supernatant comprises the following steps:

taking macrophage Raw264.7 in logarithmic phase, discarding the old culture medium, washing once with 3mL PBS, and discarding the supernatant; adding 3mL of PBS into each dish, and repeatedly blowing and beating the cells to ensure that the cells fall off to form single cell suspension; transferring the cell suspension into a 15mL centrifuge tube, and centrifuging at 1000rpm for 5 min;

discarding the supernatant, adding an appropriate amount of RPMI 1640, mixing uniformly, taking a small amount of cell suspension for dilution, sucking 60 mu L of diluted liquid, and adding the liquid into a cell counting plate for cell counting;

cell suspension density was adjusted to 12X 10⁶The individual cells were seeded in 100mm cell culture dishesThen supplementing to 6mL by RPMI 1640, and placing the culture dish into a 5% CO2 incubator at 37 ℃ for balancing for 12 h;

taking out the culture dish after 12h, discarding the supernatant, adding 4mL of RPMI 1640 culture medium into each dish of the control group, adding 4mL of RPMI 1640 culture medium containing 10 mu M ELB into each dish of the experimental group, and culturing for 24h in an incubator;

taking out the culture dish after 24h, collecting the supernatant, placing the supernatant in a 15mL centrifuge tube, centrifuging the supernatant at 1000rpm for 3min, sucking the supernatant into a 5mL centrifuge tube, writing a mark, taking a Control group as a conditioned medium S-Control, taking an experimental group as a drug-containing conditioned medium group S-ELB (10 mu M), and storing the culture dish in a refrigerator at the temperature of minus 20 ℃ for later use;

the proliferation experiment of the endothelial cells comprises the following steps:

taking HUVEC cells in logarithmic growth phase, digesting with 0.25% trypsin, counting the resuspended cells, adjusting the cell suspension density, inoculating 5000 cells per well into a 96-well plate, and inoculating 100 mu L of the cells per well; putting the 96-well plate with the well-paved cells into an incubator with the temperature of 37 ℃ and the CO2 of 5 percent for culturing overnight until the cells adhere to the wall;

discarding the supernatant, and adding 200 μ L of corresponding culture solution into each well; the experiment is divided into four groups, wherein the first group is a simple conditioned medium group S-Control; the second group is a drug-containing conditioned medium group S-ELB; the third group is blank Control group Control; the fourth group is a simple medicine adding group ELB;

incubating in an incubator for 24h and 48h, taking out at each time point, adding 20 mu L MTT solution into each hole, and continuing to incubate for 4 h; discarding the supernatant, adding 150 μ L DMSO into each well, and shaking on a shaker for 10min to completely dissolve the bluish purple crystals; the absorbance values were measured with a microplate reader at a wavelength of 570 nm.

5. The method for verifying the anti-angiogenesis effect of ELB-mediated macrophages according to claim 4, wherein the influence of ELB stock solution on the migration of endothelial cells by the macrophages Raw264.7 is determined, and the method comprises the steps of preparing macrophage supernatant and performing endothelial cell scratching test, wherein the endothelial cell scratching test comprises the following steps:

sterilizing all experimental tools before experiment, placing the ruler and the marking pen in an ultra-clean bench before operation, and performing ultraviolet irradiation for 30 min;

taking a 6-hole plate, drawing a line on the back surface of the plate at an average interval of 0.5-1 cm by using a ruler, and drawing at least 5 lines in each hole transversely passing through the holes;

taking HUVEC cells in logarithmic growth phase, digesting with 0.25% trypsin, counting the number of the resuspended cells, adjusting the cell suspension density, inoculating 8 × 105 cells per well into a 6-well plate, inoculating 2mL of the cells per well, and incubating in an incubator at 37 ℃ and 5% CO2 overnight;

taking out the 6-well plate the next day, scribing with a 10-microliter gun head perpendicular to the transverse line on the back surface, discarding the supernatant, and washing off the scribed cells with PBS;

adding 2mL of corresponding culture solution into each hole; the experiment was divided into four groups: the first group is a simple conditioned medium group S-Control; the second group is a drug-containing conditioned medium group S-ELB; the third group is blank Control group Control; the fourth group is a simple medicine adding group ELB;

culturing in incubator, taking out at 24 hr, and taking picture; the width of the scratch was measured, and the mobility was calculated.

6. The method for verifying the anti-angiogenesis effect of the ELB-mediated macrophages according to claim 5, wherein the influence of ELB stock solution on the formation of endothelial cells by the mouse mononuclear macrophage Raw264.7 is determined, and the method comprises the steps of preparing macrophage supernatant and performing a tube forming experiment on the endothelial cells, and the tube forming experiment on the endothelial cells comprises the following steps:

placing Matrigel in a refrigerator at 4 ℃ one day before the experiment, melting the Matrigel overnight, and pre-cooling a 200 mu L gun head and a 96 pore plate at-20 ℃;

centrifuging for several minutes after Matrigel melts on the next day; adding matrigel into a 96-well plate pre-cooled in advance, adding 60 mu L of matrigel into each well, and then placing the plate in an incubator at 37 ℃ and 5% CO2 for incubation for 1 h;

during incubation, HUVEC cells in logarithmic growth phase are taken, digested by 0.25% trypsin, the number of the resuspended cells is counted, and the cell suspension density is adjusted by RPMI 1640 culture medium and conditioned medium;

taking out after the matrigel is solidified, inoculating 2.5 multiplied by 104 cells in each hole into a 96-hole plate, wherein each hole is 200 mu L, each group comprises 3 auxiliary holes, then placing the plate into an incubator to continue incubation, forming blood vessels after 6h, taking out the cell culture plate, observing and taking pictures, and randomly taking 3 visual fields in each hole; the images were processed using Image-J angiogenesis software, GraphPad Prism 5.01 for mapping.

Technical Field

The invention relates to the field of medicine detection, and in particular relates to a method for verifying anti-angiogenesis effect of eltrombopag-mediated macrophages.

Background

Breast cancer is a common malignant tumor in women, the incidence rate of the breast cancer is increased year by year, and the breast cancer presents a trend of youthfulness and seriously harms the health of the women. Currently, drug therapy (chemotherapy) is still the primary treatment for breast cancer, but chemotherapy drugs, when treated, reduce the platelet content of patients. Therefore, it is important to find a drug that both inhibits breast cancer growth and does not affect platelet levels. Research finds that the growth and metastasis of tumors depend on new blood vessels to provide oxygen and nutrients for the tumors, and the inhibition of tumor angiogenesis is an effective strategy for treating the tumors. Meanwhile, in a tumor microenvironment, a great number of macrophages can release a plurality of angiogenesis promoting factors to further promote angiogenesis of tumors, so that a medicine for inhibiting angiogenesis through macrophages can be found.

Eltrombopag (ELB) is a thrombopoietin receptor agonist and is used as a drug for chronic immune thrombocytopenia. The research shows that ELB has cytotoxicity to breast cancer cells, but no relevant report is found about the application of ELB in the field of anti-angiogenesis therapeutic drugs at present.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a verification method for the antiangiogenic effect of ELB-mediated macrophages, and the specific scheme is as follows:

a method for verifying the anti-angiogenesis effect of ELB mediated macrophages comprises the following steps:

preparing reagents at least comprising cell culture solution, cell cryopreservation solution, ELB mother solution and MTT solution; performing cell culture on a macrophage Raw264.7, wherein the cell culture comprises cell recovery, cell passage, cell cryopreservation and cell counting;

the MTT method is used for detecting the influence of ELB mother liquor with different concentrations on the proliferation of the macrophage Raw264.7 cells, and comprises the following steps:

taking macrophage Raw264.7 in logarithmic phase, discarding the old culture medium, washing once with 3mL PBS, and discarding the supernatant; adding 3mL of PBS into each dish, repeatedly blowing and beating macrophage Raw264.7 to enable the macrophage to fall off into single cell suspension, and transferring the cell suspension into a 15mL centrifuge tube; centrifuging at 1000rpm for 5 min;

discarding the supernatant, adding a proper amount of complete culture solution, mixing uniformly, taking a small amount of cell suspension for dilution, sucking 60 mu L of diluted liquid, and adding the liquid into a cell counting plate for cell counting; adjusting the density of cell suspension, inoculating 6000 cells per hole into a 96-hole plate, wherein each hole is 100 mu L, and a circle of PBS is added at the outermost circle of the 96-hole plate;

putting the 96-well plate with the well-paved cells into a cell culture box with the temperature of 37 ℃ and the CO2 of 5 percent for culture overnight until the cells adhere to the wall; discarding the supernatant, adding 200 μ L of cell culture solution containing drugs with different concentrations into each well, and setting 6 multiple wells for each group of concentration;

incubating in a cell culture box at 37 deg.C and 5% CO2 for 48 h; after 48 hours, adding 20 mu L of MTT solution into each hole, and continuously incubating for 4 hours; discarding the supernatant, adding 150 μ L DMSO into each well, and shaking on a shaker for 10min to completely dissolve the bluish purple crystals; measuring absorbance value at 570nm wavelength by using an enzyme-labeling instrument;

calculating the cell survival rate according to the following formula, drawing survival curves under different concentrations, and calculating the IC50 value after the ELB acts on macrophage Raw264.748h; cell viability (%) × (experimental OD-blank OD/control OD-blank OD) × 100%.

Based on the above, the q-PCR method is used for detecting the expression of mRNA (messenger ribonucleic acid) of a macrophage Raw264.7 angiogenesis related factor, and comprises a cell administration experiment, the extraction of RNA in cells, the measurement of RNA concentration, the reverse transcription of RNA and a q-PCR amplification reaction; the cell administration experiment comprises:

taking macrophage Raw264.7 in logarithmic phase, discarding the old culture medium, washing once with 3mL PBS, and discarding the supernatant; adding 3mL of PBS into each dish to repeatedly blow and beat the macrophages, so that the macrophages fall off to form single cell suspension; transferring the cell suspension into a 15mL centrifuge tube; centrifuging at 1000rpm for 5 min;

discarding the supernatant, adding a proper amount of PBS, mixing uniformly, taking a small amount of cell suspension for dilution, sucking 60 mu L of diluted liquid, and adding the liquid into a cell counting plate for cell counting; cell suspension density was adjusted to inoculate 2mL per dish in 60mm cell culture dishes at 1.5X 106 cells per dish; placing the culture dish in a cell culture box for culturing for 3 hours to make the culture dish adhere to the wall;

taking out cell culture dishes after 3h, discarding the supernatant, adding 2mL of RPMI culture medium containing drugs with different concentrations into each dish, wherein the drug concentrations are 5 mu M and 10 mu M respectively, and placing the culture dishes in an incubator for incubation for 24 h;

the culture dish was removed, the supernatant was discarded, 500. mu. LTrizol reagent was added to each dish, cells were repeatedly blown on ice to completely lyse them, the lysates were transferred to 1.5mL EP tubes, numbered, and quickly stored in a-80 ℃ freezer overnight for subsequent experiments.

Based on the above, the effect of ELB mother liquor on the release of VEGF protein of the macrophage Raw264.7 cell is analyzed, which comprises the following steps:

taking macrophage Raw264.7 in logarithmic phase, discarding the old culture medium, washing once with 3mL PBS, and discarding the supernatant; adding 3mL of PBS into each dish, and repeatedly blowing and beating the cells to ensure that the cells fall off to form single cell suspension; transferring the cell suspension into a 15mL centrifuge tube, and centrifuging at 1000rpm for 5 min;

discarding the supernatant, adding an appropriate amount of RPMI 1640, mixing uniformly, taking a small amount of cell suspension for dilution, sucking 60 mu L of diluted liquid, and adding the liquid into a cell counting plate for cell counting; cell suspension density was adjusted to 12X 10⁶Inoculating each cell in a cell culture dish with the diameter of 100mm, supplementing the cell culture dish to 6mL by RPMI 1640, and balancing the culture dish in a 5% CO2 incubator at the temperature of 37 ℃ for 12 hours;

taking out the culture dish after 12h, discarding the supernatant, adding 4mL of RPMI 1640 culture medium into each dish of the control group, adding 4mL of RPMI 1640 culture medium containing 10 mu M ELB into each dish of the experimental group, and culturing for 24h in an incubator;

taking out the culture dish after 24h, collecting the supernatant, placing the supernatant in a 15mL centrifuge tube, centrifuging the supernatant at 1000rpm for 3min, sucking the supernatant into a 5mL centrifuge tube, and writing a mark; the content of VEGF protein was detected by ELISA.

Based on the above, the influence of the ELB mother liquor on the proliferation of endothelial cells by the macrophage Raw264.7 is analyzed, and the preparation of macrophage supernatant and the proliferation experiment of the endothelial cells are included, wherein the preparation of the macrophage supernatant comprises the following steps:

taking macrophage Raw264.7 in logarithmic phase, discarding the old culture medium, washing once with 3mL PBS, and discarding the supernatant; adding 3mL of PBS into each dish, and repeatedly blowing and beating the cells to ensure that the cells fall off to form single cell suspension; transferring the cell suspension into a 15mL centrifuge tube, and centrifuging at 1000rpm for 5 min;

discarding the supernatant, adding an appropriate amount of RPMI 1640, mixing uniformly, taking a small amount of cell suspension for dilution, sucking 60 mu L of diluted liquid, and adding the liquid into a cell counting plate for cell counting;

cell suspension density was adjusted to 12X 10⁶Inoculating each cell in a cell culture dish with the diameter of 100mm, supplementing the cell culture dish to 6mL by RPMI 1640, and balancing the culture dish in a 5% CO2 incubator at the temperature of 37 ℃ for 12 hours;

taking out the culture dish after 12h, discarding the supernatant, adding 4mL of RPMI 1640 culture medium into each dish of the control group, adding 4mL of RPMI 1640 culture medium containing 10 mu M ELB into each dish of the experimental group, and culturing for 24h in an incubator;

taking out the culture dish after 24h, collecting the supernatant, placing the supernatant in a 15mL centrifuge tube, centrifuging the supernatant at 1000rpm for 3min, sucking the supernatant into a 5mL centrifuge tube, writing a mark, taking a Control group as a conditioned medium S-Control, taking an experimental group as a drug-containing conditioned medium group S-ELB (10 mu M), and storing the culture dish in a refrigerator at the temperature of minus 20 ℃ for later use;

the proliferation experiment of the endothelial cells comprises the following steps:

taking HUVEC cells in logarithmic growth phase, digesting with 0.25% trypsin, counting the resuspended cells, adjusting the cell suspension density, inoculating 5000 cells per well into a 96-well plate, and inoculating 100 mu L of the cells per well; putting the 96-well plate with the well-paved cells into an incubator with the temperature of 37 ℃ and the CO2 of 5 percent for culturing overnight until the cells adhere to the wall;

discarding the supernatant, and adding 200 μ L of corresponding culture solution into each well; the experiment is divided into four groups, wherein the first group is a simple conditioned medium group S-Control; the second group is a drug-containing conditioned medium group S-ELB; the third group is blank Control group Control; the fourth group is a simple medicine adding group ELB;

incubating in an incubator for 24h and 48h, taking out at each time point, adding 20 mu L MTT solution into each hole, and continuing to incubate for 4 h; discarding the supernatant, adding 150 μ L DMSO into each well, and shaking on a shaker for 10min to completely dissolve the bluish purple crystals; the absorbance values were measured with a microplate reader at a wavelength of 570 nm.

Based on the above, the influence of the ELB mother liquor on the migration of endothelial cells by the macrophage Raw264.7 is determined, including the preparation of macrophage supernatant and the scratching test of the endothelial cells, wherein the scratching test of the endothelial cells comprises the following steps: sterilizing all experimental tools before experiment, placing the ruler and the marking pen in an ultra-clean bench before operation, and performing ultraviolet irradiation for 30 min;

taking a 6-hole plate, drawing a line on the back surface of the plate at an average interval of 0.5-1 cm by using a ruler, and drawing at least 5 lines in each hole transversely passing through the holes;

taking HUVEC cells in logarithmic growth phase, digesting with 0.25% trypsin, counting the number of the resuspended cells, adjusting the cell suspension density, inoculating 8 × 105 cells per well into a 6-well plate, inoculating 2mL of the cells per well, and incubating in an incubator at 37 ℃ and 5% CO2 overnight;

taking out the 6-well plate the next day, scribing with a 10-microliter gun head perpendicular to the transverse line on the back surface, discarding the supernatant, and washing off the scribed cells with PBS;

adding 2mL of corresponding culture solution into each hole; the experiment was divided into four groups: the first group is a simple conditioned medium group S-Control; the second group is a drug-containing conditioned medium group S-ELB; the third group is blank Control group Control; the fourth group is a simple medicine adding group ELB;

culturing in incubator, taking out at 24 hr, and taking picture; the width of the scratch was measured, and the mobility was calculated.

Based on the above, the effect of the ELB mother liquor on endothelial cell tubule formation by the mouse mononuclear macrophage Raw264.7 is determined, including preparation of macrophage supernatant and endothelial cell tube forming experiment, wherein the endothelial cell tube forming experiment comprises:

placing Matrigel in a refrigerator at 4 ℃ one day before the experiment, melting the Matrigel overnight, and pre-cooling a 200 mu L gun head and a 96 pore plate at-20 ℃;

centrifuging for several minutes after Matrigel melts on the next day; adding matrigel into a 96-well plate pre-cooled in advance, adding 60 mu L of matrigel into each well, and then placing the plate in an incubator at 37 ℃ and 5% CO2 for incubation for 1 h;

during incubation, HUVEC cells in logarithmic growth phase are taken, digested by 0.25% trypsin, the number of the resuspended cells is counted, and the cell suspension density is adjusted by RPMI 1640 culture medium and conditioned medium;

taking out after the matrigel is solidified, and taking out the matrigel at a rate of 2.5 multiplied by 10 per hole⁴Inoculating each cell into a 96-well plate, wherein each well is 200 mu L, each group comprises 3 auxiliary wells, then placing the plate in an incubator for continuous incubation, after 6h, blood vessel formation can be seen, taking out the cell culture plate, observing and taking a picture, and randomly shooting 3 visual fields in each well; the images were processed using Image-J angiogenesis software, GraphPad Prism 5.01 for mapping.

Compared with the prior art, the invention has outstanding substantive characteristics and remarkable progress, and particularly has the following advantages:

the invention firstly detects the influence of ELB on macrophage proliferation, mRNA expression of angiogenesis related factors (VEGF-A, bFGF and MMP-9) and VEGF protein through MTT experiment, q-PCR and ELISA; and then the influence of the ELB on the proliferation and migration of endothelial cells and the formation of a lumen through macrophages is observed through an MTT experiment, a cell scratch experiment and a Matrigel cell tube forming experiment.

According to the invention, on an in vitro level, ELB can inhibit the proliferation of macrophages, inhibit the expression of mRNA of angiogenesis related factors (VEGF-A, bFGF and MMP-9) and the release of VEGF protein, and meanwhile, ELB acting on the supernatant of the macrophages can obviously inhibit the proliferation, migration and tubule formation of endothelial cells, which indicates that the ELB can inhibit the functions of the endothelial cells by acting on the macrophages.

Drawings

FIG. 1 is a flow chart of the present invention;

FIG. 2 is a MTT method for detecting the effect of ELB on Raw264.7 cell proliferation;

FIG. 3 is a schematic diagram showing the quantitative analysis of VEGF-A mRNA level after ELB acts on Raw264.7 cells for 24 h;

FIG. 4 is a schematic representation of the quantitative analysis of bFGF mRNA levels 24h after ELB was applied to Raw264.7 cells;

FIG. 5 is a schematic representation of the quantitative analysis of MMP-9mRNA levels after ELB has been applied to Raw264.7 cells for 24 h;

FIG. 6 is a graph showing that ELB inhibits the release of Raw264.7 cellular VEGF protein;

FIG. 7 is a schematic diagram showing the MTT method for detecting the cell proliferation of the supernatant of ELB-affected macrophages after 24h of the action on endothelial cells;

FIG. 8 is a schematic diagram showing the MTT method for detecting the cell proliferation of the supernatant of ELB-affected macrophages after 48h of the action on endothelial cells;

FIG. 9 is a schematic diagram showing cell proliferation after detecting ELB directly acting on endothelial cells for 24h by MTT method;

FIG. 10 is a schematic diagram showing the cell proliferation of the endothelial cells after 48h of direct effect of ELB on the endothelial cells by MTT method;

FIG. 11 is a schematic illustration of scoring after 0h and 24h of ELB drug action;

FIG. 12 is a graph showing the relative mobility of supernatants of ELB-affected macrophages after 24h exposure to endothelial cells;

FIG. 13 is a graph showing the relative mobility of ELB after 24h of direct effect on endothelial cells;

FIG. 14 is a graph showing that supernatants of ELB-activated macrophages inhibited tubule formation by endothelial cells for 6 h.

Detailed Description

The technical solution of the present invention is further described in detail by the following embodiments.

ELB is researched through macrophage-mediated in-vitro anti-angiogenesis effect, macrophages are the primary inflammatory cells in a tumor microenvironment and can secrete various vascular growth factors such as VEGF-A, bFGF, MMPs, TGF-alpha and IL-1 beta, and the angiogenesis of tumors is promoted. The ELB can obviously inhibit the growth of the breast cancer cells 4T1 and the expression of the angiogenesis-related factors, so the influence of the ELB on the expression of macrophage angiogenesis-related factors and the influence on the function of downstream vascular endothelial cells mediated by macrophages are further discussed. Firstly, detecting the influence of ELB on macrophage proliferation, angiogenesis related factor expression and VEGF protein by MTT experiment, q-PCR method and ELISA; and then the influence of the ELB on the macrophage-mediated angiogenesis function is researched by detecting the influence of the ELB on the proliferation and migration of endothelial cells and the tubule forming function by the action of macrophage supernatant treated by the ELB on vascular endothelial cells. Fig. 1 shows a flow chart of the present invention.