CN110845641A - A kind of heparin oligosaccharide and its application in the preparation of anti-angiogenesis drugs - Google Patents

Abstract

The invention discloses a heparin oligosaccharide and its application in the preparation of anti-angiogenesis drugs. It belongs to the field of biomedicine. The present invention uses basic fibroblast growth factor bFGF to stimulate human umbilical vein endothelial cells to establish a cell abnormal model, and studies the effect and mechanism of heparin oligosaccharide on endothelial cells in vitro angiogenesis through experiments such as in vitro angiogenesis and co-immunoprecipitation. The results show that heparin Oligosaccharides disrupt the tube formation of vascular endothelial cells and inhibit cell proliferation and migration, of which 1 μM heparin oligosaccharide has the best effect; at the same time, heparin oligosaccharide significantly down-regulates the mRNA and protein expression levels of cell surface receptor FGFR2, and inhibits bFGF and FGFR2 , indicating that FGFR2 is one of the important anti-angiogenic targets of heparin oligosaccharides. This study may provide a new way to treat tumor growth and metastasis caused by abnormal neovascularization.

Description

A kind of heparin oligosaccharide and its application in the preparation of anti-angiogenesis drugs

technical field

The invention relates to a heparin oligosaccharide with a molecular weight of 3200 D, which has the effect of inhibiting the formation of blood vessels in vascular endothelial cells in vitro, provides the application of the heparin oligosaccharide for preparing anti-angiogenesis drugs, and belongs to the field of biomedicine.

Background technique

Blood vessels can provide nutrients and catabolite exchange to cancer cells and facilitate communication between primary and metastatic tumors. Therefore, angiogenesis is critical for cancer development and represents a potential targeted approach for cancer therapy. Studies have found that heparin and low molecular weight heparin can prolong the survival of cancer patients and have a significant anti-tumor metastasis effect in animals; and the anti-tumor activity of heparin does not depend on its anticoagulant activity, but by inhibiting angiogenesis and blocking tumors. cell adhesion, etc. Vascular endothelial cell proliferation and migration is an important part of angiogenesis, and preventing the proliferation and migration of vascular endothelial cells may inhibit the growth and metastasis of malignant solid tumors.

The Chinese patent patent number is CN201010139398.9, the patent name is: a heparin dodecose, its preparation method and its use in resisting the proliferation of vascular smooth muscle cells; the preparation method of heparin oligosaccharide is described in detail.

In this study, heparin oligosaccharide is a heparin dodecose with a molecular weight of about 3200D obtained by cleaving heparin by nitrous acid cleavage method and separating and purifying by Bio-gel P6 molecular sieve chromatography. Its target organ in vivo is the vascular endothelium. Experiments have shown that heparin oligosaccharide with this molecular weight has an inhibitory effect on the effect of basic fibroblast growth factor (bFGF) on the proliferation of vascular endothelial cells. However, the anti-angiogenesis effect of heparin oligosaccharide has not been studied, which will help to clarify the anti-tumor metastasis mechanism of heparin oligosaccharide.

SUMMARY OF THE INVENTION

The purpose of the present invention is to study the mechanism of heparin oligosaccharide inhibiting endothelial cell angiogenesis in vitro, and to provide the application of heparin oligosaccharide as an anti-angiogenesis drug.

The technical scheme of the invention is as follows: a heparin oligosaccharide, the heparin is cleaved by a nitrous acid cleavage method, and the heparin dodecose with a molecular weight of 3200 D is obtained by separation and purification by Bio-gel P6 molecular sieve chromatography.

The application of a heparin oligosaccharide in the preparation of anti-angiogenesis drugs; the heparin oligosaccharide can inhibit the in vitro lumen formation, proliferation and migration of endothelial cells in blood vessels.

Further, the heparin oligosaccharide acts as an FGFR2 inhibitor during anti-angiogenesis, which can down-regulate the mRNA and protein expression levels of fibroblast growth factor receptor FGFR2.

Further, the heparin oligosaccharide can inhibit the binding effect of fibroblast growth factor bFGF and its receptor FGFR2.

Further, the heparin oligosaccharide is prepared into various clinically required preparations by adding a pharmaceutically acceptable carrier.

Beneficial effects of the present invention: the present invention uses different concentrations of heparin oligosaccharide to act on human umbilical vein endothelial cell HUVEC, selects bFGF growth factor to stimulate abnormal proliferation and migration of endothelial cells, and finds that heparin oligosaccharide can inhibit endothelial cells in a dose-dependent manner In vitro angiogenesis, proliferation and migration, significantly down-regulated the mRNA and protein expression levels of basic fibroblast growth factor receptor FGFR2, and inhibited the binding of bFGF to its receptor FGFR2. This study may provide a new way to treat tumor growth and metastasis caused by abnormal neovascularization.

Description of drawings

Fig. 1 is the cell diagram of determining the effect of heparin oligosaccharide on HUVEC cell lumen formation by in vitro angiogenesis experiment in the present invention;

Fig. 2 is a graph showing the results of statistical analysis of data on the effect of heparin oligosaccharide on the proliferation of HUVEC cells determined by MTT experiment in the present invention;

Fig. 3 is the cell diagram of the cell scratch test in the present invention to determine the effect of heparin oligosaccharide on the migration of HUVEC cells;

Fig. 4 is the data statistical analysis result figure of the migration distance of each experimental group in the cell scratch experiment in the present invention;

Figure 5 is a data statistical analysis diagram of the Cell-based ELISA experiment in the present invention to determine the effect of heparin oligosaccharides on FGFR2 protein expression;

Figure 6 is a data statistical analysis diagram of the effect of heparin oligosaccharide on FGFR2 mRNA level determined by qPCR experiment in the present invention;

Fig. 7 is the protein band diagram of the binding effect of heparin oligosaccharide on bFGF and FGFR2 determined by co-immunoprecipitation experiment in the present invention;

Figure 8 is a statistical analysis diagram of the data of the co-immunoprecipitation experiment to determine the binding effect of heparin oligosaccharides on bFGF and FGFR2.

Detailed ways

The technical solutions of the invention are described in detail below in conjunction with examples and accompanying drawings:

A heparin oligosaccharide, the heparin is cleaved by the nitrous acid cracking method, and the heparin dodecose with a molecular weight of 3200 D is obtained by separation and purification by Bio-gel P6 molecular sieve chromatography.

The application of a heparin oligosaccharide in the preparation of anti-angiogenesis drugs; the heparin oligosaccharide can inhibit the in vitro lumen formation, proliferation and migration of endothelial cells in blood vessels.

Further, the heparin oligosaccharide acts as an FGFR2 inhibitor during anti-angiogenesis, which can down-regulate the mRNA and protein expression levels of fibroblast growth factor receptor FGFR2.

Further, the heparin oligosaccharide can inhibit the binding effect of fibroblast growth factor bFGF and its receptor FGFR2.

Further, the heparin oligosaccharide is prepared into various clinically required preparations by adding a pharmaceutically acceptable carrier.

Specific examples:

(1) In vitro angiogenesis assay to detect the effect of heparin oligosaccharides on the formation of HUVEC cell lumen:

The aliquoted growth factor reduced Matrigel gel was thawed in a 4°C refrigerator 1 hour in advance, and the pipette tip and 96-well plate were placed at -20°C to pre-cool; Add 50 μl of Matrigel gel to each well of the 96-well plate to avoid air bubbles. Place the gel in a 37°C incubator for 1 h; the HUVEC cells in the cell flask are about 90% full and in good condition, add 3 mL of medium to digest, take 1 mL of centrifugation to obtain cell pellet, add 3 mL of serum-free medium to resuspend, and take 400 μl of cells The suspension was added to a new EP tube and divided into 6 groups, namely the blank (Control) group, the model group (Model, containing 10ng/mL bFGF) and the HDO group with different concentrations (0.01μM, 0.1μM, 1μM+10ng/mL bFGF) ); add corresponding drugs to make drug-containing cell suspension. Add 200 μl of cell resuspension solution to each well, repeat three wells; incubate at 37°C for 12 hours, observe the formation of blood vessels and take pictures; the results are shown in Figure 1.

(2) MTT assay to detect the effect of heparin oligosaccharide on the proliferation of HUVEC cells:

Human umbilical vein endothelial cells were cultured, digested with 0.25% trypsin, and seeded into a 96-well plate at a density of 8×10 ³ cells/well; 100 μL of medium was added to each well, and cultured at 37°C for 24 hours; the cells were divided into blanks (Control) Group, model group (Model, containing 10ng/mL bFGF) and HDO groups with different concentrations (0.01μM, 0.1μM, 1μM+10ng/mL bFGF), a total of six groups, each group set 6 duplicate wells, and each group of culture medium Change to DMEM medium containing 0.5% FBS, culture at 37 °C for 24 h; add 20 μL of 5 mg/mL MTT solution to each well, incubate at 37 °C for 4 h, carefully aspirate the medium, add 150 μL of DMSO to each well to dissolve the crystals, and mix well at 490 nm. The absorbance value was measured at the same place; the quantitative analysis results obtained by Graph Pad Prism 5.0 software are shown in Figure 2.

(3), cell scratch test to detect the effect of heparin oligosaccharide on the migration of vascular endothelial cells:

Cultivate human umbilical vein endothelial cells, inoculate 2 ml/well of endothelial cell suspension with appropriate density in six-well plates, and culture at 37°C for 24 hours. Line marking; cells were divided into blank (Control) group, model group (Model, containing 10ng/mL bFGF) and HDO groups with different concentrations (0.01μM, 0.1μM, 1μM+10ng/mL bFGF); Use a 100 μL pipette tip perpendicular to the well plate to make cell scratches, and use the same pipette tip between different wells; aspirate the cell culture medium, and wash away the cell debris produced by the scratch with PBS. Add low-serum administration medium and take pictures for recording. Put the culture plate into the incubator for 24 hours, take pictures and record; analyze the experimental results according to the collected picture data; use Image J software to open the picture, draw 6 to 8 horizontal lines at random, and calculate the average value of the distance between cells; the software analysis calculates the upper and lower The length between the two black lines (6 vertical lines were randomly selected to calculate the average length) and the area between the black lines were quantitatively analyzed by Graph Pad Prism 5.0 software; the results are shown in Figures 3 and 4.

(4), Cell-based ELISA to detect the effect of heparin oligosaccharide on the expression of receptor protein FGFR2:

Cells were seeded into 96-well plates at appropriate density, and incubated in groups for 24 hours; washed with PBS, dried, and then added with 4% paraformaldehyde, fixed at room temperature for 20-30 min; rinsed with PBS-TritonX100, 5 min × 3 times, added with 0.6% Incubate with H2O2-PBS-TritonX100 for 20-30min; add PBS-TritonX100 to rinse, 5min×3 times, add 10% BSA, block at 37°C for 100min; add 50μL of 5% BSA-diluted primary antibody to the 6-well plate, overnight at 4°C; discard Liquid, rinsed with PBS-TritonX100 for 5 min×3 times, rinsed with PBS for 5 min×3 times, and finally added 200 μL of TMB color developing solution to each well for 30 min in the dark at 37°C; 50 μL of stop solution (1M H ₂ SO ₄ ) was added to each well to stop After the reaction, the absorbance at 450 nm was measured immediately; the quantitative analysis results obtained by the Graph Pad Prism 5.0 software are shown in Figure 5 .

(5), qPCR detection of the effect of heparin oligosaccharide on the receptor protein FGFR2 mRNA level:

The cells were seeded into six-well plates at an appropriate density, and incubated in groups for 24 hours; after the cells were treated with drugs, total RNA was extracted with TRIzol; SYBR green fluorescence detection kit was used for Real-time PCR analysis; Prepare the PCR reaction solution (on ice) according to the following components in an eight-row thin-walled tube; each template is simultaneously amplified for the target gene and the internal reference gene, and each template is repeated at least twice, set to 2 wells, each Each PCR plate was set with a calibration template (positive control) and blank control (deionized water).

Prepare PCR reaction solution according to the following components:

The above reaction solution was prepared on ice and prepared within 1 hour as much as possible to prevent the attenuation of the fluorescent dye. After a short period of centrifugation, the reaction tubes were placed in sequence on the qPCR system for PCR. GAPDH was used as the internal reference. The quantitative analysis results obtained by Graph Pad Prism 5.0 software are shown in Figure 6 .

(6) Co-immunoprecipitation experiment to detect the effect of heparin oligosaccharide on the binding of bFGF and FGFR2:

The cells were seeded into a six-well plate at an appropriate density, and incubated in groups for 24 hours; after administration, the cultured cells were rinsed with cold 0.01mol/L PBS, 150 μl IP lysis solution was added, and the cells were lysed on ice for half an hour and centrifuged to take the supernatant. The total protein was obtained, the protein content was determined by BCA, and the interaction protein was enriched; 20 μl protein-A-G-beads magnetic beads were centrifuged to remove the supernatant, and 100 μl lysis buffer was added to pretreat the magnetic beads for several hours; every 50 μl protein solution was added 1.5 μl FGFR2 antibody was mixed, shaken at 4°C for 1 h; the protein-A-G-beads magnetic bead solution was centrifuged to remove the supernatant, the protein solution containing the antibody was added, resuspended by gently pipetting, and rotated at 4°C overnight; the next day at 2000r at 4°C Centrifuge at 2000 r/min for 2 min, take the precipitate, wash it twice with PBS+0.1% Triton, flick the bottom of the tube with fingers or flip it up and down, centrifuge at 2000 r/min for 2 min, take the precipitate and add 30 μL of 2× protein loading buffer, and bath in boiling water for 5 min; The upper sample was prepared; the bFGF and FGFR2 proteins were detected by Western Blotting; the protein samples were separated by polyacrylamide denaturing gel electrophoresis, the protein was transferred to PVDF membrane by wet electroporation, blocked with 5% nonfat milk powder, and incubated with primary antibody at 4°C overnight , after washing the membrane, the horseradishase-labeled goat anti-rabbit secondary antibody was incubated at room temperature for 2 h, developed by chemiluminescence, and recorded the results by exposure; the quantitative analysis results obtained by IPP6 and Graph Pad Prism 5.0 software are shown in Figures 7 and 8.

Claims (5)

1. A heparin oligosaccharide, which is characterized in that: cracking heparin by a nitrous acid cracking method, and separating and purifying by Bio-gel P6 molecular sieve chromatography to obtain the heparin dodecasaccharide with the molecular weight of 3200D.

2. The application of heparin oligosaccharide in preparing anti-angiogenesis drugs is characterized in that: the heparin oligosaccharide can inhibit the in vitro luminal generation, proliferation and migration of endothelial cells in blood vessels.

3. The application of heparin oligosaccharide in preparing anti-angiogenesis drugs is characterized in that: the heparin oligosaccharide is used as an FGFR2 inhibitor in antiangiogenesis, and can down-regulate the mRNA and protein expression level of fibroblast growth factor receptor FGFR 2.

4. The application of heparin oligosaccharide in preparing anti-angiogenesis drugs is characterized in that: the heparin oligosaccharide can inhibit the binding action of fibroblast growth factor bFGF and a receptor FGFR2 thereof.

5. The application of heparin oligosaccharide in preparing anti-angiogenesis drugs is characterized in that: the heparin oligosaccharide is added with a pharmaceutically acceptable carrier to prepare various preparations required clinically.

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