Application of ITGB1 in hemangioma treatment
Using ITGB1 as a therapeutic target, drugs containing ITGB1 inhibitors were prepared, which solved the problem of limitations in hemangioma treatment methods and achieved effective inhibition of hemangioma.
Patent Information
- Application Number
- CN202510340018.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-21
- Publication Date
- 2025-07-04
AI Technical Summary
In the prior art, the pathogenesis of hemangioma is not fully understood, resulting in limitations in the treatment methods.
ITGB1 is used as a therapeutic target to treat hemangioma by preparing drugs containing ITGB1 inhibitors such as GLPG0187, including dosage forms such as oral preparations, injections, powders or transdermal patches, combined with CRISPR-Cas9 technology to construct ITGB1 stable knockout cells to verify the efficacy of the drug.
The expression of ITGB1 in hemangioma endothelial cells was verified through in vitro experiments, and it was proved that inhibiting ITGB1 can significantly inhibit the proliferation, migration and duct formation ability of hemangiomas. GLPG0187 small molecule inhibitor can effectively inhibit the occurrence of hemangiomas.
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Figure CN120241733A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of ITGB1 in the treatment of hemangiomas. Background Art
[0002] Hemangioma is a kind of benign tumor originating from blood vessels, which mostly occurs in infants and young children, and can appear in various tissues and organs of the body, including internal organs, and is commonly found in the skin of the face, scalp, chest and back. Hemangiomas occurring on the skin are bright red, and the redundant blood vessels protrude from the skin like a spongy or rubbery elevation. Most infantile hemangiomas will naturally regress by the age of 5, and at most by 10 years old. Therefore, generally, no treatment is required. However, if the hemangioma interferes with functions such as breathing and vision or shows symptoms such as rupture and bleeding, artificial intervention and treatment are needed.
[0003] Hemangiomas have pathological characteristics of a proliferation phase and a regression phase clinically. In the proliferation phase, the blood flow taken in by the hemangioma increases rapidly and is accompanied by the formation of immature blood vessels. The tumor body grows rapidly and its volume increases significantly, thus causing symptoms such as compression of surrounding tissues and organs. In the regression phase, the endothelium of the hemangioma tends to mature, stops growing or even atrophies, and regresses spontaneously. However, about 5 - 10% of infantile hemangiomas are destructive, can form scars and ulcers, and the hemangiomas in the proliferation phase will have complications such as rupture and bleeding, and in severe cases, it can even endanger life safety. Currently, the treatment methods for infantile hemangiomas include laser surgery, anti-tumor drug treatment, hormone treatment, interferon treatment, and oral propranolol, etc. However, since the pathogenesis of hemangiomas has not been clearly studied yet, there are still limitations in treatment. Further research on the pathogenesis of hemangiomas will provide new solutions for the clinical treatment of hemangiomas. Summary of the Invention
[0004] The purpose of the present invention is to solve the problem that there are still limitations in treatment due to the unclear pathogenesis of hemangiomas in the prior art.
[0005] In order to achieve the above purpose, the present invention adopts the following technical solutions:
[0006] The application of ITGB1 as a therapeutic target in the preparation of a drug for treating hemangiomas, and the drug is used to inhibit the expression of ITGB1 to treat hemangiomas.
[0007] Preferably, the hemangiomas include infantile hemangiomas.
[0008] Preferably, the drug includes the small molecule inhibitor GLPG0187.
[0009] The present application also provides a drug for treating hemangioma, which contains an effective amount of an ITGB1 inhibitor and a pharmaceutically acceptable carrier.
[0010] Preferably, the ITGB1 inhibitor is GLPG0187.
[0011] Preferably, the drug dosage form is selected from at least one of oral preparations, injections, powders, ointments or transdermal patches.
[0012] The present application also provides a verification method for verifying the efficacy of a drug for treating hemangioma, which comprises the following steps:
[0013] S1: Extract the RNA of hemangioma tissue and adjacent normal tissue, and quantitatively analyze the mRNA level of ITGB1 by qPCR;
[0014] S2: In human hemangioma endothelial cells, verify the migration ability, proliferation rate and tube formation ability of ITGB1 knockout in HemECs;
[0015] S3: In human hemangioma endothelial cells, overexpress the migration ability, proliferation rate and tube formation ability of TGB1 in HemECs;
[0016] S4: In human hemangioma endothelial cells, verify the addition of the ITGB1 small molecule inhibitor GLPG0187, and observe its migration ability, proliferation rate and tube formation ability in HemECs.
[0017] Preferably, in S2, an ITGB1 stable knockout cell is successfully constructed by CRISPR-Cas9 technology.
[0018] Preferably, the specific steps of S4 are as follows:
[0019] (4-1) Successfully construct a hemangioma endothelial cell (HemECs) strain;
[0020] (4-2) Treat HemECs with the ITGB1 small molecule inhibitor GLPG0187;
[0021] (4-3) Perform CCK8 experiments, cell scratch-healing experiments and tubule formation experiments with different concentrations of GLPG0187 respectively;
[0022] (4-4) Observe the cell proliferation, migration rate and tube formation ability of HemECs.
[0023] Preferably, in item 4-4, the concentrations of GLPG0187 in the CCK-8 proliferation assay, cell injury-healing assay, and tubule formation assay are all 0, 2 nM, 4 nM, 6 nM, 8 nM, and the treatment time is 24 hours.
[0024] Beneficial effects:
[0025] In the present invention, through specific verification experiments, it is verified by qPCR that the expression of ITGB1 is increased in infantile hemangiomas; it is verified that knocking out ITGB1 can inhibit the proliferation, tube formation, and migration abilities of HemECs; it is verified that overexpressing ITGB1 can promote the proliferation, tube formation, and migration abilities of human hemangioma endothelial cells; it is verified that applying the ITGB1 small molecule inhibitor GLPG0187 can inhibit the proliferation, tube formation, and migration abilities of human hemangioma endothelial cells. Thus, the present invention confirms the expression of ITGB1 in clinical hemangioma tissues, and in hemangioma endothelial cells, through a series of in vitro experiments, it is confirmed that inhibiting ITGB1 can strongly inhibit the occurrence of hemangiomas. Description of the drawings
[0026] Figure 1 It is a comparison diagram of mRNA expression of ITGB1 in infantile hemangioma tissues and normal tissues adjacent to hemangiomas in one embodiment of the present invention;
[0027] Figure 2 It is a diagram for verifying the knockout efficiency of ITGB1 in HemECs and examining the proliferation, migration, and tube formation abilities of HemECs after knocking out ITGB1 in one embodiment of the present invention; among them, Figure 2 A is the ITGB1 expression level detected by Western Blot in the stable cell line after screening to verify the successful knockout of ITGB1 in HemECs, Figure 2 B is a comparison diagram of CCK-8 proliferation assays in the stable transfected cells of four groups of HemECs sgNC / sgITGB1-1 / sgITGB1-2 / sgITGB1-3; Figure 2 C is the injury-healing assay performed in the stable transfected cells of four groups of HemECs sgNC / sgITGB1-1 / sgITGB1-2 / sgITGB1-3, and the width of injury-healing is marked at 0 h, 8 h, and 24 h respectively; Figure 2 D is Figure 2 A statistical chart of the injury-healing rate at 24 h for each group in C, Figure 2 E is the tube formation assay performed in the four groups of stably transfected cells of HemECs sgNC / sgITGB1-1 / sgITGB1-2 / sgITGB1-3; Figure 2 F is Figure 2 A statistical chart for analyzing and calculating the total tube length of the four groups in E.
[0028] Figure 3 In one embodiment of the present invention, after overexpressing ITGB1, Western Blot was used to verify the overexpression efficiency of HemECs and to examine the proliferation, migration and tube formation abilities of HemECs. Among them, Figure 3 A shows the ITGB1 expression level of the stable cell line screened by Western Blot to verify the successful overexpression of ITGB1 in HemECs. Figure 3 B is a comparison graph of CCK-8 proliferation experiments in the stable transfected cells of two groups of HemECs PLV3-VECTOR / PLV3-ITGB1. Figure 3 C shows the scratch-wound healing experiment in the stable transfected cells of two groups of HemECs PLV3-VECTOR / PLV3-ITGB1, and the width of the scratch-wound healing was marked at 0 h, 24 h, and 48 h respectively. Figure 3 D is Figure 3 A statistical graph of the scratch-wound healing rate of each group in C at 48 h. Figure 3 E is a comparison graph of tube formation experiments in the stable transfected cells of two groups of HemECs PLV3-VECTOR / PLV3-ITGB1. Figure 3 F is Figure 3 A statistical graph of the total tube length analysis and calculation of the two groups in E.
[0029] Figure 4 In one embodiment of the present invention, HemECs were treated with the small molecule inhibitor GLPG0187 of ITGB1 to inhibit the proliferation, migration and tube formation abilities of HemECs. Among them, Figure 4 A is a comparison graph of CCK-8 proliferation experiments after treating HemECs with different concentrations of GLPG0187 (0, 2 nM, 4 nM, 6 nM, 8 nM) for 24 hours. Figure 4 B is the scratch-wound healing experiment of treating HemECs with different concentrations of GLPG0187 (0, 2 nM, 4 nM, 6 nM, 8 nM) for 24 hours, and the width of the scratch-wound healing at 0 h, 10 h, and 24 h was photographed and recorded. Figure 4 C is Figure 4 The width of the scratch-wound healing of different drug-added groups in B was marked, and a statistical graph of the scratch-wound healing rate at 24 h of each group was made with the non-drug-added group as the standard value of 1. Figure 4 D is a comparison graph of tube formation experiments after treating HemECs with different concentrations of GLPG0187 (0, 2 nM, 4 nM, 6 nM, 8 nM) for 24 hours. Figure 4 E is Figure 4 A statistical comparison graph of the total tube length of each group in D. Detailed implementation method
[0030] The present invention will be further described in detail below in conjunction with specific embodiments.
[0031] Use of ITGB1 as a therapeutic target in the preparation of a drug for treating hemangioma, wherein the drug is used to inhibit the expression of ITGB1 to treat hemangioma.
[0032] The hemangioma includes infantile hemangioma.
[0033] In one embodiment, the drug includes the small molecule inhibitor GLPG0187.
[0034] A drug, which is used to treat hemangioma, and the drug contains an effective amount of an ITGB1 inhibitor and a pharmaceutically acceptable carrier. Preferably, the ITGB1 inhibitor is GLPG0187.
[0035] The pharmaceutical dosage form is selected from at least one of oral preparations, injections, powders, ointments or transdermal patches.
[0036] In addition, the present application also provides a verification method, which is used to verify the efficacy of a drug for treating hemangioma, and includes the following steps:
[0037] S1: Extract the RNA of infantile hemangioma tissue and adjacent normal tissue, and quantitatively analyze the mRNA level of ITGB1 by qPCR
[0038] S2: In human hemangioma endothelial cells, verify the migration ability, proliferation rate and tube formation ability of knocking out ITGB1 in HemECs;
[0039] In one embodiment, the CRISPR-Cas9 technology is used to successfully construct ITGB1 stable knockout cells.
[0040] S3: In human hemangioma endothelial cells, overexpress the migration ability, proliferation rate and tube formation ability of TGB1 in HemECs;
[0041] S4: In human hemangioma endothelial cells, verify the addition of the ITGB1 small molecule inhibitor GLPG0187, and observe its migration ability, proliferation rate and tube formation ability in HemECs.
[0042] In one embodiment, the specific steps of S4 are as follows:
[0043] (4-1) Successfully construct a hemangioma endothelial cell (HemECs) strain;
[0044] (4-2) Treat HemECs with the ITGB1 small molecule inhibitor GLPG0187;
[0045] (4-3) CCK8 experiments, cell injury-healing experiments, and tubule formation experiments were respectively carried out with different concentrations of GLPG0187;
[0046] In one embodiment, the concentrations of GLPG0187 in the CCK-8 proliferation experiment, cell injury-healing experiment, and tubule formation experiment were all 0, 2 nM, 4 nM, 6 nM, 8 nM, and the treatment time was 24 hours.
[0047] (4-4) Observe the cell proliferation, migration rate, and tube formation ability of HemECs.
[0048] The above content is elaborated below in conjunction with specific embodiments:
[0049] Experimental materials and sources used in the application
[0050]
[0051] Experimental methods
[0052] 1. Tissue RNA extraction
[0053] (1) Sampling: Within half an hour after resection of the hemangioma tissue, take about soybean-sized hemangioma tissue and adjacent normal skin tissue, place them in a cryopreservation tube and store them in a -80 °C refrigerator.
[0054] (2) Take out the hemangioma tissue and adjacent tissue from the -80 °C refrigerator, cut them into pieces and put them into labeled 1.5 mL RNase-free EP tubes respectively, add 1 mL of Trizol to each tube, homogenize with a homogenizer and let it stand on ice until the tissue is completely lysed, at room temperature, for 5 min.
[0055] (3) Add 200 μL of chloroform and shake the EP tube, let it stand at room temperature for 10 min, and centrifuge at a high speed of 13000 rpm / min at 4 °C for 20 min under low-temperature conditions.
[0056] (4) Avoid contaminating the middle white liquid layer, transfer the upper liquid phase (about 300 - 500 μL) to a new RNase-free EP tube, add 500 μL of isopropanol and mix gently. At this time, RNA has precipitated, let it stand for 20 min at room temperature, centrifuge at a high speed of 13000 rpm, 4 °C, for 20 min. After completion, there is a white precipitate at the bottom of the tube. Remove the waste liquid and retain the precipitate.
[0057] (5) Add 1 mL of pre-cooled 75% ethanol, centrifuge at a high speed of 9000 rpm / min, 4 °C, for 12 min, and repeat to wash the RNA precipitate.
[0058] (6) Mark the position of the white precipitate on the EP tube with a marker pen, discard the supernatant, invert and air-dry for 15 min, and dissolve the transparent precipitate (the position has been marked) with an appropriate amount of DEPC water.
[0059] (7) 1 μL of each group was pipetted onto the detector, and the concentration and purity were measured using a Nano Drop ultraviolet spectrophotometer (which needs to be calibrated with DEPC water before and after use).
[0060] (8) After completion, label and store in a -80 °C refrigerator.
[0061] 2. Injury-healing experiment:
[0062] (1) HemECs with ITGB1 knocked out and overexpressed, HemECs after drug treatment, and the control group were seeded in 6-well plates at a confluence of 50% and cultured in a 37 °C, 5% CO2 incubator for 24 h.
[0063] (2) Sterilize the ruler. When the cell confluence reaches 80% under the microscope, take out the culture plate. Use a 10 μL pipette to draw lines perpendicular to the bottom of the plate and close to the ruler. Make three injury-healing lines in each well. Add PBS to rinse and remove cell debris. Remove the waste liquid. Depending on the cell state, it can be rinsed once more. Add DMEM and observe the injury-healing under an inverted phase contrast microscope.
[0064] (3) Take pictures of the initial injury-healing of each group at 4× magnification, three pictures for each well. After completion, put it back into the cell culture incubator and let it stand. Then take pictures again at the appropriate time.
[0065] 3. Tube formation assay:
[0066] (1) Prepare one day in advance: Thaw the Matrigel in a 4 °C refrigerator, pre-cool the ice box and pipette tips.
[0067] (2) Mix DMEM and Matrigel at a ratio of 1:1 and add 50 μL / well to a 96-well plate. Avoid generating bubbles. Place it flat in a 37 °C incubator for at least 30 min to wait for the Matrigel to solidify.
[0068] (3) Digest the prepared HemECs with ITGB1 knocked down, knocked out or overexpressed, HemECs after drug treatment, and the control group, count them respectively, and gently add 26,000 cells / well onto the Matrigel and continue to culture for 4 - 6 hours. The tube formation can be observed during this period.
[0069] (4) Observe the tube formation of each well under a microscope at 4× magnification and take pictures for recording. Measure the relevant index parameters using ImageJ.
[0070] 4. GLPG0187 drug addition experiment:
[0071] (1) Dilute the GLPG0187 stock solution with DMEM to prepare the drug master solution, and add 2 nM, 4 nM, 6 nM, and 8 nM of GLPG0187 to HemECs respectively.
[0072] (2) Incubate for another 24 hours, followed by further CCK-8 assay, wound-healing assay and tube formation assay.
[0073] 5. Western blot
[0074] Prepare the gel, 12% separating gel and 5% stacking gel. Denature the protein sample at 100 °C for 5 min and load 50 μg. Run the gel at a constant voltage of 80 v until the bromophenol blue front just enters the separating gel, then run the gel at a constant voltage of 120 v. Transfer the proteins using a transfer tank at a constant current for 90 min. Block the membrane with 5% non-fat milk solution at room temperature for 2 h. Dilute the primary antibody (1:1000) with TBST solution containing 3% bovine serum albumin and incubate with the PVDF membrane containing the target protein overnight at 4 °C. Immerse the membrane in TBST once for 10 min. Immerse the membrane in TBST twice, 10 min each time. Dilute the horseradish peroxidase-labeled secondary antibody (1:5000) with TBST and incubate at room temperature for 1.5 h. Immerse the membrane in TBST three times, 10 min each time, and develop the color with ECL and analyze the scanned results.
[0075] Example 1: The mRNA expression of ITGB1 is increased in infantile hemangioma tissues
[0076] In this example, first, qPCR was used to detect the mRNA in infantile hemangioma tissue and adjacent normal tissue.
[0077] The qPCR results showed that the mRNA expression level in IH (hemangioma tissue) was significantly increased compared with that in normal tissue (Control) ( Figure 1 ). This indicates that ITGB1 plays an important role in the occurrence of hemangioma.
[0078] Example 2: Knocking out ITGB1 inhibits the proliferation, tube formation and migration of human hemangioma endothelial cells
[0079] To further study the role of ITGB1 in hemangioma, in this example, first, the sg plasmid was constructed using the crisp-cas9 technology, and the HemECs cells with stable ITGB1 knockout were established, as shown in Figure 2 the Western result diagram in A. In HemECs, the stable ITGB1 knockout cells were successfully constructed using the Crisp-cas9 technology, as shown in Figure 2 A. The HemECs cells with stable ITGB1 overexpression were successfully constructed. Then, using these cells, CCK8 cell proliferation assay, tube formation assay and wound-healing assay were performed (the specific experimental procedures refer to the above experimental methods).
[0080] The results of this example showed:
[0081] Please refer to Figure 2B. The results of the CCK8 cell proliferation assay showed that the proliferation ability of HemECs stably transfected with ITGB1 (sgITGB1-1, sgITGB1-2, sgITGB1-3) was weaker than that of the control group (sgNC).
[0082] As Figure 2 Shown in C and D, the results of the wound-healing assay showed that the wound-healing ability of HemECs stably transfected with ITGB1 (sgITGB1-1, sgITGB1-2, sgITGB1-3) was weaker than that of the control group (sgNC).
[0083] Please refer to Figure 2 E and F. The results of the tube formation assay showed that the tube formation ability of HemECs with stable knockout of ITGB1 (sgITGB1-1, sgITGB1-2, sgITGB1-3) was weaker than that of the control group (sgNC) cells. Therefore, compared with the control group (sgNC) cells, the CCK8 proliferation assay, wound-healing ability, and tube formation ability were also weaker.
[0084] The above results indicate that knocking out ITGB1 can inhibit the proliferation, tube formation, and migration of hemangioma endothelial cells and play a certain role in hemangiomas.
[0085] Example 3: Overexpressing ITGB1 inhibits the proliferation, tube formation and migration of human hemangioma endothelial cells
[0086] In this example, an ITGB1 overexpression plasmid was first constructed, and then ITGB1-stable overexpressing HemECs cells were established. As Figure 3 Shown in A, HemECs cells stably transfected with ITGB1 were successfully constructed. Then, the CCK8 cell proliferation assay, tube formation assay, and wound-healing assay were performed on these cells (the specific experimental procedures refer to the above experimental methods).
[0087] The results in this example showed that:
[0088] Please refer to Figure 3 B. The results of the CCK8 cell proliferation assay showed that the proliferation ability of HemECs stably transfected with ITGB1 (PLV3-ITGB1) was stronger than that of the control group (PLV3-VECTOR).
[0089] Please refer to Figure 3 C and D. The results of the wound-healing assay showed that the wound-healing ability of HemECs stably transfected with ITGB1 (PLV3-ITGB1) was stronger than that of the control group (PLV3-VECTOR).
[0090] Please refer to Figure 3E and F. The results of the tube formation experiment showed that HemECs with stable overexpression of ITGB1 (PLV3-ITGB1) had enhanced tube formation ability compared with the control group (PLV3-VECTOR) cells. Therefore, compared with the control group (PLV3-VECTOR) cells, overexpression of ITGB1 promoted the proliferation ability, injury-healing ability, and tube formation ability of HemECs ( Figure 3 B-F).
[0091] The above results indicated that overexpression of ITGB1 could promote the proliferation, tube formation, and migration of hemangioma endothelial cells, and this result further supported the promoting role of ITGB1 in hemangioma.
[0092] Example 4: GLPG0187 inhibits the proliferation, migration and tube formation abilities of HemECs
[0093] In Examples 2 and 3, the experimental results were all obtained by treating HemECs. Then, what would be the impact on cell function by treating normal HemECs with drugs without changing the intracellular proteins, and whether ITGB1 also played a role in hemangioma.
[0094] Based on the above conjecture, in this example, first, HemECs were treated with the ITGB1 small molecule inhibitor GLPG0187, and the HemECs treated with the inhibitor were subjected to CCK8 cell proliferation experiments, tube formation experiments, and injury-healing experiments.
[0095] The results of the present invention showed that
[0096] Please refer to Figure 4 A. The results of the CCK8 cell proliferation experiment showed that after treatment with the ITGB1 small molecule inhibitor GLPG0187, the proliferation ability of HemECs was weakened compared with that without drug treatment;
[0097] Please refer to Figure 4 B and C. The results of the injury-healing experiment showed that the healing ability of HemECs treated with the ITGB1 small molecule inhibitor GLPG0187 was weakened compared with that without drug treatment;
[0098] Please refer to Figure 4 D and E. The results of the tube formation experiment showed that after treating HemECs cells with the ITGB1 small molecule inhibitor GLPG0187, the tube formation ability was also inhibited compared with that of cells without drug treatment.
[0099] Therefore, compared with cells without drug treatment, the CCK8 proliferation experiment, injury-healing ability, and tube formation ability were all inhibited after treatment with the ITGB1 small molecule inhibitor GLPG0187 ( Figure 4 A-E).
[0100] These results suggest that inhibiting ITGB1 can suppress the proliferation, tube formation, and migration of hemangioma endothelial cells. Therefore, ITGB1 has a therapeutic effect in the treatment of hemangioma.
[0101] In summary, the present invention has identified a new target for the treatment or inhibition of hemangioma, providing a new possibility for the treatment method or therapeutic drug for hemangioma patients. In the present invention, through specific verification experiments, it is verified by qPCR that the expression of ITGB1 is increased in infantile hemangioma; it is verified that knocking out ITGB1 can inhibit the proliferation, tube formation, and migration ability of HemECs; it is verified that overexpressing ITGB1 can promote the proliferation, tube formation, and migration ability of human hemangioma endothelial cells; it is verified that the application of the ITGB1 small molecule inhibitor GLPG0187 can inhibit the proliferation, tube formation, and migration ability of human hemangioma endothelial cells. Thus, the present invention has confirmed the expression of ITGB1 in clinical hemangioma tissues, and in hemangioma endothelial cells, through a series of in vitro experiments, it has been confirmed that inhibiting ITGB1 can strongly inhibit the occurrence of hemangioma.
[0102] In summary, this application has passed specific verification experiments, demonstrating that inhibiting ITGB1 can treat hemangioma, thus providing a new treatment method or therapeutic drug for hemangioma patients and becoming a new target for the treatment of hemangioma, with strong applicability.
Claims
1. The use of ITGB1 as a therapeutic target in the preparation of drugs for treating hemangioma, characterized in that: The drug is used to treat hemangioma by inhibiting the expression of ITGB1.
2. Use of ITGB1 as a therapeutic target in the preparation of a drug for treating hemangioma according to claim 1, characterized in that: The hemangioma includes infantile hemangioma.
3. Use of ITGB1 as a therapeutic target in the preparation of a medicament for treating hemangioma according to claim 1, characterized in that: The drug includes the small molecule inhibitor GLPG0187.
4. A drug, which is used for treating hemangioma, and is characterized in that: The drug contains an effective amount of an ITGB1 inhibitor and a pharmaceutically acceptable carrier.
5. A drug according to claim 4, characterized in that: The ITGB1 inhibitor is GLPG0187.
6. A drug according to claim 4, characterized in that: The drug dosage form is selected from at least one of oral preparations, injections, powders, ointments or transdermal patches.
7. A verification method, characterized in that: The verification method is used to verify the efficacy of drugs for treating hemangioma, and the verification method comprises the following steps: S1: Extract the RNA of hemangioma tissue and adjacent normal tissue, and quantitatively analyze the mRNA level of ITGB1 by qPCR; S2: In human hemangioma endothelial cells, verify the migration ability, proliferation rate and tube formation ability of HemECs after knocking out ITGB1; S3: In human hemangioma endothelial cells, overexpress TGB1 and observe its migration ability, proliferation rate and tube formation ability in HemECs; S4: In human hemangioma endothelial cells, verify the addition of the ITGB1 small molecule inhibitor GLPG0187, and observe its migration ability, proliferation rate and tube formation ability in HemECs.
8. The verification method according to claim 7, wherein: In the above S2, a stable ITGB1 knockout cell line is successfully constructed by CRISPR-Cas9 technology first.
9. A verification method according to claim 7, characterized in that: The specific steps of the above S4 are as follows: (4-1) Successfully construct a hemangioma endothelial cell (HemECs) line; (4-2) Treat HemECs with the ITGB1 small molecule inhibitor GLPG0187; (4-3) Perform CCK8 experiments, cell scratch-wound healing experiments and tube formation experiments with different concentrations of GLPG0187 respectively; (4-4) Observe the cell proliferation, migration rate and tube formation ability of HemECs.
10. According to the method for treating miasma described in claim 9, in one embodiment of the above (4-4), the concentrations of GLPG0187 in the CCK-8 proliferation experiment, cell scratch-wound healing experiment and tube formation experiment are all 0, 2 nM, 4 nM, 6 nM, 8 nM, and the treatment time is 24 hours.