Construction method and application of a micro-tumor model of infantile hemangioma

Abstract

The invention belongs to the field of biomedicine, and discloses a method for constructing an infantile hemangioma CD31+HemECs 3D micro-tumor model and its application, comprising: under sterile conditions, using surgical scissors to cut up a hemangioma sample, and using collagenase A to digest the cut tissue; using a flow cytometer to aseptically sort CD31+ cells from a primary culture, and culturing CD31+HemECs in a humidified environment containing 5% CO2 at 37°C; soaking a fresh porcine aorta in phosphate buffered saline (PBS) containing 100U / ml penicillin and 100g / ml streptomycin, placing it on ice and immediately transferring it to a laboratory to decellularize the porcine aorta and perform vascular-specific DAM characteristics; analyzing the characteristics of a decellularized porcine aorta extracellular matrix hydrogel; preparing a micropattern array printed 3D culture dish to form a CD31+HemEC micro-tumor; and evaluating the applicability of the model to propranolol, a first-line therapeutic drug for IH. The present invention provides a more stable and efficient experimental model for mechanism exploration and drug screening of IH, which is beneficial to maintaining tumor heterogeneity and simulating tumor tissue in vivo.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a construction method and application of an infantile hemangioma CD31+HemECs 3D micro-tumor model. Background Art

[0002] Currently, infantile hemangioma (IH) is the most common vascular tumor in infants. It is a non-congenital benign tumor with an incidence of about 4%-5% in newborns. The incidence of IH seems to have increased significantly in the past 40 years, which may be related to the increase in low birth weight and premature infants.

[0003] The development of IH presents a unique life cycle. The lesion does not exist at birth. It usually begins when the child is 3 to 7 weeks old, proliferates until 12 months old, and the regression period begins around 1 year old and lasts for 3-5 years. About 50% to 70% of the tumors have residual (capillary dilation, fibrofatty tissue, skin excess, edema, pigmentation or scars). About 10%-15% of infantile hemangiomas may have complications, such as tumor ulceration and bleeding, which may also cause cosmetic problems and even serious disfigurement, airway obstruction, serious complications that endanger life, visual impairment, and in some cases congestive heart failure. Our previous studies have confirmed that IH has different degrees of impact on the quality of life of children and parents.

[0004] The pathogenesis of IH is still unclear. To date, the pathogenesis of IH is still unclear, and there are many hypotheses for the cause. One hypothesis suggests that it may be of embryonic origin. It is reported that hypoxia and angiotensin are considered to play an important role in the occurrence and development of hemangiomas as independent factors, and the two have a synergistic effect. At present, there is no unified safe and effective treatment for IH. In a landmark paper published in 2008, Leaute-Labreze et al. found that two infants with IH had remission of IH after taking propranolol for heart disease. Since then, propranolol, a non-selective beta-blocker, has become the first-line drug for the treatment of IH. However, the mechanism of action of propranolol on IH has not been elucidated yet. It is reported that the possible causes include vasoconstriction, inhibition of angiogenesis, induction of apoptosis, inhibition of nitric oxide production, and antagonism of the renin-angiotensin axis. Propranolol is considered safe and effective, but the safety of propranolol in infants has not been fully demonstrated, and it can cause a variety of adverse reactions, leading to termination of treatment and recurrence of IH. In addition, the first patients being treated are only now entering adolescence, and the potential long-term effects of propranolol on neurocognition in infants and young children are unknown. Developing safer and more effective treatments and drugs is critical.

[0005] There is no standard and stable model for IH research at present. There is no stable cell line for IH related research, and primary cells need to be isolated, mainly IH-derived endothelial cells and IH endothelial progenitor cells. This two-dimensional cell culture has huge differences from the tissue structure and physiology in vivo, and spontaneous regression often occurs during the tumor formation process in nude mice, which seriously restricts the research on the mechanism related to the occurrence and development of IH.

[0006] At present, many medical studies still rely on traditional two-dimensional in vitro cell culture or animal models for drug testing and pathogenesis exploration. However, both models have insurmountable shortcomings. Although the two-dimensional cell culture system is derived from human tissue, it is very different from the in vivo tissue structure and physiology. These models lack the complex microenvironment of human tumors, including cell-cell and cell-extracellular matrix interactions, which are key factors affecting cell fate and will cause the failure of drug clinical transformation. Two-dimensional cells also cannot represent the heterogeneity of the disease because they are originally derived from homogeneous cell lines. Three-dimensional (3D) tumor cell culture can significantly improve the viability, tissue morphology, genotype stability, function and drug metabolism of tumor cells in vitro. Their cell aggregates are wrapped by the natural extracellular matrix (ECM), which better replicates the microenvironment of solid tumors in vivo. In addition, animal models inevitably face many problems, such as high cost, ethical issues, heterogeneity, etc.

[0007] 3D cell culture methods include hanging drop method, matrix encapsulation culture, transfer culture flask, ultra-low attachment plate, rocking suspension culture technology, microporous mesh, microfluidics, magnetic levitation and 3D printing technology. In recent years, some scholars have formed a pattern arrangement of a certain shape and size on the culture dish substrate by covalently or non-covalently coating a micropattern array formed by carbohydrates, peptides and proteins. Cells are restrictedly adhered to the micropattern. Cells will spontaneously assemble into spheres with 3D multicellular structures through cell proliferation and cell-cell adhesion ability, so that 3D multicellular spheres with controllable size and orderly arrangement can be cultured, which is conducive to high-throughput drug screening. Proteins adsorbed by polydimethylsiloxane (PDMS) (as bio-ink) are used to create protein-specific microarrays on non-attached culture dishes through microcontact printing technology, which has been widely used in cytology, drug screening and tissue engineering. Fibronectin, collagen I, collagen IV and laminin are commonly used in traditional micropattern arrays as bio-inks. However, a single ECM protein component is not sufficient to improve and regulate the viability and function of differentiated cells derived from specific cell lines, primary cells or induced pluripotent stem cells. In recent years, recent progress has been made in organ or tissue decellularization, making it possible to obtain ECM with unique structure, composition, and biological and organ specificity. Compared with a single ECM protein component, such as collagen I, which is widely used in liver tissue engineering, liver-specific ECM containing various biomacromolecules can interact with tumor cells through discoidin domain receptors and transmembrane proteoglycans or through integrins, regulate signaling pathways related to cell proliferation, migration and differentiation, and thus affect the biological behavior of cells.

[0008] Through the above analysis, the problems and defects of the prior art are as follows:

[0009] (1) The pathogenesis of IH is still unclear;

[0010] (2) Currently, there is no standard and stable model for the study of IH. Summary of the invention

[0011] In view of the problems existing in the prior art, the present invention provides a method for constructing a CD31+HemECs 3D micro-tumor model of infantile hemangioma and its application.

[0012] The present invention is implemented as follows: a method for constructing a CD31+HemECs 3D micro-tumor model of infantile hemangioma, the method for constructing a CD31+HemECs 3D micro-tumor model of infantile hemangioma comprising:

[0013] Step 1: Under sterile conditions, the hemangioma sample is minced with surgical scissors, and the minced tissue is digested with collagenase A;

[0014] Step 2: Aseptically sort CD31+ cells from primary culture using flow cytometry, and culture CD31+HemECs in a humidified environment at 37°C with 5% CO2;

[0015] Step 3: The fresh porcine aorta was immersed in phosphate buffered saline (PBS) containing 100 U / ml penicillin and 100 g / ml streptomycin, placed on ice and immediately transferred to the laboratory, and the porcine aorta was decellularized using the Triton-SDS-Triton continuous oscillation method to obtain vascular-specific DAM;

[0016] Step 4, freeze-drying DAM and grinding it into powder to prepare a DAM solution;

[0017] Step 5, preparing micropattern array printed 3D culture dishes and culturing CD31+HemEC microtumors;

[0018] Step six, evaluate the applicability of the CD31+HemECs microtumor model to propranolol, a first-line treatment for IH.

[0019] Furthermore, the first step is to culture CD31+HemECs primary cells using endothelial cell culture medium, wherein the endothelial cell culture medium comprises EBM-2, supplemented with 20% fetal bovine serum, 100 units / ml penicillin and 100 μg / ml streptomycin.

[0020] Furthermore, the specific process of decellularization of the porcine aorta in step 3 is as follows:

[0021] First, blood cells were lysed by washing with distilled water on an orbital shaker at a speed of 100 r / min for 24 h;

[0022] Then, the aorta was immersed in a 0.5% TritonX-100 solution and continuously shaken for 24 hours. After washing with PBS for 2 hours, the aorta was immersed in a 0.25% SDS solution and continuously shaken for 72 hours. In order to remove the residual detergent, the decellularized aorta was shaken with PBS for 72 hours.

[0023] Finally, the decellularized porcine aorta and fresh porcine aorta were fixed with paraformaldehyde, embedded in paraffin and sectioned, and then HE staining was performed to evaluate the degree of decellularization.

[0024] Furthermore, the specific process of the vascular-specific DAM feature in step 3 is as follows:

[0025] First, the obtained decellularized blood vessels were stored at −20°C for further use, and all steps were performed under a sterile environment;

[0026] Then, the freeze-dried DAM was ground into powder using WileyMill and stirred with 10% pepsin and 0.01 M HCl at room temperature for 48 h to obtain vascular decellularized ECM hydrogel;

[0027] Finally, 0.1 M NaOH was added to neutralize the DAM solution to a pH of 7.2 to 7.4, and the final concentration of the DAM solution was adjusted to 10 mg / mL using 1× PBS (PBS can be used to adjust the concentration).

[0028] Further, the specific process of step 4 is:

[0029] First, a PDMS chip was obtained by etching a characteristic pattern on a silicon wafer using a laser, with diameters of 50, 100, 150, and 200 μm, respectively, and a template spacing of 50 μm. A layer of 0.1 mg / mL DAM solution and 2 μg of fluorescein isothiocyanate isomers was coated on the surface of the PDMS chip and allowed to react at room temperature for 20 minutes.

[0030] Secondly, the excess solution was removed, dried at 37° for 10 min, and the chip was placed on a non-adhesive culture dish with a diameter of 35 mm and stacked at a force of 0.2 N for 10 min;

[0031] Then, the shape of the microarray was observed under a fluorescence microscope, and the treated culture dishes were treated with 10 g / L pluronic F-127 aqueous solution at room temperature for 1 h to prevent nonspecific cell adhesion;

[0032] Finally, sterilize with ultraviolet light for 2 h.

[0033] Further, the specific process of step five is:

[0034] Add 3 ml of EBM-2 medium to the treated micropatterned culture dish and plant 2x10 5 CD31+HemECs were cultured in a cell culture incubator for 6 h, the culture medium was removed, and the cells were washed three times with 1xPBS to remove the cells that had not yet attached. The morphology of the cell aggregates cultured for 7 days was observed and imaged using EVOSXLCore.

[0035] Cell aggregates grown on micropatterned culture dishes were fixed with 4% formaldehyde for 20 min at room temperature and stained with 100 nM phalloidin solution for 45 min at room temperature;

[0036] The cells were counterstained with DAPI, and images were collected by two-photon confocal microscopy. Tumors cultured at 50, 100, 150, and 200 μm were collected, RNA was extracted, and the expression of VEGFA and MMP2 was detected by q-PCR.

[0037] Further, the specific process of step six is:

[0038] The cells were treated with propranolol at concentrations of 50, 100, 150, and 200 μg / mL for 24 and 48 h, respectively, and 0.25% DMSO was used as a negative control;

[0039] The viability of spheroids was assessed using fluorescence quenching staining, and images were taken under a confocal microscope, and the fluorescence intensity was measured and analyzed as the mean gray value;

[0040] ImageJ software was used for quantification to evaluate the reliability of the model for drug screening.

[0041] Another object of the present invention is to provide an application of the 3D micro-tumor model of infantile hemangioma CD31+HemECs in the field of IH mechanism exploration and drug screening.

[0042] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0043] The present invention uses CD31+HemECs microtumors for research, providing a more stable and efficient experimental model for mechanism exploration and drug screening of IH.

[0044] The vascular-specific ECM pattern microarray created using PDMC micropattern printing technology shortened the in vitro expansion culture time of CD31+HemECs, effectively controlled the particle size and arrangement of CD31+HemECs microtumors, and improved the repeatability and stability of drug screening.

[0045] Vascular decellularized ECM provides a tumor microenvironment for CD31+HemECs microtumor culture, which is beneficial to maintain tumor heterogeneity and simulate in vivo tumor tissue.

[0046] The PDMC micropattern printing technology uses a laser-etched silicon wafer with a specific pattern as a template. The etching accuracy reaches 1μm and the maximum etching area reaches 12 inches. The PDMS stamp is obtained by inverting the mold. Then, the PDMC micropattern printing technology is used to lay the extracellular matrix gel pattern microarray on the bottom of the cell culture dish treated with non-attached cells. Pluronic F-127 seals the space on the remaining culture substrate so that the added cells can only grow by attaching to the extracellular matrix gel pattern.

[0047] Preparation of vascular decellularized ECM: The porcine aorta decellularized scaffold was prepared by the Triton-SDS-Triton portal vein perfusion method. The decellularized scaffold was further freeze-dried, ball-milled and pepsin-hydrolyzed to obtain vascular decellularized ECM hydrogel.

[0048] Cultivation of CD31+HemECs microtumors: Using vascular decellularized ECM pattern microarray chips to culture CD31+HemECs microtumors can shorten their in vitro expansion culture time and effectively control their particle size and arrangement.

[0049] The technical solution of the present invention fills the technical gap in the industry at home and abroad: cutaneous infantile hemangioma is the most common benign vascular tumor in infants, with an incidence rate of about 4%-5% in newborns. About 10%-15% of infantile hemangiomas may develop serious complications. Our previous studies have confirmed that IH has varying degrees of impact on the quality of life of children and parents. To date, the pathogenesis of IH is still unclear, and there is no unified, safe and effective treatment for IH. The mechanism of action of propranolol, the current first-line drug for the treatment of IH, on IH has not yet been elucidated, and the safety of propranolol for infants has not been fully demonstrated. It can pass through the blood-brain barrier and may have side effects on the central nervous system. It is crucial to develop safer and more effective treatments and drugs.

[0050] Currently, there is no stable cell line for IH research, and primary cells need to be isolated, mainly IH-derived endothelial cells and IH endothelial progenitor cells. This two-dimensional cell culture has huge differences from the in vivo tissue structure and physiology, and spontaneous regression often occurs during the tumor formation process in nude mice, which seriously restricts the research on the related mechanisms of the occurrence and development of IH.

[0051] The starting point of the present invention is that the pathogenesis of IH is still unclear and a standard and stable research model is urgently needed. By using vascular-specific ECM and combining it with a micropattern array, a high-throughput IH-derived CD31+HemECs microtumor model of uniform size is constructed, laying an important foundation for further in-depth research on the IH mechanism and drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a flow chart for constructing an infantile hemangioma CD31+HemECs 3D micro-tumor model provided by an embodiment of the present invention;

[0053] Figure 2 This is a flow chart of the separation and culture of IH-derived CD31+HemECs provided in an embodiment of the present invention;

[0054] Figure 3 The decellularization and vascular-specific DAM characteristic diagram of the porcine aorta provided by the embodiments of the present invention, (A) H&E staining diagram, (B) MT staining diagram of the porcine aorta and decellularized porcine aorta, (C) relative DNA content diagram of the porcine aorta and decellularized porcine aorta, (D) type I collagen diagram of the porcine aorta and decellularized porcine aorta, (E) type III collagen diagram, (F) type IV collagen staining;

[0055] Figure 4Schematic diagram of the properties of the decellularized porcine aorta extracellular matrix hydrogel provided in an embodiment of the present invention, (A) Live / dead staining of CD31+HemECs cultured on different hydrogels for 1, 3 and 5 days, (B) Adhesion rate of CD31+HemECs in different hydrogels 10 minutes after cell seeding, (C) Proliferation rate of CD31+HemECs on different hydrogels 24 hours after cell seeding;

[0056] Figure 5 It is a schematic diagram of micro-pattern array printing, 3D culture dish preparation and micro-tumor formation provided by an embodiment of the present invention;

[0057] Figure 6 3. The two-photon confocal microscope stereoscopic imaging and VGGF and MMP-2 expression result diagram provided by the embodiments of the present invention, (A) a three-dimensional view of a CD31+HemEC micro-tumor with a diameter of 50 μm under a confocal microscope, (B) a three-dimensional view of a CD31+HemEC micro-tumor with a diameter of 50 μm under a confocal microscope, (C) a three-dimensional view of a CD31+HemEC micro-tumor with a diameter of 50 μm under a confocal microscope, (D) a three-dimensional view of a CD31+HemEC micro-tumor with a diameter of 200 μm under a confocal microscope;

[0058] Figure 7 This is the drug screening result provided by the embodiment of the present invention. DETAILED DESCRIPTION

[0059] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0060] In order to enable those skilled in the art to fully understand how to implement the present invention in detail, this section is an explanatory embodiment that expands and describes the technical solution of the claims.

[0061] The method for constructing the infantile hemangioma CD31+HemECs 3D micro-tumor model provided in the embodiment of the present invention is as follows Figure 1 As shown:

[0062] Under sterile conditions, hemangioma samples were minced with surgical scissors, the minced tissue was digested with collagenase A, and primary cells were cultured in endothelial cell culture medium (EBM-2, supplemented with 20% fetal bovine serum, penicillin (100 units / ml) and streptomycin (100 μg / ml). CD31+ cells were aseptically sorted from primary cultures using flow cytometry, and CD31+HemECs were cultured in a humidified environment at 37°C containing 5% CO2.

[0063] Fresh porcine aorta was immersed in phosphate buffered saline (PBS) containing 100U / ml penicillin and 100g / ml streptomycin, placed on ice and immediately transferred to the laboratory. First, it was rinsed with distilled water on an orbital shaker (100r / min) for 24h to lyse blood cells; then it was immersed in 0.5% TritonX-100 solution and continuously shaken for 24h (100r / min); after washing with PBS for 2h, the artery was immersed in 0.25% SDS solution and continuously shaken for 72h (100r / min); in order to remove residual detergent, the decellularized artery was shaken with PBS for 72h (100r / min). The decellularized porcine artery and fresh porcine aorta were fixed with paraformaldehyde, embedded in paraffin and sectioned, and HE staining was performed to evaluate the degree of decellularization. Finally, the obtained decellularized blood vessels were stored at -20℃ for further use. All steps were performed under sterile conditions. The freeze-dried DAM was ground into powder using WileyMill, and stirred with 10% pepsin and 0.01M HCl at room temperature for 48 hours. Then 0.1M NaOH was added to neutralize the DAM solution to pH 7.2-7.4, and the final concentration of the DAM solution was adjusted to 10 mg / mL with 1×PBS.

[0064] The PDMS chips were obtained by laser etching the characteristic patterns on the silicon wafer, with diameters of 50, 100, 150, and 200 μm, respectively, and the template spacing was 50 μm (working basis Figure 4 A), apply a layer of 0.1mg / mL DAM solution and 2μg fluorescein isothiocyanate isomer on the surface of the chip, act at room temperature for 20min, remove excess solution, dry at 37° for 10min, place the chip on a non-adhesive culture dish with a diameter of 35mm, and stack it with a force of 0.2N for 10min. Then observe the shape of the microarray under a fluorescence microscope. Treat the treated culture dish with 10g / L pluronicF-127 aqueous solution at room temperature for 1h to prevent nonspecific cell adhesion, and then sterilize it with ultraviolet irradiation for 2h.

[0065] Add 3 ml of EBM-2 medium to the treated micropatterned culture dish and plant 2x10 5CD31+HemECs were cultured in a cell culture incubator for 6 hours, the culture medium was removed, and the cells that had not yet attached were washed three times with 1xPBS. The morphology of the cell aggregates cultured for 7 days was observed and imaged using EVOS XL Core. The cell aggregates grown on the micropatterned culture dishes were fixed with 4% formaldehyde for 20 minutes at room temperature, stained with phalloidin solution (100nM) at room temperature for 45 minutes, and then counterstained with DAPI, and images were collected by two-photon confocal microscopy. Tumors cultured at 50, 100, 150, and 200μm were collected, RNA was extracted, and the expression of VEGFA and MMP2 was detected by q-PCR.

[0066] The cells were treated with different concentrations of propranolol (50, 100, 150 and 200 μg / mL) for 24 and 48 h, respectively, and 0.25% DMSO was used as a negative control. Fluorescence quenching staining was used to evaluate the viability of the spheroids, and images were taken under a confocal microscope. The fluorescence intensity was measured and analyzed by the mean gray value, and quantified using ImageJ software to evaluate the reliability of the model for drug screening.

[0067] The embodiments of the present invention have achieved some positive effects during the development or use process, and indeed have great advantages over the prior art. The following content is described in conjunction with data, charts, etc. of the experimental process.

[0068] Under sterile conditions, the hemangioma samples were minced with surgical scissors, and the minced tissue was digested with collagenase A. CD31+ cells were aseptically sorted from the primary culture using flow cytometry, and CD31+HemECs were cultured in EBM-2 medium, as follows: Figure 2 shown.

[0069] Porcine aorta decellularization and vessel-specific DAM characteristics Figure 3 Shown: (A) H&E staining; (B) MT staining of porcine aorta and decellularized porcine aorta; (C) relative DNA content of porcine aorta and decellularized porcine aorta; (D) type I collagen, (E) type III collagen, and (F) type IV collagen staining of porcine aorta and decellularized porcine aorta.

[0070] Characterization of decellularized porcine aorta extracellular matrix hydrogels: Using DAM-based hydrogels as surface coating matrix can improve the adhesion, proliferation, and survival of CD31+HemECs, such as Figure 4 As shown; (A) Live / dead staining of CD31+HemECs cultured on different hydrogels at 1, 3, and 5 days; 10 minutes after cell seeding; (B) Adhesion rate of CD31+HemECs in different hydrogels; 24 hours after cell seeding (C) Proliferation rate of CD31+HemECs on different hydrogels.

[0071] Preparation of 3D culture dishes printed with micropattern arrays and formation of CD31+HemEC microtumors: Microarray patterns obtained from PDMS seals with diameters of 50, 100, 150, and 200 μm, e.g. Figure 5 shown.

[0072] The ideal diameter of the pattern is 100-150 μm; (A), (B), (C), and (D) Three-dimensional views of CD31+HemEC microtumors with diameters of 50, 100, 150, and 200 μm under confocal microscopy. Figure 6 (E) MMP-2 and VEGF-A gene expression levels in CD31+HemEC microtumors of different diameters.

[0073] The present invention evaluates the applicability of the model to propranolol, a first-line treatment drug for IH. A more significant effect is obtained when the concentration of propranolol is greater than 100 ug / ml within 24 hours of action. The drug screening results are as follows Figure 7 When propranolol was applied for 48 hours, the concentration was lower than 100ug / ml and there was some effect.

[0074] It should be noted that the embodiments of the present invention can be implemented by hardware, software or a combination of software and hardware. The hardware part can be implemented using dedicated logic; the software part can be stored in a memory and executed by an appropriate instruction execution system, such as a microprocessor or dedicated hardware. A person of ordinary skill in the art will understand that the above-mentioned devices and methods can be implemented using computer executable instructions and / or contained in a processor control code, such as a carrier medium such as a disk, CD or DVD-ROM, a programmable memory such as a read-only memory (firmware), or a data carrier such as an optical or electronic signal carrier. Such code is provided on a carrier medium such as a disk, CD or DVD-ROM, a programmable memory such as a read-only memory (firmware), or a data carrier such as an optical or electronic signal carrier. The device and its modules of the present invention can be implemented by hardware circuits such as very large-scale integrated circuits or gate arrays, semiconductors such as logic chips, transistors, etc., or programmable hardware devices such as field programmable gate arrays, programmable logic devices, etc., can also be implemented by software executed by various types of processors, or can be implemented by a combination of the above-mentioned hardware circuits and software, such as firmware. The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any modification, equivalent substitution and improvement made by any technician familiar with the technical field within the technical scope disclosed by the present invention, within the spirit and principle of the present invention, should be covered within the protection scope of the present invention.

Claims

1. A method for constructing an infantile hemangioma CD31+HemECs 3D micro-tumor model, characterized in that: The method for constructing the infantile hemangioma CD31+HemECs 3D micro-tumor model comprises: Step 1: Under sterile conditions, the hemangioma sample is minced with surgical scissors, and the minced tissue is digested with collagenase A; Step 2: Aseptically sort CD31+ cells from primary culture using flow cytometry, and culture CD31+HemECs in a humidified environment at 37°C with 5% CO2; Step 3: The fresh porcine aorta was immersed in phosphate buffered saline (PBS) containing 100 U / ml penicillin and 100 g / ml streptomycin, placed on ice and immediately transferred to the laboratory, and the porcine aorta was decellularized using the Triton-SDS-Triton portal vein perfusion method to obtain vascular-specific DAMs; Step 4, freeze-drying the vascular-specific DAM and grinding it into powder to prepare vascular decellularized ECM hydrogel; Step 5, preparing a micropattern array printed 3D culture dish to form CD31+HemEC microtumors; Step six, evaluate the applicability of the model to propranolol, the first-line treatment for infantile hemangioma IH; The specific process of decellularization of the porcine aorta in step 3 is as follows: (1.1) Rinse with distilled water on an orbital shaker at a speed of 100 r / min for 24 h to lyse blood cells; (1.2) Immerse in a 0.5% TritonX-100 solution and shake continuously for 24 hours; after washing with PBS for 2 hours, immerse the porcine aorta in a 0.25% SDS solution and shake continuously for 72 hours; in order to remove the residual detergent, shake the decellularized porcine aorta with PBS for 72 hours; (1.3) The decellularized porcine aorta and fresh porcine aorta were fixed with paraformaldehyde, embedded in paraffin and sectioned, and then stained with HE to evaluate the degree of decellularization; The specific process of step 4 is as follows: (2.1) The obtained decellularized blood vessels were stored at -20°C for further use. All steps were performed under a sterile environment. (2.2) The freeze-dried vascular-specific DAM was ground into powder using a Wiley Mill, and stirred with 10% pepsin and 0.01 M HCl at room temperature for 48 h to obtain a vascular decellularized ECM hydrogel; (2.3) adding 0.1 M NaOH to neutralize the vascular decellularized ECM hydrogel to a pH of 7.2-7.4, and adjusting the final concentration of the vascular decellularized ECM hydrogel to 10 mg / mL with 1× PBS; The specific process of step five is as follows: (3.1) A PDMS chip was obtained by etching a characteristic pattern on a silicon wafer using a laser, with diameters of 50, 100, 150, and 200 μm, respectively, and a template spacing of 50 μm. A layer of 0.1 mg / mL vascular decellularized ECM hydrogel and 2 μg of fluorescein isothiocyanate isomers was coated on the surface of the PDMS chip and allowed to react at room temperature for 20 min; (3.2) Remove excess solution, dry at 37° for 10 min, place the chip on a non-adhesive culture dish with a diameter of 35 mm, and stack it at a force of 0.2 N for 10 min; (3.3) The shape of the microarray was observed under a fluorescence microscope. The treated culture dishes were treated with 10 g / L pluronic F-127 aqueous solution at room temperature for 1 h to prevent nonspecific cell adhesion; (3.4) Sterilize with ultraviolet light for 2 hours; (3.5) Add 3 ml of EBM-2 medium to the treated micropatterned culture dish and plant 2x10 5 CD31+HemECs were cultured in a cell culture incubator for 6 h, the culture medium was removed, and the cells were washed three times with 1xPBS to remove the cells that had not yet attached. The morphology of the cell aggregates cultured for 7 days was observed and imaged using EVOS XL Core. (3.6) Cell aggregates grown on micropatterned culture dishes were fixed with 4% formaldehyde at room temperature for 20 min and stained with 100 nM phalloidin solution at room temperature for 45 min; (3.7) The cells were counterstained with DAPI, and images were acquired using a two-photon confocal microscope. Tumors cultured at 50, 100, 150, and 200 μm were collected, RNA was extracted, and the expression of VEGFA and MMP2 was detected using q-PCR.

2. The method for constructing the infantile hemangioma CD31+HemECs 3D micro-tumor model according to claim 1, characterized in that: The first step is to culture primary cells using endothelial cell culture medium, wherein the endothelial cell culture medium comprises EBM-2, supplemented with 20% fetal bovine serum, 100 units / ml penicillin, and 100 μg / ml streptomycin.

3. The method for constructing the infantile hemangioma CD31+HemECs 3D micro-tumor model according to claim 1, characterized in that: The specific process of step six is ​​as follows: (4.1) CD31+HemECs3D microtumors were treated with 50, 100, 150 and 200 μg / mL propranolol for 24 and 48 h, respectively, and 0.25% DMSO was used as a negative control; (4.2) Fluorescence quenching staining was used to assess spheroid viability, images were taken under a confocal microscope, and fluorescence intensity was measured and analyzed as mean gray value; (4.3) Use ImageJ software for quantification and evaluate the reliability of the model for drug screening.

4. Application of the model constructed by the method for constructing an infantile hemangioma CD31+HemECs 3D micro-tumor model as described in any one of claims 1 to 3 in the field of mechanism exploration and drug screening of infantile hemangioma IH.

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