CN115651912A - Construction method and application of infantile hemangioma micro-tumor model - Google Patents

Abstract

The invention belongs to the field of biomedicine, and discloses a construction method and application of a baby hemangioma CD31+ HemECs3D micro-tumor model, which comprises the following steps: cutting the hemangioma sample by using a surgical scissors under a sterile condition, and digesting the cut tissue by adopting collagenase A; aseptically sorting CD31+ cells from the primary culture using a flow cytometer, culturing the CD31+ HemECs in a humidified environment containing 5% CO2 at 37 ℃; soaking fresh pig aorta in Phosphate Buffered Saline (PBS) containing 100U/ml penicillin and 100g/ml streptomycin, placing on ice, immediately transferring into a laboratory, and performing pig aorta decellularization and blood vessel specific DAM characteristics; analyzing the characteristics of the acellular porcine aortic extracellular matrix hydrogel; preparing a 3D culture dish for micropattern array printing to form a CD31+ HemEC micro-tumor; the model was evaluated for its suitability for the first-line treatment of IH drug propranolol. The invention provides a more stable and efficient experimental model for IH mechanism exploration and drug screening, which is beneficial to maintaining tumor heterogeneity and simulating in vivo tumor body tissues.

Description

Construction method and application of infantile hemangioma micro-tumor model

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a construction method and application of a baby hemangioma CD31+ HemECs3D micro-tumor model.

Background

Currently, infantile Hemangioma (IH) is the most common vascular tumor in infants, a non-congenital benign tumor with an incidence of about 4% -5% in newborns, the incidence of IH appears to increase significantly over the past 40 years, which may be associated with low birth weight and increased preterm infants.

The development of IH presents a unique life cycle, with lesions not present at birth, typically beginning at 3 to 7 weeks in infants, proliferating to 12 months, beginning at about 1 year of age and continuing for 3-5 years, with approximately 50% to 70% of the tumor remaining (telangiectasia, fibroadipose tissue, skin excrescences, edema, pigmentation or scarring). Approximately 10% to 15% of infantile hemangiomas present with complications such as tumour ulceration bleeding, can cause cosmetic problems, can even cause severe disfigurement, can cause airway obstruction, can develop severe complications life threatening, can cause visual impairment, and in some cases congestive heart failure.

The pathogenesis of IH is unclear. To date, the pathogenesis of IH is unknown, with a large number of etiology hypotheses suggesting possible embryonic origins. It is reported that hypoxia and angiotensin are considered to play important roles in the development of hemangiomas as independent factors, and that there is a synergistic effect of both. Currently, there is no uniform, safe and effective treatment for IH. In a milestone-meaningful paper published in 2008 by Leaute-Labreze et al, 2 infants with IH were found to have had a regression of IH after heartburn due to heart disease. Since then, propranolol, a non-selective beta blocker, has become the first line drug for IH treatment. However, the mechanism of action of propranolol on IH has not been elucidated at present, and its possible causes have been reported to 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 administration to infants is not fully demonstrated, and various adverse reactions can be caused, so that treatment is stopped, and IH relapse is caused. Furthermore, the first patients currently receiving treatment now enter puberty, and the potential long-term effects of propranolol on neurocognition in infants is unknown. It is important to develop safer and more effective treatments and drugs.

Currently there is no standard stable model for IH research. IH has no stable cell line for relevant research, primary cells, mainly IH-derived endothelial cells and IH endothelial progenitor cells, need to be isolated, and the two-dimensional cell culture has great difference with the in vivo tissue structure and physiology, and spontaneous regressions often exist in the process of nude mouse tumorigenesis, thus seriously restricting the research on the mechanism related to the generation and development of IH.

Currently, many medical studies still rely on traditional two-dimensional in vitro cell culture or animal models for drug testing and pathogenesis exploration. However, both of these models have insurmountable disadvantages, although the two-dimensional cell culture system is derived from human tissues, it has great differences from the in vivo tissue structure and physiology, and 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 can cause failure of clinical transformation of drugs. Two-dimensional cells also do not represent disease heterogeneity, as they are originally from homogeneous cell lines. And the three-dimensional (3D) tumor cell culture can obviously improve the activity, the tissue morphology, the genotype stability, the function and the drug metabolism of the in vitro tumor cells. Their cell aggregates are encapsulated by the native extracellular matrix (ECM), which better replicates the microenvironment of solid tumors in vivo. In addition, animal models inevitably face a number of problems, such as high cost, ethical issues, heterogeneity, etc.

The 3D cell culture method comprises a hanging drop method, matrix encapsulation culture, a rotary culture flask, an ultra-low attachment plate, a swing suspension culture technology, a micropore net, microfluidics, magnetic suspension and a 3D printing technology. In recent years, researchers have formed patterned arrays of certain shapes and sizes on a culture dish substrate by covalently or non-covalently coating micropattern arrays of carbohydrates, peptides and proteins, cells are restrictively adhered to the micropatterns, and the cells spontaneously assemble into spheres with 3D multicellular structures through cell proliferation and cell-cell adhesion capabilities, thereby being capable of culturing 3D multicellular spheres of controlled sizes and ordered arrays, and facilitating high-throughput drug screening. Polydimethylsiloxane (PDMS) adsorbed proteins (as bio-ink) have been widely used in cytology, drug screening, and tissue engineering by creating protein-specific microarrays on non-attached culture dishes by microcontact printing techniques. Fibronectin, collagen I, collagen IV, and laminin are commonly used as bio-inks in conventional micropatterned arrays. However, the single ECM protein component is not sufficient to improve and regulate the viability and function of a particular cell line, primary cell or differentiated cell derived induced pluripotent stem cell. Recent advances in organ or tissue decellularization have made it possible in recent years to obtain ECMs with unique structures, compositions, and biological and organ specificities. In contrast to single ECM protein components, such as collagen I, which is widely used in liver tissue engineering, liver-specific ECMs containing various biological macromolecules may modulate signaling pathways associated with cell proliferation, migration, and differentiation through discotic domain receptors and transmembrane proteoglycans or through integrin interactions with tumor cells, thereby affecting the biological behavior of the cells.

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

(1) The pathogenesis of IH is currently unknown;

(2) Currently there is no standard stable model for IH research.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a construction method and application of a baby hemangioma CD31+ HemECs3D micro-tumor model.

The invention is realized in such a way that the construction method of the infant hemangioma CD31+ HemECs3D micro-tumor model comprises the following steps:

cutting a hemangioma sample by using surgical scissors under an aseptic condition, and digesting the cut tissue by adopting collagenase A;

step two, aseptically sorting CD31+ cells from the primary culture using a flow cytometer, culturing the CD31+ HemECs in a humidified environment containing 5% co2 at 37 ℃;

step three, soaking fresh pig aorta in Phosphate Buffered Saline (PBS) containing 100U/ml penicillin and 100g/ml streptomycin, placing the pig aorta in ice, immediately transferring the pig aorta into a laboratory, and performing pig aorta decellularization by adopting a Triton-SDS-Triton continuous oscillation method to obtain vascular specific DAM;

step four, freeze-drying the DAM and grinding the DAM into powder to prepare a DAM solution;

preparing a micro-pattern array printing 3D culture dish, and culturing CD31+ HemEC micro-tumor;

and sixthly, evaluating the applicability of the CD31+ HemECs micro-tumor model to the propranolol serving as the first-line IH treatment drug.

Further, the step is to culture the CD31+ HemECs primary cells with endothelial cell culture medium comprising EBM-2 supplemented with 20% fetal bovine serum, penicillin at a concentration of 100 units/ml and streptomycin at a concentration of 100. Mu.g/ml.

Further, the specific process of the porcine aorta decellularization in the third step is as follows:

firstly, washing for 24 hours on a track vibrating screen with the rotating speed of 100r/min by using distilled water, and cracking blood cells;

then, immersing the mixture into TritonX-100 solution with the concentration of 0.5 percent, and continuously oscillating for 24 hours; after washing with PBS for 2h, the porcine aorta was immersed in 0.25% SDS solution and shaken continuously for 72h; to remove residual detergent, the decellularized porcine aorta was shaken with PBS for 72h;

and finally, fixing the decellularized pig aorta and the fresh pig aorta by using paraformaldehyde, embedding the fixed aorta into paraffin, slicing, performing HE staining, and evaluating the degree of decellularization.

Further, the specific process of vascular-specific DAM characterization in step three is as follows:

firstly, the obtained decellularized blood vessel is stored at-20 ℃ for further use, and all the steps are carried out in a sterile environment;

then, lyophilized DAM was pulverized using WileyMill and stirred at room temperature with pepsin at a concentration of 10% and 0.01MHCl for 48 hours to obtain a vascular decellularized ECM hydrogel;

finally, the DAM solution was neutralized to pH 7.2-7.4 by adding 0.1M NaOH and adjusted to a final concentration of 10mg/mL with 1XPBS (PBS can be used to adjust the concentration).

Further, the specific process of the fourth step is as follows:

firstly, etching characteristic patterns on a silicon wafer by using laser to obtain a PDMS chip, wherein the diameters of the PDMS chip are respectively 50 micrometers, 100 micrometers, 150 micrometers and 200 micrometers, the distance between templates is 50 micrometers, coating a layer of solution with the concentration of 0.1mg/mLDAM and 2 micrometers g of fluorescein isothiocyanate isomers on the surface of the PDMS chip, and acting for 20 minutes at room temperature;

second, excess solution was removed, dried at 37 ° for 10min, and the chips were placed on a 35mm diameter non-adherent culture dish and stacked at a force of 0.2N for 10min;

then, the shape of the microarray was observed under a fluorescent microscope, and the treated culture dish was treated with 10g/Lpluronic F-127 aqueous solution at room temperature for 1 hour to prevent non-specific cell adhesion;

finally, sterilization was performed by ultraviolet irradiation for 2 hours.

Further, the concrete process of the fifth step is as follows:

3ml of EBM-2 medium was added to the treated micro-patterned culture dishes, and 2X10 medium was plated on each dish ⁵ CD31+ HemECs, after 6h incubation in a cell incubator, medium was removed, washed 3 times with 1xPBS, unattached cells were removed, and morphology of cell aggregates cultured for 7 days was observed and imaged using evoxlcore;

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

counterstaining with DAPI, collecting images by two-photon confocal microscope, collecting tumor bodies cultured at 50, 100, 150 and 200 μm, extracting RNA, and detecting expression of VEGFA and MMP2 by q-PCR.

Further, the specific process of the sixth step is as follows:

treatment with propranolol concentrations of 50, 100, 150 and 200 μ g/mL for 24 and 48h, respectively, with 0.25% dmso as a negative control;

evaluating the activity of the sphere by adopting a fluorescence quenching staining method, imaging under a confocal microscope, measuring the fluorescence intensity by using an average gray value and analyzing;

quantification was performed using ImageJ software to evaluate the reliability of the model for drug screening.

The invention also aims to provide an application of the 3D micro-tumor model for implementing the infant hemangioma CD31+ HemECs in the fields of IH mechanism exploration and drug screening.

By combining the technical scheme and the technical problem to be solved, the technical scheme to be protected by the invention has the advantages and positive effects that:

the CD31+ HemECs micro-tumor is used for research, and a more stable and efficient experimental model is provided for IH mechanism exploration and drug screening.

The vascular specific ECM pattern microarray created by the PDMC micropattern printing technology shortens the in-vitro amplification culture time of CD31+ HemECs, effectively controls the particle size and arrangement of CD31+ HemECs micro-tumors, and improves the repeatability and stability of drug screening.

The vascular decellularized ECM provides a tumor microenvironment for culturing CD31+ HemECs micro tumors, is beneficial to maintaining tumor heterogeneity and simulates in vivo tumor body tissues.

The PDMC micro-pattern printing technology uses a laser etching specific pattern silicon wafer as a template, the etching precision reaches 1 mu m, the maximum etching area reaches 12 inches, a PDMS stamp is obtained by reversing the template, then an extracellular matrix glue pattern microarray is laid at the bottom of a cell culture dish which is not subjected to cell attachment treatment by using the PDMC micro-pattern printing technology, and Pluronic F-127 seals the space on the rest culture substrate, so that added cells can only grow attached to the extracellular matrix glue pattern.

Preparation of vascular decellularized ECM: the porcine aorta decellularized scaffold is prepared by adopting a Triton-SDS-Triton portal vein infusion method. The obtained decellularized scaffold is further lyophilized, crushed by a ball mill and enzymolyzed by pepsin to obtain the vascular decellularized ECM hydrogel.

Culture of CD31+ HemECs micro-tumors: the vascular decellularization ECM pattern microarray chip is adopted to culture the CD31+ HemECs micro tumor, the in-vitro amplification culture time is shortened, and the particle size and the arrangement can be effectively controlled.

The technical scheme of the invention fills the technical blank in the industry at home and abroad: cutaneous infantile hemangiomas are the most common benign vascular tumors in infants, with an incidence of about 4% -5% in newborns. Severe complications can occur in about 10% -15% of infantile hemangiomas. Our previous studies demonstrated that IH affects the quality of life of children and parents to varying degrees. To date, the pathogenesis of IH is unknown and there is no uniform, safe and effective treatment for IH. At present, the action mechanism of propranolol for IH, which is a first-line medicament for treating IH, is not clarified, the safety of propranolol used by infants is not fully demonstrated, the propranolol can pass through a blood brain barrier, side effects on the central nervous system are possibly generated, and the development of a safer and more effective treatment method and medicament is very important.

Currently, IH has no stable cell line for research, primary cells, mainly including IH-derived endothelial cells and IH endothelial progenitor cells, need to be isolated, and the two-dimensional cell culture has great difference with the in vivo tissue structure and physiology, and spontaneous regression often exists in the process of nude mouse tumorigenesis, which severely restricts the research of the mechanism related to the generation and development of IH.

The starting point of the invention is based on the facts that the IH pathogenesis is not clear, a standard and stable research model is urgently needed, the blood vessel specific ECM is utilized, and the micro-pattern array is combined to construct a high-flux IH-derived CD31+ HemECs micro-tumor model with uniform size, so that an important foundation is laid for further deep research of the IH pathogenesis and drug screening.

Drawings

FIG. 1 is a flow chart of construction of a CD31+ HemECs3D micro-tumor model of infantile hemangioma according to an embodiment of the present invention;

FIG. 2 is a flow chart of the IH-derived CD31+ HemECs isolation and culture according to the present invention;

FIG. 3 is a diagram of decellularization and vascular-specific DAM characteristics of porcine aorta provided by an embodiment of the present invention, (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) collagen I of porcine aorta and decellularized porcine aorta, (E) collagen III, (F) collagen IV staining;

FIG. 4 is a characteristic diagram of acellular porcine aortic extracellular matrix hydrogels provided by the present invention, (A) live/dead staining patterns of CD31+ HemECs cultured on different hydrogels for days 1, 3, and 5, (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;

FIG. 5 is a schematic diagram of micro-pattern array printing and 3D culture dish preparation and micro-tumor formation according to an embodiment of the present invention;

FIG. 6 is a graph showing the results of two-photon confocal microscope stereo imaging and VGGF and MMP-2 expression provided in 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, and (D) a three-dimensional view of a CD31+ HemEC micro-tumor with a diameter of 200 μm under a confocal microscope;

figure 7 is a drug screening result provided by an embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.

This section is an illustrative example developed to explain the claims in order to enable those skilled in the art to fully understand how to implement the present invention.

The construction method of the infant hemangioma CD31+ HemECs3D micro-tumor model provided by the embodiment of the invention is shown in figure 1:

hemangioma specimens were minced with surgical scissors under sterile conditions, the minced tissue digested with collagenase A, and primary cells were cultured with endothelial cell medium (EBM-2 supplemented with 20% fetal bovine serum, penicillin (100 units/ml) and streptomycin (100 μ g/ml). CD31+ cells were aseptically sorted from the primary culture using a flow cytometer, and cultured in a humidified environment containing 5% CO2 at 37 ℃ for CD31+ HemECs.

Fresh porcine aorta was soaked in Phosphate Buffered Saline (PBS) containing 100U/ml penicillin and 100g/ml streptomycin and placed on ice immediately for transfer to the laboratory. Firstly, washing for 24 hours on a track vibrating screen (100 r/min) by using distilled water, and cracking blood cells; then immersing into 0.5 percent TritonX-100 solution, and continuously oscillating for 24h (100 r/min); after washing with PBS for 2h, the artery was immersed in 0.25% SDS solution, continuously shaken for 72h (100 r/min); to remove residual detergent, the decellularized artery was shaken with PBS for 72h (100 r/min). Fixing decellularized pig artery and fresh pig aorta with paraformaldehyde, embedding and slicing with paraffin, performing HE staining, evaluating the degree of decellularization, and finally storing the obtained decellularized blood vessel at-20 ℃ for further use, wherein all steps are performed in a sterile environment. Lyophilized DAM was ground to a powder using WileyMill and stirred with 10% pepsin and 0.01MHCl at room temperature for 48h. The DAM solution was then neutralized to pH 7.2-7.4 by the addition of 0.1M NaOH and adjusted to a final concentration of 10mg/mL with 1 XPBS.

Etching characteristic patterns on a silicon wafer by laser to obtain a PDMS chip with diameters of 50 μm, 100 μm, 150 μm and 200 μm respectively, a template interval of 50 μm (figure 4A), coating a layer of 0.1mg/mL DAM solution and 2 μ g fluorescein isothiocyanate isomer on the surface of the chip, acting at room temperature for 20min, removing the redundant solution, drying at 37 ℃ for 10min, placing the chip on a non-adhesive culture dish with a diameter of 35mm, and stacking at a force of 0.2N for 10min. The shape of the microarray was then observed under a fluorescence microscope. The treated culture dish was treated with 10g/L aqueous solution of pluronic F-127 at room temperature for 1 hour to prevent non-specific cell adhesion, and then sterilized by ultraviolet irradiation for 2 hours.

3ml of EBM-2 medium was added to the treated micro-patterned culture dishes, and 2X10 medium was plated on each dish ⁵ After 6h of incubation in the cell incubator, the media was removed, washed 3 times with 1xPBS, the cells that had not yet attached were removed, and the morphology of cell aggregates was observed and imaged using the EVOS XL Core for 7 days of incubation. Cell aggregates grown on micropatterned dishes were fixed with 4% formaldehyde at room temperature for 20min, stained with phallodin solution (100 nM) at room temperature for 45min, then counterstained with DAPI, and images were collected by two-photon confocal microscopy. Tumor bodies cultured at 50, 100, 150 and 200 mu m are respectively collected, RNA is extracted, and the expression conditions of VEGFA and MMP2 are detected by using q-PCR.

Treatment with different concentrations of propranolol (50, 100, 150 and 200. Mu.g/mL) for 24 and 48h, respectively, 0.25% DMSO as a negative control. And evaluating the activity of the spheres by adopting a fluorescence quenching staining method, imaging under a confocal microscope, measuring fluorescence intensity by using an average gray value, analyzing, quantifying by using ImageJ software, and evaluating the reliability of the model for screening the drugs.

The embodiment of the invention has some positive effects in the process of research and development or use, and indeed has great advantages compared with the prior art, and the following contents are described by combining data, graphs and the like in the experimental process.

Hemangioma specimens were minced with surgical scissors under sterile conditions, and the minced tissue was digested with collagenase a. CD31+ cells were aseptically sorted from the primary cultures using flow cytometry, and CD31+ HemECs were obtained and cultured using EBM-2 medium, as shown in fig. 2.

Porcine aortic decellularization and vascular-specific DAM characteristics are shown in fig. 3: (A) H & E staining; (B) MT staining of pig aorta and decellularized pig aorta; (C) Relative DNA content of porcine aorta and decellularized porcine aorta; (D) Pig aorta and acellular pig aorta type I collagen, (E) type III collagen, and (F) type IV collagen.

Characteristics of acellular porcine aortic extracellular matrix hydrogel: using DAM-based hydrogels as the surface coating matrix, adhesion, proliferation, survival of CD31+ HemECs could be improved, as shown in fig. 4; (A) Live/dead staining of CD31+ HemECs on different hydrogels cultured on days 1, 3 and 5; 10 minutes after cell seeding; (B) adhesion rate of CD31+ HemECs in different hydrogels; proliferation rates of CD31+ HemECs on different hydrogels 24 hours after cell seeding (C).

Preparing a 3D culture dish for micro-pattern array printing and forming CD31+ HemEC micro-tumor: microarray patterns obtained from PDMS seals with diameters of 50, 100, 150 and 200 μm, as shown in FIG. 5.

The ideal diameter of the pattern is 100-150 μm; (A), (B), (C) and (D) three-dimensional views of CD31+ HemEC micro-tumors with diameters of 50, 100, 150 and 200 μm under confocal microscopy, as shown in FIG. 6. (E) CD31+ HemEC micro-tumor MMP-2 and VEGF-A gene expression levels with different diameters.

The invention evaluates the applicability of the model to the IH first-line treatment drug propranolol, and when propranolol acts for 24 hours, the concentration is more than 100ug/ml, a more remarkable effect is obtained, and the drug screening result is shown in figure 7. When propranolol is used for 48 hours, the concentration of propranolol is lower than 100ug/ml, and the propranolol also has some effects.

It should be noted that the embodiments of the present invention can be realized by hardware, software, or a combination of software and hardware. The hardware portion may be implemented using dedicated logic; the software portions may be stored in a memory and executed by a suitable instruction execution system, such as a microprocessor or specially designed hardware. Those skilled in the art will appreciate that the apparatus and methods described above may be implemented using computer executable instructions and/or embodied in processor control code, such code being provided on a carrier medium such as a disk, CD-or DVD-ROM, programmable memory such as read only memory (firmware), or a data carrier such as an optical or electronic signal carrier, for example. The apparatus and its modules of the present invention may 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., or by software executed by various types of processors, or by a combination of hardware circuits and software, e.g., firmware. The above description is only for the specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made by those skilled in the art within the technical scope of the present invention disclosed in the present invention should be covered within the scope of the present invention.

Claims (8)

1. A construction method of an infant hemangioma CD31+ HemECs3D micro-tumor model is characterized by comprising the following steps:

cutting a hemangioma sample by using surgical scissors under an aseptic condition, and digesting the cut tissue by adopting collagenase A;

step two, aseptically sorting CD31+ cells from the primary culture using a flow cytometer, culturing the CD31+ HemECs in a humidified environment containing 5% co2 at 37 ℃;

step three, soaking fresh pig aorta in Phosphate Buffered Saline (PBS) containing 100U/ml penicillin and 100g/ml streptomycin, placing on ice, immediately transferring into a laboratory, and performing pig aorta decellularization and vascular specific DAM characteristics by using a Triton-SDS-Triton portal vein infusion method;

step four, performing characteristic analysis on the acellular porcine aortic extracellular matrix hydrogel;

preparing a micro-pattern array printing 3D culture dish to form a CD31+ HemEC micro-tumor;

and sixthly, evaluating the applicability of the model to the first-line IH treatment drug propranolol.

2. The method for constructing a CD31+ HemECs3D micro-tumor model of infantile hemangioma according to claim 1, wherein said step is to culture primary cells with endothelial cell culture medium comprising EBM-2 supplemented with 20% fetal bovine serum, penicillin at a concentration of 100 units/ml and streptomycin at a concentration of 100 μ g/ml.

3. The method for constructing the infantile hemangioma CD31+ HemECs3D micro-tumor model according to claim 1, wherein the specific process of the porcine aorta decellularization in the third step comprises:

(1.1) washing the blood cells for 24 hours by using distilled water on an orbital shaker with the rotating speed of 100r/min, and cracking the blood cells;

(1.2) immersing the mixture into TritonX-100 solution with the concentration of 0.5%, and continuously oscillating for 24h; after washing with PBS for 2h, the porcine aorta was immersed in 0.25% SDS solution and shaken continuously for 72h; to remove residual detergent, the decellularized porcine aorta was shaken with PBS for 72h;

(1.3) fixing the decellularized pig aorta and fresh pig aorta with paraformaldehyde, embedding the slices in paraffin, performing HE staining, and evaluating the degree of decellularization.

4. The method for constructing the infant hemangioma CD31+ HemECs3D micro-tumor model according to claim 1, wherein the specific process of vascular specific DAM characterization in step three is as follows:

(2.1) storing the obtained decellularized blood vessel at-20 ℃ for further use, all steps being performed under sterile conditions;

(2.2) grinding lyophilized DAM into powder using Wiley Mill, and stirring with pepsin at 10% concentration and 0.01M HCl at room temperature for 48h to obtain vascular decellularized ECM hydrogel;

(2.3) the DAM solution was neutralized to pH 7.2-7.4 by adding 0.1M NaOH, and the final concentration of the DAM solution was adjusted to 10mg/mL with 1 XPBS.

5. The method for constructing the infant hemangioma CD31+ HemECs3D micro-tumor model according to claim 1, wherein the specific process of the fourth step is as follows:

(3.1) etching characteristic patterns on a silicon wafer by using laser to obtain a PDMS chip, wherein the diameters of the PDMS chip are respectively 50 μm, 100 μm, 150 μm and 200 μm, the distance between templates is 50 μm, the surface of the PDMS chip is coated with a DAM solution with the concentration of 0.1mg/mL and 2 μ g of fluorescein isothiocyanate isomer, and the PDMS chip acts for 20min at room temperature;

(3.2) removing excess solution, drying at 37 ℃ for 10min, placing the chips on a 35mm diameter non-stick petri dish, stacking at a force of 0.2N for 10min;

(3.3) observing the shape of the microarray under a fluorescence microscope, treating the treated petri dish with 10g/L pluronic F-127 aqueous solution at room temperature for 1h to prevent non-specific cell adhesion;

(3.4) sterilizing by ultraviolet irradiation for 2h.

6. The method for constructing the infant hemangioma CD31+ HemECs3D micro-tumor model according to claim 1, wherein the concrete process of the fifth step is as follows:

(4.1) 3ml of EBM-2 medium was added to the treated micro-patterned culture dishes, and 2X10 medium was plated on each dish ⁵ CD31+ HemECs, after 6h incubation in a cell incubator, medium was removed, washed 3 times with 1xPBS, cells that had not attached were removed, and morphology of cell aggregates was observed and imaged using EVOS xlcore for 7 days of incubation;

(4.2) fixing the cell aggregate grown on the micro-pattern culture dish with 4% formaldehyde at room temperature for 20min, and staining with 100 nMalvanidin solution at room temperature for 45min;

(4.3) counterstaining with DAPI, collecting images by a two-photon confocal microscope, collecting tumor bodies cultured at 50, 100, 150 and 200 mu m respectively, extracting RNA, and detecting the expression condition of VEGFA and MMP2 by using q-PCR.

7. The method for constructing the infantile hemangioma CD31+ HemECs3D micro-tumor model according to claim 1, wherein the specific process of the sixth step is as follows:

(5.1) treatment with propranolol concentrations of 50, 100, 150 and 200 μ g/mL for 24 and 48h, respectively, with 0.25% dmso as a negative control;

(5.2) evaluating the activity of the spheres by adopting a fluorescence quenching staining method, imaging under a confocal microscope, measuring the fluorescence intensity by using an average gray value and analyzing;

(5.3) ImageJ software is used for quantification, and the reliability of the model for screening the drugs is evaluated.

8. Use of the infant hemangioma CD31+ HemECs3D micro-tumor model according to any of claims 1-7 in the field of mechanism exploration and drug screening of IH.

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