A method for constructing an animal model of hemangioma

Abstract

The application provides a method for constructing a hemangioma animal model, and belongs to the field of animal models. The method is to inject hemangioma endothelial cells and hemangioma pericytes into subcutaneous tissues of an animal, and to obtain a hemangioma animal model after 5-30 days. The application successfully constructs a hemangioma animal model based on hemangioma endothelial cells and hemangioma pericytes, the hemangioma animal model has a short construction time, consistent pathological characteristics with those of hemangioma, and can simulate the characteristics of in-vivo hemangioma from proliferation to regression evolution, thereby providing an important animal model carrier platform for in-vitro mechanism research of hemangioma and screening of new targeted drugs.

Description

Technical Field

[0001] This invention belongs to the field of animal models, specifically relating to a method for constructing an animal model of hemangioma based on hemangioma endothelial cells and hemangioma peritumoral cells. Background Technology

[0002] Infantile hemangioma (IH) is the most common skin tumor in infants, a vascular tumor formed by the abnormal activation and proliferation of endothelial cells. It exhibits typical growth characteristics: rapid proliferation followed by slow regression. The exact pathogenesis of IH remains unclear, but pathological angiogenesis is closely related to the pathogenesis of hemangiomas. Angiogenesis refers to the process by which endothelial cells, under hypoxia or stimulation by growth factors such as VEGF, are activated from a resting state to an active state with proliferative and migratory capabilities, ultimately fusing to form new blood vessels. Currently, multiple signaling pathways, including the VEGF / VEGFR pathway and the Notch pathway, are involved in the regulation of angiogenesis, thus affecting the occurrence and development of IH. Furthermore, influencing HemEC angiogenesis is also one of the mechanisms of action of receptor blockers, including propranolol, in the treatment of hemangiomas. However, current basic research on IH is mostly focused on the cellular level, which greatly limits the exploration of the pathogenesis of IH and the development and use of new targeted drugs. Therefore, the construction of mature and stable animal models of IH is particularly important.

[0003] Currently, the reported methods for constructing IH animal models are mainly divided into tissue transplantation and cell injection. A significant drawback of tissue transplantation is the long time required for tumor formation after transplantation; tumors typically begin to appear 30 days after transplantation and reach their maximum size around 60 days later. Due to the substantial increase in research time costs, coupled with a high failure rate and the inability to create large-scale models, researchers both domestically and internationally have abandoned tissue transplantation for constructing IH animal models. Cell injection typically involves subcutaneously injecting hemangioma-derived stem cells (HemSC) into nude mice to construct the model (Research Progress on Establishing Nude Mouse Hemangioma Models by Cell Injection, Journal of Clinical Pediatric Surgery, April 2014, Vol. 13, No. 2). However, on the one hand, because HemSCs account for a very low proportion of cells in proliferating hemangioma tissue, only 0.2%, it is difficult to control the positive sorting rate and the tumor formation efficiency is low; on the other hand, this method only begins to form some blood vessels 7 days after subcutaneous injection in nude mice, forms a large number of blood vessels 30 days after injection, and some blood vessels begin to shrink 60 days after injection, which also takes a long time to establish the model and increases the time cost required for the research.

[0004] To overcome the above problems, it is urgent to develop a method for constructing IH animal models with high tumor formation efficiency and short modeling time. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing an animal model of hemangioma based on hemangioma endothelial cells and hemangioma peritumoral cells.

[0006] This invention provides a method for constructing an animal model of hemangioma, wherein hemangioma endothelial cells and hemangioma peritumoral cells are injected into the subcutaneous tissue of an animal, and the hemangioma animal model is obtained after 5-30 days.

[0007] Furthermore, the hemangioma is an infantile hemangioma.

[0008] Furthermore, the ratio of the number of hemangioma endothelial cells to hemangioma pericytes is 1:(0.8-1.2).

[0009] Furthermore, the ratio of the number of hemangioma endothelial cells to hemangioma pericytes is 1:1.

[0010] Furthermore, the hemangioma endothelial cells are CD31-positive hemangioma endothelial cells.

[0011] Furthermore, the CD31-positive hemangioma endothelial cells were prepared by the following method: hemangioma tissue fragments were prepared into a single-cell suspension, cultured, and purified by CD31 immunomagnetic bead sorting to obtain CD31-positive hemangioma endothelial cells.

[0012] Furthermore, the hemangioma animal model was obtained 7-28 days after injecting hemangioma endothelial cells and hemangioma peritumoral cells into the subcutaneous tissue of an animal.

[0013] Furthermore, the hemangioma endothelial cells and hemangioma peritumoral cells are mixed with Matrigel matrix gel and then injected into the subcutaneous tissue of the animal.

[0014] Furthermore, the animal is a mammal, preferably a nude mouse.

[0015] The present invention also provides an animal model of hemangioma constructed by the above method.

[0016] Hemangioma-derived endothelial cells (HemEC) and hemangioma-derived pericyte cells (HemPC).

[0017] Based on HemEC and HemPC, this invention successfully constructed an animal model of infantile hemangioma. The animal model was constructed in a short time, and its pathological features were consistent with those of IH. It can simulate the evolution of IH from proliferation to regression in vivo, providing an important animal model platform for the study of the in vitro mechanism of IH and the screening of new targeted drugs.

[0018] Compared with existing methods for constructing infantile hemangioma models in nude mice by subcutaneous injection of hemangioma stem cells, the construction method of this invention based on HemEC and HemPC has the following advantages: Firstly, HemEC used in this invention is the cell with the highest proportion in proliferating IH tissue, making the positive sorting rate easier to control and resulting in high tumor formation efficiency; secondly, the construction method of this invention can successfully construct an infantile hemangioma animal model 14 days after injection, significantly shortening the modeling time and reducing the time cost required for research.

[0019] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0020] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0021] Figure 1 Clinical specimens and histopathological verification of hemangiomas. A. Proliferative IH in the right apex of a 4-month-old female; B. Proliferative IH in the left abdominal wall of a 3-month-old male; C. Proliferative IH in the left forearm of a 3-month-old female; D. Incisional image of the tumor in case C, showing bright red proliferative IH tissue with rich blood supply; E. HE staining of proliferative IH tissue, showing a large number of endothelial cells and vascular networks; F. GLUT-1 staining positive in proliferative IH tissue.

[0022] Figure 2 Results of primary HemEC culture and identification. AC. On day 3 of primary hemangioma cell culture, the cells are elongated spindle-shaped under a microscope; DF. Hemangioma endothelial cells exhibit characteristic "tube-forming" growth; GI. The morphology of the cells is more clearly visible after changing the medium; JL. On day 7, the cells cover the bottom of the dish, showing multilayer growth. Figure 3Results of sorting and identification of primary HemEC cells. AB. Cell morphology before sorting: cells are long spindle-shaped, can grow in multiple layers, and show no obvious contact or compression; CF. Cell morphology after sorting: cells are short spindle-shaped (CD) or oval / round (EF); GI. Cell immunofluorescence staining identification: blue represents DAPI (G), green represents vWF (H), composite image (I); JL. Flow cytometry identification: control group (J), after CD31... + Synthetic diagram (L) of experimental group (K) after magnetic bead sorting.

[0023] Figure 4 Construction of an infantile hemangioma animal model and its pathological characteristics. A. Schematic diagram of the nude mouse animal model construction process; BC. Subcutaneous formation of oval-shaped "masses" in nude mice after cell injection; DF. External image of the tumor in nude mice taken on day 14; GI. Pathological staining results of nude mice taken on day 14: HE staining shows a large number of neovascular cavities with residual red blood cells (G); CD31 staining positive (H); GLUT1 staining positive (I); JL. External image of the tumor in nude mice taken on day 28, the redness of the tumor is weaker than that on day 14; MO. Pathological staining results of nude mice taken on day 28: HE staining shows a reduction in neovascular cavities with residual red blood cells (M); the degree of CD31 staining positive (N) and GLUT1 staining positive (O) is weaker than that on day 14. Detailed Implementation

[0024] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0025] Main materials and reagent sources:

[0026] Fetal bovine serum, EBM-2 medium (Gibco, USA), rabbit anti-human Glut-1 polyclonal antibody (Wuhan Sanying Biotechnology Co., Ltd.), rabbit anti-human CD31 polyclonal antibody (Abcam, USA), CD31 immunomagnetic beads and tumor cell dissociation kit (Miltenyi, USA), Matrigel (Corning, USA).

[0027] The surgically removed proliferative IH tissue specimens were obtained from three cases of IH that underwent surgical resection at the Department of Pediatric Surgery, West China Hospital of Sichuan University between March and August 2021 and had not received treatment. Pathological examination confirmed that these were proliferative IH. Figure 1 All patients' guardians voluntarily chose surgical resection and refused oral propranolol and other treatment options.

[0028] HemPCs were isolated and identified according to the methods described in the existing technology (Regulation of Notch signaling pathway on pericyte differentiation of infantile hemangiomas, Chinese Journal of Pediatric Surgery, September 2016, Vol. 37, No. 9).

[0029] BALB / c nude mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., consisting of 12 female mice randomly divided into two groups, and were uniformly raised by the Animal Experiment Base of West China Hospital, Sichuan University. This invention experiment was approved by the Ethics Committee of West China Hospital, Sichuan University (March 10, 2021), and all patient guardians signed informed consent forms.

[0030] Example 1: A method for constructing an infantile hemangioma animal model based on hemangioma endothelial cells and hemangioma peritumoral cells

[0031] 1. Extraction of primary HemEC

[0032] Hemangioma tissue specimens in the proliferative phase, obtained through surgical resection, were placed in sterile centrifuge tubes containing serum-free EBM-2 medium. After transfer to a clean bench, the tissue was cut into 2mm × 2mm × 2mm fragments. Following the manufacturer's instructions, a human tumor cell dissociation kit was used to prepare a single-cell suspension from the hemangioma tissue fragments. Following centrifugation, filtration, and resuspending of the cell pellet, the cell suspension was seeded into 6cm culture dishes. Once the cells adhered and reached 80% cell density at the bottom of the dish, they were passaged and sorted for identification.

[0033] 2. Cultivation of primary HemECs

[0034] Cells were seeded in 10cm cell culture dishes and cultured in EBM-2 medium containing 10% fetal bovine serum. The dishes were then incubated at 37°C with 5% CO2. The medium was changed every 2-3 days. When the cells reached 80%-90% confluence in the culture dish, they were passaged.

[0035] 3. Sorting of primary HemEC

[0036] When the cells reached 80%–90% confluence in the culture dish, the adherent cells were digested with trypsin, and the cell pellet was collected. Following the manufacturer's instructions, HemECs were sorted using a magnetic rack with CD31 immunomagnetic beads, yielding CD31-positive cells. + HemECs were sorted. A portion of the sorted cells were used for flow cytometry and immunofluorescence identification, while the remainder were cultured for subsequent animal experiments.

[0037] 4. Construction of a nude mouse animal model

[0038] At a 1:1 ratio, 4×10 6 One HemPC and 4×10 6After sorting, each HemEC was mixed with 200 μL of L Atrigel matrix gel and injected subcutaneously into nude mice using a syringe. After implantation, the maximum and minimum transverse diameters (a and b) of the tumor were measured using calipers on days 7, 14, and 28. The result was calculated using the formula V = (π / 6) × a × b. 2 Estimate the changes in tumor volume.

[0039] On days 14 and 28 after implantation, nude mice were euthanized by spinal dislocation. The tumor was then completely separated from the subcutaneous tissue of the mice, and its size was routinely measured by photography. After fixation in 4% formaldehyde, paraffin embedding, and sectioning, HE staining and immunohistochemical staining were performed, and microvessel density (MVD) was calculated. The MVD calculation method involved randomly selecting four fields of view in the HE section and quantifying the microvascular structure containing erythrocytes, expressed as vessels / mm². 2 calculate.

[0040] Statistical methods: SPSS 23.0 software was used. Quantitative data were analyzed using... The results indicate that t-tests or ANOVA were used for comparisons between groups, with P < 0.05 considered statistically significant. All experiments were repeated three times.

[0041] The following are the experimental results data.

[0042] 1. Results of primary HemEC culture and identification

[0043] On the 3rd day after inoculation of single-cell suspension, cells with elongated spindle-shaped and oval morphologies were observed under a microscope, with a density reaching approximately 40% of the dish bottom area. Figure 2 AC); simultaneously, cells exhibiting characteristic "tube-forming" growth can be observed. Figure 2 DF). On the first day after the medium change, the amount of floating matter in the cells decreased significantly, while the number and density of cells continued to increase, and their morphology became more clearly visible. Figure 2 GI). Continue growing until day 6 or 7, when cells cover the entire bottom of the dish, with some cells exhibiting overlapping multilayer growth. Figure 2 JL).

[0044] 2. Sorting and identification results of primary HemEC

[0045] Primary cells that had grown to 80%–90% of the dish bottom area were digested with trypsin and then sorted using magnetic beads. Before sorting, after the cells reached confluence, there was some mutual compression between the cells, which could result in an elongated spindle shape but did not exhibit contact inhibition. Figure 3 AB). After sorting, the adherent cells mainly exhibited a short spindle shape ( ). Figure 3 CD), oval or nearly circular ( Figure 3EF). Cell immunofluorescence showed positive vWF staining ( Figure 3 GI), CD31 + The proportion of endothelial cells reached 96.4% ( Figure 3 JL). The above results indicate that the sorted and purified endothelial cells were identified as CD31. + The HemEC can be used for subsequent animal experiments.

[0046] 3. Validation of the infantile hemangioma animal model

[0047] HemPC and sorted HemEC were mixed in Matrigel and then subcutaneously injected into nude mice using a cell injection method. Figure 4 A) An oval-shaped "mass" can be observed under the skin immediately. Figure 4 BC). Subcutaneous tumors formed on day 7 after vaccination; the tumors reached their largest size on day 14. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) Figure 4 DF) and day 28 ( Figure 4 Tumors were removed subcutaneously from nude mice by JL and then analyzed by HE staining and immunohistochemical staining. The tumor volume on day 14 was significantly larger than that on day 28 (214±27 vs 86±18, P=0.0024).

[0048] 4. Pathological characteristics of tumors in nude mice

[0049] Compared with the normal subcutaneous tissue next to the tumor in nude mice, the tumor in nude mice appeared more reddish on day 14. Figure 4 DF), HE microscopy revealed numerous neovascularizations, with residual red blood cells visible within the lumens. Figure 4 G), GLUT1 and CD31 staining positive and highly expressed ( Figure 4 HI). Compared to the tumors on day 14, the tumors in nude mice on day 28 showed a reduced redness, and the density and number of vascular cavities were reduced under the microscope. Figure 4 M), both GLUT1 and CD31 expression were decreased (M), Figure 4 NO). The microvascular density of the tumor on day 14 was higher than that on day 28, and the difference was significant (0.17 cm). 3 ±0.02cm 3 vs 0.10cm 3 ±0.03cm 3 (P = 0.03).

[0050] The above experimental results show that the method of Example 1 of the present invention successfully constructed an animal model of infantile hemangioma. The animal model was constructed in a short time, and its pathological characteristics were consistent with those of IH. It can simulate the evolution of IH from proliferation to regression in vivo, providing an important animal model platform for the study of the in vitro mechanism of IH and the screening of new targeted drugs.

Claims

1. A method for constructing an animal model of infantile hemangioma, characterized in that: The method involves injecting hemangioma endothelial cells and hemangioma peritumoral cells into the subcutaneous tissue of an animal, and obtaining an infantile hemangioma animal model after 14 days; the animal is a mammal; the ratio of the number of hemangioma endothelial cells to hemangioma peritumoral cells is 1:(0.8-1.2).

2. The method according to claim 1, characterized in that: The ratio of hemangioma endothelial cells to hemangioma pericytes is 1:

1.

3. The method according to claim 1, characterized in that: The hemangioma endothelial cells are CD31-positive hemangioma endothelial cells.

4. The method according to claim 3, characterized in that: The CD31-positive hemangioma endothelial cells were prepared by the following method: hemangioma tissue fragments were prepared into a single-cell suspension, cultured, and purified by CD31 immunomagnetic bead sorting to obtain CD31-positive hemangioma endothelial cells.

5. The method according to claim 1, characterized in that: The hemangioma endothelial cells and hemangioma peritumoral cells are mixed with Matrigel and then injected into the subcutaneous tissue of the animal.

6. The method according to claim 1, characterized in that: The animal in question is a nude mouse.

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