Method for promoting human amniotic mesenchymal stem cells to differentiate into vascular endothelial cells

Abstract

The invention discloses a method for promoting human amniotic mesenchymal stem cells to differentiate into vascular endothelial cells. The method comprises the following steps: preparing an electrostatic spinning film as a cell culture substrate; planting human amniotic mesenchymal stem cells (hAMSC) on the electrostatic spinning film; adding a vascular endothelial growth factor (VEGF) into the culture medium to induce hAMSC differentiation; and maintaining the cell culture until the characteristic expression of the vascular endothelial cells. The polylactic acid-polycaprolactone copolymer (PLCL) is used as the material of the electrostatic spinning film, and the film prepared by the electrostatic spinning technology has a unique three-dimensional reticular fiber structure, so that a growth environment closer to a natural extracellular matrix is provided for cells. The structure can effectively promote adhesion and proliferation of human amniotic mesenchymal stem cells (hAMSC), and differentiates to vascular endothelial cells under the induction of vascular endothelial growth factors (VEGF), so that the differentiation efficiency is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering technology, and specifically relates to stem cells, stem cell fate regulation, stem cell tissue engineering and other technologies, especially a method for promoting the differentiation of human amniotic mesenchymal stem cells into vascular endothelial cells. Background Art

[0002] Cardiovascular disease is one of the leading causes of death worldwide, especially ischemic cardiovascular diseases, such as coronary heart disease and stroke, which seriously threaten the life and health of patients by affecting blood oxygen supply and tissue healing. In the field of cell therapy and regenerative medicine, the infusion of vascular endothelial cells (VEC) or the implantation of artificial cardiovascular tissue has been shown to significantly improve the blood supply to ischemic areas, providing new hope for the treatment of these diseases. However, one of the main challenges currently faced is the severe shortage of functional VECs, and a safe, stable and efficient method for obtaining VECs is urgently needed.

[0003] Traditional methods for obtaining VECs are mainly divided into two categories: the isolation and expansion of autologous VECs, and the directed differentiation of stem cells. The autologous VEC method is limited by limited sample size, invasive operations, and functional degradation, and it is difficult to meet clinical needs. Although the stem cell differentiation method has the potential for self-renewal and multidirectional differentiation, the current differentiation system has defects such as low efficiency, long time, and large toxic side effects, which limit its clinical application. In addition, although embryonic stem cells and induced pluripotent stem cells have powerful expansion and differentiation capabilities, there are ethical controversies, tumorigenic risks, and genetic safety issues, which limit their widespread application.

[0004] Adult stem cells (ASCs) are considered as a potential stem cell resource due to their low immunogenicity and low ethical risks, but their content in the body is low, the acquisition process is invasive, and there are problems with low separation and purification efficiency. In recent years, human amniotic mesenchymal stem cells (hAMSCs) have attracted attention due to their unique advantages. They are derived from the placenta, are multipotent, have no immune rejection reaction, have low tumorigenicity, and are easy to obtain and expand. Studies have shown that hAMSCs can differentiate into VECs in vivo and in vitro, and exhibit the characteristics of promoting angiogenesis and anti-thrombosis in artificial vascular models.

[0005] Although hAMSCs have shown great potential as a source of VECs, the traditional induction system for stem cell differentiation into vECs mainly relies on biomacromolecules such as vascular endothelial growth factor (VEGF). However, these macromolecular active substances are involved in complex cell physiology and signal transduction processes, have multiple regulatory capabilities, and their differentiation induction effects are low and need to be improved urgently. Therefore, developing a new differentiation system to improve the efficiency and function of hAMSC differentiation into VECs while reducing potential toxic side effects is of great significance for promoting the clinical application of stem cells in the treatment of cardiovascular diseases. Summary of the invention

[0006] The purpose of the present invention is to provide a method for promoting the differentiation of human amniotic mesenchymal stem cells into vascular endothelial cells, which optimizes the differentiation system, improves the differentiation efficiency, and reduces the potential risks in the differentiation process, so as to achieve a safer and more effective treatment of cardiovascular diseases.

[0007] The invention discloses a method for promoting the differentiation of human amniotic mesenchymal stem cells into vascular endothelial cells, comprising the following steps: preparing an electrospun film as a cell culture substrate; planting human amniotic mesenchymal stem cells hAMSC on the electrospun film; adding vascular endothelial growth factor VEGF to the culture medium to induce hAMSC differentiation; and maintaining the cell culture until the vascular endothelial cell characteristics are expressed.

[0008] The method of the present invention utilizes a film prepared by electrospinning technology as a cell culture substrate, which can provide a microenvironment similar to the extracellular matrix, thereby facilitating cell adhesion, proliferation and differentiation. By adding VEGF to the culture medium, hAMSCs can be specifically induced to differentiate into vascular endothelial cells, and ultimately achieve the expression of vascular endothelial cell characteristics. The method of the present invention not only improves the differentiation efficiency, but also provides a new cell source for tissue engineering and regenerative medicine, which helps to solve the problem of insufficient number of vascular endothelial cells in the prior art.

[0009] As a further preferred embodiment, the electrospinning film is prepared from polylactic acid-polycaprolactone copolymer PLCL. By using polylactic acid-polycaprolactone copolymer (PLCL) as the material of the electrospinning film, a biocompatible and degradable cell culture substrate is provided for hAMSC. PLCL, as a biodegradable material, can gradually degrade during the cell culture process, thereby reducing long-term foreign body stimulation to the cells. In addition, the degradation products of PLCL are lactic acid and caprolactone, which can be naturally metabolized in the body, reducing the potential impact on the cell culture environment. Using PLCL film as a cell culture substrate helps to maintain the normal physiological state of cells and promote the long-term stable differentiation of cells.

[0010] As a further preferred embodiment, the preparation of the electrospinning film includes at least the following steps: dissolving PLCL particles in a solvent to form a spinning solution; preparing a film using electrospinning technology; and performing subsequent treatment on the film, including freeze drying, ultraviolet sterilization, and gelatin incubation. The present invention ensures the quality and performance of the film through a series of fine electrospinning film preparation steps, thereby providing an excellent platform for the cultivation and differentiation of hAMSC. First, the PLCL particles are dissolved in a solvent to form a spinning solution, and a film with a specific micron or nanometer fiber structure can be prepared using electrospinning technology. This structure mimics the morphology of the natural extracellular matrix and is conducive to cell adhesion and growth. Secondly, the film is freeze-dried and ultraviolet sterilized, which not only ensures the pore structure of the film, but also ensures its sterility, which is suitable for cell culture. Finally, the cell adhesion of the film is further enhanced by gelatin incubation, creating favorable conditions for the cultivation and differentiation of hAMSC. The comprehensive application of these technical steps significantly improves the performance of the electrospinning film as a cell culture substrate, providing an important guarantee for the efficient differentiation of hAMSC.

[0011] As a further preferred embodiment, the concentration of VEGF is 50 ng / mL. The concentration of VEGF is accurate to 50 ng / mL, which not only ensures the effective concentration required in the differentiation process, but also helps to maintain the cost-effectiveness of cell culture. The selection of this concentration may be based on experimental data, showing that at this level, the differentiation efficiency of hAMSC is the highest, while avoiding excessive cell proliferation or dysfunction that may be caused by excessive concentration.

[0012] As a further preferred embodiment, the culture medium containing VEGF is replaced every 2 days during the cell culture process for 14 days. By replacing the culture medium containing VEGF every 2 days for 14 days, the continued presence and effect of VEGF during the differentiation process are ensured. This periodic replacement mechanism not only ensures the renewal of nutrients in the culture environment, but also helps to remove metabolic waste or inhibitory factors that may accumulate, thereby providing a stable and optimized microenvironment for the cells. In addition, the 14-day continuous culture time may be based on experimental observations, and it is believed that this time period is sufficient to achieve effective differentiation of hAMSCs into vascular endothelial cells.

[0013] As a further preferred embodiment, it also includes detecting the expression of endothelial cell-specific marker CD31 by immunofluorescence staining and real-time fluorescence quantitative polymerase chain reaction qRT-PCR. By detecting the expression of endothelial cell-specific marker CD31 by immunofluorescence staining and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), changes in cell morphology and quantitative analysis of gene expression levels can be intuitively observed. The application of this method not only helps to monitor and verify the success of the differentiation process, but also can further understand the molecular mechanism of the differentiation process by analyzing the expression pattern of CD31. In addition, the establishment of this detection method provides a standardized evaluation method for stem cell differentiation research, which helps to improve the reliability and repeatability of experimental results.

[0014] As a further preferred embodiment, the qRT-PCR to detect the expression of CD31, a specific marker for vascular endothelial cells, includes designing target gene quantitative PCR reaction primers to amplify the CD31 gene to quantitatively analyze the efficiency of hAMSC differentiation into vascular endothelial cells; the target gene quantitative PCR reaction primers have the 16S rDNA sequence shown in SEQ ID NO:1-SEQ ID NO:4. By specifying the 16S rDNA sequence shown in SEQ ID NO:1-SEQ ID NO:4 as part of the target gene quantitative PCR reaction primer, the specificity and accuracy of the qRT-PCR experiment are ensured. The use of this specific sequence helps to avoid nonspecific amplification, thereby improving the credibility of the experimental results. In addition, clarifying the primer sequence provides clear guidance for researchers implementing the technical solution, so that the method can be replicated and verified by other researchers or laboratories, which helps to promote the accumulation of knowledge and standardization of technology in this field.

[0015] As a further preferred embodiment, the surface of the electrospun film is specially treated to increase cell adhesion, and the special treatment includes plasma treatment or coating modification. By specially treating the surface of the electrospun film, the cell adhesion of hAMSC is significantly improved. This special treatment includes plasma treatment or coating modification, which can effectively improve the surface properties of the film, thereby increasing the interaction between cells and materials. Plasma treatment can activate the surface of the material, increase surface roughness and chemical activity, while coating modification may involve covering a molecular layer with specific biological activity. These treatments not only promote initial cell adhesion, but may also affect cell proliferation and differentiation behavior, providing a more suitable growth microenvironment for hAMSC, thereby improving the efficiency and effect of the entire differentiation process.

[0016] As a further preferred embodiment, the planting density of hAMSC is 10 per square centimeter. 4 to 10 5After seeding, the cells were incubated at 37°C and 5% CO 2 The precise control of the planting density ensures that the cells have appropriate space and nutrient supply during the culture process, avoiding cell growth inhibition or adverse differentiation results caused by over-crowding or over-sparseness. In addition, 37°C and 5% CO 2 The culture conditions simulate the physiological environment in the human body and help maintain the normal metabolism and functional state of the cells. The optimization of these conditions helps to achieve the effective differentiation of hAMSCs into vascular endothelial cells and provides a standardized culture method for subsequent cell function research and application.

[0017] As a further preferred embodiment, the method further comprises the following steps: before hAMSC is implanted, the cell purity is detected by flow cytometry to ensure that the cell purity reaches more than 95%; during the induction of differentiation, the cell activity is regularly evaluated using a cell activity kit to ensure that the cells maintain high activity during the culture process. Through a series of quality control measures, the high purity and high activity of hAMSC are ensured, which is crucial for improving differentiation efficiency and obtaining functional vascular endothelial cells. The use of flow cytometry can detect the purity of hAMSC before implantation, exclude impurity cells that may affect the differentiation effect, and ensure the accuracy of the experiment. At the same time, during the induction of differentiation, the cell activity kit is regularly used to evaluate the cell activity, which can timely discover and adjust the culture conditions to prevent the cells from being damaged due to environmental changes or malnutrition. The implementation of these measures helps to maintain the stability and repeatability of the cell culture process and provides a guarantee for obtaining high-quality vascular endothelial cells.

[0018] In summary, compared with the existing technology:

[0019] First, the present invention uses polylactic acid-polycaprolactone copolymer (PLCL) as the material of the electrospinning film. This material not only has good biocompatibility and biodegradability, but also the film prepared by electrospinning technology has a unique three-dimensional reticular fiber structure, which provides a growth environment for cells that is closer to the natural extracellular matrix. This structure can effectively promote the adhesion and proliferation of human amniotic mesenchymal stem cells (hAMSCs), and differentiate into vascular endothelial cells under the induction of vascular endothelial growth factor (VEGF), thereby significantly improving the differentiation efficiency.

[0020] Secondly, the present invention adopts a method of precisely controlling the VEGF concentration and periodically replacing the culture medium during the cell culture process, which ensures the continuous supply of nutrients and growth factors required in the differentiation process, while avoiding the accumulation of metabolic waste, and provides optimized culture conditions for the stable differentiation of hAMSC. In addition, through detection methods such as immunofluorescence staining and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), the present invention can accurately evaluate the effect of hAMSC differentiation into vascular endothelial cells, ensuring the reliability and repeatability of the experimental results.

[0021] Finally, the present invention has made innovations in technical implementation, including special treatment of the surface of the electrospun film to increase cell adhesion, precise control of the planting density and culture conditions of hAMSC, and regular evaluation of cell purity and activity during the differentiation process. These measures further improve the differentiation efficiency, ensure that the obtained vascular endothelial cells have high purity and high activity, and provide a new cell source for the treatment of cardiovascular diseases and tissue engineering. Through these substantial technical innovations, the present invention not only solves the problems existing in the prior art, but also opens up a new path for stem cell differentiation and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0023] Figure 1 FIG. 4 shows a schematic diagram of CD31 endothelial differentiation fluorescence staining in one embodiment of the present invention (scale bar = 100 μm); Figure 1 In the figure, (A) and (B) are the fluorescence results of TCP without adding VEGF and with adding VEGF, respectively; (C) and (D) are the fluorescence results of PLCL spinning membrane without adding VEGF and with adding VEGF, respectively.

[0024] Figure 2 Figure 2 shows the expression level of hAMSC marker genes on the PLCL spinning membrane in one embodiment of the present invention ( n=3,*P﹤0.05,****P﹤0.0001) Figure 2In the figure, TCP: tissue culture plate without any stimulation factor; PLCL: PLCL spinning membrane without any stimulation factor; TCP(VEGF): tissue culture plate with VEGF stimulation; PLCL(VEGF): PLCL spinning membrane with VEGF stimulation. Note: All groups were seeded with hAMSCs. DETAILED DESCRIPTION

[0025] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings so that the purpose, features and advantages of the present invention can be more clearly understood. It should be understood that the embodiments shown in the accompanying drawings are not intended to limit the scope of the present invention, but are only intended to illustrate the essential spirit of the technical solution of the present invention.

[0026] In the following description, certain specific details are set forth for the purpose of illustrating the various disclosed embodiments to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the relevant art will recognize that the embodiments may be practiced without one or more of these specific details. In other cases, well-known devices, structures, and techniques associated with the present application may not be shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0027] References throughout the specification to "one embodiment" or "an embodiment" indicate that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of "in one embodiment" or "in an embodiment" in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any manner in one or more embodiments.

[0028] Embodiment 1:

[0029] In this embodiment, the following English abbreviations may be involved. The following Table 1 gives the full English names and Chinese names of these abbreviations.

[0030] Table 1: Chinese and English abbreviations

[0031]

[0032]

[0033]

[0034] This embodiment is implemented according to the following steps:

[0035] 1. PLCL film preparation

[0036] (1) Preparation of PLCL thin film by electrospinning

[0037] Spinning sample with an average diameter of 0.36 μm: PLCL particles (Jinan Daigang, the molar ratio of PLA and PCL is 50:50, molecular weight 80000 Da) were dissolved in hexafluoroisopropanol at a mass concentration of 10%, the receiving distance was 15 cm, the receiving voltage was 12-14 kV, and the spinning solution flow rate was 1 mL / h.

[0038] (2) Post-processing of materials

[0039] After spinning, the membrane was peeled off from the substrate and immersed in a small amount of deionized water. Subsequently, it was frozen at -80°C for 24 hours and then dried overnight in a freeze dryer. Before use, the material was sterilized by ultraviolet irradiation and immersion in 75% alcohol for 1 hour, and incubated in a 37°C incubator with 0.1% gelatin solution for 60 minutes to promote cell adhesion on the scaffold surface. For the culture dish substrate used for control, it was also incubated in a 37°C incubator with 0.1% gelatin solution for 60 minutes.

[0040] 2. Key morphology analysis of PLCL tubular stent

[0041] The microstructure of the artificial blood vessel was analyzed by SEM images. The SEM sample preparation process was as follows: freeze-dry the sample, spray gold for 60 seconds, and observe under SEM with a scanning voltage of 10 kV.

[0042] Fiber diameter of PLCL electrospun: The average diameter of PLCL artificial blood vessels was measured in SEM images. 100 fibers were manually measured and analyzed using ImageJ software.

[0043] 3. Implantation of amniotic mesenchymal stem cells

[0044] (1) Cut the electrospun membrane into appropriate size and shape and sterilize. For 96-well plates, cut the membrane into a 6 mm diameter circle; for 6-well plates, cut the membrane into a 30 mm circle. Sterilize by washing twice with 75% ethanol, then soaking in 75% ethanol for 30 min, air-drying in a clean bench, and storing for later use.

[0045] (2) Before seeding cells, wash twice in PBS, incubate in 0.1% gelatin, and soak the culture dish and spinning membrane in culture medium for about 20 minutes.

[0046] (3) Digestion of amniotic stem cells, trypan blue counting, and implantation.

[0047] 4. Evaluation of hAMSC-PLCL artificial blood vessels on endothelial differentiation of hAMSC

[0048] In order to detect whether the presence of PLCL material affects the ability of hAMSC to differentiate into EC, the present invention uses CD31 immunofluorescence experiment and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) for analysis.

[0049] (1) Spinning membrane preparation

[0050] Cut the spinning membrane of appropriate size, soak it in 75% alcohol and sterilize it under UV light for 1 hour, soak it with culture medium and remove the excess culture medium, place it in an incubator to dry and store it for later use. Soak it in 0.1% gelatin solution in a 37℃ incubator for 60 minutes before use.

[0051] (2) Cell implantation

[0052] For cell digestion and counting, 2 × 10 cells were seeded per well in a 6-well plate. 5 The cells were placed in an incubator and cultured for 1 day.

[0053] (3) Endothelial differentiation induction

[0054] The L-DMEM medium containing 50 ng / mL VEGF and 2% FBS was replaced every 2 days to induce differentiation. After 14 days, samples were collected for immunofluorescence staining and qRT-PCR analysis.

[0055] 5.CD31 Immunofluorescence Staining and qRT-PCR Detection

[0056] After differentiation was completed, CD31 immunofluorescence staining and qRT-PCR were used to detect the differentiation of ECs.

[0057] (1) Immunofluorescence staining

[0058] 1.1) Fixation

[0059] First, the 6-well plate was taken out and washed three times with PBS. Then, it was fixed with 4% paraformaldehyde at room temperature for 15 minutes. After fixation, it was washed again with PBS for 3 minutes each time, for a total of three times.

[0060] 1.2) Closure

[0061] The tissue was evenly covered with 5% BSA solution and blocked at room temperature of 37°C for 30 min.

[0062] 1.3) Primary Antibody Incubation

[0063] Gently shake off the blocking solution, add CD31 antibody prepared in a certain proportion to the slices, place the slices flat in a humidified box, and incubate at 4°C overnight.

[0064] 1.4) Secondary Antibody Incubation

[0065] Shake the slices dry, add the secondary antibody of the same species as the primary antibody in the circle, cover the tissue, and incubate at room temperature for 60 minutes in the dark.

[0066] 1.5) DAPI staining

[0067] Place the slide in PBS and wash on a shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add DAPI staining solution in the circle and incubate at room temperature for 5 minutes away from light.

[0068] 1.6) Washing and sealing

[0069] Place the slide in PBS and wash on a shaker for 3 times, 5 minutes each time. After the slices are slightly dried, they are sealed with anti-fluorescence quenching sealing medium.

[0070] 1.7) Observation and interpretation of results

[0071] Use fluorescence microscope for observation and imaging system for image acquisition and analysis.

[0072] (2) qRT-PCR

[0073] 2.1) RNA extraction and purification

[0074] Aspirate the culture medium in the 6-well plate, wash with PBS three times, add 500μL RNAiso Plus and lyse at room temperature for 2 minutes. After blowing the cells evenly, transfer to a 1.5mL EP tube and let stand for 5 minutes. Add 100μL nucleic acid extraction solution, shake vigorously for 10 seconds to ensure mixing, let stand for 5 minutes, and centrifuge at 12000rpm below 4℃ for 15 minutes.

[0075] Transfer 200 μL of the upper aqueous phase to a new EP tube, add an equal volume of isopropanol, shake vigorously 10 times, let stand for 10 minutes, and centrifuge at 12,000 rpm for 10 minutes at 4°C. Pour out excess water from the EP tube, add 1 mL of 75% ethanol, mix well, and centrifuge at 12,000 rpm for 10 minutes at 4°C.

[0076] Invert the EP tube on filter paper in a fume hood and dry for 30 minutes. Add 20 μL of DEPC water to each tube and mix well.

[0077] 2.2) RNA quantification and reverse transcription

[0078] Measure RNA concentration and purity: After pre-washing the system, add 1 μL of DEPC water for calibration, then add sample for measurement. Change the tip and clean the measuring point each time. The ideal 260 / 280 value is 1.8-2.0.

[0079] The concentration of the extracted RNA was detected and diluted to 100 ng / μL with DEPC water; 2 μL of each sample was added with the following reagent mixture (as shown in Table 2).

[0080] Table 2: Reverse transcription reaction system

[0081]

[0082]

[0083] The reverse transcription reaction conditions were as follows: 37°C for 15 min, 85°C for 5 s, and 4°C.

[0084] 2.3) Real-Time PCR

[0085] The primers for the quantitative PCR reaction of the target gene are shown in Table 3. Each cDNA sample was diluted 5 times on ice with DEPC water and then prepared according to the following system (as shown in Table 4).

[0086] Table 3: Primer sequences for Real-Time PCR (SEQ ID NO: 1-SEQ ID NO: 4)

[0087]

[0088] Table 4: Real-Time PCR reaction system

[0089]

[0090] After the above system is mixed, the sample is placed in a real-time fluorescence quantitative PCR instrument. Amplification is performed according to the following program: 95°C for 30s; 95°C for 5s, 60°C for 30s, for a total of 40 cycles. The internal reference is GAPDH, 2 -ΔΔCt The expression level of the target gene in each sample was calculated by the PCR method (the Ct value is the cycle number that reaches the cycle threshold).

[0091] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for promoting the differentiation of human amniotic mesenchymal stem cells into vascular endothelial cells, characterized in that: The method comprises the following steps: preparing an electrospun film as a cell culture substrate; planting human amniotic mesenchymal stem cells (hAMSC) on the electrospun film; adding vascular endothelial growth factor (VEGF) to the culture medium to induce hAMSC differentiation; and maintaining cell culture until vascular endothelial cell characteristics are expressed.

2. The method for promoting differentiation of human amniotic mesenchymal stem cells into vascular endothelial cells according to claim 1, characterized in that: The electrospinning film is prepared from polylactic acid-polycaprolactone copolymer PLCL.

3. The method for promoting differentiation of human amniotic mesenchymal stem cells into vascular endothelial cells according to claim 1, characterized in that: The preparation of the electrospinning film at least comprises the following steps: dissolving PLCL particles in a solvent to form a spinning solution; preparing the film by using an electrospinning technique; and performing subsequent treatment on the film, including freeze drying, ultraviolet sterilization and gelatin incubation.

4. The method for promoting differentiation of human amniotic mesenchymal stem cells into vascular endothelial cells according to claim 1, characterized in that: The concentration of VEGF is 50 ng / mL.

5. The method for promoting differentiation of human amniotic mesenchymal stem cells into vascular endothelial cells according to claim 4, characterized in that: During the cell culture process, the culture medium containing VEGF was replaced every 2 days for 14 days.

6. The method for promoting differentiation of human amniotic mesenchymal stem cells into vascular endothelial cells according to claim 1, characterized in that: It also includes detecting the expression of vascular endothelial cell-specific marker CD31 by immunofluorescence staining and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR).

7. The method for promoting differentiation of human amniotic mesenchymal stem cells into vascular endothelial cells according to claim 6, characterized in that: The qRT-PCR to detect the expression of the vascular endothelial cell-specific marker CD31 includes designing target gene quantitative PCR reaction primers to amplify the CD31 gene to quantitatively analyze the efficiency of hAMSC differentiation into vascular endothelial cells; the target gene quantitative PCR reaction primers have 16S rDNA sequences shown in SEQ ID NO:1-SEQ ID NO:

4.

8. The method for promoting differentiation of human amniotic mesenchymal stem cells into vascular endothelial cells according to claim 1, characterized in that: The surface of the electrospun film is specially treated to increase cell adhesion, and the special treatment includes plasma treatment or coating modification.

9. The method for promoting differentiation of human amniotic mesenchymal stem cells into vascular endothelial cells according to claim 1, characterized in that: The planting density of hAMSC is 10 per square centimeter. 4 to 10 5 The cells were seeded and cultured at 37°C and 5% CO2.

10. The method for promoting differentiation of human amniotic mesenchymal stem cells into vascular endothelial cells according to claim 1, characterized in that: The method further comprises the following steps: before hAMSC planting, using a flow cytometer to detect the purity of the cells to ensure that the cell purity reaches more than 95%; during the differentiation induction process, regularly using a cell activity kit to evaluate the cell activity to ensure that the cells maintain high activity during the culture process.

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