Preparation method and treatment device of silk membrane loaded with recombinant human epidermal growth factors

Abstract

The invention discloses a preparation method and a treatment device of a silk membrane loaded with recombinant human epidermal growth factors, and relates to the technical field of biological products. According to the scheme, the transgenic epidermal growth factor silkworm strain NSECS is spun on a two-dimensional plane of a mold to prepare flat silk, and then the flat silk is subjected to the technical processes of hot pressing, sterilization and the like to prepare the transgenic epidermal growth factor silk biological membrane dressing. The transgenic epidermal growth factor silk biological membrane prepared by using the technology can greatly maintain the content and activity of the growth factor, meanwhile, the time required for preparing the silk structure biological membrane dressing is greatly saved, the natural structure and mechanical property of silk are maintained, and the preparation process is simpler. Silk degumming and silk fibroin extraction do not need to be carried out, so that the treatment cost of a degumming solution and a silk fibroin extracting solution can be reduced, the pollution to the environment is reduced, and the problem of maintaining the natural structure of silk and the activity of epidermal growth factors in the traditional silk membrane preparation process is solved. The human epidermal growth factor silk membrane prepared by the method can be applied to the medical fields of wound repair, tissue repair and the like, and has good application potential and value.

Description

Technical Field

[0001] The invention belongs to the technical field of biological products, and in particular, relates to a preparation method and a processing device for a silk membrane loaded with recombinant human epidermal growth factor. Background Art

[0002] In the past few years, silk has expanded from the textile industry with thousands of years of history to various emerging fields, such as electronics, photonics, energy, environmental science and biomedicine. After verification by many laboratories at home and abroad, silk has good biocompatibility, non-immunogenicity, high cell attachment, easy processing, stability, biodegradability, water permeability, oxygen permeability and good mechanical strength, becoming an ideal biomaterial.

[0003] Epidermal growth factor is the earliest discovered polypeptide growth factor. Domestic and foreign research teams have proven that it has a promoting effect on the proliferation of skin fibroblasts, cell migration, angiogenesis, keratinocyte regeneration, etc., and can help wound healing.

[0004] At present, growth factor-loaded silk dressings are mainly prepared by the method of regenerating silk fibroin. For example, transgenic epidermal growth factor cocoons are cut into 1 cm² sizes, degummed, and dissolved in a high-concentration lithium bromide solution. The silk fibroin solution is dialyzed for 3 days. Then, the regenerated silk fibroin biofilm is prepared through processes such as thermoplastic molding and electrospinning.

[0005] However, the current process of preparing transgenic epidermal growth factor biofilm dressings using silk fibroin regeneration methods involves high temperature, high pressure, high salt and other conditions, which seriously affect the activity of transgenic epidermal growth factor and cause serious damage to the natural structure of silk. The main reason for this problem is that when raising silkworms, mature silkworms are usually placed in a mold with a three-dimensional structure so that the silkworms can form an elliptical cocoon after spinning silk. It is necessary to go through silk fibroin extraction or reeling processes to produce a two-dimensional biofilm dressing.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] In order to solve the above technical problems, the basic concept of the technical solution adopted by the present invention is: The preparation method of the silk membrane loaded with recombinant human epidermal growth factor comprises the following method: Rearing transgenic epidermal growth factor silkworm strain N S EC S At the fifth instar, 3-5 days later, the prepared silkworms are obtained; The prepared silkworms were placed under a stereofluorescence microscope, and red fluorescence was used to observe the fluorescence of the silkworm eyes. The fifth-instar silkworms with red fluorescence in the eyes were selected and continued to be raised to obtain mature silkworms. After the mature silkworms are put into the cluster to defecate, a certain number of mature silkworms are selected and put into the mold; Making the mature silkworms spin silk on the two-dimensional plane of the mold to obtain flat silk; The flat wire is peeled off from the mold and subjected to heat pressing treatment; Finally, beta irradiation sterilization was performed; The silk biofilm is obtained by cutting into a specified size and attaching to the surface of a substrate.

[0008] Preferably, the number of mature silkworms in the mold is 25-35, and silkworms with similar growth conditions are selected.

[0009] Preferably, the spinning situation on the two-dimensional plane of the mold is checked every 1-2 hours, and the position of the silkworms is manually adjusted according to the spinning situation to obtain uniform flat silk, and the silkworm feces need to be cleaned during the inspection.

[0010] Preferably, the hot pressing treatment includes: a hot pressing time of 1-10 min, a hot pressing temperature of 60° C., a hot pressing pressure of 5-15 MPa, and a β irradiation sterilization treatment dose of 15 kGy.

[0011] Preferably, the mold comprises a spinning plane made of plastic or paper material, the spinning plane is arranged in a disc shape, and a pillar for supporting the spinning plane is arranged on the lower surface thereof.

[0012] Preferably, the recombinant human epidermal growth factor loaded silk membrane treatment device comprises a base, the inner wall of the base is installed with a hydraulic component, the hydraulic component is connected to a hydraulic pump, the movable end of the hydraulic component is fixedly connected to a tension frame, and the upper surface of the tension frame is fixedly connected to a holding component; The upper surface of the base is fixedly connected with a retaining frame, the inner wall of the retaining frame is slidably provided with two hot pressing components, and one side of the hot pressing component and the inner wall of the tension frame are jointly installed with a pressure component; A feeding assembly is installed on the surface of the pressing assembly. The feeding assembly includes a transmission frame fixed on the inner wall of the pressing assembly. A conveyor belt is arranged on the inner wall of the transmission frame. A hook is arranged on the surface of the conveyor belt.

[0013] Preferably, the hot pressing assembly comprises a movable seat connected to the pressure-applying assembly, a rear seat is fixedly connected to one side of the movable seat, a support pad is fixedly connected to one side of the rear seat, a heat insulation board is fixedly connected to one side of the support pad, pressure sensors are arranged at the four corners of the heat insulation board, and the pressure sensors are fixedly connected to the surface of the rear seat; A heating plate is fixedly connected to one side of the heat insulation plate, and a temperature uniforming plate is symmetrically arranged on the other side of the heating plate; A heating wire is arranged on one side of the heating plate, and the heating wire is electrically connected to a controller.

[0014] Preferably, the hydraulic assembly comprises a hydraulic cylinder fixed on the lower surface of the retaining frame, a hydraulic piston is fixed on the surface of the hydraulic cylinder, a hydraulic chamber is sealed and attached to the surface of the hydraulic piston, and the hydraulic chamber is slidably arranged on the arc-shaped side wall of the hydraulic cylinder; A slide is slidably penetrated through the surface of the hydraulic warehouse, and the slide is fixedly connected to the inner wall of the base. The top of the hydraulic warehouse is fixedly connected to the bottom of the tension frame. An oil guide hole is penetrated through the inner wall of the hydraulic cylinder, and a connecting pipe for connecting the hydraulic pump and the oil guide hole is opened on the arc-shaped side wall of the hydraulic cylinder.

[0015] Preferably, the pressure-applying assembly comprises a main shaft, two cross seats are slidably penetrated through the surface of the main shaft, opposite ends of the two cross seats are commonly fixedly connected with a tension spring, the tension spring is spirally coiled and located on the surface of the main shaft, connecting columns are fixed on both sides of the cross seat, one end of the connecting column is movably connected to two deflection plates, one end of the two deflection plates on the same side of the two cross seats are respectively provided with a first connecting part and a second connecting part, the first connecting part is movably connected to one side of the rear seat, and the second connecting part is movably connected to the top end of the tension frame.

[0016] Preferably, the pressing assembly includes a pressing frame fixedly connected to the top of the tension frame, a connecting block is fixed to the inner wall of the pressing frame, a slide is movably connected to the surface of the connecting block, a sliding hole is provided on the surface of the slide for sliding with the connecting block, a bonding plate is fixedly connected to the bottom end of the slide, two guide shafts are fixedly connected to the upper surface of the bonding plate, the guide shafts penetrate and slide on the surface of the pressing frame, a spring is arranged on the surface of the guide shaft, and the transmission frame is fixedly connected to the opposite surfaces of the two slides.

[0017] Beneficial effects: This protocol involves the transgenic epidermal growth factor silkworm line N S EC S The flat silk is obtained by spinning on the two-dimensional plane of the mold, and then the transgenic epidermal growth factor silk biofilm dressing is obtained through technical processes such as hot pressing and sterilization. The transgenic epidermal growth factor silk biofilm prepared by this technology can greatly maintain the content and activity of the growth factor, while greatly saving the time required for the preparation of the silk structure biofilm dressing to maintain the natural structure and mechanical properties of silk. The preparation process is also simpler. Since there is no need for silk degumming and silk fibroin extraction, the processing cost of the degumming liquid and the silk fibroin extract can be reduced, the pollution to the environment can be reduced, and the problem of maintaining the natural structure of silk and the activity of epidermal growth factor in the preparation process of traditional silk dressings can be solved.

[0018] At the same time, this solution improves and optimizes the hot pressing process. Through linear pressure changes and temperature coordination, it can provide a better hot pressing effect for the flat wire. At the same time, the calibration and alignment of the flat wire can maintain sufficient hot pressing operation and avoid local waste.

[0019] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In the attached picture: Figure 1 It is a schematic diagram of the process structure of the preparation method of the present invention; Figure 2 It is a three-dimensional structural schematic diagram of the present invention; Figure 3 It is a three-dimensional structural schematic diagram of the present invention from another viewing angle; Figure 4 It is a schematic structural diagram of the present invention; Figure 5 It is a three-dimensional structural schematic diagram of the hot pressing assembly of the present invention; Figure 6 It is a three-dimensional structural schematic diagram of the pressure-applying assembly of the present invention; Figure 7 It is a schematic diagram of the explosion structure of the hot pressing assembly of the present invention; Figure 8 It is a schematic diagram of a three-dimensional cross-sectional structure of a hydraulic assembly of the present invention; Fig. 9 It is a schematic diagram of the structure of the cooperation between the holding component and the feeding component of the present invention; Fig.10 It is a schematic diagram of the mold structure of the present invention; Fig.11 For the present invention S EC S Schematic diagram of the observation of the red fluorescent environment of the silkworm strains under a stereofluorescence microscope; Fig.12 This is a schematic diagram comparing the flat silk and silk of the present invention; Fig.13 This is a schematic diagram comparing the void structures of the flat silk and the silk of the present invention taken under an electron microscope; Fig.14 The common silkworm strains 932 and N S EC S Schematic diagram of the mechanical properties of strain silk fiber tensile test; Fig.15 The common strains of silkworm and N S EC S Schematic diagram of the contact angle between the strain and pure water.

[0021] In the figure: 1, base; 2, tension frame; 3, hydraulic assembly; 31, hydraulic chamber; 32, hydraulic piston; 33, hydraulic cylinder; 34, oil guide hole; 35, connecting pipe; 36, slide; 4, retaining frame; 5, hot pressing assembly; 51, rear seat; 52, support pad; 53, heat insulation board; 54, heating plate; 55, uniform temperature board; 56, pressure sensor; 57, movable seat; 6, pressure assembly; 61, main shaft; 62, cross seat; 63, tension spring; 64. Deflection plate; 65. Connecting column; 66. First connecting part; 67. Second connecting part; 7. Pressing assembly; 71. Pressing frame; 72. Connecting block; 73. Slide plate; 74. Sliding hole; 75. Guide shaft; 76. Spring; 77. Laminating plate; 8. Feeding assembly; 81. Transmission frame; 82. Conveyor belt; 83. Hook; 9. Hydraulic pump; 10. Controller; 11. Heating wire; 12. Flat wire; 13. Spinning plane; 14. Pillar. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention.

[0023] This scheme is based on an existing invention patent (for example, authorization announcement number CN 112852876 B) and proposes a recombinant expression vector of silkworm silk gland expressing human epidermal growth factor and its preparation method and application. The patent "constructs a recombinant expression vector of silkworm silk gland expressing human epidermal growth factor and then prepares transgenic silkworms". Based on the silkworm, this scheme further proposes the "preparation method of silk membrane loaded with recombinant human epidermal growth factor" of this scheme, and based on the drawbacks of this method, discusses, optimizes and expands it, and proposes a "silk membrane processing device loaded with recombinant human epidermal growth factor" with optimization effect, which realizes the final preparation of silk biofilm. The specific contents are as follows: like Figures 1 to 10 As shown, the preparation method of the silk membrane loaded with recombinant human epidermal growth factor comprises the following method: Rearing transgenic epidermal growth factor silkworm strain N S EC S At the fifth instar, 3-5 days later, the prepared silkworms are obtained; The prepared silkworms were placed under a stereofluorescence microscope, and red fluorescence was used to observe the fluorescence of the silkworm eyes. The fifth-instar silkworms with red fluorescence in the eyes were selected and continued to be raised to obtain mature silkworms. After the mature silkworms are put into the cluster to defecate, a certain number of mature silkworms are selected and put into the mold; Making the mature silkworms spin silk on the two-dimensional plane of the mold to obtain flat silk 12; The flat wire 12 is peeled off from the mold and subjected to heat pressing treatment; Finally, beta irradiation sterilization was performed; The silk biofilm is obtained by cutting into a specified size and attaching to the surface of a substrate.

[0024] Among them Fig.11 As shown, after selection, it can be seen from the results that the prepared silkworms are placed under a stereofluorescence microscope, and red fluorescence is used to observe the fluorescence of the silkworm eyes. The fifth-instar silkworms with red fluorescence in the eyes are selected and continued to be raised to adults (silk moths). The selected silkworms have red fluorescence in the eyes of both larvae and adults.

[0025] Further, such as Fig.12 The flat wire 12 and N shown on the left side S EC S strains of silkworm cocoons, using Fig.10 The mold shown in FIG. 1 (composed of a spinning plane 13 and a column) is prepared, wherein the flat wire 12, N S EC S - Results of electron microscope scanning of NSC and common 932 strain silk, see Fig.13 It can be seen that compared with the cocoon silk group, the flat silk 12 has a looser structure and a higher porosity. The high porosity makes the flat silk 12 have better air permeability and impact resistance.

[0026] Based on the measurement of electron microscope scanning results, we can get Table 1 N S EC S Variety of cocoon silk and flat silk 12 diameter: Table 1 Diameters of common silk and flat silk

[0027] Based on Table 1 N S EC S The diameters of cocoon silk and flat silk 12 are known, and the flat silk 12 is larger than N S EC S The silk of the cocoon is finer, making the flat silk 12 softer and the overall comprehensive performance more superior; The number of mature silkworms in the mold includes 25-35, and silkworms with similar growth conditions are selected during selection.

[0028] During the spinning process on the two-dimensional plane of the mold, the spinning situation is checked every 1-2 hours, and the position of the silkworms is manually adjusted according to the spinning situation to obtain uniform flat silk 12, and the silkworm feces need to be cleaned during the inspection.

[0029] The hot pressing treatment includes: hot pressing time of 1-10 min, hot pressing temperature of 60° C., hot pressing pressure of 5-15 MPa, and beta irradiation sterilization treatment dosage of 15 kGy.

[0030] Tensile test: The common silkworm strains 932, N S EC S The silk fiber samples of the strains were transferred to a paper template with a gauge length of 10 mm, and the two ends of the samples were fixed to the frame by double-sided tape and adhesive. The tensile test was carried out using a universal tensile testing machine at room temperature of 25°C. The mechanical properties of silk of different forms are shown in Table 2.

[0031] Table 2 Mechanical properties of silk with different forms

[0032] Further, the silk stress-strain curve (such as Fig.14 as shown).

[0033] Depend on Fig.14 932 and N shown S EC S The stress-strain curves of the strains show that all three strains are increasing, with no obvious yield point, and the slope of the curves decreases with increasing strain. S EC S Silk, N S EC S Flat wire 12 exhibits better mechanical properties.

[0034] Contact angle test: Depend on Fig.15 The common strains of silkworm and N S EC S The contact angles of the three strains with pure water are all greater than 90°. S EC S The contact angles of -FSC with pure water at each time point were smaller than those of 932-NSC and N S EC S -NSC, showing better hydrophilicity. The contact angles of silk with different morphologies are shown in Table 3.

[0035] Table 3 Contact angles of silk with different morphologies

[0036] The transgenic epidermal growth factor silk biofilm prepared using this technology can significantly maintain the content and activity of the growth factor (the content is increased to 2.75%), while significantly saving the time required for the preparation of the silk structure biofilm dressing, maintaining the natural structure and mechanical properties of silk, and making the preparation process simpler. Since silk degumming and silk fibroin extraction are not required, the processing cost of the degumming solution and silk fibroin extract can be reduced.

[0037] Based on the above method, the flat wire 12 also needs to be hot pressed and β-irradiated for sterilization. During the hot pressing process, according to the defects of the current hot pressing equipment, including the hot pressing, the pressure is quickly applied after the direct hydraulic force contacts. Based on this, this solution provides an elastic clamping force that can provide a continuously increasing elastic clamping force through elasticity during the extrusion process. In this process, preheating is performed. When the temperature rises to a suitable temperature, direct pressurization is performed. Compared with the traditional method of slowly increasing the temperature after direct pressurization, this solution maintains sufficient clamping force before heating, and directly pressurizes after the temperature is suitable, which has a significant improvement on the thermoforming quality of the flat wire 12, as shown in more detail below: Specifically, Fig.10 As shown, the mold includes a spinning plane 13 made of plastic or paper material, the spinning plane 13 is arranged in a disc shape, and a support 14 is arranged on the lower surface of the spinning plane 13 to support it.

[0038] The silkworm flat silk 12 spinning mold consists of two parts: a spinning plane 13 and a support 14. The spinning plane 13 is the core part of the flat silk 12 mold, and is composed of a plastic or paper disc with a certain curvature. This structure provides a two-dimensional plane of the mold for the silkworm to spin silk. The curvature setting provides a certain support for the silkworm to spin silk, preventing the silkworm from falling too much during the spinning process. The support 14 is the supporting part of the silk spinning mold, and is composed of a hollow cylinder with a certain curvature. This structure can support the spinning plane 13 to prevent the silkworm from crawling out of the spinning plane 13.

[0039] Specifically, Figures 2 to 9 As shown: a recombinant human epidermal growth factor loaded silk membrane treatment device comprises a base 1, a hydraulic component 3 is installed on the inner wall of the base 1, the hydraulic component 3 is connected to a hydraulic pump 9, a tension frame 2 is fixedly connected to the movable end of the hydraulic component 3, and a holding component 7 is fixedly connected to the upper surface of the tension frame 2; The upper surface of the base 1 is fixedly connected with a holder 4, the inner wall of the holder 4 is slidably provided with two hot pressing components 5, and one side of the hot pressing component 5 and the inner wall of the tension frame 2 are jointly installed with a pressure component 6; A feeding assembly 8 is installed on the surface of the pressing assembly 7. The feeding assembly 8 includes a transmission frame 81 fixed to the inner wall of the pressing assembly 7. A conveyor belt 82 is arranged on the inner wall of the transmission frame 81. A hook 83 is arranged on the surface of the conveyor belt 82.

[0040] Specifically, Figure 5 and Figure 7As shown: the hot pressing assembly 5 includes a movable seat 57 connected to the pressure-applying assembly 6, a rear seat 51 is fixed to one side of the movable seat 57, a support pad 52 is fixed to one side of the rear seat 51, a heat insulation board 53 is fixed to one side of the support pad 52, and pressure sensors 56 are arranged at the four corners of the heat insulation board 53, and the pressure sensors 56 are fixedly connected to the surface of the rear seat 51; A heating plate 54 is fixedly connected to one side of the heat insulation plate 53, and a temperature-uniform plate 55 is symmetrically arranged on the other side of the heating plate 54 and the heat insulation plate 53; A heating wire 11 is disposed on one side of the heating plate 54 , and the heating wire 11 is electrically connected to a controller 10 .

[0041] By setting the rear seat 51, the rear seat 51 can be movably connected with the pressure-applying component 6, and the supporting force can be applied to the heat insulation plate 53 through the support pad 52. The heat insulation plate 53 is in contact with the heating plate 54, so that the side of the heating plate 54 can maintain heat insulation. At the same time, the heat of the heating plate 54 can be transferred to the surface of the temperature-uniform plate 55, so that the plane of the temperature-uniform plate 55 forms a uniform heat distribution. At the same time, the support pad 52 can be slightly deformed under the action of pressure, so that the pressure data can be detected by the pressure sensor 56 while it is deformed, so that feedback data of the pressure can be obtained.

[0042] Specifically, Figure 8 As shown: the hydraulic assembly 3 includes a hydraulic cylinder 33 fixed on the lower surface of the retainer 4, a hydraulic piston 32 is fixed on the surface of the hydraulic cylinder 33, a hydraulic chamber 31 is sealed and attached to the surface of the hydraulic piston 32, and the hydraulic chamber 31 is slidably arranged on the arc-shaped side wall of the hydraulic cylinder 33; A slide 36 slides through the surface of the hydraulic warehouse 31, and the slide 36 is fixedly connected to the inner wall of the base 1. The top of the hydraulic warehouse 31 is fixedly connected to the bottom of the tension frame 2. An oil guide hole 34 is opened through the inner wall of the hydraulic cylinder 33, and a connecting pipe 35 for connecting the hydraulic pump 9 and the oil guide hole 34 is opened on the arc-shaped side wall of the hydraulic cylinder 33.

[0043] This solution can transmit hydraulic pressure to the oil guide hole 34 in the hydraulic cylinder 33 through the hydraulic pump 9 and the connecting pipe 35, so that the oil forms a pressure space at the bottom of the oil guide hole 34. The pressure can cooperate with the hydraulic piston 32 to move the hydraulic chamber 31 downward, so that the hydraulic chamber 31 slides on the inner wall of the slide 36, and then the hydraulic chamber 31 can move downward with the tension frame 2 to achieve stable hydraulic control.

[0044] Specifically, Figure 6As shown: the pressure-applying assembly 6 includes a main shaft 61, and two cross seats 62 are slidably penetrated through the surface of the main shaft 61, and opposite ends of the two cross seats 62 are commonly fixedly connected with tension springs 63, and the tension springs 63 are spirally coiled and located on the surface of the main shaft 61, and connecting columns 65 are fixed on both sides of the cross seats 62, and one end of the connecting column 65 is movably connected to two deflection plates 64, and one end of the two deflection plates 64 on the same side of the two cross seats 62 is respectively provided with a first connecting portion 66 and a second connecting portion 67, and the first connecting portion 66 is movably connected to one side of the rear seat 51, and the second connecting portion 67 is movably connected to the top of the tension frame 2.

[0045] The pressure-applying component 6 serves as a transfer node for pressure during the hot pressing process. The hydraulic pressure moves the tension frame 2, causing the misalignment force between the tension frame 2 and the retaining frame 4 to change. The misalignment force is provided by the hydraulic displacement of the hydraulic chamber 31, so that the pressure-applying component 6 can convert the misalignment force into the pressure of the hot pressing component 5.

[0046] Specifically, Fig. 9 As shown: the pressing assembly 7 includes a pressing frame 71 fixedly connected to the top of the tension frame 2, a connecting block 72 is fixed to the inner wall of the pressing frame 71, a slide plate 73 is movably connected to the surface of the connecting block 72, a sliding hole 74 is provided on the surface of the slide plate 73 for sliding with the connecting block 72, a fitting plate 77 is fixedly connected to the bottom end of the slide plate 73, two guide shafts 75 are fixedly connected to the upper surface of the fitting plate 77, the guide shafts 75 penetrate and slide on the surface of the pressing frame 71, a spring 76 is provided on the surface of the guide shaft 75, and the transmission frame 81 is fixedly connected to the opposite surfaces of the two slide plates 73.

[0047] The transmission component can be supported by the pressing component 7, and the pressing component 7 can be located on the surface of the retaining frame 4 for bonding.

[0048] At the same time, the retaining frame 4 can maintain the hot pressing assembly 5 to slide in a fitted manner and maintain the stable displacement of the hot pressing assembly 5 .

[0049] When in use, the hydraulic pump 9 is connected to the oil guide hole 34 in the hydraulic cylinder 33 through the connecting pipe 35. When the oil is injected into the oil guide hole 34 through the hydraulic pump 9, high pressure is formed under the hydraulic piston 32, causing it to move the hydraulic chamber 31 downward. When the hydraulic chamber 31 moves, it slides on the inner wall of the slide 36 and moves downward with the tension frame 2. Then, the tension frame 2 moves downward while pressing the second connection part 67 of the pressure component 6 downward. Based on the action of the tension spring 63 on the inner wall of the pressure component 6, the pressure component 6 directly applies force to the hot pressing component 5, causing the hot pressing component 5 to slide on the inner wall of the retaining frame 4 until the two hot pressing components 5 press the flat wire together. 12 is clamped, the tension frame 2 continuously applies pressure to the pressure assembly 6, and the height and horizontal distance between the second connection part 67 and the first connection part 66 change. In this process, the opposite ends of the two deflection plates 64 on the same side are slidably connected to one end of the connecting column 65. In this process, the connecting column 65 slides the cross seat 62 on the surface of the main shaft 61, so that the distance between the two cross seats 62 changes, and the tension spring 63 is stretched. The force of the tension spring 63 is consistent with the clamping force of the two hot pressing components 5, until the cross seat 62 is limited with one end of the main shaft 61, so that the elastic force of the tension spring 63 reaches the limit value, and then the direct hot pressing operation is performed; During the above-mentioned pressure process, the two hot pressing components 5 slide on the inner wall of the retaining frame 4 and clamp the flat wire 12 with each other. At this time, the electric heating wire 11 in the heating plate 54 is heated, so that its heat is evenly transferred to the surface of the flat wire 12 through the uniform temperature plate 55, and then under the elastic force of the tension spring 63, the pressure is in a linear continuous increase. The temperature in this process also changes accordingly. The temperature can be gradually increased to 60°C while maintaining the pressure change, so that a better hot pressing surface can be formed. After the cross seat 62 slides on the surface of the main shaft 61 to reach the maximum stroke, the pressure suddenly rises to 15 MPa, and is maintained for 10 minutes to complete the hot pressing operation.

[0050] The above-mentioned hot pressing steps can well form the surface molding of the flat wire 12. The method of gradually increasing the pressure and heating the temperature, and suddenly increasing the pressure at the end, can achieve a better hot pressing surface. At the same time, the feeding component 8 is cooperated with the feeding component 8 to horizontally transfer the flat wire 12 so that the flat wire 12 moves between the two hot pressing components 5. When the tension frame 2 moves downward, the pressing frame 71 of the holding component 7 moves downward accordingly until the flat wire 12 is completely placed between the two hot pressing components 5. At this time, the bonding plate 77 contacts the upper surface of the retaining frame 4, so that the flat wire 12 is at a constant height, the guide shaft 75 slides on the surface of the pressing frame 71, and the sliding hole 74 of the slide plate 73 slides on the surface of the connecting block 72, so that the flat wire 12 moves downward to a certain height, and the feeding component 8 can be at a constant height. After the hot pressing is completed and the tension frame 2 is reset, the feeding component 8 can move upward. In this way, automatic loading and unloading operations can be realized, which is more convenient and safe to use, and can move downward for alignment, so that the flat wire 12 is fully hot-pressed and waste is avoided.

[0051] In summary, the present solution provides a device and method for hot pressing which can provide a continuously increasing elastic clamping force through elasticity during the extrusion process, performs preheating during the process, and performs direct pressurization when the temperature rises to a suitable temperature. Compared with the traditional method of slowly increasing the temperature after direct pressurization, the present solution maintains sufficient clamping force and then performs heating, and directly performs pressurization when the temperature is suitable. This device and method have a significant improvement on the hot forming quality of the flat wire 12 prepared by the present solution.

[0052] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a silk membrane loaded with recombinant human epidermal growth factor, characterized in that: The method comprises the following steps: raising transgenic epidermal growth factor silkworm strain N S EC S At the fifth instar, 3-5 days later, the prepared silkworms are obtained; The prepared silkworms were placed under a stereofluorescence microscope, and red fluorescence was used to observe the fluorescence of the silkworm eyes. The fifth-instar silkworms with red fluorescence in the eyes were selected for further breeding to obtain mature silkworms. After the mature silkworms are put into the cluster to defecate, a certain number of mature silkworms are selected and put into the mold; Allowing mature silkworms to spin silk on a two-dimensional plane of a mold to obtain flat silk (12); The flat wire (12) is peeled off from the mold and subjected to heat pressing treatment; Finally, beta irradiation sterilization was performed; The silk biofilm is obtained by cutting into a specified size and attaching to the surface of a substrate.

2. The method for preparing the recombinant human epidermal growth factor loaded silk membrane according to claim 1, characterized in that: The number of mature silkworms in the mold includes 25-35, and silkworms with similar growth conditions are selected during selection.

3. The method for preparing the silk membrane loaded with recombinant human epidermal growth factor according to claim 1, characterized in that: During the spinning process on the mold, the spinning situation is checked every 1-2 hours, and the position of the silkworms is manually adjusted according to the spinning situation to obtain uniform flat silk (12), and the silkworm feces need to be cleaned during the inspection.

4. The method for preparing the recombinant human epidermal growth factor loaded silk membrane according to claim 1, characterized in that: The hot pressing treatment includes: hot pressing time of 1-10 minutes, hot pressing temperature of 60° C., hot pressing pressure of 5-15 MPa, and beta irradiation sterilization treatment dosage of 15 kGy.

5. The method for preparing the silk membrane loaded with recombinant human epidermal growth factor according to claim 1, characterized in that: The mould comprises a spinning plane (13) made of plastic or paper material. The spinning plane (13) is arranged in a disc shape, and a support (14) for supporting the spinning plane (13) is arranged on the lower surface of the spinning plane (13).

6. A device for processing silk membranes loaded with recombinant human epidermal growth factor, according to the method for preparing silk membranes loaded with recombinant human epidermal growth factor according to any one of claims 1 to 5, characterized in that: It comprises a base (1), the inner wall of the base (1) is mounted with a hydraulic assembly (3), the hydraulic assembly (3) is connected to a hydraulic pump (9), the movable end of the hydraulic assembly (3) is fixedly connected to a tension frame (2), and the upper surface of the tension frame (2) is fixedly connected to a holding assembly (7); The upper surface of the base (1) is fixedly connected to a retaining frame (4), the inner wall of the retaining frame (4) is slidably provided with two hot pressing components (5), and one side of the hot pressing component (5) and the inner wall of the tension frame (2) are jointly installed with a pressure component (6); A feeding assembly (8) is installed on the surface of the pressing assembly (7), and the feeding assembly (8) comprises a transmission frame (81) fixed to the inner wall of the pressing assembly (7), a conveyor belt (82) is arranged on the inner wall of the transmission frame (81), and a hook (83) is arranged on the surface of the conveyor belt (82).

7. The recombinant human epidermal growth factor loaded silk membrane processing device according to claim 6, characterized in that: The hot pressing assembly (5) comprises a movable seat (57) connected to the pressure-applying assembly (6); a rear seat (51) is fixedly connected to one side of the movable seat (57); a support pad (52) is fixedly connected to one side of the rear seat (51); a heat insulating plate (53) is fixedly connected to one side of the support pad (52); pressure sensors (56) are arranged at the four corners of the heat insulating plate (53); and the pressure sensors (56) are fixedly connected to the surface of the rear seat (51); A heating plate (54) is fixedly connected to one side of the heat insulation plate (53), and a temperature-uniform plate (55) is symmetrically arranged on the other side of the heating plate (54) and the heat insulation plate (53); A heating wire (11) is provided on one side of the heating plate (54), and the heating wire (11) is electrically connected to a controller (10).

8. The recombinant human epidermal growth factor loaded silk membrane processing device according to claim 6, characterized in that: The hydraulic assembly (3) comprises a hydraulic cylinder (33) fixed on the lower surface of the retaining frame (4), a hydraulic piston (32) being fixed on the surface of the hydraulic cylinder (33), a hydraulic chamber (31) being sealingly attached to the surface of the hydraulic piston (32), and the hydraulic chamber (31) being slidably arranged on the arc-shaped side wall of the hydraulic cylinder (33); A slide (36) is slidably penetrated through the surface of the hydraulic chamber (31), and the slide (36) is fixedly connected to the inner wall of the base (1). The top of the hydraulic chamber (31) is fixedly connected to the bottom of the tension frame (2). An oil guide hole (34) is penetrated through the inner wall of the hydraulic cylinder (33), and a connecting pipe (35) for connecting the hydraulic pump (9) and the oil guide hole (34) is provided on the arc-shaped side wall of the hydraulic cylinder (33).

9. The recombinant human epidermal growth factor loaded silk membrane processing device according to claim 7, characterized in that: The pressure-applying assembly (6) comprises a main shaft (61), and two cross seats (62) are slidably penetrated through the surface of the main shaft (61); opposite ends of the two cross seats (62) are commonly fixedly connected with a tension spring (63); the tension spring (63) is spirally wound and located on the surface of the main shaft (61); connecting columns (65) are fixed on both sides of the cross seat (62); one end of the connecting column (65) is movably connected to two deflection plates (64); one end of the two deflection plates (64) on the same side of the two cross seats (62) are respectively provided with a first connecting portion (66) and a second connecting portion (67); the first connecting portion (66) is movably connected to one side of the rear seat (51); and the second connecting portion (67) is movably connected to the top of the tension frame (2).

10. The recombinant human epidermal growth factor loaded silk membrane processing device according to claim 6, characterized in that: The pressing assembly (7) comprises a pressing frame (71) fixedly connected to the top of the tension frame (2); a connecting block (72) is fixedly provided on the inner wall of the pressing frame (71); a slide plate (73) is movably connected to the surface of the connecting block (72); a sliding hole (74) is provided on the surface of the slide plate (73) for sliding with the connecting block (72); a bonding plate (77) is fixedly connected to the bottom end of the slide plate (73); two guide shafts (75) are fixedly connected to the upper surface of the bonding plate (77); the guide shafts (75) penetrate and slide on the surface of the pressing frame (71); a spring (76) is provided on the surface of the guide shafts (75); and the transmission frame (81) is fixedly connected to the opposite surfaces of the two slide plates (73).

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