Preparation method and processing device of recombinant human epidermal growth factor loaded silk membrane

Abstract

The application discloses a preparation method and a treatment device of recombinant human epidermal growth factor silk membrane, relates to the technical field of biological products, and is characterized in that a transgenic epidermal growth factor silkworm strain N S EC S The transgenic epidermal growth factor silk biological membrane dressing is prepared through the technical processes of hot pressing and sterilization after the flat plate silk is spun on the two-dimensional plane of a mold. The transgenic epidermal growth factor silk biological membrane prepared by using the technology can greatly maintain the content and activity of the growth factor, greatly saves the time required for preparing the silk structure biological membrane dressing, maintains the natural structure and mechanical properties of the silk, and the preparation process is simpler. Since degumming and silk fibroin extraction are not needed, the treatment cost of the degumming liquid and the silk fibroin extraction liquid is reduced, the pollution to the environment is reduced, and the problem of maintaining the natural structure of the silk and the activity of the epidermal growth factor in the preparation process of the traditional silk membrane is solved. The human epidermal growth factor silk membrane prepared by the method can be applied to the medical fields of wound repair and tissue repair, and has good application potential and value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological products, and particularly relates to a preparation method and a treatment device of recombinant human epidermal growth factor silk film. BACKGROUND

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

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

[0004] The current growth factor-loaded silk dressing is mainly prepared by the method of silk fibroin regeneration. For example: the transgenic epidermal growth factor cocoon is cut into 1 cm2 size, and after degumming treatment, it is dissolved in a high-concentration lithium bromide solution. Dialysis for 3 days to obtain a silk fibroin solution. Then a regenerated silk fibroin biomembrane is prepared by hot plastic forming, electrospinning and the like process.

[0005] However, the process of preparing the transgenic epidermal growth factor biomembrane dressing by the method of silk fibroin regeneration at present involves high temperature, high pressure, high salt and the like conditions, which seriously affects the activity of the transgenic epidermal growth factor and seriously damages the natural structure of the silk. The main reason for this problem is that when the silkworms are raised, the mature silkworms are usually put into a mold with a three-dimensional structure to make the silkworms spin silk to form an oval cocoon, and a two-dimensional biomembrane dressing can be prepared only by silk fibroin extraction or reeling process.

[0006] Therefore, the present application is proposed. SUMMARY

[0007] To solve the above technical problems, the basic idea of the technical solution of the present application is:

[0008] The preparation method of the recombinant human epidermal growth factor silk film comprises the following methods:

[0009] Raising a transgenic epidermal growth factor silkworm strain N S EC S to obtain a prepared silkworm at 3-5 days of the fifth instar;

[0010] The prepared silkworms are placed under a stereoscopic fluorescence microscope, red fluorescence is used to observe the eye fluorescence of the silkworms, and the fifth instar silkworms with red fluorescence in the eyes are selected for further rearing to obtain mature silkworms;

[0011] After the mature silkworms are arranged and defecated, a certain number of mature silkworms are selected and placed in a mold;

[0012] The mature silkworms are made to spin silk on the two-dimensional plane of the mold to obtain a flat silk;

[0013] The flat silk is peeled off from the mold and subjected to heat pressing treatment;

[0014] Finally, β irradiation sterilization treatment is performed;

[0015] The silk biomembrane is obtained by cutting to a specified size and attaching a substrate surface.

[0016] Preferably, the number of mature silkworms in the mold includes 25-35, and the selection follows the selection of silkworms with similar growth conditions.

[0017] Preferably, the spinning on the two-dimensional plane of the mold is checked every 1-2 hours to adjust the position of the silkworms to obtain uniform flat silk, and the silkworm sand needs to be cleaned when checking.

[0018] Preferably, the heat pressing treatment includes a heat pressing time of 1-10 min, a heat pressing temperature of 60°C, a heat pressing pressure of 5-15 MPa, and a β irradiation sterilization treatment dose of 15 kGy.

[0019] Preferably, the mold includes a spinning plane made of plastic or paper material, the spinning plane is disc-shaped, and the lower surface of the spinning plane is provided with a support column supporting it.

[0020] Preferably, the recombinant human epidermal growth factor silk membrane treatment device includes a base, a hydraulic assembly is installed on the inner wall of the base, the hydraulic assembly is communicated with a hydraulic pump, the movable end of the hydraulic assembly is fixedly connected with a tension frame, and a pressure holding assembly is fixedly connected to the upper surface of the tension frame.

[0021] The upper surface of the base is fixedly connected with a retaining frame, two heat pressing assemblies are slidably arranged on the inner wall of the retaining frame, and a pressure applying assembly is jointly installed on one side of the heat pressing assembly and the inner wall of the tension frame.

[0022] A material conveying assembly is installed on the surface of the pressure holding assembly, the material conveying assembly includes a transmission frame fixedly arranged on the inner wall of the pressure holding assembly, a conveyor belt is arranged on the inner wall of the transmission frame, and hooks are arranged on the surface of the conveyor belt.

[0023] Preferably, the hot pressing assembly comprises a movable seat connected with the pressure applying assembly, one side of the movable seat is fixed with a rear seat, one side of the rear seat is fixedly connected with a support pad, one side of the support pad is fixedly connected with a heat insulation plate, the heat insulation plate is provided with a pressure sensor at each corner, and the pressure sensor is fixedly connected to the surface of the rear seat.

[0024] One side of the heat insulation plate is fixedly connected with a heating plate, and the other side of the heating plate is symmetrically provided with a uniform temperature plate.

[0025] One side of the heating plate is provided with an electric heating wire, and the electric heating wire is electrically connected with a controller.

[0026] Preferably, the hydraulic assembly comprises a hydraulic cylinder fixed to the lower surface of the holder, a hydraulic piston fixed to the surface of the hydraulic cylinder, and a hydraulic chamber sealingly attached to the surface of the hydraulic piston, wherein the hydraulic chamber is slidingly arranged in the arc-shaped side wall of the hydraulic cylinder.

[0027] The surface of the hydraulic chamber is slidingly penetrated by a sliding frame, the sliding frame is fixedly connected to the inner wall of the base, the top end of the hydraulic chamber is fixedly connected to the bottom of the tension frame, the inner wall of the hydraulic cylinder is penetratingly provided with an oil guide hole, and the arc-shaped side wall of the hydraulic cylinder is provided with a connecting pipe for communication between the hydraulic pump and the oil guide hole.

[0028] Preferably, the pressure applying assembly comprises a main shaft, two cross seats slidingly penetrating the surface of the main shaft, a tension spring fixedly connected to the opposite ends of the two cross seats, the tension spring being in the form of a spiral coil on the surface of the main shaft, a connecting column fixed to the two sides of the cross seat, two deflection plates movably connected to one end of the connecting column, a first connecting part and a second connecting part provided at one end of the two deflection plates on the same side of the two cross seats, the first connecting part movably connected to one side of the rear seat, and the second connecting part movably connected to the top end of the tension frame.

[0029] Preferably, the pressure holding assembly comprises a pressing frame fixedly connected to the top end of the tension frame, a connecting block fixed to the inner wall of the pressing frame, a sliding plate movably connected to the surface of the connecting block, a sliding hole in the surface of the sliding plate for sliding cooperation with the connecting block, a fitting plate fixedly connected to the bottom end of the sliding plate, two guide shafts fixedly connected to the upper surface of the fitting plate, the guide shafts slidingly penetrating the surface of the pressing frame, springs provided on the surface of the guide shafts, and a transmission frame fixedly connected to the opposite surfaces of the two sliding plates.

[0030] Beneficial effects:

[0031] The present application makes the transgenic epidermal growth factor silkworm strain N S EC SThe flat plate silk is prepared by spinning on the two-dimensional plane of the mold, and the transgenic epidermal growth factor silk biomembrane dressing is prepared through the technical processes of hot pressing, sterilization and the like. The transgenic epidermal growth factor silk biomembrane prepared by the technology can greatly maintain the content and activity of the growth factor, greatly saves the time required for preparing the silk structure biomembrane dressing, maintains the natural structure and mechanical properties of the silk, and the preparation process is simpler. Since degumming and silk fibroin extraction are not required, the treatment cost of the degumming liquid and the silk fibroin extraction liquid is reduced, the pollution to the environment is reduced, and the problem of maintaining the natural structure of the silk and the activity of the epidermal growth factor in the preparation process of the traditional silk dressing is solved.

[0032] Meanwhile, the hot pressing treatment is improved and optimized, a better flat plate silk hot pressing effect can be provided through linear pressure change and temperature cooperation, and sufficient hot pressing operation can be maintained through the calibration and alignment of the flat plate silk, so that local waste is avoided.

[0033] The specific embodiments of the present application will be described in further detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0034] In the drawings:

[0035] Figure 1 The flow structure schematic diagram of the preparation method of the present application is shown in the figure;

[0036] Figure 2 The three-dimensional structure schematic diagram of the present application is shown in the figure;

[0037] Figure 3 The three-dimensional structure schematic diagram of the present application is shown in the figure;

[0038] Figure 4 The front view structure schematic diagram of the present application is shown in the figure;

[0039] Figure 5 The three-dimensional structure schematic diagram of the hot pressing assembly of the present application is shown in the figure;

[0040] Figure 6 The three-dimensional structure schematic diagram of the pressure applying assembly of the present application is shown in the figure;

[0041] Figure 7 The exploded structure schematic diagram of the hot pressing assembly of the present application is shown in the figure;

[0042] Figure 8 The three-dimensional cross-sectional structure schematic diagram of the hydraulic assembly of the present application is shown in the figure;

[0043] Figure 9 The structure schematic diagram of the pressure holding assembly and the material conveying assembly of the present application is shown in the figure;

[0044] Figure 10 The mold structure schematic diagram of the present application is shown in the figure;

[0045] Figure 11 The N S EC S The observation schematic diagram of the red fluorescent environment of the Bombyx mori in the stereoscopic fluorescence microscope;

[0046] Figure 12 The schematic diagram of the comparison between the flat filament and the silk of the present application;

[0047] Figure 13 The schematic diagram of the comparison between the flat filament and the silk in the electron microscope of the present application;

[0048] Figure 14 The Bombyx mori of the present application and the N S EC S The schematic diagram of the mechanical property of the silk fiber tensile test of the Bombyx mori;

[0049] Figure 15 The Bombyx mori of the present application and the N S EC S The schematic diagram of the contact angle of the Bombyx mori and pure water.

[0050] 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, sliding frame; 4, retaining frame; 5, hot pressing assembly; 51, rear seat; 52, support pad; 53, heat insulation plate; 54, heating plate; 55, uniform temperature plate; 56, pressure sensor; 57, movable seat; 6, pressure applying 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, pressure holding assembly; 71, pressure frame; 72, connecting block; 73, sliding plate; 74, sliding hole; 75, guide shaft; 76, spring; 77, fitting plate; 8, material conveying assembly; 81, transmission frame; 82, conveying belt; 83, hook; 9, hydraulic pump; 10, controller; 11, electric heating wire; 12, flat filament; 13, spinning plane; 14, support column. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical scheme and advantages of the embodiments of the present application more clear, the technical scheme in the embodiments will be described clearly and completely below with reference to the drawings in the embodiments of the present application, and the following embodiments are used to illustrate the present application.

[0052] The present scheme is based on the existing invention patent (for example, authorized announcement number CN 112852876 B) proposes a recombinant expression vector for expressing human epidermal growth factor in silkworm silk gland and its preparation method and application.The patent through “constructing a recombinant expression vector for expressing human epidermal growth factor in silkworm silk gland, and preparing transgenic silkworm”, the present scheme based on the silkworm as the basis further puts forward the preparation method of “recombinant human epidermal growth factor silk membrane loading device” with optimization effect, realizes the preparation of final silk biomembrane, the specific content is as follows:

[0053] As shown in Figures 1 to 10 , the preparation method of recombinant human epidermal growth factor silk membrane loading device, comprising the following methods:

[0054] Raising transgenic epidermal growth factor silkworm strain N S EC S To five age 3-5 days, obtain the pre-prepared silkworm;

[0055] The pre-prepared silkworm is placed under a stereoscopic fluorescence microscope, red fluorescence is used to observe the fluorescence of the eyes of the silkworm, and the five age silkworm with red fluorescence in the eyes is selected for further rearing to obtain the mature silkworm;

[0056] After the mature silkworm is arranged to defecate, a certain number of mature silkworms are selected and placed in a mold;

[0057] Make the mature silkworms spin silk on the two-dimensional plane of the mold to obtain the flat plate silk 12;

[0058] The flat plate silk 12 is peeled off from the mold and subjected to heat pressing treatment;

[0059] Finally, β irradiation sterilization treatment is carried out;

[0060] Cut to the specified size and attach the surface of the substrate to obtain the silk biomembrane.

[0061] As shown in Figure 11 , after selection, it can be seen from the results that the pre-prepared silkworm is placed under a stereoscopic fluorescence microscope, red fluorescence is used to observe the fluorescence of the eyes of the silkworm, and the five age silkworm with red fluorescence in the eyes is selected for further rearing to obtain the mature silkworm;

[0062] Further, as shown in Figure 12 , the flat plate silk 12 and N S EC S strain cocoon, the mold (spinning plane 13 and column) shown in Figure 10 is used for preparation, wherein the flat plate silk 12, NS EC S -NSC, ordinary 932 strain cocoon silk electron microscope scanning results, see Figure 13 It can be seen that compared with the cocoon silk group, the flat silk 12 structure is looser, and has higher porosity. High porosity makes the flat silk 12 have better air permeability and impact resistance.

[0063] Based on the measurement of the electron microscope scanning results, Table 1 N S EC S Strain cocoon silk and flat silk 12 diameter:

[0064] Table 1 ordinary silk and flat silk diameter

[0065]

[0066] Based on Table 1 N S EC S The flat silk 12 is thinner than N S EC S Strain cocoon silk, making the flat silk 12 more flexible, and the overall comprehensive performance is more superior;

[0067] The number of silkworms in the mold includes 25-35, and the selection follows the selection of silkworms with similar growth conditions.

[0068] The spinning situation is checked every 1-2 hours during the spinning on the two-dimensional plane of the mold, and the position of the silkworm is adjusted manually according to the spinning situation to obtain uniform flat silk 12. The silkworm sand needs to be cleaned when checking.

[0069] The hot pressing treatment includes: hot pressing time is 1-10 min, hot pressing temperature is 60°C, hot pressing pressure is 5-15 MPa, and beta irradiation sterilization treatment dose includes 15 kGy.

[0070] Tensile test:

[0071] The ordinary strain Bombyx mori 932, N S EC S The cocoon silk fiber sample of the strain is transferred to a paper template with a gauge length of 10 mm, and the two ends of the sample are fixed on the frame by double-sided tape and adhesive. The tensile test is carried out at room temperature 25°C using a universal tensile testing machine, and the mechanical properties of different forms of silk are shown in Table 2.

[0072] Table 2 Mechanical properties of different forms of silk

[0073]

[0074] Further and obtain the silk stress-strain curve (as Figure 14as shown).

[0075] by Figure 14 932 and N S EC S The stress-strain curves of the three strains can be seen that all of them showed an increasing state, no obvious yield point was found, and the curve slope gradually decreased with the increase of strain. Compared with the ordinary strain silkworm and N S EC S cocoon silk, N S EC S The flat silk 12 showed better mechanical properties.

[0076] Contact angle experiment:

[0077] by Figure 15 ordinary strain silkworm and N S EC S The contact angles of the three strains with pure water can be seen that the contact angles of the three strains are all greater than 90°. Among them, N S EC S -FSC at each time point with pure water are all less than 932-NSC and N S EC S -NSC, showing better hydrophilicity. The contact angles of different forms of silk are shown in Table 3.

[0078] Table 3 Contact angles of different forms of silk

[0079]

[0080] The transgenic epidermal growth factor silk biomembrane prepared by the present technology can greatly maintain the content and activity of the growth factor (the content is increased to 2.75%), while greatly saving the time required for preparing the silk structure biomembrane dressing, maintaining the natural structure and mechanical properties of the silk, and the preparation process is also simpler. Since degumming and silk fibroin extraction are not required, the treatment cost of degumming liquid and silk fibroin extraction liquid can be reduced.

[0081] Based on the above method, the flat silk 12 also needs to be subjected to heat pressing and beta irradiation sterilization treatment. In the heat pressing process, according to the defects of the current heat pressing equipment, including that the heat pressing is directly contacted by hydraulic force and then the pressure is quickly applied. Based on this, the present scheme provides an elastic clamping force that is continuously increased during the extrusion process. In this process, preheating is performed, and when the temperature is raised to a suitable temperature, direct pressing is performed. Compared with the traditional way of slowly increasing the temperature after direct pressing, the present scheme maintains sufficient clamping force for heating, and when the temperature is suitable, direct pressing is performed, which has a significant improvement effect on the heat forming quality of the flat silk 12. More specifically, as shown in the following contents:

[0082] Specifically, as Figure 10As shown: the mold includes a plastic or paper material made of spinning plane 13, the spinning plane 13 is disc-shaped, and the lower surface of the spinning plane 13 is provided with a support column 14.

[0083] The Bombyx mori flat filament 12 spinning mold is composed of two parts of spinning plane 13 and support column 14. The spinning plane 13 is the core part of the flat filament 12 mold, which is composed of a plastic or paper disc with a certain curvature. This structure provides a two-dimensional plane for the silkworm to spin, and the curvature provides certain support for the silkworm spinning to prevent the silkworm from falling too much during the spinning process. The support column 14 is the support part of the spinning mold, which is composed of a hollow column with a certain curvature. This structure can support the spinning plane 13 and prevent the silkworm from climbing out of the spinning plane 13.

[0084] Specifically, as shown in Figures 2 to 9 As shown: the recombinant human epidermal growth factor silk membrane treatment device includes a base 1, the inner wall of the base 1 is provided with a hydraulic assembly 3, the hydraulic assembly 3 is communicated with a hydraulic pump 9, the movable end of the hydraulic assembly 3 is fixedly connected with a tension frame 2, the upper surface of the tension frame 2 is fixedly connected with a pressing assembly 7;

[0085] The upper surface of the base 1 is fixedly connected with a retaining frame 4, the inner wall of the retaining frame 4 is slidably provided with two heat pressing assemblies 5, one side of the heat pressing assembly 5 is jointly provided with a pressure applying assembly 6 on the inner wall of the tension frame 2;

[0086] The surface of the pressing assembly 7 is provided with a material conveying assembly 8, the material conveying assembly 8 includes a transmission frame 81 fixedly arranged on the inner wall of the pressing assembly 7, the inner wall of the transmission frame 81 is provided with a conveyor belt 82, and the surface of the conveyor belt 82 is provided with a hook 83.

[0087] Specifically, as shown in Figure 5 And Figure 7 As shown: the heat pressing assembly 5 includes a movable seat 57 connected with the pressure applying assembly 6, one side of the movable seat 57 is fixedly provided with a rear seat 51, one side of the rear seat 51 is fixedly connected with a support pad 52, one side of the support pad 52 is fixedly connected with a heat insulation plate 53, the heat insulation plate 53 is provided with a pressure sensor 56 at the four corners, and the pressure sensor 56 is fixedly connected on the surface of the rear seat 51;

[0088] One side of the heat insulation plate 53 is fixedly connected with a heating plate 54, and the other side of the heating plate 54 is symmetrically provided with a uniform temperature plate 55 with the heat insulation plate 53;

[0089] One side of the heating plate 54 is provided with an electric heating wire 11, and the electric heating wire 11 is electrically connected with a controller 10.

[0090] Through the setting of the rear seat 51, the rear seat 51 can be movably connected with the pressing assembly 6, and the supporting force can be applied to the heat insulation plate 53 through the supporting 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 keep heat insulation, and the heat of the heating plate 54 can be conducted to the surface of the uniform temperature plate 55, so that the plane of the uniform temperature plate 55 forms uniform heat distribution, and meanwhile the supporting pad 52 can be deformed slightly under the action of the pressure, so that the deformation can be detected by the pressure sensor 56 at the same time, so that the feedback data of the pressure can be obtained.

[0091] Specifically, as shown in the figure: Figure 8 The hydraulic assembly 3 comprises a hydraulic cylinder 33 fixed on the lower surface of the holder 4, the surface of the hydraulic cylinder 33 is fixed with a hydraulic piston 32, the surface of the hydraulic piston 32 is tightly fitted with a hydraulic chamber 31, and the hydraulic chamber 31 is slidably arranged on the arc-shaped side wall of the hydraulic cylinder 33.

[0092] The surface of the hydraulic chamber 31 penetrates and slides a sliding frame 36, the sliding frame 36 is fixedly connected to the inner wall of the base 1, the top end of the hydraulic chamber 31 is fixedly connected to the bottom of the tension frame 2, the inner wall of the hydraulic cylinder 33 penetrates and is provided with an oil guide hole 34, and the arc-shaped side wall of the hydraulic cylinder 33 is provided with a connecting pipe 35 for communication between the hydraulic pump 9 and the oil guide hole 34.

[0093] The scheme can transmit the oil in 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 end of the oil guide hole 34, and 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 sliding frame 36, and then the hydraulic chamber 31 can move downward with the tension frame 2, realizing stable hydraulic control.

[0094] Specifically, as shown in the figure: Figure 6 The pressing assembly 6 comprises a main shaft 61, two cross seats 62 are slidably arranged on the surface of the main shaft 61, the opposite ends of the two cross seats 62 are fixedly connected with a tension spring 63, the tension spring 63 is in a spiral winding shape on the surface of the main shaft 61, the two sides of the cross seat 62 are fixedly connected with a connecting column 65, one end of the connecting column 65 is movably connected with two deflection plates 64, one end of the two deflection plates 64 on the same side of the cross seat 62 is provided with a first connecting part 66 and a second connecting part 67 respectively, the first connecting part 66 is movably connected with one side of the rear seat 51, and the second connecting part 67 is movably connected with the top end of the tension frame 2.

[0095] The pressing assembly 6 is a transfer node of the pressure in the hot pressing process, the hydraulic force moves with the tension frame 2, the dislocation force of the tension frame 2 and the holder 4 changes, the dislocation force is provided by the hydraulic displacement of the hydraulic chamber 31, so that the pressing assembly 6 can convert the dislocation force into the pressure of the hot pressing assembly 5.

[0096] Specifically, such as Figure 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 sliding plate 73 is movably connected to the surface of the connecting block 72. A sliding hole 74 is opened on the surface of the sliding plate 73 to slide in cooperation with the connecting block 72. A bonding plate 77 is fixedly connected to the bottom end of the sliding plate 73. Two guide shafts 75 are fixedly connected to the upper surface of the bonding plate 77. The guide shafts 75 slide through the surface of the pressing frame 71. A spring 76 is provided on the surface of the guide shafts 75. The transmission frame 81 is fixedly connected to the opposite surfaces of the two sliding plates 73.

[0097] The holding component 7 can support the transmission component and keep the holding component 7 in contact with the surface of the retainer 4.

[0098] Meanwhile, the retainer 4 can maintain the hot pressing assembly 5 in a close sliding motion, thus maintaining the stable displacement of the hot pressing assembly 5.

[0099] In use, the hydraulic pump 9 connects to the oil guide hole 34 inside the hydraulic cylinder 33 via the connecting pipe 35. When oil is injected into the oil guide hole 34 through the hydraulic pump 9, high pressure is formed below the hydraulic piston 32, causing it to move the hydraulic chamber 31 downward. As the hydraulic chamber 31 moves, it slides on the inner wall of the slide 36, and moves the tension frame 2 downward. Simultaneously, the tension frame 2 moves downward and presses the second connecting part 67 of the pressure application component 6 downward. Based on the action of the tension spring 63 on the inner wall of the pressure application component 6, the pressure application 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 retainer 4 until the two hot pressing components 5 press the flat wire. 12 clamps, and the tension frame 2 continuously applies pressure to the pressure application component 6. The height and horizontal distance between the second connecting part 67 and the first connecting part 66 change. During 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. During this process, the connecting column 65 carries the cross seat 62 to slide on the surface of the main shaft 61, causing the distance between the two cross seats 62 to change, and the tension spring 63 is stretched. The force of this tension spring 63 is consistent with the clamping force of the two hot pressing components 5 until the cross seat 62 and one end of the main shaft 61 are limited, so that the elastic force of the tension spring 63 reaches the limit value. At this time, the direct hot pressing operation is performed.

[0100] In the above pressure process, the two hot pressing assemblies 5 slide on the inner wall of the retainer 4 and clamp the flat wire 12, at this time the heating wire 11 in the heating plate 54 is heated, the heat is uniformly conducted to the surface of the flat wire 12 through the uniform temperature plate 55, and under the action of the elastic force of the tension spring 63, the pressure is in a linear and continuous increase, the temperature also changes, the pressure change can be maintained, and the temperature is gradually increased to 60°C, a better hot pressing surface can be formed, and after the cross seat 62 slides on the surface of the main shaft 61 to the maximum stroke, the pressure is suddenly increased to 15 MPa, and the hot pressing operation is completed.

[0101] The above hot pressing step can well form the surface forming of the flat wire 12, and the pressure is gradually increased and the temperature is heated, and finally the pressure is suddenly increased, so that a better hot pressing surface can be realized, and the flat wire 12 is horizontally transmitted by the feeding assembly 8, so that the flat wire 12 moves between the two hot pressing assemblies 5, when the tension frame 2 moves downward, the pressure frame 71 of the pressing assembly 7 moves downward, until the flat wire 12 is completely placed between the two hot pressing assemblies 5, at this time the contact plate 77 contacts the upper surface of the retainer 4, so that the flat wire 12 is at a constant height, the guide shaft 75 slides on the surface of the pressure frame 71, the sliding hole 74 of the sliding 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 assembly 8 can be at a constant height, after the hot pressing is completed and the tension frame 2 is reset, the feeding assembly 8 can move upward, in this way, the automatic feeding and discharging operation can be realized, which is more convenient and safe to use, and can be aligned downward, so that the flat wire 12 can be fully hot pressed, avoiding local exposure and waste caused by not being hot pressed.

[0102] As can be seen from the above, the present application provides a hot pressing device and method which can provide a continuously increasing elastic clamping force by elasticity during extrusion, preheating during the process, and direct pressing when the temperature is suitable. Compared with the traditional method of slowly increasing the temperature after direct pressing, the present application maintains sufficient clamping force for heating, and directly presses when the temperature is suitable, which significantly improves the hot forming quality of the flat wire 12 prepared by the present application.

[0103] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can make equivalent replacement or change according to the technical scheme and inventive concept of the present application within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A device for treating recombinant human epidermal growth factor-loaded silk membranes, characterized by, Including base (1), the inner wall of base (1) is provided with hydraulic assembly (3), hydraulic assembly (3) is communicated with hydraulic pump (9), the movable end of hydraulic assembly (3) is fixedly connected with tension frame (2), the upper surface of tension frame (2) is fixedly connected with pressure holding assembly (7); The upper surface of base (1) is fixedly connected with retainer (4), the inner wall of retainer (4) is slidably provided with two hot pressing assemblies (5), one side of hot pressing assembly (5) is provided with pressure applying assembly (6) in common with the inner wall of tension frame (2);The hot pressing assembly (5) includes a movable seat (57) connected with the pressure applying assembly (6), one side of the movable seat (57) is fixed with a rear seat (51), one side of the rear seat (51) is fixedly connected with a support pad (52), one side of the support pad (52) is fixedly connected with a heat insulation plate (53), the heat insulation plate (53) is provided with a pressure sensor (56) at the four corners, and the pressure sensor (56) is fixedly connected to the surface of the rear seat (51); One side of the heat insulation plate (53) is fixedly connected with a heating plate (54), and the other side of the heating plate (54) is symmetrically provided with a uniform temperature plate (55) with the heat insulation plate (53); One side of the heating plate (54) is provided with an electric heating wire (11), and the electric heating wire (11) is electrically connected with a controller (10); The pressure applying assembly (6) includes a main shaft (61), two cross seats (62) are slidably arranged on the surface of the main shaft (61), the opposite ends of the two cross seats (62) are fixedly connected with a tension spring (63) in common, the tension spring (63) is in spiral winding on the surface of the main shaft (61), the two sides of the cross seat (62) are fixedly connected with a connecting column (65), one end of the connecting column (65) is movably connected with two deflection plates (64), one end of the two deflection plates (64) on the same side of the two cross seats (62) is provided with a first connecting part (66) and a second connecting part (67) respectively, the first connecting part (66) is movably connected with one side of the rear seat (51), and the second connecting part (67) is movably connected to the top end of the tension frame (2); The surface of the pressure holding assembly (7) is provided with a material conveying assembly (8), the material conveying assembly (8) includes a transmission frame (81) fixedly connected to the inner wall of the pressure holding assembly (7), the inner wall of the transmission frame (81) is provided with a conveyor belt (82), and the surface of the conveyor belt (82) is provided with a hook (83).

2. The recombinant human epidermal growth factor-loaded silk film treatment device according to claim 1, wherein, The hydraulic assembly (3) includes a hydraulic cylinder (33) fixedly connected to the lower surface of the retainer (4), the surface of the hydraulic cylinder (33) is fixedly connected with a hydraulic piston (32), the surface of the hydraulic piston (32) is sealingly attached with a hydraulic chamber (31), and the hydraulic chamber (31) is slidably arranged on the arc-shaped side wall of the hydraulic cylinder (33). The surface of the hydraulic bin (31) is slid through a sliding frame (36) which is fixedly connected to the inner wall of the base (1), the top end of the hydraulic bin (31) is fixedly connected to the bottom of the tension frame (2), the inner wall of the hydraulic cylinder (33) is provided with an oil guide hole (34), and the arc-shaped side wall of the hydraulic cylinder (33) is provided with a connecting pipe (35) for communication between the hydraulic pump (9) and the oil guide hole (34). 3.The device for treating recombinant human epidermal growth factor loaded silk fibroin membrane according to claim 1, wherein, The pressing assembly (7) includes a pressing frame (71) fixedly connected to the top end of the tension frame (2), the inner wall of the pressing frame (71) is fixedly connected with a connecting block (72), the surface of the connecting block (72) is movably connected with a sliding plate (73), the surface of the sliding plate (73) is provided with a sliding hole (74) matched with the connecting block (72), the bottom end of the sliding plate (73) is fixedly connected with a matching plate (77), the upper surface of the matching plate (77) is fixedly connected with two guide shafts (75), the guide shafts (75) are slid through the surface of the pressing frame (71), the surface of the guide shafts (75) is provided with springs (76), and the transmission frame (81) is fixedly connected to the opposite surfaces of the two sliding plates (73).

4. A method for preparing a load recombined human epidermal growth factor silk membrane using the device of any one of claims 1-3, comprising the following steps: Rearing of transgenic epidermal growth factor Bombyx mori strain N S EC S To five age 3-5 days, get ready to silkworm; Preparation of the body silkworm under a stereoscopic fluorescence microscope, using red fluorescence to observe the fluorescence of the silkworm eye, selecting five-year-old silkworms with red fluorescence in the eye for further feeding, and obtaining mature silkworms; After the mature silkworms are clustered and defecated, a certain number of mature silkworms are selected and placed in a mold; The mature silkworms are made to spin silk on the two-dimensional plane of the mold to obtain a flat silk (12); The flat silk (12) is peeled off from the mold and subjected to heat pressing treatment; Finally, β irradiation sterilization treatment is performed; Cut to a specified size and attach to the surface of the substrate to obtain a silk biomembrane.

5. The method for preparing a silk membrane loaded with recombinant human epidermal growth factor according to claim 4, characterized in that, The number of mature silkworms in the mold includes 25-35, and the selection follows the selection of silkworms with similar growth conditions.

6. The method for preparing a silk membrane loaded with recombinant human epidermal growth factor according to claim 4, characterized in that, During the spinning on the mold, the spinning situation is checked every 1-2 hours, and the position of the silkworms is adjusted manually according to the spinning situation to obtain uniform flat silk (12), and the silkworm sand needs to be cleaned when checking.

7. The method for preparing a silk membrane loaded with recombinant human epidermal growth factor according to claim 4, characterized in that, The heat pressing treatment includes a heat pressing time of 1-10 min, a heat pressing temperature of 60℃, a heat pressing pressure of 5-15 MPa, and a β irradiation sterilization treatment dose of 15 kGy.

8. The method for preparing a silk membrane loaded with recombinant human epidermal growth factor according to claim 4, characterized in that, The mold includes a spinning plane (13) made of plastic or paper material, which is disc-shaped, and the lower surface of the spinning plane (13) is provided with a support pillar (14).

Images