A recombinant human fibronectin freeze-dried sponge and its preparation method and application

Abstract

The present invention discloses a recombinant human fibronectin freeze-dried sponge and its preparation method and application. A recombinant human fibronectin freeze-dried sponge, which is formed by freeze-drying a mixed solution containing recombinant human fibronectin at a concentration of 0.1-6 mg / mL, carboxymethyl chitosan at a mass concentration of 1-5%, and metal ions at a molar concentration of 0.1-0.4 mM; the amino acid sequence of the recombinant human fibronectin is shown in SEQ ID NO.1. The recombinant human fibronectin freeze-dried sponge prepared by the present invention is an auxiliary material that can protect wounds from infection and promote wound healing. The preparation method of the present invention has no effect on the functional activity of recombinant human fibronectin, and can enable the protein to be stored at room temperature, no longer limited to low temperature, and when used as a dressing, the recombinant human fibronectin still has the functions of promoting cell adhesion and proliferation. When used, after being treated with alcohol and water in sequence, a recombinant human fibronectin dressing with certain ductility and viscosity can be obtained, and the protein activity can be maintained.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to a recombinant human fibronectin freeze-dried sponge and a preparation method and application thereof. Background Art

[0002] Fibronectin (FN) is a high molecular weight glycoprotein in the extracellular matrix. It exists mainly in three forms, namely plasma fibronectin produced by hepatocytes or endothelial cells, cellular fibronectin synthesized by fibroblasts and early mesenchymal cells, and fetal fibronectin in placenta and amniotic tissue. The interaction between fibronectin and the corresponding integrin is the central link in the regulation of cell adhesion and migration. FN can connect cells to the extracellular matrix. In order to facilitate the transportation and storage of fibronectin, most of the freeze-dried products of fibronectin are freeze-dried powder preparations.

[0003] Chitosan is the product of N-deacetylation of chitin. Chitin and chitosan have many unique properties such as biodegradability, cell affinity and biological effects. In particular, chitosan containing free amino groups is the only alkaline polysaccharide among natural polysaccharides. Therefore, chitosan is often used in the preparation of hydrogels and freeze-dried sponges. However, due to the property that chitosan is a solution in an acidic environment and forms an insoluble gel after adding alkali, the application of chitosan in the freeze-drying or hydrogel preparation of acid-sensitive proteins such as fibronectin is limited. Carboxymethyl chitosan is an important water-soluble chitosan derivative with many medical effects, such as promoting wound healing, hemostasis, inhibiting scars, analgesia and antibacterial effects. In addition, it is also widely used in chemical industry, environmental protection and health care products. Carboxymethyl chitosan improves the water solubility of chitosan, but the preparation of carboxymethyl chitosan hydrogel is usually achieved by physical and chemical crosslinking (Liu Qunfeng et al., Study on polyvinyl pyrrolidone-carboxymethyl chitosan in situ hydrogel [J]. Materials Research and Application. 2016, 10(3): 209-212.) or photocrosslinking (Hou Bingna et al., Preparation of photocrosslinked carboxymethyl chitosan hydrogel and study of drug sustained release performance [J]. Materials Engineering, 2020, 48(11): 76-84.). However, carboxymethyl chitosan is rarely used in protein freeze-drying process.

[0004] Although freeze-dried sponges provide convenience for protein transportation and retention, they dissolve into liquid state after rehydration in application, and solid medium carriers are required to be added to wound dressings. The preparation method and process of hydrogels used as wound dressings have certain damage to protein stability, especially large molecular proteins such as fibronectin, which may break or degrade under ultraviolet or significant pH changes, and are not as convenient as freeze-dried sponges in storage and transportation. Summary of the invention

[0005] In view of the problems existing in the above-mentioned prior art, the present invention provides a recombinant human fibronectin freeze-dried sponge and a preparation method and application thereof.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] In a first aspect, a recombinant human fibronectin freeze-dried sponge is formed by freeze-drying a mixed solution containing recombinant human fibronectin at a concentration of 0.1-6 mg / mL, carboxymethyl chitosan at a mass concentration of 1-5%, and metal ions at a molar concentration of 0.05-0.4 mM.

[0008] Furthermore, the amino acid sequence of the recombinant human fibronectin is shown in SEQ ID NO.1.

[0009] Furthermore, the metal ion is Ca 2+ , Fe 3+ and Mn 2+ Any of.

[0010] In a second aspect, a method for preparing a recombinant human fibronectin freeze-dried sponge comprises the following steps: mixing recombinant human fibronectin, a carboxymethyl chitosan solution and a metal ion-containing solution to obtain a mixed solution, and freeze-drying to obtain a recombinant human fibronectin freeze-dried sponge; in the mixed solution, the concentration of recombinant human fibronectin is 0.1-6 mg / mL, the concentration of carboxymethyl chitosan is 1-5% (W / V), and the concentration of metal ions is 0.05-0.4 mM.

[0011] Furthermore, the amino acid sequence of the recombinant human fibronectin is shown in SEQ ID NO.1.

[0012] Furthermore, the metal ion is Ca 2+ , Fe 3+ and Mn 2+ Furthermore, the metal ion is preferably Ca 2+ .

[0013] Furthermore, the preparation steps of recombinant human fibronectin are as follows: clone the recombinant human fibronectin sequence gene with an amino acid sequence as shown in SEQ ID NO.1 into a vector and transform it into an Escherichia coli expression strain; induce the expression of the recombinant human fibronectin, lyse the Escherichia coli, purify and diafiltration concentrate, and harvest the recombinant human fibronectin. Among them, the purification is specifically a single affinity chromatography or a secondary anion exchange chromatography of Ni-NTA filler with polyacrylate microspheres as the matrix material, and the fillers used are: DEAE Sepharose FF, Q sepharose FF, Capto Q or Capto Q Impres. Diafiltration concentration refers to the use of ultrafiltration membrane bags for solution replacement and concentration, pre-concentration before ultrafiltration replacement, and the replacement solution volume is 5-10 times the volume of the protein solution.

[0014] Further, the freeze drying comprises the steps of front box pre-freezing, primary drying, analytical drying and pressure reduction, wherein the front box pre-freezing: set temperature 4.0°C, set time 1min, hold time 60min; -20.0°C set time 1min, hold 10min; -45.0°C set time 1min, hold 180min; pre-vacuum 0.100mbar; primary drying: -45.0°C, set time 1min, hold time 30min, control pressure 0.1mbar; -22.0°C, set time 60min, Holding time 1200min, control pressure 0.100mbar; pressure rise setting value 0.030mbar, setting time 1min, setting number 2, interval time 30; analytical drying setting: 0.0℃, setting time 30min, holding time 60min, control pressure 0.100mbar; 30.0℃, setting time 60min, holding time 180min, control pressure 0.100mbar; 30.0℃, setting time 1min, holding time 60min, control pressure 0.010mbar.

[0015] Furthermore, the buffer in the carboxymethyl chitosan solution is a phosphate buffer or a citrate buffer.

[0016] Furthermore, the degree of substitution of carboxymethyl chitosan in the carboxymethyl chitosan solution is ≥90%.

[0017] Thirdly, the application of the above-mentioned recombinant human fibronectin freeze-dried sponge in promoting skin wound healing and repair dressings.

[0018] Further, the application steps include: placing the recombinant human fibronectin freeze-dried sponge in a clean container and rewetting it with alcohol to obtain a moist dressing with a certain degree of ductility; or by adding an appropriate amount of purified water for reconstitution and aseptic filtration to obtain a recombinant human fibronectin solution of the required concentration; or, the application steps include: placing the recombinant human fibronectin freeze-dried sponge in a clean container and rewetting it with alcohol to obtain a moist dressing; after the alcohol evaporates, rehydrating it, such as dripping or spraying an appropriate amount of water, to obtain a further expanded but insoluble sustained-release dressing with appropriate ductility and viscosity, and can be gradually dissolved for a long time for more than 1 day. When other water-soluble chitosans such as chitosan hydrochloride are used, although they can be used for freeze-drying, the dressing obtained after rehydration is not ductile.

[0019] Furthermore, the alcohol is medical alcohol with a concentration of 20%-75%.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The recombinant human fibronectin freeze-dried sponge prepared by the present invention is a dressing that can protect wounds from infection and promote wound healing. The preparation method of the present invention has no effect on the functional activity of recombinant human fibronectin, and enables the protein to be stored at room temperature, no longer limited to low temperature, and when used as a dressing, the recombinant human fibronectin still has the functions of promoting cell adhesion and proliferation. When used, after being treated with alcohol and water in sequence, a recombinant human fibronectin dressing with certain ductility and viscosity can be obtained, and the protein activity is no different from that before freeze-drying, and alcohol treatment or no treatment has no significant effect on the protein activity.

[0022] The prepared freeze-dried sponge can form a dressing with certain tension and ductility when sprayed or soaked with an alcohol solution of appropriate concentration, compared with direct rehydration with sterile water. Moreover, after the alcohol evaporates and then rehydrates, it can be further extended and has a certain viscosity but does not dissolve into a liquid state. When used as a dressing, it can be slowly dissolved to protect the wound when covered on a wound. It is an easy-to-use, slowly dissolving, sustained-release dressing that can be gradually released and dissolved for a long time for more than 1 day. It has excellent application value in the fields of trauma surgery, dermatology, etc., including skin injury healing, skin in situ regeneration and repair, and in vitro skin tissue engineering construction.

[0023] Alcohol can also play a certain disinfecting role on the sponge, and due to the protection of chitosan and the short alcohol treatment time, the activity of recombinant human fibronectin is not affected. In addition, adding CaCl 2 The extensibility of the sustained-release dressing is significantly improved after rehydration. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the solutions in the present application or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 Comparison of the effects of recombinant human fibronectin (rhFN) on cell proliferation after reconstitution with water;

[0026] Figure 2 The appearance of the recombinant human fibronectin freeze-dried sponge in different application scenarios, wherein (a)-(c) respectively show the appearance of the recombinant human fibronectin freeze-dried sponge after cutting, after being sprayed with alcohol, and after being immersed in an alcohol solution;

[0027] Figure 3 The appearance of the recombinant human fibronectin freeze-dried sponge in different application scenarios, wherein (a)-(c) respectively show the appearance of the recombinant human fibronectin freeze-dried sponge after freeze-drying, after spraying with alcohol, and after spraying with pure water after the alcohol evaporates;

[0028] Figure 4 Comparison of the status of recombinant human fibronectin after treatment with different concentrations of alcohol;

[0029] Figure 5 The cell proliferation promoting function of the recombinant human fibronectin sequence SEQ ID NO.1 was compared with that of the commercial recombinant human fibronectin after freeze-drying and re-dissolving;

[0030] Figure 6 Comparison of the appearance of the recombinant human fibronectin freeze-dried sponge after spraying with water and alcohol;

[0031] Figure 7 Comparison of the cell proliferation promoting activity of recombinant human fibronectin after lyophilization and reconstitution or without lyophilization;

[0032] Figure 8 The effects of alcohol treatment or no treatment on cell proliferation after reconstitution of two different batches of recombinant human fibronectin freeze-dried sponges;

[0033] Fig. 9 The effect of different metal ions on the ductility of the recombinant human fibronectin freeze-dried sponge after rehydration in PBS buffer system;

[0034] Fig.10 The effect of different metal ions on the ductility of the recombinant human fibronectin freeze-dried sponge after rehydration with sodium citrate as the buffer system;

[0035] Fig.11Comparison of the ductility of the recombinant human fibronectin freeze-dried sponge after rehydration using PBS or sodium citrate as the buffer system;

[0036] Fig.12 The effects of different metal ions and buffer systems on cell proliferation after reconstitution of recombinant human fibronectin freeze-dried sponge with water;

[0037] Fig.13 The morphology and reconstitution of the recombinant human fibronectin freeze-dried sponge after freeze-drying with different chitosan matrices. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0039] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0040] Example 1

[0041] In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0042] The preparation method of the recombinant human fibronectin freeze-dried sponge of the present embodiment comprises the following steps: mixing the recombinant human fibronectin, carboxymethyl chitosan solution and metal ion-containing solution to obtain a mixed solution, and freeze-drying to obtain the recombinant human fibronectin freeze-dried sponge. Specifically, the steps include:

[0043] S1: Using an E. coli expression system, the recombinant human fibronectin sequence gene represented by SEQ ID NO.1 is cloned into a pET28 vector and transformed into an E. coli expression strain;

[0044] The amino acid sequence of SEQ ID NO.1 recombinant human fibronectin is as follows:

[0045]

[0046] S2: inducing the expression of the recombinant human fibronectin, lysing the Escherichia coli, and purifying by affinity chromatography and ultrafiltration solution replacement to harvest the recombinant human fibronectin with a purity greater than 95%;

[0047] The purification method is a single affinity chromatography of Ni-NTA filler with polyacrylate microspheres as the matrix material; the purified recombinant human fibronectin is subjected to solution replacement and concentration using an ultrafiltration membrane package to harvest recombinant protein with a purity greater than 95%, wherein pre-concentration is required before ultrafiltration replacement, and the replacement solution volume is 8 times the volume of the protein solution;

[0048] S3: The purified sterile filtered recombinant human fibronectin was mixed with carboxymethyl chitosan solution, CaCl 2 The solutions were mixed to form a clear and transparent solution, so that the final concentration of recombinant human fibronectin was 3 mg / mL, the final concentration of carboxymethyl chitosan solution was 2% (W / V), and the CaCl 2 The final concentration is 0.05 mM;

[0049] The carboxymethyl chitosan solution is obtained by completely dissolving carboxymethyl chitosan powder (substitution degree ≥ 90%) with PBS;

[0050] S4: Pour the prepared and mixed stock solution into a freeze-drying plate to make the liquid thickness about 4-6 mm, and freeze-dry it using a vacuum freeze-drying device;

[0051] (1) The freeze-vacuum drying process includes pre-freezing in the front box, primary drying, analytical drying and pressure reduction steps;

[0052] Front box pre-freezing: set temperature 4.0℃, set time 1min, hold time 60min; -20.0℃ set time 1min, hold 10min; -45.0℃ set time 1min, hold 180min; pre-vacuum 0.100mbar;

[0053] Primary drying: -45.0℃, set time 1min, hold time 30min, control pressure 0.1mbar; -22.0℃, set time 60min, hold time 1200min, control pressure 0.100mbar; pressure rise set value 0.030mbar, set time 1min, set times 2, interval time 30;

[0054] Analysis and drying settings: 0.0℃, setting time 30min, holding time 60min, control pressure 0.100mbar; 30.0℃, setting time 60min, holding time 180min, control pressure 0.100mbar; 30.0℃, setting time 1min, holding time 60min, control pressure 0.010mbar;

[0055] (2) After freeze-drying, cut the freeze-dried sponge into the required size.

[0056] Example 2

[0057] The implementation steps of this embodiment are the same as steps S1, S2, and S4 of embodiment 1, except that the preparation process of the recombinant human fibronectin lyophilized mixture used is as follows: the purified sterile filtered recombinant human fibronectin and carboxymethyl chitosan solution are mixed with sterile CaCl 2 The solutions were mixed to form a clear and transparent solution, with a final concentration of 3 mg / mL for recombinant human fibronectin, 2% for carboxymethyl chitosan solution, and 1% for CaCl 2 The final concentration is 0.1 mM.

[0058] Example 3

[0059] The implementation steps of this embodiment are the same as steps S1, S2, and S4 of embodiment 1, except that the preparation process of the recombinant human fibronectin lyophilized mixture used is as follows: the purified sterile filtered recombinant human fibronectin and carboxymethyl chitosan solution are mixed with sterile CaCl 2 The solutions were mixed to form a clear and transparent solution, with a final concentration of 5 mg / mL for recombinant human fibronectin, 2% for carboxymethyl chitosan solution, and 1% for CaCl 2 The final concentration is 0.2 mM.

[0060] Example 4

[0061] The implementation steps of this embodiment are the same as steps S1, S2, and S4 of embodiment 1, except that the preparation process of the recombinant human fibronectin lyophilized mixture used is as follows: the purified sterile filtered recombinant human fibronectin and carboxymethyl chitosan solution are mixed with sterile CaCl 2 The solutions were mixed to form a clear and transparent solution, with a final concentration of 2 mg / mL for recombinant human fibronectin, 3% for carboxymethyl chitosan solution, and 1% for CaCl 2 The final concentration is 0.375 mM.

[0062] The ductility of the freeze-dried sponge samples obtained in Examples 1-4 was measured and statistically analyzed, and the statistical results are shown in Table 1.

[0063] Table 1 Measurement of ductility of freeze-dried sponge samples

[0064]

[0065] The recombinant human fibronectin freeze-dried sponge obtained in Examples 1-4 was reconstituted with water to prepare a solution with a protein concentration of 1 mg / mL and filtered with a 0.22 μm filter membrane. Human keratinocytes HaCat were revived and cultured, and after adaptive passage for 2 times, the cells were passaged after growing to 80%-90% coverage, and the cells were transferred to a 96-well plate, with 4000 cells per well. After the cells were adaptively cultured overnight after passage, the reconstituted solution of the recombinant human fibronectin freeze-dried sponge harvested in the corresponding example was added, and after culturing for 48 hours, CCK8 reagent was added, and the cells were cultured in a 37°C incubator for 2-4 hours. The absorbance of each well was read using an ELISA reader A450, as shown in FIG. Figure 1 Compared with PBS control, freeze-dried fibronectin has a better ability to promote cell proliferation, p < 0.01.

[0066] The recombinant human fibronectin freeze-dried sponge obtained in Example 4 was cut and sprayed or soaked with 75% medical alcohol to obtain a recombinant human fibronectin moist dressing with ductility. Figure 2 , shown is the freeze-dried sponge after the recombinant human fibronectin is freeze-dried in Example 4 (such as Figure 2 (a) in the figure) and spray with alcohol (as shown in Figure 2 (b) in the figure) or immersed in an alcohol solution (such as Figure 2 The recombinant human fibronectin freeze-dried sponge in (c) still appears as a moist dressing with a block structure rather than a liquid state.

[0067] The recombinant human fibronectin freeze-dried sponge obtained in Example 2 was sprayed or soaked with 75% medical alcohol, and quickly extended to obtain a wet dressing. After the alcohol was basically evaporated, pure water was added, and the wet dressing was further expanded and extended to obtain a sustained-release dressing with a certain viscosity. Figure 3 , which shows the morphology of the sample in Example 2 after the above treatment.

[0068] The recombinant human fibronectin freeze-dried sponge obtained in Example 2 was treated with 10%, 20%, 50% and 75% alcohol and then treated with purified water. The results are as follows: Figure 4 As shown in the figure, after being treated with 20%-75% alcohol and then purified water, the sustained-release dressing can basically maintain a good shape, while after being treated with 10% alcohol and purified water in sequence, the sustained-release dressing showed a more obvious dissolution phenomenon. Therefore, the preferred concentration range of the alcohol solution for treating the recombinant human fibronectin freeze-dried sponge is 20%-75%.

[0069] Comparative Example 1

[0070] The implementation steps of this comparative example are the same as those of Example 2, except that commercial recombinant human fibronectin (full-length sequence expressed in rice cells) (rhFN-commercial 1) and commercial GMP-grade recombinant human fibronectin (expressed in yeast, including three functional regions, cell binding region, high-affinity heparin binding region and CS1 site, protein size 63KD) (rhFN-commercial 2) are used for comparison, and the other component ratios are consistent with the freeze-drying conditions. The obtained freeze-dried sponge is reconstituted with water, prepared into a solution with a protein concentration of 1 mg / mL and filtered with a 0.22 μm filter membrane. After adaptive passage for 2 times, human keratinocytes HaCat were grown to 80%-90% coverage, and then the cells were transferred into a 96-well plate, with 4000 cells per well. After adaptive culture overnight, the reconstituted solution of the recombinant human fibronectin freeze-dried sponge harvested in the corresponding example was added, and after culture for 48 hours, CCK8 reagent was added, and cultured in a 37°C incubator for 2-4 hours. The absorbance of each well was read using an ELISA reader A450, as shown in FIG. Figure 5 After the freeze-dried recombinant human fibronectin was reconstituted, they all had the effect of promoting cell proliferation, among which the protein characterized by SEQ ID NO.1 had a significantly higher ability to promote cell proliferation than the commercial recombinant human fibronectin, p < 0.01.

[0071] Comparative Example 2

[0072] This comparative example is a comparative experiment of Example 4, in which the obtained recombinant human fibronectin freeze-dried sponge is sprayed with water to dissolve. Since the recombinant human fibronectin freeze-dried sponge is easily soluble in water, the ductility and uniformity of the dressing obtained by spraying with pure water are not as good as those obtained by spraying or soaking with alcohol. When the amount of water is large, the freeze-dried sponge will dissolve into a paste. Figure 6 shown.

[0073] Comparative Example 3

[0074] The implementation steps of this comparative example are the same as those of Example 2. The recombinant human fibronectin freeze-dried sponge harvested by this method is reconstituted with water to prepare a solution with a protein concentration of 1 mg / mL and filtered with a 0.22 μm filter membrane. Before freeze-drying, the recombinant human fibronectin stock solution is diluted with PBS to a solution with a protein concentration of 1 mg / mL. After two adaptive passages of human keratinocytes HaCat, when the cells grow to a coverage rate of 80%-90%, the cells are transferred into a 96-well plate with 4,000 cells per well. After the cells are adaptively cultured overnight after the passage, the reconstituted solution of the recombinant human fibronectin freeze-dried sponge harvested in the corresponding example is added. After culturing for 48 hours, CCK8 reagent is added, and the cells are cultured in a 37°C incubator for 2-4 hours. The absorbance of each well is read using an ELISA reader A450. The results are as follows Figure 7As shown, both recombinant human fibronectin solution and PBS control promoted the proliferation of HaCat cells (p < 0.001), but there was no significant difference in the promoting effect of freeze-dried or non-freeze-dried recombinant human fibronectin on cell proliferation (p > 0.1).

[0075] Comparative Example 4

[0076] The implementation steps of this comparative example are basically the same as steps S1-S4 of Example 1, except that when re-dissolving the two batches of recombinant human fibronectin, it is directly re-dissolved with water (untreated group) or treated with alcohol to form a ductile wet dressing and then re-dissolved with water (alcohol-treated group). The protein concentration after re-dissolution is 1 mg / mL and filtered with a 0.22 μm filter membrane. Stably cultured human keratinocytes HaCat are introduced into a 96-well plate, with 4,000 cells per well. After the cells are adaptively cultured overnight after passage, the re-dissolved solution of the recombinant human fibronectin freeze-dried sponge harvested in the corresponding example is added. After culturing for 48 hours, CCK8 reagent is added, and the cells are cultured in a 37°C incubator for 2-4 hours. The absorbance of each well is read using an ELISA reader A450, as shown in FIG. Figure 8 Both batches promoted cell proliferation for PBS (p < 0.01), and there was no significant difference between alcohol treatment and no treatment (p > 0.1).

[0077] Comparative Example 5

[0078] The implementation steps of this comparative example are the same as steps S1, S2, and S4 of Example 1, except that the preparation process of the recombinant human fibronectin freeze-dried mixed solution used is: the purified sterile filtered recombinant human fibronectin and carboxymethyl chitosan solution, sterile CaCl 2 、FeCl 3 、MnCl 2 or ZnCl 2 The solutions were mixed to form a clear and transparent solution, with a final concentration of 2 mg / mL for recombinant human fibronectin, 3% for carboxymethyl chitosan solution, and 1% for CaCl 2 、FeCl 3 、MnCl 2 、ZnCl 2 The final concentration was 0.2 mM, and the solution without recombinant fibronectin and without metal ions was used as the control. 2 The fibronectin will precipitate and the precipitate is insoluble, so it cannot be used in the present invention. After freeze-drying, the corresponding recombinant human fibronectin freeze-dried sponge was rehydrated with alcohol and then rehydrated with pure water. Fig. 9 As shown, Ca 2+ The ductility of freeze-dried sponge with ions as auxiliary materials was the best, significantly higher than that of the control group, p < 0.05.

[0079] Comparative Example 6

[0080] The implementation steps of this comparative example are the same as steps S1, S2, and S4 of Example 1, except that the preparation process of the recombinant human fibronectin freeze-dried mixed solution used is as follows: carboxymethyl chitosan is resuspended in sodium citrate solution (pH value is 7.4), the purified sterile filtered recombinant human fibronectin and carboxymethyl chitosan citrate solution are mixed, and sterile CaCl 2 、FeCl 3 、MnCl 2 The solutions were mixed to form a clear and transparent solution, with a final concentration of 2 mg / mL for recombinant human fibronectin, 3% for carboxymethyl chitosan solution, and 1% for CaCl 2 、FeCl 3 、MnCl 2 The final concentration was 0.2 mM. After freeze-drying, the corresponding recombinant human fibronectin freeze-dried sponge was tested after being rehydrated in alcohol and then in pure water. Fig.10 As shown, Ca 2+ The ductility of the ion group has been significantly improved.

[0081] Comparative Example 2 and Comparative Example 3 Ca 2+ The ion groups were compared with the control group, such as Fig.11 As shown in Figure 2, the group with PBS as buffer was significantly better than the group with sodium citrate as buffer, p < 0.05. Therefore, the PBS buffer system and Ca 2+ ion.

[0082] Comparative Example 7

[0083] The recombinant human fibronectin freeze-dried sponge with different ions as auxiliary groups was fully dissolved in water to prepare a 1 mg / mL protein solution and sterilized and filtered. Similar to the HaCat cell treatment method in Comparative Example 3, the protein solution was added to the cells to stimulate cell proliferation for 48 hours, and then CCK8 reagent was added for 2 hours, and the A450 absorption value was read using an ELISA reader. Fig.12 Different formulas of recombinant human fibronectin freeze-dried sponges all had a promoting effect on cell proliferation after reconstitution, and the p values ​​were all less than 0.05. 2+ The freeze-dried sponge with recombinant human fibronectin as a cofactor has a better effect.

[0084] Comparative Example 8

[0085] The implementation steps of this comparative example are the same as steps S1, S2, and S4 of Example 1, except that the preparation process of the recombinant human fibronectin freeze-dried mixed solution used is as follows: two different brands of chitosan hydrochloride (McLean (brand A) and Aladdin (brand B), substitution degree 80%-90%) are dissolved in PBS solution, and an appropriate amount of NaOH is added to adjust the pH. The purified sterile filtered recombinant human fibronectin and sterile CaCl 2 The solution and chitosan hydrochloride solution formed a clear and transparent solution, so that the final concentration of recombinant human fibronectin was 2 mg / mL, the final concentration of chitosan hydrochloride solution was 3%, and CaCl 2 The final concentration is 0.2 mM. Fig.13 After spraying 75% alcohol, the recombinant human fibronectin freeze-dried sponge based on chitosan hydrochloride did not dissolve, but it was also not ductile. After adding purified water, the recombinant human fibronectin freeze-dried sponge based on chitosan hydrochloride broke into pieces, but compared with the carboxymethyl chitosan recombinant human fibronectin freeze-dried sponge, it was very difficult to dissolve. After re-dissolving for 24 hours, there were still a lot of insoluble flocs.

[0086] Comparative Example 9

[0087] The implementation steps of this comparative example are the same as those of Example 1, except that the CaCl 2 The concentration is 0.75 mM. At this time, the lyophilized stock solution becomes white and turbid, which should be the precipitation of recombinant human fibronectin and cannot be used for preparation.

[0088] Comparative Example 10

[0089] The implementation steps of this comparative example are the same as those of Example 1, except that CaCl was not used in the preparation of the recombinant human fibronectin lyophilized mixture. 2 , while adding 2% sucrose as a freeze-drying protective agent, the freeze-dried protein product is in the form of a thin cookie with a yellow color. Sucrose is not suitable for this product under this freeze-drying condition and cannot form the effect achieved by the formula in the present invention.

[0090] Obviously, the embodiments described above are only some embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this application.

Claims

1. A method for preparing a recombinant human fibronectin freeze-dried sponge, characterized in that: The method comprises the following steps: mixing the purified sterile filtered recombinant human fibronectin with a carboxymethyl chitosan solution and a metal ion-containing solution to obtain a mixed solution, and freeze-drying to obtain a recombinant human fibronectin freeze-dried sponge; in the mixed solution, the recombinant human fibronectin concentration is 0.1-6 mg / mL, the carboxymethyl chitosan concentration is 1-5% (W / V), and the metal ion concentration is 0.1-0.4 mM; the amino acid sequence of the recombinant human fibronectin is shown in SEQ ID NO.1; The metal ion is Ca 2+ ; The buffer in the carboxymethyl chitosan solution is a phosphate buffer or a citrate buffer; When the freeze-dried sponge is used, it is first re-moistened with alcohol having a concentration of 20%-75% to obtain a dressing with ductility.

2. The method for preparing the recombinant human fibronectin freeze-dried sponge according to claim 1, characterized in that: The freeze drying comprises the steps of pre-freezing in a front box, primary drying, analytical drying and pressure reduction, wherein the pre-freezing in the front box: setting temperature at 4.0°C, setting time at 1 min, holding time at 60 min; setting time at -20.0°C for 1 min, holding time at 10 min; setting time at -45.0°C for 1 min, holding time at 180 min; pre-vacuuming at 0.100 mbar; primary drying: -45.0°C, setting time at 1 min, holding time at 30 min, and controlling pressure at 0.1 mbar; -22.0°C, setting time at 60 min, holding time at 1200min, control pressure 0.100mbar; pressure rise setting value 0.030mbar, setting time 1min, setting number 2, interval time 30; analytical drying setting: 0.0℃, setting time 30min, holding time 60min, control pressure 0.100mbar; 30.0℃, setting time 60min, holding time 180min, control pressure 0.100mbar; 30.0℃, setting time 1min, holding time 60min, control pressure 0.010mbar.

3. A recombinant human fibronectin freeze-dried sponge, characterized in that: It is prepared by the preparation method as claimed in claim 1 or 2.

4. A dressing, characterized in that: The invention comprises a recombinant human fibronectin freeze-dried sponge prepared by the method according to claim 1.

5. The method for using the dressing according to claim 4, characterized in that: The method of use comprises the following steps: placing the recombinant human fibronectin freeze-dried sponge in a clean container and rewetting it with alcohol to obtain a moist dressing; or, comprising: placing the recombinant human fibronectin freeze-dried sponge in a clean container and rewetting it with alcohol to obtain a moist dressing; rehydrating the recombinant human fibronectin freeze-dried sponge after the alcohol evaporates to obtain a sustained-release dressing; the alcohol is medical alcohol with a concentration of 20%-75%.

Images