CN118725082B

CN118725082B - Recombinant human type I collagen, preparation method and application in preparation of medical cosmetic filler

Info

Publication number: CN118725082B
Application number: CN202410699371.7A
Authority: CN
Inventors: 请求不公布姓名
Original assignee: Shanghai Baiyue Youth Medical Technology Group Co ltd
Current assignee: Shanghai Baiyue Youth Medical Technology Group Co ltd
Priority date: 2024-05-31
Filing date: 2024-05-31
Publication date: 2025-01-21
Anticipated expiration: 2044-05-31
Also published as: CN119529061A; CN118725082A

Abstract

The present invention discloses a recombinant human type I collagen, a preparation method and an application thereof in the preparation of medical aesthetic fillers. The recombinant human type I collagen with a high content of isopeptide and nanocellulose are prepared into a composite hydrogel, which has high stability, low swelling, high weight retention rate, strong adhesion and hemostasis, and has the potential to be a skin tissue repair material. In addition, the composite hydrogel can be used to repair burned skin, significantly reduce the inflammatory response of burned skin, and promote wound healing. In addition, the composite hydrogel can also be used as a filler or inhibitor to be filled in mice, has a lower inflammatory response, and can promote collagen synthesis in mice, promote angiogenesis and bone cell generation in the long term, and then promote tissue regeneration, providing a technical basis for it as a medical aesthetic filling material.

Description

Recombinant type I human collagen, preparation method and application thereof in preparation of medical filler

Technical Field

The invention relates to the technical field of I-type human collagen, in particular to recombinant I-type human collagen, a preparation method and application thereof in preparation of medical filler.

Background

Type I collagen is the only component constituting collagen fibers in dermis, and has a proportion of 80% or more in skin, and is the most abundant component in dermal connective tissue. The type I collagen is a heterotrimer molecule, each chain is composed of more than 1000 amino acids, the length of the type I collagen molecule is about 300nm, the width is about 1-5nm, and the collagen trimer has high tensile strength. In most cases, it consists of two α1 chains and one α2 chain, whereas α1 homotrimers exist in minor form.

The type I collagen provides a mechanical supporting function for organisms, maintains the integrity of organs and tissues, ensures the normal function of the organs and tissues, and is very important for the parenchymal organs. Type I collagen is the main component of the basal membrane of a solid organ, more than 80% of organic matters in bones are type I collagen, the main mode of action of type I collagen in bones and connective tissues is to form and maintain the integrity of the bones, and besides, type I collagen plays a main role in forming specific extracellular microenvironments, and the microenvironments play a very key role in maintaining the integrity of cells and transmitting extracellular signals.

The initial use of type I collagen in medicine was traced back to the last 175 s.a., the Galen doctor used collagen for the first time as an absorbable enteric suture. Along with the development of process improvement and modification technology, collagen is prepared into hemostatic powder, burn dressing, drug carrier, heart ban membrane, vascular, tube, tracheal and other substitute materials, surgical suture and the like, and the collagen is matched with various purposes for use, thus having good application prospect in clinical medicine. The collagen has wide application in the industries of medicine, cosmetics, food and the like.

Disclosure of Invention

In view of the above, the invention provides a recombinant type I human collagen, a preparation method and application thereof in preparing medical filler.

The invention provides a preparation method of recombinant type I human collagen, which comprises the following steps:

Preparing a solution of human type I procollagen amino terminal peptide with the same concentration and a solution of cross-linked peptide, wherein the human type I procollagen amino terminal peptide is shown as SEQ ID NO.1, and the cross-linked peptide is shown as SEQ ID NO. 2;

mixing the solution of the human type I procollagen amino terminal peptide with the solution of the cross-linked peptide according to the volume ratio of (1-2): 1-4;

adding 5-40U/g of glutaminase;

after fully and uniformly mixing, placing the mixture in a 50 ℃ for reaction for 0.5-6 h;

after the reaction is finished, taking out a sample, placing the sample in a boiling water bath to inactivate enzyme, and cooling the sample to room temperature;

adjusting the pH of the solution to 4.6, precipitating the modified product and removing the cross-linked peptide which is not mixed and cross-linked, centrifuging 15 min at 10000 Xg, and discarding the supernatant;

The precipitate was washed with water at pH 4.6 and the resulting precipitate was recombinant type I human collagen.

Specifically, the solution of human type I procollagen amino terminal peptide is mixed with the solution of cross-linked peptide in a volume ratio of 1:1, 1:2, 1:3, 1:4 or 2:1.

Specifically, the glutaminase is added in an amount of 5U/g, 10U/g, 20U/g, 30U/g or 40U/g protein.

Specifically, the reaction time is 0.5h, 1h, 2h, 4h or 6h.

The invention provides a preparation method of composite hydrogel, which comprises the following steps:

Preparing an ionic liquid containing 50g/L of 1, 5-diazabicyclo [4.3.0] -5-nonene and 50g/L of levulinic acid under an ice bath at 0 ℃;

adding nanocellulose and the recombinant type I human collagen prepared by the preparation method into the ionic liquid at the same time under the mechanical stirring of a 100 ℃ oil bath pot, rapidly stirring the mixture, and fully dissolving;

Placing the reactant in a culture dish, standing at normal temperature for 1 d, and repeatedly cleaning with absolute ethyl alcohol.

Specifically, the final concentration of the nanocellulose is 1.25g/L, and the final concentration of the recombinant type I human collagen is 1.25g/L.

Specifically, the diameter of the nanocellulose is 4-10 nm, and the length of the nanocellulose is 200nm.

The invention provides the recombinant type I collagen prepared by the method.

The invention provides an application of the recombinant type I collagen prepared by the method in preparing skin tissue repair materials.

The invention provides an application of the recombinant type I collagen prepared by the method in preparation of biological filling materials.

Advantageous effects

The recombinant type I human collagen prepared by the method provided by the invention has high content of isopeptid bonds. The recombinant type I human collagen with high content of isopeptid bonds and nanocellulose are prepared into composite hydrogel, so that the composite hydrogel has the advantages of high stability, low swelling, high weight retention, strong adhesion and hemostatic capability, and has the potential of being used as a skin tissue repair material. The composite hydrogel can be used for repairing burn skin, remarkably reduces inflammatory response of the burn skin and promotes wound healing. In addition, the composite hydrogel can be used as a filler or inhibitor to be filled in a mouse body, has lower inflammatory reaction, can promote collagen synthesis in the mouse body, promotes angiogenesis and bone cell generation after long-term action, and further promotes tissue regeneration, thereby providing a technical foundation for the composite hydrogel serving as a medical filling material.

Drawings

Fig. 1 is a picture of experimental, control 1, control 2 and control 3 at day 1, day 20, day 40, day 60 in an inversion test experiment.

FIG. 2 shows the results of the in vitro swelling and degradation experiments for the experimental, control 1, control 2 and control 3 groups.

Fig. 3 shows the results of the experimental, control 1, control 2 and control 3 groups in the in vitro adhesion experiment.

Fig. 4 shows the results of experimental, control 1, control 2 and control 3 groups in the cell experiment.

Fig. 5 is a 14 day mouse White Blood Cell (WBC), neutrophil (Neu) and lymphocyte levels for model group, test group 1 (Test 1), test group 2 (Test 2), test group 3 (Test 3), test group 4 (Test 4), test group 5 (Test 5) in skin burn experiments.

Fig. 6 shows the results of 7, 14 and 21 day mouse burn areas for model group, test group 1 (Test 1), test group 2 (Test 2), test group 3 (Test 3), test group 4 (Test 4), test group 5 (Test 5) in the skin burn Test.

Fig. 7 shows the results of relative amounts of collagen fibers in 7 days, 14 days and 21 days of mice in model group, test group 1 (Test 1), test group 2 (Test 2), test group 3 (Test 3), test group 4 (Test 4), test group 5 (Test 5) in skin burn experiments.

FIG. 8 shows the relative expression amounts of TNF-. Alpha.in mice of Blank (Blank), test group 1 (Test 1), test group 2 (Test 2), test group 3 (Test 3), test group 4 (Test 4), and Test group 5 (Test 5) in the injection experiment.

FIG. 9 shows the results of the relative expression amounts of IL-6 in mice of Blank (Blank), test group 1 (Test 1), test group 2 (Test 2), test group 3 (Test 3), test group 4 (Test 4), and Test group 5 (Test 5) in the injection experiment.

FIG. 10 shows the results of the relative expression amounts of type I collagen in mice of Blank (Blank), test group 1 (Test 1), test group 2 (Test 2), test group 3 (Test 3), test group 4 (Test 4), and Test group 5 (Test 5) in the injection experiment.

FIG. 11 shows the results of the relative expression amounts of type III collagen in mice of Blank (Blank), test group 1 (Test 1), test group 2 (Test 2), test group 3 (Test 3), test group 4 (Test 4), and Test group 5 (Test 5) in the injection experiment.

FIG. 12 shows the relative expression levels of VEGF in mice in Blank (Blank), test group 1 (Test 1), test group 2 (Test 2), test group 3 (Test 3), test group 4 (Test 4), and Test group 5 (Test 5) of the injection experiments.

FIG. 13 shows the relative expression level results of mice AKP in Blank (Blank), test group 1 (Test 1), test group 2 (Test 2), test group 3 (Test 3), test group 4 (Test 4), and Test group 5 (Test 5) in the injection experiment.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. The reagents which are not specifically described in the present invention are conventional reagents and are commercially available, and the methods which are not specifically described in the present invention are conventional experimental methods and are known from the prior art.

1. Preparation of recombinant type I human collagen

Human type I procollagen amino terminal peptide (MSST 0034, sigma-Aldrich, shown in SEQ ID NO. 1) and cross-linked peptide (SEQ ID NO. 2) were subjected to catalytic cross-linking by using transglutaminase (MTG, CAS number 80146-85-6) with a solubility in water of 1.0g/L, beijing Soy Corp technology Co., ltd.) to obtain a recombinant type I human collagen. The method comprises the following specific steps:

influence of the reaction conditions

Crosslinking ratio

Human type I procollagen amino terminal peptide (shown as SEQ ID NO. 1) solution and cross-linked peptide (shown as SEQ ID NO. 2) solution with protein mass concentration of 50 g/L are respectively prepared, pH is respectively regulated to 7.5, and the human type I procollagen amino terminal peptide solution and the cross-linked peptide solution are respectively mixed in volume ratios of 1:1, 1:2, 1:3, 1:4 and 2:1. The adding amount of the glutaminase is 30U/g protein respectively, the reaction system is fully and evenly mixed, and the mixture is placed in a 50 ℃ constant temperature incubator for shaking reaction by a horizontal shaking table for 4h, and each sample is parallel to the other 3 samples. After the reaction is finished, the sample is taken out, put in a boiling water bath to inactivate enzyme 15 min, and cooled to room temperature. The pH of the solution was adjusted to 4.6, the modified product was precipitated and the unmixed cross-linked peptide was removed, centrifuged at 10000 Xg at 15 min, and the supernatant was discarded. Washing the precipitate with water of pH 4.6, centrifuging again to remove supernatant, repeating the washing process for 2 times to obtain recombinant type I human collagen, and lyophilizing.

2) Enzyme addition amount

Preparing human type I procollagen amino terminal peptide (shown as SEQ ID NO. 1) solution and cross-linked peptide (SEQ ID NO. 2) solution with protein mass concentration of 50 g/L respectively, regulating pH to 7.5 respectively, and mixing the human type I procollagen amino terminal peptide solution and the cross-linked peptide solution at a volume ratio of 1:3. The added amount of glutaminase is 5U/g, 10U/g, 20U/g, 30U/g and 40U/g protein respectively, and after the reaction system is fully mixed, the mixture is placed in a 50 ℃ constant temperature incubator for shaking reaction by a horizontal shaking table for 4h, and each sample is subjected to 3 parallels. After the reaction is finished, the sample is taken out, put in a boiling water bath to inactivate enzyme 15 min, and cooled to room temperature. The pH of the solution was adjusted to 4.6, the modified product was precipitated and the unmixed cross-linked peptide was removed, centrifuged at 10000 Xg at 15 min, and the supernatant was discarded. Washing the precipitate with water of pH 4.6, centrifuging again to remove supernatant, repeating the washing process for 2 times, and lyophilizing to obtain recombinant type I human collagen.

3) Reaction time

Human type I procollagen amino terminal peptide and cross-linked peptide solution with protein mass concentration of 50 g/L are respectively prepared, pH is respectively adjusted to 7.5, and the human type I procollagen amino terminal peptide solution and the cross-linked peptide solution are mixed in a volume ratio of 1:3. The addition amount of the transglutaminase is 30U/g protein, the reaction system is fully and uniformly mixed, and then the mixture is placed in a 50 ℃ constant temperature incubator for shaking by a horizontal shaking table, and the reaction time is respectively 0.5, 1,2, 4 and 6h, and each sample is 3 parallel. After the reaction, the sample was taken out and treated as above.

2. Determination of the content of isopeptidic bonds in recombinant type I human collagen

Epsilon- (gamma-Glu) Lys (isopeptide bond) content was detected using SilGreen ODS C chromatography column. The cross-linked group (recombinant type I human collagen) and the blank group (human type I procollagen amino terminal peptide and cross-linked peptide) were subjected to trypsin hydrolysis, 8000U/g of protease substrate was added, and the mixture was dissolved in 0.01 mol/L PBS at pH 7.0, and digested in a 37 ℃ water bath 48 and h to complete the enzyme inactivation in a boiling water bath 5 min. Centrifuging the digested sample 10000 r/min for 20: 20min, and lyophilizing the supernatant. Adding 100% methanol into the freeze-dried sample to deproteinize, centrifuging to obtain supernatant, blow-drying the sample by using a nitrogen blower at 60 ℃, and finally re-dissolving in a mobile phase of 0.5 mL. Samples dissolved in the mobile phase were added to an Amicon O R Ultra 3K ultrafiltration centrifuge tube and the filtrate was collected by centrifugation at 4000 Xg for 30 min. Derivatizing the treated sample filtrate, and detecting the content of the isopeptide bond. An OPA derivatization reagent consisting of 22.4 mmol/L OPA (phthalic dicarboxaldehyde, CAS:643-79-8, shanghai Ala) and 0.4 mol/L Na ₂CO₃, 50% methanol containing 2% beta-mercaptoethanol was prepared and used. 80. Mu.L of the sample was rapidly mixed with 320. Mu.L of OPA derivative reagent, vortexed 70S and then allowed to stand 2. 2 min, and 10. Mu.L was injected into a liquid chromatograph for fluorescence detection. Mobile phase A, 20 mmol/L potassium acetate (pH 5.5) contained 1% Tetrahydrofuran (THF) and mobile phase B, 1% THF was dissolved in pure methanol. 20-95% of 0-55 min mobile phase B and 95-20% of 55-60 min mobile phase B. The column temperature was 40 ℃, the flow rate was 1.0 mL/min, the excitation wavelength (EX) was 334 nm, and the emission wavelength (EM) was 440 nm. Standard epsilon- (gamma-Glu) Lys was dissolved in mobile phase and concentration gradients of 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 2.0 mmol/L were set up to prepare standard curves.

As a result, as shown in Table 1, the crosslinking ratio was 1:3 or 1:4, the enzyme addition amount was 30U/g, and when the reaction time was 4 hours, a higher content of epsilon- (gamma-Glu) Lys isopeptide bond was obtained, indicating that highly crosslinked recombinant type I human collagen was successfully prepared.

TABLE 1 epsilon- (gamma-Glu) Lys (isopeptide bond) content (. Mu.mol/100 g)

2. Preparation of hydrogels

The levulinic acid matrix ionic liquid is used as a solvent to prepare the composite hydrogel of the nanocellulose/the recombinant I-type human collagen. The method comprises the following specific steps:

1, 5-diazabicyclo [4.3.0] -5-nonene and levulinic acid were added sequentially in two vials at a weight ratio of 1:1 in an ice bath at 0 ℃ and stirred for 5min to form a yellow transparent ionic liquid (50 g/L concentration each). The nanocellulose (the final concentration is 1.25g/L, the purity is more than or equal to 99 percent, the diameter (nm) is 4-10, the length is 200nm, YXL0026, yixin laboratory) and the recombinant type I human collagen (the final concentration is 1.25g/L, the isopeptide bond content is 98.36 +/-0.72 mu mol/100 g) are simultaneously added into the ionic liquid under the mechanical stirring of a 100 ℃ oil bath pot, the mixture is rapidly stirred, about 30min, the nanocellulose and the recombinant type I human collagen can be fully dissolved, reactants are taken and placed in a culture dish, the mixture is kept stand at normal temperature for 1d, then the mixture is repeatedly washed by using absolute ethyl alcohol, and 4 formed composite gels are perforated by a puncher to prepare hydrogel columns with the diameter of 30mm and the height of 3 mm. It was then immersed in pure water 12 h during which the pure water was replaced.

The recombinant type I human collagen was replaced with normal human type I collagen (5008, advanced Biomatrix) in the same manner as control group 1. In addition, no protein was added and only nanocellulose was used for preparation as control group 2. Wherein, the recombinant type I human collagen is replaced by the human type I procollagen amino terminal peptide in the same way to prepare hydrogel as a control group 3. The recombinant type I human collagen was replaced with a cross-linked peptide in the same manner to prepare a hydrogel as control group 3.

3. Physical Property test

1. Inversion test

Gel inversion detection was performed on hydrogel cylinders prepared from each experimental group. The prepared hydrogel was placed in a vial upside down and observed on days 1, 20, 40 and 60, respectively. As shown in fig. 1, control 4 did not form a hydrogel, control 2 and control 3 did not liquefy on day 20, and neither experimental nor control 1 did liquefy.

2. In vitro swelling and degradation test

The hydrogel to be tested was placed in a 9 cm petri dish with phosphate buffered saline (PBS, 20 mM), the hydrogel was removed from the dish at intervals and the excess water remaining on the surface was removed with filter paper. The hydrogel was weighed by an analytical balance. Swelling ratio = (Wt-W0)/w0×100%, weight retention = (W0-Wt)/w0×100%, where Wt is the weight of hydrogel taken out of the culture dish at different times, and W0 is the initial weight of wet hydrogel. As shown in FIG. 2, the hydrogels of the experimental group had an upper swelling after 2 hours, whereas the swelling of the control groups 2 and 3 was evident, and the hydrogels of the experimental group had the highest weight retention rate within 60 days.

3. In vitro adhesion test

The adhesion of hydrogels to tissues was determined by simulation using fresh pigskin. Pig skin tissue was cut into a rectangle of 20 mm x 40 mm and soaked in PBS (20 mM). The surface of the pigskin was coated with a pre-polymerized hydrogel solution (100. Mu.L), buckled with another pigskin and the adhesive surface was pressed, the adhesive area being 20 mm X10 mm. The pigskin was left at room temperature for 2 minutes or 2 hours, and then the tissue adhesion of the hydrogels was measured for short and long time by a universal tensile tester.

As shown in fig. 3, the adhesion strength of the hydrogels of the experimental group was about 35kPa, which is significantly higher than that of the control groups 1,2 and 3. The skin repair hydrogel needs to have excellent adhesion to skin tissues and hemostatic capability, thus indicating that the hydrogel provided by the experimental group has potential as a skin tissue repair material.

4. Cell experiment

L929 cell lines (Shang En organism), human cardiac muscle cells (AC 16, 1X 10 ⁶, BC-C-HU-038, biochannel), rat liver parenchymal cells (CP-R033, punuocele), rat kidney cells (NRK, CL-0173, punuocele) (50000 cells per well) were added to the medium, and hydrogels (concentrations of 5 mg/mL) obtained in each of the above-mentioned experiment group, control group 1, control group 2, control group 3 and control group 4 were cultured at 37℃for 24 hours, 48 hours and 72 hours. At the set time point, 10 μl of cell counting kit (INVIGENTECH) was added to each well and incubated at 37deg.C for 2 hours, and the absorbance at 590 nm was measured by a microplate reader without hydrogel as a blank. Cell viability is the percentage of absorbance after incubation to that of the blank.

As shown in FIG. 4, the hydrogels of each group have no obvious toxicity to L929 cell strain, human cardiac muscle cells, rat liver parenchymal cells and rat kidney cells, and have the application potential as skin tissue repair of body surfaces and filling materials in vivo.

5. Animal experiment

1. Skin burn test

ICR mice (female, body weight: 20-30 g, martial arts laboratory center) were randomly assigned to Blank (Blank), model (Mod), test group 1 (Test 1), test group 2 (Test 2), test group 3 (Test 3), test group 4 (Test 4), test group 5 (Test 5).

The test article of test set 1 was a hydrogel prepared in the above experimental set. The test pieces of test group 2 were subjected to the same method as in the above experimental group, and the recombinant type I human collagen was replaced with recombinant type I human collagen having an isopeptide bond content of 88.18.+ -. 0.56. Mu. Mol/100g in Table 1 to prepare hydrogels. The test article of test group 3 was the hydrogel provided by control group 1 described above. The test article of test set 4 was the hydrogel provided by control set 2 described above. Test group 5 was the hydrogel provided by control group 3 above.

The treatment scheme of burn is mainly focused on stabilizing metabolism of organism, preventing infection and accelerating functional recovery. The deeper the burn is, the higher the risk of complications such as infection, which undoubtedly increases the difficulty of clinical treatment. The burn wound is mainly divided into three areas, namely a coagulation (necrosis) area, a congestion (ischemia) area and an outermost inflammation area. In the ischemic area, tissue may exhibit progressive ischemia within 24-48 hours, and tissue necrosis may occur, but cells in that area remain viable at this time, and if dry pretreatment is performed within 24 hours, the progression of necrosis may be impeded, maximizing the preservation of surviving skin around the burn wound area.

The experiment constructs a deep II degree burn model of mice for evaluating the therapeutic effect. Deep burns can lead to immunosuppression in mice. The model construction process comprises weighing mice, anesthetizing with chloral hydrate (4% w/v,0.1 mL/10 g), removing hair on the back of the mice after anesthesia and development, removing excessive water on the back, and attaching the mice on the skin surface with a heated metal rod to prepare circular burn with diameter of 1.5 cm. The constructed deep II burn model mice were divided into model group, test group 1 (Test 1), test group 2 (Test 2), test group 3 (Test 3), test group 4 (Test 4), test group 5 (Test 5) by average number. Test group 1 (Test 1), test group 2 (Test 2), test group 3 (Test 3), test group 4 (Test 4), test group 5 (Test 5) were each coated with the corresponding hydrogel at the skin burn site of the mice, and the model group and the blank group were not treated.

Mice were sacrificed on days 7, 14, 21 post-surgery, damaged area surface area was measured using Image J, and a ruler was placed to calibrate the magnification of the photograph of the wound area. The calculation formula of the wound surface collagen fiber is that the collagen fiber (%) =A0 [ type I collagen area ]/A1[ total tissue area ] ×100%. Blood routine testing the levels of White Blood Cells (WBCs), neutrophils (Neu) and lymphocytes (Lym) in the serum of each group of mice for 21 days.

As shown in fig. 5, the blood routine test for 14 days test model group mice showed significantly reduced levels of White Blood Cells (WBC), neutrophils (Neu) and lymphocytes, and their immunity was suppressed, and the deep II burn model mice were successfully modeled. In the figure, the mouse leukocyte levels of test groups 1 and 2 were significantly higher than that of model group (< p < 0.01), and the mouse leukocyte levels of test groups 3, 4 and 5 were not significantly different (ns) from that of model group. The levels of mouse neutrophils were significantly higher in test groups 1 and 2 than in model group (< p < 0.01), and there was no significant difference (ns) between the levels of mouse neutrophils in test groups 3, 4 and 5 and model group. The mouse lymphocyte levels of test groups 1 and 2 were significantly higher than the model group (< p < 0.05), and the mouse lymphocyte levels of test groups 3, 4 and 5 were not significantly different (ns) from the model group. From this, it was demonstrated that after treatment of skin burn sites with hydrogels provided by test groups 1 and 2, the levels of mouse White Blood Cells (WBC), neutrophils (Neu) and lymphocytes were restored in the 14 th day mice, and that there was no obvious sign of restoration in test groups 3 to 5.

As shown in fig. 6, the burn area of mice in test groups 1 and 2 was significantly lower than that of the model group on day 7 (p < 0.05), and the burn area of mice in test groups 3, 4 and 5 was not significantly different (ns) from that of the model group. The mouse burn area for test groups 1 and 2 was significantly lower than the model group on day 14 (x, p < 0.01), the mouse burn area for test group 3 was significantly lower than the model group on day 14 (x, p < 0.05), and the mouse burn areas for test groups 4 and 5 were not significantly different (ns) from the model group. The mouse burn area for test groups 1 and 2 was significantly lower than the model group on day 21 (x, p < 0.01), the mouse burn area for test group 3 was significantly lower than the model group on day 21 (x, p < 0.05), and the mouse burn areas for test groups 4 and 5 were not significantly different (ns) from the model group. From this, it is shown that the hydrogel provided by test group 1 and 2 can promote the wound healing of mice rapidly, reduce burn area rapidly, and model group and test group 3~ 5's mouse burn department is because obvious coagulability necrosis, and the epidermis ulceration leads to the dorsal muscle to expose, and test group 4 and 5 mice surface crust is intact, and the wound remains moist, does not have the chapping, inverts and is difficult to heal.

As shown in fig. 7, the relative amounts of mouse collagen fibers in test groups 1 and 2 were significantly higher than that in model group on day 7 (x, p < 0.01), and the relative amounts of mouse collagen fibers in test groups 3,4 and 5 were not significantly different (ns) from that in model group. The relative amounts of mouse collagen fibers for test groups 1 and 2 were significantly higher than that of the model group on day 14 (p < 0.01), and the relative amounts of mouse collagen fibers for test groups 3,4 and 5 were not significantly different (ns) from that of the model group. From this, it is shown that the hydrogels provided by test groups 1 and 2 can rapidly promote wound healing of mice, rapidly reduce the burn area and promote fibrous growth of skin tissue of mice, while the burn skin of mice in model group and control group may be irreversibly denatured and coagulated necrotic, resulting in failure of rapid recovery of collagen fibers thereof.

2. Injection experiment

ICR mice were randomly divided into Blank (Blank), test group 1 (Test 1), test group 2 (Test 2), test group 3 (Test 3), test group 4 (Test 4), test group 5 (Test 5).

The test article of test set 1 was a hydrogel prepared in the above experimental set. The test pieces of test group 2 were subjected to the same method as in the above experimental group, and the recombinant type I human collagen was replaced with recombinant type I human collagen having an isopeptide bond content of 88.18.+ -. 0.56. Mu. Mol/100g in Table 1 to prepare hydrogels. The test article of test group 3 was the hydrogel provided by control group 1 described above. The test article of test set 4 was the hydrogel provided by control set 2 described above. Test group 5 was the hydrogel provided by control group 3 above. The blank group was not injected.

ICR mice were subjected to one week after IVC conditioning, each mouse was weighed, anesthetized with 50mg/kg body weight dorsal spinal injection of 0.5% sodium pentobarbital solution, dehairing was performed on the back, freshly prepared 0.1mL each set of test samples were injected clockwise at the dehairing site, one dehairing zone per mouse was square at four injection sites, and after injection was completed. Injection sites (including injectate, peri-capsule and full skin) were surgically removed at weeks 1, 4 and 16 for relevant detection analysis.

1) RT-PCR of transplanted tissue

Total RNA of the transplanted tissue is extracted. Specifically, 100mg of transplanted tissue is precooled, ground at 4 ℃, centrifuged at 4 ℃ and 12000rpm for 10min to obtain supernatant, added with 400 mu L of chloroform, uniformly mixed, kept stand for 3min, centrifuged at 4 ℃ and 12000rpm for 10min to obtain supernatant, added with 550 mu L of isopropanol, uniformly mixed, kept stand for 15min, centrifuged at 4 ℃ and 12000rpm for 10min to obtain precipitate, added with 1.5mL of 75% ethanol for 2 times, centrifuged at 4 ℃ and 12000rpm for 5min, removed of ethanol, dried in an ultra-clean bench for 5-10 min to make the precipitate transparent, dissolved in 15 mu L of double-distilled coreless enzyme water, the purity of the precipitate is measured to be 1.8-2.0, and diluted to 100-500 ng/mu L with double-distilled sterile water.

The total RNA extracted was reverse transcribed. The reverse transcription reaction system is 4μL 5×Reaction Buffer,0.5μL Oligo (dT)18 Primer (100μM),0.5μL Random Hexamer primer(100μM),1μL Servicebio®RT Enzyme Mix,2μg total RNA, the double distilled water is added to 20 mu L, the mixture is centrifuged after being uniformly mixed, the reverse transcription is carried out on a PCR instrument by setting a reverse transcription program at 25 ℃ for 5 minutes and 45 ℃ for 30 minutes and 85 ℃ for 5s, and the obtained cDNA is reserved at-20 ℃.

Fluorescent PCR. A fluorescent PCR reaction system was prepared, 7.5. Mu.L of 2 XSYBR GREEN QPCR MASTER Mix, 1.5. Mu.L of the upstream primer (2.5. Mu.M), 1.5. Mu.L of the downstream primer (2.5. Mu.M), 2. Mu.L of cDNA, and 4. Mu.L of nuclease-free water. The PCR reaction procedure, pre-denaturation at 95℃for 30s, denaturation at 95℃for 15 s/annealing extension at 60℃for 30s, 95℃for 10s,65℃for 1 min, and rise to 95 ℃. The relative expression levels of the genes were calculated from the instrument-derived Ct values using DeltaDeltaCT, and each sample was repeated 3 times.

GAPDH-F: cctcgtcccgtagacaaaatg,SEQ ID NO.3; GAPDH-R: tgaggtcaatgaaggggtcgt,SEQ ID NO.4;

TNF-α-F: ccctcacactcacaaaccacc,SEQ ID NO.5; TNF-α-R: ctttgagatccatgccgttg,SEQ ID NO.6

IL-6-F: catagctacctggagtacatgaagaa,SEQ ID NO.7; IL-6-R: gactccagcttatctcttggttga,SEQ ID NO.8

Colla1-F: gagaggtgaacaaggtcccg,SEQ ID NO.9; Colla1-R: aaacctctctcgcctcttgc,SEQ ID NO.10

Col3a1-F: tttcttctcacccttcttcatcc,SEQ ID NO.11; Col3a1-R: catatttgacatggttctggcttc,SEQ ID NO.12

VEGF-F: aggagtaccccgacgagataga,SEQ ID NO.13; VEGF-R: cacatctgctgtgctgtaggaa,SEQ ID NO.14

AKP-F: gagatggacaagttcccttacg,SEQ ID NO.15; AKP-R: tcgtggtggtcacaatgc,SEQ ID NO.16。

2) Results

As shown in fig. 8, the relative expression levels of mouse TNF- α in test groups 1 and 2 were significantly different (ns) between week 1 and the blank, and the relative expression levels of mouse TNF- α in test groups 3, 4 and 5 were significantly higher (x, p <0.05; (x, p < 0.01)) in week 1 than in the blank. The relative amounts of mouse TNF- α expressed in test groups 1 and 2 were significantly higher at week 4 than in the blank groups (p <0.05; p < 0.01) without significant difference (ns) between the 4 th week and the blank groups. The relative amounts of mouse TNF- α expressed in test groups 1 and 2 were significantly higher at week 16 than in the blank groups (p <0.05; p < 0.01) without significant difference (ns) between the test groups 3, 4 and 5.

As shown in fig. 9, the relative expression levels of mouse IL-6 in test groups 1 and 2 were significantly different (ns) between week 1 and the blank, and the relative expression levels of mouse IL-6 in test groups 3, 4 and 5 were significantly higher than those in the blank at week 1 (p < 0.05;. P < 0.01). The relative amounts of mouse IL-6 expressed in test groups 1 and 2 were significantly higher at week 4 than in the blank groups (p <0.05; p < 0.01) without significant difference (ns) between the 4 th week and the blank groups. The relative amounts of mouse IL-6 expressed in test groups 1 and 2 were significantly higher at week 16 than in the blank groups (p <0.05; p < 0.01) without significant difference (ns) between the test groups 3, 4 and 5.

Thus, although the TNF- α and IL-6 expression levels of each group of mice increased within one week after the transplantation, producing a significant inflammatory response, the TNF- α and IL-6 expression levels of the mice of test groups 1 and 2 gradually decreased and were not significantly different from the blank groups throughout 16 weeks after the injection, indicating that the hydrogels of test groups 1 and 2 as the test samples did not produce a significant inflammatory response in the mice as a filler or graft.

As shown in fig. 10, the relative expression amount of mouse type I collagen in test group 2 was significantly higher than that in the blank group at week 1 (x, p < 0.05), and the relative expression amounts of mouse type I collagen in test groups 1, 3, 4, and 5 were not significantly different (ns) from that in the blank group at week 1. The relative expression levels of mouse type I collagen in test groups 1,2, 3 and 4 were significantly higher at week 4 than in the blank group (p <0.05; p < 0.01), and there was no significant difference (ns) between the test group 5 and the blank group at week 4. The relative expression of type I collagen was significantly higher in mice from test groups 1 to 5 than in the blank group at week 16 (p <0.05; p < 0.01).

As shown in fig. 11, the relative expression amount of mouse type III collagen in test group 2 was significantly higher than that in the blank group at week 1 (p < 0.05), and the relative expression amounts of mouse type III collagen in test groups 1, 3,4 and 5 were not significantly different (ns) from that in the blank group at week 1. The relative expression of mouse type III collagen for test groups 1 and 2 was significantly higher at week 4 than for the blank group (p < 0.01), and there was no significant difference (ns) between the mice type III collagen for test groups 3,4 and 5 at week 4. The relative expression levels of mouse type III collagen in test groups 1-4 were significantly higher at week 16 than in the blank group (p <0.05; p < 0.01), and there was no significant difference (ns) between the test groups and the 5 mice type III collagen at week 16.

From this, it was revealed that each group of mice was tested for an increase in the relative expression amount of type I collagen and type III collagen only in group 2 mice after 1 week of hydrogel injection. In addition, although the relative expression amounts of type I collagen and type III collagen were increased to different extents in each group of mice at week 16 of injection, the relative expression amounts of type I collagen and type III collagen were increased significantly in test groups 1 and 2 of mice. It was thus shown that hydrogels of test groups 1 and 2 as test samples were able to promote collagen expression in mice as a filler or graft.

As shown in fig. 12, the relative expression amounts of VEGF in mice of test groups 1 to 5 were not significantly different (ns) between week 1 and the blank group. The relative expression of the mouse VEGF was significantly higher in test groups 1 and 2 than in the blank group at week 4 (p < 0.05), and there was no significant difference (ns) between the test groups 3, 4 and 5. The relative expression of the mouse VEGF was significantly higher in test groups 1 and 2 than in the blank group at week 16 (p < 0.01), and there was no significant difference (ns) between test groups 3, 4 and 5 at week 16 and the blank group. VEGF is vascular endothelial growth factor, is vital to angiogenesis, and can specifically act on vascular endothelial cells, promote mitosis and cell migration, and increase vascular permeability. From the results of fig. 12, it was shown that hydrogels of test groups 1 and 2 as test samples as a filler or graft were able to promote VEGF expression in mice and promote angiogenesis.

As shown in fig. 13, the relative expression amounts of mouse AKP in test groups 1 to 5 were not significantly different (ns) between week 1 and the blank group. The relative expression levels of mouse AKP in test groups 1-5 were not significantly different (ns) between week 4 and the blank group. The relative expression levels of mouse AKP for test groups 1 and 2 were significantly higher at week 16 than for the blank group (p < 0.05), and there was no significant difference (ns) between the mice AKP for test groups 3,4 and 5 at week 16. AKP (alkaline phosphatase) is a specific marker of bone cell synthesis, and its high or low expression level or presence of expression often determines whether there is a marker for bone cell formation. As shown in fig. 13, hydrogels of test groups 1 and 2 as test samples as a filler or implant can promote AKP expression in mice and promote bone cell formation.

In summary, a recombinant type I human collagen having a high content of isopeptidic bonds is produced by the method provided by the invention. The recombinant type I human collagen with high content of isopeptid bonds and nanocellulose are prepared into composite hydrogel, so that the composite hydrogel has the advantages of high stability, low swelling, high weight retention, strong adhesion and hemostatic capability, and has the potential of being used as a skin tissue repair material. The composite hydrogel can be used for repairing burn skin, remarkably reduces inflammatory response of the burn skin and promotes wound healing. In addition, the composite hydrogel can be used as a filler or inhibitor to be filled in a mouse body, has lower inflammatory reaction, can promote collagen synthesis in the mouse body, promotes angiogenesis and bone cell generation after long-term action, and further promotes tissue regeneration, thereby providing a technical foundation for the composite hydrogel serving as a medical filling material.

The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.

Claims

1. A method for preparing recombinant human type I collagen, comprising:

A solution of human type I procollagen amino terminal peptide and a solution of cross-linked peptide of the same concentration are prepared, wherein the human type I procollagen amino terminal peptide is as shown in SEQ ID NO.1, and the cross-linked peptide is as shown in SEQ ID NO.2;

Mixing the solution of the human type I procollagen amino terminal peptide and the solution of the cross-linked peptide in a volume ratio of (1-2):(1-4);

Add glutaminase 5-40 U/g protein;

After thorough mixing, place at 50℃ for 0.5~6 h;

After the reaction, the sample was taken out, placed in a boiling water bath to inactivate the enzyme, and cooled to room temperature;

The solution pH was adjusted to 4.6 to precipitate the modified product and remove the unmixed cross-linked peptides, and the mixture was centrifuged at 10,000 × g for 15 min, and the supernatant was discarded;

The precipitate was washed with water at pH 4.6, and the obtained precipitate was recombinant type I human collagen.

2. The preparation method according to claim 1, characterized in that the solution of the human type I procollagen amino-terminal peptide and the solution of the cross-linked peptide are mixed in a volume ratio of 1:1, 1:2, 1:3, 1:4 or 2:1.

3. The preparation method according to claim 1, characterized in that the added amount of glutaminase is 5U/g, 10U/g, 20U/g, 30U/g or 40U/g protein.

4. The preparation method according to claim 1, characterized in that the reaction time is 0.5h, 1h, 2h, 4h or 6h.

5. A method for preparing a composite hydrogel, comprising:

In an ice bath at 0 °C, an ionic liquid containing 50 g/L 1,5-diazabicyclo[4.3.0]-5-nonene and 50 g/L levulinic acid was prepared;

Under mechanical stirring in an oil bath at 100° C., nanocellulose and recombinant human type I collagen prepared by the preparation method of claim 1 are simultaneously added to the ionic liquid, and the mixture is rapidly stirred to fully dissolve;

Place the reactants in a culture dish, let it stand at room temperature for 1 day, and then wash it repeatedly with anhydrous ethanol.

6. The preparation method according to claim 5, characterized in that the final concentration of the nanocellulose is 1.25 g/L, and the final concentration of the recombinant human type I collagen is 1.25 g/L.

7. The preparation method according to claim 5, characterized in that the diameter of the nanocellulose is 4-10 nm and the length is 200 nm.

8. Recombinant human type I collagen obtained by the method according to claim 1.

9. Use of the recombinant human type I collagen obtained by the method according to claim 1 in preparing skin tissue repair materials.

10. Use of the recombinant human type I collagen prepared by the method according to claim 1 in preparing biological filling materials.