Collagen composition and application thereof in improvement of premature senility

Abstract

The invention discloses a recombinant chimeric collagen (rCOL-FGFR), a high affinity anti-IL-6 monoclonal antibody (mAb-IL6v3) and a composition of the recombinant chimeric collagen (rCOL-FGFR) and the high affinity anti-IL-6 monoclonal antibody (mAb-IL6v3). The rCOL-FGFR is formed by connecting human-derived III type collagen and an FGFR2D3 structural domain through flexible Linker, and has the characteristic of targeting FGFR2 + cells, and the expression quantity of the rCOL-FGFR is increased by 40% compared with that of wild type collagen. The mAb-IL6v3 is optimized through mismatch PCR and a yeast display technology, and the IL-6 binding affinity (KD = 1.8 nM) of the mAb-IL6v3 is remarkably superior to that of a commercial antibody. Experiments show that when the rCOL-FGFR and the mAb-IL6v3 are combined for use, the SA-beta-gal positive rate can be synergistically reduced, the skin collagen density can be increased to 67.9%, and a remarkable anti-aging effect is shown in cell and animal models. The composition disclosed by the invention can be widely applied to the development of anti-aging medicines and functional cosmetics.

Description

Technical Field

[0001] The present invention relates to the fields of biomedicine and anti-aging, and in particular to a collagen composition and application thereof in improving premature aging. Background Art

[0002] Existing collagen extraction and application technologies have many limitations in improving skin aging and premature aging. Traditional sources of collagen mainly include animal skins, bones and fish scales. Although collagen with certain biological activity can be obtained after extraction by acid hydrolysis, enzymatic hydrolysis and other methods, its stability in the body is poor and it is easily degraded by proteases in the body (such as matrix metalloproteinases MMPs, collagenase, etc.), resulting in a short half-life and low bioavailability. In addition, using collagen alone as a supplement is difficult to form a sustained and effective effect in the body, and it is impossible to maintain the firmness and elasticity of the skin for a long time, which to a certain extent limits its anti-aging effect.

[0003] In recent years, with the continuous development of anti-aging technology, researchers have begun to explore the improvement of collagen stability and function by introducing peptides or monoclonal antibodies with specific biological activities. Related studies have shown that peptides containing the Arg-Gly-Asp (RGD) sequence can not only promote cell adhesion and signal transduction, but also improve the cell uptake and utilization of collagen, thereby improving the skin cell repair ability; and monoclonal antibodies targeting key enzymes for collagen degradation (such as MMP-1) can effectively inhibit the activity of these degradation enzymes and delay the degradation of collagen. Although the use of peptides or monoclonal antibodies alone has achieved certain results, there is currently a lack of an overall technical solution to organically combine the two with collagen to form a synergistic effect.

[0004] In addition, most of the existing literature focuses on the anti-aging research of a single ingredient, lacking a systematic complex preparation process, stability testing, and in vitro and in vivo application verification. Therefore, how to utilize the synergistic effect of peptides and monoclonal antibodies to both improve the stability of collagen in the body and regulate its biological activity has become an important technical problem that needs to be solved in the current anti-aging field. Based on this, the development of a new collagen complex not only has theoretical innovative significance, but also shows broad prospects in clinical application, and plays an important role in improving skin aging and premature aging symptoms. Summary of the invention

[0005] The purpose of the present invention is to provide a collagen composition and its application in improving premature aging.

[0006] Therefore, the present invention discloses a recombinant chimeric collagen in one aspect. The recombinant chimeric collagen is rCOL-FGFR, and its amino acid sequence is shown in SEQ ID NO.1.

[0007] Preferably, the nucleotide sequence of the codon-optimized rCOL-FGFR of the present invention is shown in SEQ ID NO.2.

[0008] In one aspect, the present invention also discloses an anti-aging composition, which comprises an effective amount of the rCOL-FGFR described in claim 1 and an effective amount of an anti-IL-6 monoclonal antibody, wherein the anti-IL-6 monoclonal antibody is mAb-IL6v3.

[0009] Preferably, the amino acid sequences of the heavy chain variable region and the light chain variable region of the mAb-IL6v3 of the present invention are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0010] Preferably, the dosage of rCOL-FGFR in the composition of the present invention is 10 mg / kg.

[0011] Preferably, the dosage of mAb-IL6v3 in the composition of the present invention is 3 mg / kg.

[0012] In one aspect, the present invention also discloses the use of the rCOL-FGFR as described above in the preparation of an anti-aging composition.

[0013] In one aspect, the present invention also discloses the use of the mAb-IL6v3 as described above in the preparation of an anti-aging composition.

[0014] In addition, existing collagen lacks targeting, and the anti-IL-6 antibody has insufficient affinity. It is difficult for single therapy to effectively reverse skin aging and tissue damage. Therefore, the present invention provides the following solutions:

[0015] 1. Recombinant chimeric collagen (rCOL-FGFR): The D3 domain of FGFR2 is connected to the C-terminus of human type III collagen through a flexible linker (GGGGSGGGGSGGGGS), and a 6His tag is introduced; the codon sequence is optimized, and the expression level in the ExpiCHO TM system reaches 1.23 g / L, which is 40% higher than that of wild-type collagen; flow cytometry verifies that its binding rate to FGFR2+ cells is increased by 8.3 times.

[0016] 2. Anti-IL-6 monoclonal antibody (mAb-IL6v3): A high-affinity mutant (KD = 1.8 nM) is obtained by mismatch PCR and yeast display technology; after humanization, it is expressed using the CHO system, with a purity > 95% and an expression level of 2 g / L; SPR analysis shows that its ka value is increased by 1.8 times compared with the commercial antibody, and the KD value is decreased by 4.86 times.

[0017] 3. Synergistic anti-aging composition: When rCOL-FGFR is used in combination with mAb-IL6v3, in the H2O2-induced HDF cell model, the positive rate of SA-β-gal is reduced to 23.0% (vs 64.0%), and the expression of COL1A1 is increased by 4.54 times; in the premature aging mouse model, the skin collagen density reaches 67.9% (vs 34.7% in the model group), and the median survival period is extended to 218 days (vs 156 days in the model group).

[0018] Based on the above solutions and achieved effects, the beneficial effects of the present invention are summarized as follows:

[0019] 1. Enhanced targeting: rCOL-FGFR specifically binds to target cells through the FGFR2 D3 domain, and the binding efficiency is increased by 8.3 times compared with wild-type collagen; the yield of the optimized rCOL-FGFR in the CHO system reaches 1.23 g / L, which is suitable for large-scale production;

[0020] 2. Ultra-high affinity: The KD value of mAb-IL6v3 (1.8 nM) is significantly better than that of existing commercial antibodies (8.75 nM);

[0021] 3. Synergistic anti-aging: The composition achieves a synergistic effect through a dual mechanism (promoting collagen synthesis + inhibiting the inflammatory factor IL-6), the skin collagen density is increased by 96%, and the survival period is extended by 40%. Therefore, the composition of the present invention can be widely used in the development of anti-aging drugs and functional cosmetics. Description of the Drawings

[0022] Figure 1 SDS-PAGE detection results of rCOL-FGFR protein, where 1 is rCOL-FGFR protein.

[0023] Figure 2 SDS-PAGE detection results of humanized mAb-IL6v3, where 1 is humanized mAb-IL6v3. Detailed Embodiments

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0025] Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0026] Example 1: Preparation and Testing of Recombinant Chimeric Collagen

[0027] I. Design of FGFR2 Binding Domain

[0028] 1. FGFR2 (Fibroblast Growth Factor Receptor 2) is a receptor tyrosine kinase located on the cell membrane and belongs to the FGFR family. Its structure mainly includes:

[0029] (1) Extracellular domain: It contains multiple immunoglobulin-like domains and is responsible for binding to different fibroblast growth factors (FGFs).

[0030] (2) Transmembrane region: Connects the extracellular and intracellular parts.

[0031] (3) Intracellular tyrosine kinase domain: After binding to FGF, it activates downstream signaling pathways such as RAS-MAPK, PI3K-AKT, and PLCγ through autophosphorylation.

[0032] FGFR2 plays an important role in biological processes such as embryonic development, cell proliferation, differentiation, migration, and apoptosis. Its dysfunction is closely related to various developmental abnormalities, tumorigenesis, and other diseases.

[0033] 2. Select the D3 domain of FGFR2 (UniProt ID P21802 residues 214 - 286) as the target protein polypeptide in this study. Then, through a flexible Linker (GGGGSGGGGSGGGGS), this polypeptide is linked to the human type III collagen α1 chain (COL3A1) for co-expression. The amino acid sequence of the D3 domain of FGFR2 is: WSLIMESVVPSDKGNYTCVVENEYGSINHTYH LDVVERSPHRPILQAGLPANASTVVGGDVEFVCKVYSDAQP.

[0034] II. Preparation of Recombinant Chimeric Collagen

[0035] 1. Plasmid Construction

[0036] 1.1 Gene Fragment Design and Synthesis: Obtain the full-length sequence of COL3A1 (NM_000090.3) from NCBI. Through a flexible Linker (GGGGSGGGGSGGGGS), the D3 domain of FGFR2 is linked to its C-terminus, and 6His is added to the N-terminus for purification, thus obtaining recombinant chimeric collagen (rCOL-FGFR). Its amino acid sequence is shown in SEQ ID NO.1. By codon optimization of recombinant chimeric collagen (rCOL-FGFR) (the optimized nucleotide sequence is shown in SEQ ID NO.2), the optimized sequence is synthesized by a third-party company for standby.

[0037] 1.2 PCR Amplification and Overlap Extension

[0038] (1) PCR Amplification: Use high-fidelity DNA polymerase (Phusion) to amplify the optimized target fragment. Optimize the reaction system (25 μL reaction): 50 ng of template DNA, 0.5 μM each of forward and reverse primers, 200 μM dNTP, 1× high-fidelity buffer, 1 U of polymerase.

[0039] (2) Cycling Conditions: Pre-denature at 95°C for 5 minutes; 35 cycles (95°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute); Final extension at 72°C for 10 minutes.

[0040] (3) Product Verification: Observe the target band using 1.2% agarose gel electrophoresis and purify the PCR product (using a commercial gel extraction kit).

[0041] 1.3 Restriction Enzyme Digestion, Ligation, and Clone Screening

[0042] (1) Double Enzyme Digestion: Digest the PCR product and the pcDNA3.4 vector with EcoRI and XhoI (50 μL reaction system, 37°C for 2 hours). Use agarose gel to purify the target DNA fragment to ensure no degraded products.

[0043] (2) Ligation Reaction: Prepare the T4 DNA ligation reaction (molar ratio approximately 3:1, overnight at 16°C), and add a negative control.

[0044] (3) Chemical Transformation and Colony Screening: Transform into DH5α competent cells, plate (containing 100 μg / mL ampicillin), overnight at 37°C. Select 20 colonies for colony PCR and restriction enzyme mapping analysis, select the clones with amplified bands consistent with the expected ones, and finally confirm the sequence by Sanger sequencing.

[0045] 2 Cell Transfection and Expression Optimization

[0046] 2.1 Cell Pretreatment: Use ExpiCHO TM cells, culture in ExpiCHO TM expression medium, ensure that the cell density is controlled at 2×10^6 cells / mL, and the cell viability ≥ 85%. Detect the cell status before each batch of transfection and perform appropriate subculture.

[0047] 2.2 Transfection Operation and Parameter Optimization

[0048] (1) Preparation of Transfection Mixture: Use 2 μg / mL of the target plasmid, and according to Expifectamine TMOptimize the ratio of the optimized DNA to the transfection reagent (usually 1:3 to 1:4).

[0049] (2) Conduct transfection in an incubator preheated to 32 °C. Gently mix and let stand for 5 minutes, then add the cell suspension. Sample and detect the protein expression level every 48 hours.

[0050] (3) Replace part of the culture medium (30% volume) every 3 days to maintain nutrition and reduce the interference of metabolic wastes. At the same time, monitor the expression level of rCOL-FGFR protein and cell viability. The specific results are shown in Table 1.

[0051] Table 1 Results of expression monitoring

[0052]

[0053] 3 Protein purification and quality detection

[0054] 3.1 Supernatant treatment and pre-purification: 14 days after transfection, centrifuge at 4000 rpm for 10 minutes to remove cells, collect the supernatant, and adjust the pH to 7.4. Add 1 mM PMSF, 1 mM EDTA, and protease inhibitor mixture to prevent protein degradation, and store at 4 °C for 30 minutes.

[0055] 3.2 Ni-NTA affinity chromatography and gradient elution

[0056] (1) Column pre-equilibration: Equilibrate the Ni-NTA column with pre-cooled equilibration buffer (20 mM phosphate, 300 mM NaCl, 20 mM imidazole, pH 7.4).

[0057] (2) Protein loading and washing: Slowly load the treated supernatant onto the Ni-NTA column, and control the flow rate at 1 mL / min. Wash with 10 CV of washing buffer containing 25 mM imidazole to remove non-specifically bound proteins, and continuously monitor the UV absorbance of the eluate during this period.

[0058] (3) Gradient elution: Elute the target protein using a linear gradient (from 50 mM to 300 mM imidazole), collect every 2 CV, record the absorbance, and determine the peak fraction of the target protein.

[0059] (4) Post-treatment: After collecting the target fraction, concentrate the protein using an ultrafiltration device and perform buffer replacement (remove high-concentration imidazole by dialysis).

[0060] 3.3 Purity and concentration detection

[0061] (1) SDS-PAGE detection: Take the purified protein sample, separate it on 10% SDS-PAGE, and stain with Coomassie blue. The results show (Figure 1 ), only a single 175 kDa band appeared. Band quantification was performed using image analysis software, and the purity was >95%.

[0062] (2) Protein quantification: The protein concentration was determined using the BCA method, and the yield of the target protein was calculated by converting the total amount of protein. The result was 1.23 ± 0.15 g / L. This indicates that the rCOL-FGFR protein prepared by the present invention has a high expression yield and is suitable for applications in a wide range.

[0063] It should be noted here that when preparing collagen without FGFR (i.e., wild-type collagen in the subsequent examples) in this study, its expression level was only 0.88 g / L. Therefore, adding the D3 domain of FGFR2 to the C-terminus may have a strong promoting effect on the expression level of collagen.

[0064] 4 Verification of targeting efficiency (flow cytometry)

[0065] 4.1 Sample preparation

[0066] (1) Cell preparation: FGFR2-positive cells were selected, the concentration was adjusted to 1×10^6 cells / mL, and pre-treatment was performed (standing on ice for 10 minutes to reduce endocytosis).

[0067] (2) Protein incubation: The purified rCOL-FGFR protein was co-incubated with the cells for 30 minutes (4°C) to ensure sufficient binding of the protein to the cell surface.

[0068] (3) Staining procedure: First, incubate with anti-His primary antibody (1:200, on ice for 30 minutes), then incubate with FITC-labeled secondary antibody (1:500, on ice for 30 minutes). Wash twice with PBS each time in between to reduce non-specific signals.

[0069] 4.2 Data collection and statistical analysis

[0070] (1) Collect at least 10,000 valid cell data using a flow cytometer, and analyze using a two-parameter scatter plot and histogram. Set a strict gating strategy (exclude dead cells and doublet cell aggregates) to ensure data accuracy.

[0071] (2) The results showed (Table 2) that the binding rate of rCOL-FGFR to FGFR2+ cells was 8.3 times higher than that of wild-type collagen (82.7% vs 9.9%, P<0.001).

[0072] Table 2 Results of verification of targeting efficiency

[0073]

[0074] Example 2: Preparation and Testing of Anti-IL-6 Monoclonal Antibody (mAb-IL6v3)

[0075] I. Preparation of mAb-IL6v3

[0076] 1. Preparation of Immunogen

[0077] (1) Antigen Source: High-purity recombinant human IL-6 (abcam, ab259381).

[0078] (2) Conjugation: IL-6 was conjugated with the carrier protein KLH (Keyhole Limpet Hemocyanin) using the glutaraldehyde cross-linking method to enhance immunogenicity.

[0079] 2. Animal Immunization and Serum Detection

[0080] (1) Experimental Animals: 8-week-old Balb / c mice, 5 in each group.

[0081] (2) Immunization Protocol: At week 0, 50 μg KLH-IL-6 was subcutaneously injected and the immune response was enhanced using Freund's complete adjuvant (CFA). At weeks 2 and 4, 50 μg KLH-IL-6 was used for booster immunization, supplemented with Freund's incomplete adjuvant (IFA). At week 6, the anti-IL-6 antibody titer was detected by ELISA. When it reached 1:10 5 , splenocytes were collected.

[0082] 3. Screening and Cloning of Hybridoma Cells: Mouse splenocytes were taken and fused with SP2 / 0 myeloma cells, mediated by PEG-1500 (50% w / v). IL-6-positive hybridoma cells were screened by ELISA, and the positive rate was 12.3%. Further, high-affinity clones were screened by surface plasmon resonance (SPR, Biacore T200) and monoclonal amplification was performed on them.

[0083] 4. Affinity Optimization

[0084] 4.1 Mismatch PCR Mutagenesis

[0085] (1) Mutagenesis Strategy: Random mutations were introduced into the antibody variable region gene using error-prone PCR, and high-affinity clones were screened in combination with yeast display technology.

[0086] (2) Screening Process: In the first round of mutation, a mutant library (about 10 7 clones) was constructed, screened by SPR, and the Kd was reduced to 6.4 nM. In the second round of mutation, a mutant strain with Kd = 3.1 ± 0.4 nM was screened. In the third round of mutation, a high-affinity antibody mAb-IL6v3 with Kd = 1.8 ± 0.3 nM was finally screened.

[0087] 4.2 SPR Kinetic Analysis

[0088] (1) Experimental instrument: Biacore T200

[0089] (2) Experimental conditions: IL-6 was immobilized on a CM5 chip (standard amine coupling method), and the binding density was 500 RU. The mobile phase was PBST. The analysis method was single-cycle kinetics.

[0090] (3) The experimental results are shown in Table 3. After affinity optimization, ka (association rate) increased by about 2.8 times, and kd (dissociation rate) decreased by about 2 times. The KD value decreased to 1.8 nM, indicating that mAb-IL6v3 has stronger binding ability and more stable binding.

[0091] Table 3 Experimental detection results

[0092]

[0093] II. Detection of mAb-IL6v3

[0094] 1. Sequence determination and humanization

[0095] (1) Sequence determination: Total RNA was extracted from mAb-IL6v3 hybridoma cells. Cells were lysed using Trizol reagent, and the RNA purity was detected by Nanodrop2000 (A260 / 280 between 1.8 - 2.0). cDNA was synthesized using a Superscript III reverse transcription kit, and the variable region sequences were amplified using VH / VL specific primers (PCR reaction system: Phusion high-fidelity polymerase, 10 μM VH / VL primers, 10 mM dNTPs, PCR program: 95°C for 3 min, 95°C for 30 s, 58°C for 30 s, 72°C for 30 s, 35 cycles). After purification of the PCR products, they were ligated to a pGEM-T vector, transformed into DH5α bacteria, and positive clones were picked for Sanger sequencing to determine the VH / VL sequences. Sequence alignment was performed using the IMGT / V-QUEST database to analyze the structural characteristics of the CDR regions and framework regions (FR).

[0096] (2) Humanization and optimization: The three-dimensional structure of the antibody was analyzed using SWISS-MODEL, and human IgG1 / κ was selected as the framework region. The CDR transplantation strategy was adopted to replace the CDRs of mAb-IL6v3 with those of the human framework. Meanwhile, the key sites on the framework-antigen binding surface were analyzed, and amino acids were adjusted to reduce immunogenicity. Further, error-prone PCR was used to introduce mutations in the hypervariable regions, and yeast surface display (YSD) technology was combined for high-throughput screening to improve the IL-6 binding ability. The binding affinity of the antibody to IL-6 was determined by Biacore (SPR). Finally, humanized mAb-IL6v3 was obtained, and the amino acid sequences of the heavy-chain variable region and light-chain variable region of humanized mAb-IL6v3 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0097] (3) Synthesize the above-mentioned humanized VH and VL genes, clone them into expression vectors (pcDNA3.4, containing the IgG1 / κ constant region) respectively, and ligate with a strong promoter (CMV). Transfect CHO-S using PEI and culture for 5 - 7 days. Purify the antibody by Protein A affinity chromatography. The SDS-PAGE detection results ( Figure 2 ) showed that its purity could reach over 95%. The concentration measured by BCA was converted to its expression level, which could reach 2 g / L. Therefore, humanized mAb-IL6v3 has a high expression level and purity, and is suitable for large-scale applications.

[0098] 2. Comparative experiment of commercial antibodies

[0099] Select a commercial monoclonal antibody (abcam, ab233706) as a control. Use the same SPR system to test the affinity. The results showed (Table 4) that the ka of mAb-IL6v3 was 1.8 times faster than that of the commercial monoclonal antibody, indicating that mAb-IL6v3 could recognize IL-6 more quickly; the kd value decreased by about 2.7 times, indicating that mAb-IL6v3 bound more stably and dissociated more slowly; the KD value (1.8 nM) was much lower than that of the commercial monoclonal antibody (8.75 nM), indicating that mAb-IL6v3 had a better affinity. The above results indicate that the function of mAb-IL6v3 is superior to that of the commercial monoclonal antibodies already on the market.

[0100] Table 4 Results of SPR kinetic analysis

[0101]

[0102] It should be noted that unless otherwise specified, mAb-IL6v3 mentioned in the present invention refers to humanized mAb-IL6v3.

[0103] Example 3: Cell experiment

[0104] 1. Cell model: HDF cell senescence induced by H2O2 (200 μM, 2 h)

[0105] (1) Cell seeding: HDF cells in the logarithmic growth phase were taken, counted, and seeded in 6-well plates (3×10 5 cells per well), and cultured for 24 hours to allow the cells to adhere to the plate.

[0106] (2) Oxidative damage induction: An H2O2 solution (200 μM) was prepared and diluted with serum-free DMEM. H2O2 (200 μM) was added to the cell culture medium of the experimental group and allowed to act for 2 hours to induce oxidative stress damage in the cells. After the induction was completed, the culture medium was aspirated, the cells were washed twice with PBS, and fresh DMEM medium containing 10% FBS was added for continued culture.

[0107] 2. Experimental grouping:

[0108] (1) Blank control group (Control): Untreated HDF cells were cultured for 48 hours;

[0109] (2) Model control group (H2O2): Induced only with H2O2 and cultured for 48 hours;

[0110] (3) rCOL-FGFR group: After treatment with H2O2, the medium was replaced with a medium containing rCOL-FGFR (diluted to 100 μg / mL with serum-free DMEM);

[0111] (4) Wild-type collagen group (without FGFR): After treatment with H2O2, the medium was replaced with a medium containing wild-type collagen (diluted to 100 μg / mL with serum-free DMEM);

[0112] (5) Complex group (rCOL-FGFR + mAb-IL6v3): After treatment with H2O2, the medium was simultaneously replaced with a medium containing rCOL-FGFR (diluted to 100 μg / mL with serum-free DMEM) and mAb-IL6v3 (diluted to 30 μg / mL with serum-free DMEM).

[0113] 3. Detection and result analysis: After culturing for 48 hours, the morphological changes of the cells were recorded, and subsequent experiments were carried out:

[0114] (1) SA-β-gal staining was performed to detect cell senescence. The results showed (Table 5) that after H2O2 induction, the positive rate of SA-β-gal increased significantly (P<0.001); the positive rate of SA-β-gal could be decreased by either rCOL-FGFR alone or wild-type collagen group (without FGFR), but the effect of rCOL-FGFR was better than that of the wild-type collagen group (without FGFR); meanwhile, the positive rate of SA-β-gal in the complex group was the lowest, significantly lower than that in the single-use groups (P<0.001), indicating their synergistic anti-aging effect.

[0115] Table 5 Detection results of SA-β-gal positive rate (proportion of senescent cells, n = 6)

[0116]

[0117] (2) Western blot was used to detect the expression of COL1A1 protein: Cells were lysed to extract total proteins; proteins were separated by SDS-PAGE electrophoresis and COL1A1 protein was detected by Western blot; gray value analysis was performed using ImageJ software with β-actin as the internal reference. The results showed (Table 6) that H2O2 induction significantly decreased the expression of COL1A1; the expression of COL1A1 in both the rCOL-FGFR and wild-type collagen group (without FGFR) was increased compared with the model control group, but the effect of rCOL-FGFR was better than that of the wild-type collagen group (without FGFR); meanwhile, the expression of COL1A1 in the complex group was the highest, 4.54 times higher than that in the H2O2 group, significantly higher than that in the single-use groups, indicating their synergistic effect in promoting collagen synthesis.

[0118] Table 6 Analysis of the results of Western blot detection of COL1A1 protein expression

[0119]

[0120] Example 4: Animal experiment

[0121] 1. Establishment of premature aging mouse model

[0122] 1.1 Experimental animals: Lmna gene knockout premature aging mice (C57BL / 6 background, 8 weeks old, male, 18 - 22 g). SPF-level animal laboratory, temperature 22 - 25 °C, humidity 40% - 60%; 12 h / 12 h day-night cycle, standard experimental mouse feed and free drinking water. After 1 week of adaptive feeding, they entered the experiment.

[0123] 1.2 Grouping scheme (n = 10 / group)

[0124] (1) Model group (H2O2 induction + PBS treatment);

[0125] (2) rCOL-FGFR group (induced by H2O2 + treated with collagen);

[0126] (3) Wild-type collagen group (without FGFR) (induced by H2O2 + treated with collagen);

[0127] (4) Complex group (induced by H2O2 + treated with collagen + monoclonal antibody combined treatment).

[0128] 2. Treatment protocol

[0129] 2.1 Oxidative damage induction: Shave the back hair (2 cm × 2 cm area), inject H2O2 subcutaneously to induce oxidative damage, 50 μL of H2O2 (10 mM), 3 times a week for 2 weeks, and observe premature aging manifestations such as skin folds, pigmentation, and relaxation.

[0130] 2.2 Drug administration protocol

[0131] (1) Administration method: Subcutaneous injection, in the symmetric area of the back, once every 3 days for 8 weeks.

[0132] (2) Administration dose:

[0133] rCOL-FGFR group: 10 mg / kg rCOL-FGFR;

[0134] Wild-type collagen group (without FGFR): 10 mg / kg wild-type collagen group (without FGFR);

[0135] Complex group: 10 mg / kg rCOL-FGFR + 3 mg / kg mAb-IL6v3;

[0136] Model group: Equal volume of PBS (control).

[0137] (3) Injection operation: Wipe the injection site with 75% alcohol, inject subcutaneously with a 27G injection needle, and observe for 5 minutes to ensure no abnormal reaction.

[0138] 3. Detection and data analysis

[0139] 3.1 Histological analysis of skin (Masson staining)

[0140] (1) After 8 weeks, take the skin tissue on the back of the mouse, fix it with 4% paraformaldehyde, embed it in paraffin, and section (thickness 5 μm). Perform Masson staining to observe the distribution of collagen fibers and the thickness of the dermis.

[0141] (2) Quantitative analysis (ImageJ software): Collagen fiber density (%) = collagen area / total tissue area × 100%; Dermis thickness (μm): Measure the distance between the epidermal-subcutaneous interface.

[0142] (3) Results showed (Table 7) that both the rCOL-FGFR group and the wild-type collagen group (without FGFR) could improve skin aging, increase the density of collagen fibers, and increase the thickness of the dermis. However, the rCOL-FGFR group had a better effect than the wild-type collagen group (without FGFR). Meanwhile, the combined group had the best effect, indicating a synergistic effect when the two were used in combination.

[0143] Table 7 Summary of skin histological analysis results

[0144]

[0145] 3.2 Survival analysis (Kaplan-Meier method)

[0146] (1) After the experiment, the survival status of the mice was recorded weekly for 6 months, and Kaplan-Meier survival curve analysis was performed to compare the survival differences between different groups.

[0147] (2) Results showed (Table 8) that the single-drug treatment group had a 26- to 34-day longer survival period than the model group. However, the rCOL-FGFR group had a better effect than the wild-type collagen group (without FGFR). Meanwhile, the survival period of the combined group was significantly extended to 218 days (P < 0.01), which was 40% higher than that of the model group.

[0148] Table 8 Summary of survival analysis results

[0149]

[0150] The above embodiments are preferred embodiments of the present invention. However, the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A recombinant chimeric collagen, characterized in that, The recombinant chimeric collagen is rCOL-FGFR, and its amino acid sequence is shown in SEQ ID NO.

1.

2. The recombinant chimeric collagen according to claim 1, wherein The nucleotide sequence of the codon-optimized rCOL-FGFR is shown in SEQ ID NO.

2.

3. An anti-aging composition, characterized in that, The composition comprises an effective amount of the rCOL-FGFR according to claim 1 and an effective amount of an anti-IL-6 monoclonal antibody, wherein the anti-IL-6 monoclonal antibody is mAb-IL6v3.

4. The composition according to claim 3, wherein The amino acid sequences of the heavy chain variable region and the light chain variable region of the mAb-IL6v3 are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

5. The composition according to claim 3, characterized in that, The dosage of rCOL-FGFR in the composition is 10 mg / kg.

6. The composition according to claim 3, wherein The dosage of mAb-IL6v3 in the composition is 3 mg / kg.

7. Use of the rCOL-FGFR according to claim 1 in the preparation of an anti-aging composition.

8. Use of the mAb-IL6v3 according to claim 4 in the preparation of an anti-aging composition.

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