Application of fibroblast growth factor 7 in inhibiting tissue and / or cell fibrosis and drug

Abstract

The present invention provides the application of fibroblast growth factor 7 in inhibiting tissue and / or cell fibrosis, and verifies the effect of fibroblast growth factor 7 in inhibiting tendon and / or stem cell fibrosis. In addition, the present invention also provides the application of fibroblast growth factor 7 in preparing drugs for the above aspects, as well as the application in preparing drugs for treating tendon injuries. The present invention verifies through experiments the effect of fibroblast growth factor 7 (FGF7) in inhibiting the expression of fibrotic proteins in tendon stem cells, and further verifies the application prospect of fibroblast growth factor 7 (FGF7) as a drug for treating tendon-related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of fibroblast growth factor 7 in inhibiting tissue and / or cell fibrosis and the related drugs. Background Art

[0002] Tendon injury is a common injury of the locomotor system. At present, there is no ideal treatment method for tendon injury or rupture. Clinical treatment usually focuses on symptomatic treatment, including drug treatment, physical therapy and surgical treatment, such as direct suture, autologous or allogeneic transplantation. However, the healing ability is poor, the incidence of complications is high and the long-term efficacy is unstable. Problems such as tendon adhesion, calcification, reduced strength and even re-rupture are likely to occur.

[0003] At present, growth factors have been widely used in promoting tissue repair and regeneration. Among them, the fibroblast growth factor (FGF) family is a highly complex and functionally diverse group of protein molecules, which play key roles in regulating cell growth, differentiation, migration and tissue repair. There are many members in the FGF family, including multiple subtypes, such as FGF-1, FGF-2, FGF-4, FGF-5, FGF-7 (fibroblast growth factor 7, also known as KGF, keratinocyte growth factor), FGF-8, FGF-9, FGF-10, etc. Each subtype has a unique structure and function, and there are certain differences in amino acid sequence, spatial structure and biological activity. They achieve complex regulation of cell behavior through a fine-tuning network in vivo. Among them, FGF7 consists of 194 amino acids and belongs to a secreted extracellular protein. Its main functional domain is located in the 32-194 segment and contains an N-type glycosylation modification. At present, it has been reported that FGF7 can act on keratinocytes, promote their proliferation and differentiation, which is crucial for the healing and regeneration of skin wounds. In addition, Palifermin, as a recombinant form of FGF7, has been applied clinically and can effectively reduce the duration and severity of oral mucositis in patients with hematological cancers receiving intensive chemotherapy and radiotherapy, and help oral mucosa regeneration.

[0004] Although some growth factors and their applications in some tissues and cells are disclosed in the prior art, due to the complex variability of organisms, different growth factors often exhibit different performances and effects in different biological tissues. For example, the applicant of the present invention disclosed the application of FGF7 in the preparation of stem cell amplification and phenotype maintenance reagents in the previously filed Chinese patent application (document number CN113717933A). In this disclosure, it was verified that adding FGF7 can promote the in vitro proliferation of tendon stem cells and maintain their stem cell phenotype, thus inferring that FGF7 can provide the possibility for the treatment of tendon injuries.

[0005] However, in fact, this article only demonstrated the effects of FGF7 in promoting stem cell phenotype and maintenance in vitro, and there was no detailed verification on whether FGF7 can act as a drug in the body. In fact, in the treatment of a certain disease, the in vitro stem cell amplification therapy is still a relatively expensive and less universal treatment method in terms of the current scientific and technological development and living standards. People hope to directly act on the body and obtain effects quickly and efficiently.

[0006] Therefore, studying the mechanism of action of FGF7 in the body and whether it can be directly used as a therapeutic drug has important scientific value and social significance for the innovation of drugs and treatment strategies for tendon-related diseases. Summary of the Invention

[0007] The technical objective of the present invention is to provide the application and drug of fibroblast growth factor 7 in inhibiting tissue and / or cell fibrosis in view of the problems existing in the background technology.

[0008] To achieve the above technical objective,

[0009] The first aspect of the present invention lies in providing the application of a fibroblast growth factor 7 in inhibiting tissue and / or cell fibrosis.

[0010] On this basis, the present invention further provides the application of a fibroblast growth factor 7 in the preparation of a drug for inhibiting tissue and / or cell fibrosis.

[0011] The second aspect of the present invention lies in providing the application of a fibroblast growth factor 7 in inhibiting tendon tissue fibrosis.

[0012] On this basis, the present invention further provides the application of a fibroblast growth factor 7 in the preparation of a drug for inhibiting tendon tissue fibrosis.

[0013] The third aspect of the present invention lies in providing the application of a fibroblast growth factor 7 in inhibiting the fibrotic differentiation of tendon stem cells.

[0014] On this basis, the present invention further provides an application of fibroblast growth factor 7 in the preparation of a drug for inhibiting the fibrotic differentiation of tendon stem cells.

[0015] The fourth aspect of the present invention lies in providing an application of fibroblast growth factor 7 in inhibiting the fibrosis of tendon explants.

[0016] On this basis, the present invention further provides an application of fibroblast growth factor 7 in the preparation of a drug for inhibiting the fibrosis of tendon explants.

[0017] The fifth aspect of the present invention lies in providing a drug for inhibiting tissue and / or cell fibrosis, and the drug comprises fibroblast growth factor 7.

[0018] Furthermore, the drug further comprises a pharmaceutically acceptable GelMA sustained-release system, and the GelMA sustained-release system is preferably a GelMA-c-rFGF7 sustained-release system, and the GelMA-c-rFGF7 sustained-release system is configured to release the fibroblast growth factor 7 in vivo.

[0019] Preferably, the drug comprises fibroblast growth factor 7 (FGF7) released by the GelMA-c-rFGF7 system.

[0020] The sixth aspect of the present invention lies in providing an application of fibroblast growth factor 7 in the preparation of a drug for treating tendinopathy.

[0021] Preferably, the drug comprises fibroblast growth factor 7 and a pharmaceutically acceptable carrier GelMA sustained-release system.

[0022] Preferably, the drug is a topical preparation.

[0023] Preferably, the tendon-related diseases include: tendinitis, tendon rupture, tendon injury, tendon fibrosis, tendon degeneration, etc.

[0024] The beneficial effects of the present invention are as follows:

[0025] (1) The present invention verifies the significant therapeutic effect of fibroblast growth factor 7 (FGF7) in the in vivo treatment of tendon-related diseases, providing the possibility and scientific basis for using fibroblast growth factor 7 (FGF7) to prepare drugs for treating tendon-related diseases.

[0026] (2) The present invention verifies the effect of fibroblast growth factor 7 (FGF7) in inhibiting the expression of fibrotic proteins in tendon stem cells through experiments, further verifying the application prospect of fibroblast growth factor 7 (FGF7) as a drug for treating tendon-related diseases.

[0027] (3) The present invention provides a GelMA-c-rFGF7 system for sustained release of fibroblast growth factor 7 (FGF7), which has a better repair effect on tendon tissue compared to the ordinary GelMA system. Description of the Drawings

[0028] Figure 1 It is a result diagram of the immunofluorescence analysis experiment of Example 1 of the present invention.

[0029] Figure 2 It is a treatment plan diagram for tendon injury model using FGF7 in Example 2 of the present invention.

[0030] Figure 3A It is a footprint diagram obtained from the gait analysis experiment of Example 3 of the present invention.

[0031] Figure 3B It is a step length result chart obtained from the gait analysis experiment of Example 3 of the present invention.

[0032] Figure 3C It is a step width result chart obtained from the gait analysis experiment of Example 3 of the present invention.

[0033] Figure 3D It is a step length AI result chart obtained from the gait analysis experiment of Example 3 of the present invention.

[0034] Figure 3E It is a toe spread AI result chart obtained from the gait analysis experiment of Example 3 of the present invention.

[0035] Figure 3F It is a claw length AI result chart obtained from the gait analysis experiment of Example 3 of the present invention.

[0036] Figure 4A It is the HE and Masson staining diagrams of the histopathological experiment of Example 4 of the present invention.

[0037] Figure 4B It is the histological scoring diagram of the histopathological experiment of Example 4 of the present invention.

[0038] Figure 5A It is the detection diagram of transmission electron microscope (TEM) of Example 5 of the present invention.

[0039] Figure 5B It is the collagen diameter distribution diagram of the regenerated tissue observed by electron microscope in Example 5 of the present invention.

[0040] Figure 6A It is a schematic diagram of the maximum exhaustion treadmill experiment of Example 6 of the present invention.

[0041] Figure 6BComparison result graph of the maximum exhaustion speed between FGF7 knockout (KO) mice and wild-type mice in Example 6 of the present invention.

[0042] Figure 6C Comparison result graph of the exhaustion running distance between FGF7 knockout (KO) mice and wild-type mice in Example 6 of the present invention.

[0043] Figure 6D Comparison result graph of the exhaustion time between FGF7 knockout (KO) mice and wild-type mice in Example 6 of the present invention.

[0044] Figure 7A Experimental result graph of the effect of FGF7 knockout (KO) on the tendon tissue level (Achilles tendon AT) in Example 7 of the present invention.

[0045] Figure 7B Experimental result graph of the effect of FGF7 knockout (KO) on the tendon tissue level (patellar tendon PT) in Example 7 of the present invention.

[0046] Figure 8A Experimental result graph of immunofluorescence staining and statistical data of the COL1A1 marker gene in the tendon tissues of wild-type mice and FGF7 KO mice in Example 8 of the present invention.

[0047] Figure 8B Experimental result graph of immunofluorescence staining and statistical data of the TNMD marker gene in the tendon tissues of wild-type mice and FGF7 KO mice in Example 8 of the present invention.

[0048] Figure 8C Experimental result graph of immunofluorescence staining and statistical data of the COL3 marker gene in the tendon tissues of wild-type mice and FGF7 KO mice in Example 8 of the present invention.

[0049] Figure 8D Experimental result graph of immunofluorescence staining and statistical data of the ACTA2 marker gene in the tendon tissues of wild-type mice and FGF7 KO mice in Example 8 of the present invention.

[0050] Figure 9A Schematic diagram of the experiment on the effect of FGF7 knockout (KO) on SCX-GFP expression in the tendon tissues of transgenic mice with the tendon marker gene SCX in Example 9 of the present invention.

[0051] Figure 9B Confocal fluorescence image of GFP protein expression showing the effect of FGF7 knockout (KO) on SCX-GFP expression in the tendon tissues of transgenic mice with the tendon marker gene SCX in Example 9 of the present invention.

[0052] Figure 9CThis is a statistical data graph of GFP protein expression of SCX-GFP in tendon tissue of transgenic mice with tendon marker gene SCX after FGF7 knockout (KO) in Example 9 of the present invention.

[0053] Figure 10A This is an immunofluorescence image and semi-quantitative fluorescence statistical chart (tendon-related Marker proteins MKX, EGR1) of the experiment on the effect of FGF7 on inhibiting the fibrotic differentiation of mouse tendon stem cells in Example 10 of the present invention.

[0054] Figure 10B This is an immunofluorescence image and semi-quantitative fluorescence statistical chart (fibrosis Marker proteins COL3, ACTA2) of the experiment on the effect of FGF7 on inhibiting the fibrotic differentiation of mouse tendon stem cells in Example 10 of the present invention.

[0055] Figure 11 This is an immunofluorescence image and semi-quantitative fluorescence statistical chart of the experiment on the effect of FGF7 on inhibiting the fibrotic differentiation of human tendon stem cells in Example 11 of the present invention.

[0056] Figure 12 This is the experimental result graph of the appearance of fibrotic cell subsets in tendon tissue after FGF7 knockout discovered by single-cell sequencing in Example 12 of the present invention.

[0057] Figure 13A This is the experimental result graph (failure stress) of the experiment on the effect of GelMA-c-rFGF7 releasing FGF7 on enhancing the mechanical performance of repaired tendon tissue in Example 13 of the present invention.

[0058] Figure 13B This is the experimental result graph (modulus) of the experiment on the effect of GelMA-c-rFGF7 releasing FGF7 on enhancing the mechanical performance of repaired tendon tissue in Example 13 of the present invention.

[0059] Figure 13C This is the experimental result graph (destructive force) of the experiment on the effect of GelMA-c-rFGF7 releasing FGF7 on enhancing the mechanical performance of repaired tendon tissue in Example 13 of the present invention.

[0060] Figure 14A This is an immunofluorescence image and semi-quantitative fluorescence statistical chart (THBS4) of the experiment on the effect of using rFGF7 to inhibit the fibrosis of tendon explants in Example 14 of the present invention.

[0061] Figure 14B This is an immunofluorescence image and semi-quantitative fluorescence statistical chart (EGR1) of the experiment on the effect of using rFGF7 to inhibit the fibrosis of tendon explants in Example 14 of the present invention.

[0062] Figure 14CImmunofluorescence images and semi - quantitative fluorescence statistical charts (COL3) showing the effect of rFGF7 on inhibiting tendon explant fibrosis in Example 14 of the present invention.

[0063] Figure 14D Immunofluorescence images and semi - quantitative fluorescence statistical charts (ACTA2) showing the effect of rFGF7 on inhibiting tendon explant fibrosis in Example 14 of the present invention. Detailed implementation manners

[0064] In order to enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be specifically, clearly and completely described below by way of examples in conjunction with the accompanying drawings. It should be noted that the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. And, based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts, any modifications, equivalent replacements, improvements, etc., shall be included within the protection scope of the present invention.

[0065] In addition, to better interpret the technical solutions of the present invention, some technical terms of the present invention will be explained first. In the text of the present invention, these explanations are mainly to help those skilled in the art understand and implement the technical solutions of the present invention, rather than a limitation of the present invention. And, there may be other technical understandings of these technical terms in other disclosures outside the present invention. To avoid technical ambiguities, when understanding the present invention, these explanations of the present invention should be based on.

[0066] Fibroblast growth factor 7 (FGF7)

[0067] Fibroblast growth factor 7 (FGF7), a member of the fibroblast growth factor family, also known as keratinocyte growth factor (KGF), plays an important role in the regulation of embryonic development, cell proliferation and cell differentiation, and has various biological functions such as regulating cell differentiation, promoting mitosis, and inhibiting apoptosis. This protein is an effective epithelial cell - specific growth factor, and its mitogenic activity is mainly manifested in keratinocytes, but not in fibroblasts and endothelial cells.

[0068] It has been reported and disclosed in the prior art that due to the key role of FGF family members in cell proliferation and tissue repair, they are closely related to the occurrence and development of various diseases. In cardiovascular diseases, such as myocardial infarction, certain FGF members can promote angiogenesis and myocardial repair, providing new ideas for disease treatment. In neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, FGF can protect neurons from damage, promote nerve regeneration and functional recovery, providing new strategies for the treatment of nervous system diseases. However, it is worth noting that the overexpression or abnormal activation of certain subtypes may also lead to the proliferation and metastasis of tumor cells. Therefore, when applying FGF family members for disease treatment, it is necessary to carefully evaluate their risks.

[0069] Tissue and / or cell fibrosis

[0070] Fibrosis is a medical concept. Tissue fibrosis can occur in muscle tissues and various organs. The main pathological change is the increase of fibrous connective tissue in organ tissues, the reduction of parenchymal cells, and continuous progression can lead to the destruction of organ structure and the decline of function, and even failure. Cell fibrosis refers to the situation of abnormal cell proliferation and loss of normal regulation inside cells, and then a large number of connective tissue cells are formed. These cells generally secrete a large amount of collagen and other collagen analogues, resulting in the massive aggregation of collagen, and finally the cells lose their normal functions. Cell fibrosis usually leads to pathological remodeling and has irreversible effects on certain organs and tissues.

[0071] In some disclosures of the prior art, fibroblasts are considered to possibly have a certain positive relationship with fibrosis. For example, it has been reported that in some pathological conditions, fibroblasts will be overly active, leading to abnormal fibrosis. Also, as disclosed in some literature, fibroblasts can be activated into myofibroblasts during tissue injury. Transient activation helps repair acute tissue injury, while continuous activation leads to organ scar formation (fibrosis).

[0072] In the embodiments of the present invention, there are multiple experiments to verify the effects of the present invention. Before describing these experiments and effects, first, the sources of the equipment, instruments and reagents used are described to facilitate those skilled in the art to reproduce the technical solutions of the present invention.

[0073]

Equipment and Instruments

[0074] Transmission electron microscope (TEM), model: HT7650, purchased from: Hitachi, Ltd., Japan;

[0075] Confocal fluorescence microscope, model: N-STORM / A1R, purchased from: Nikon Corporation.

[0076]

Reagents

[0077] HE and Masson staining reagents. Catalog number of HE staining solution: C0105S. Catalog number of Masson staining solution: C0189S. Purchased from: Beyotime Biotechnology Company;

[0078] The fibroblast growth factor 7 (FGF7) with catalog number: 100 - 19 - 10 was purchased from: PeproTech Company.

[0079] Example 1: Cell immunofluorescence experiment. To verify the effect of FGF7 on promoting tendon - related expression and inhibiting fibrosis in human tendon cells, in this example, human tendon cell samples were divided into two groups. One group was added with a cell culture medium solution containing 10 ng / ml of FGF7 growth factor (rFGF group), and the other group was added with a cell culture medium solution without FGF7 growth factor (control group). After separate culturing, the two groups of cell samples were subjected to cell fixation, cell membrane permeabilization, blocking, primary and secondary antibody incubation, nuclear staining, and mounting. Finally, fluorescence confocal microscopy observation was carried out, and the results are as Figure 1 shown.

[0080] It can be seen from Figure 1 that compared with the control group without FGF7, in the tendon cells (rFGF7 group) of the cell culture medium solution group added with FGF7 protein growth factor, the expression of normal tendon marker proteins such as COL1A1 and MKX decreased, while the expression of fibrosis - related marker proteins such as ACTA2 and COL3 was more obvious. Through the above experiments, it was proved that FGF7 can directly interact with tendon cells, promote the normal expression of tendon - related functions in tendon cells, and inhibit the occurrence of fibrosis.

[0081] Example 2: Treatment flowchart of FGF7 on rat tendon injury model.

[0082] To explore the effect of FGF7 on tendon - related diseases, in this example, 8 - 10 - week - old female New Zealand rats purchased from the Animal Center of Hangzhou Normal University were used for tendon injury surgery to establish a model. After establishing the tendon injury surgery model, FGF7 containing a sustained - release system was used for treatment experiments.

[0083] The experimental process is as Figure 2 shown, and all surgical procedures were performed under isoflurane (4%) anesthesia.

[0084] After removing the hair in the surgical area, the surgical area was routinely disinfected with iodophor, a lateral knee incision was made, the skin was pulled to the middle position to expose the patellar tendon, a middle fenestration was made in the patellar tendon for modeling, and the entire layer at the modeling site was resected.

[0085] Then configure the LAP initiator. Weigh 0.05 g of LAP powder and add it to 20 ml of PBS buffer. Heat it at 40 - 50 °C for 15 minutes to prepare a 0.25% (w / v) LAP solution.

[0086] Weigh 10 μg of FGF7 dry powder, add it to 1 mL of a standard solution containing 0.25% (w / v) LAP initiator, and prepare a 10 μg / mL FGF7 solution. Then incubate the FGF7 dissolved in the 0.25% LAP photoinitiator standard solution with AC - PEG - NHS for 24 hours under dark conditions at 4 °C. Weigh 1 g of GelMA powder, add it to 10 ml of a 0.25% (w / v) LAP initiator standard solution, heat it in the dark for 20 minutes, and then filter the obtained GelMA solution to get the GelMA hydrogel precursor solution. Take 1 ml of the GelMA hydrogel precursor solution and 1 ml of the polyethylene glycolated FGF7 and mix them well, then centrifuge to obtain the GelMA - c - rFGF7 hydrogel mixed solution. FGF7 can be released through the GelMA - c - rFGF7 sustained - release system. Finally, suture the wound at the modeling site. After taking good care of all the rats, other experiments are carried out during the nursing process. Sacrifice the rats 24 weeks after the operation, and the patellar tendon tissue can be taken for further evaluation during or after that.

[0087] Example 3: Gait analysis experiment

[0088] Design a walking track system with acrylic materials and set a restrictive path on which white paper is covered. Then prepare black ink and red ink to fully coat the soles of the left and right hind feet of the rats. Place the rats treated in Example 2 (2 weeks and 4 weeks after the operation) at the starting point of the restrictive path and allow them to walk freely until they complete the whole journey. At the same time, set up a sham - operation group (control group) and a wild - type healthy group for comparison. Black and red footprints will be left on the white paper. Collect the white paper covered with footprints. For the surgical group corresponding to different sustained - release systems, a total of four groups of footprints are obtained, as Figure 3A shown (taking the 4 - week footprints of a set of experimental samples as an example).

[0089] Each experimental group had 6 independent experimental samples. PS software was used to measure and analyze relevant footprint parameters, including stride length, stride width, step length, paw length, and toe spread. In order to better quantify and compare the degree of asymmetry between the sham operation group and the experimental group, we calculated the asymmetry index (AI), where NS represents the measurement value of the normal side and ES represents the measurement value of the repair side. The calculation formula is as follows: AI (%) = (NS-ES) × (NS + ES) / 0.5 × 100. The following are obtained respectively. Figure 3B and Figure 3C The stride length and stride width statistics shown in the figure are as follows: Figure 3D , Figure 3E and Figure 3F Asymmetry index graphs for stride length, toe span, and claw length are shown. Figures 3B - 3F It can be seen that the stride and step width of the treated rats gradually increased over time (2 weeks-4 weeks), and the AI ​​values ​​of step length, toe span and claw length decreased, indicating that the gait recovery after surgery was good.

[0090] Example 4: Histomorphological experiment

[0091] In order to explore whether FGF7 can improve the healing effect of tendon injury tissue, in this example, the new tendon tissue of rats 4 weeks after surgery in Example 2 was analyzed at the microscopic level, and histological staining analysis was performed in comparison with the control group. The specific steps are as follows: (1) HE staining: dewaxing, hydration, hematoxylin staining, differentiation, blueing, eosin staining, dehydration, transparency and sealing. (2) Masson staining: dewaxing, hydration, hematoxylin staining, acid fuchsin staining, phosphomolybdic acid staining, aniline blue staining, dehydration and sealing.

[0092] The staining results are shown in the figure Figure 4A As shown, Figure 4A The results showed that rFGF7 could significantly promote the functional regeneration of tendon. Figure 4B As shown, the lower the evaluation score, the better the repair effect, which shows that the GelMA-c-rFGF7 group has the best effect.

[0093] Example 5: Electron microscopy collagen diameter experiment

[0094] In order to explore whether FGF7 can inhibit the formation of fibrous scars and observe the ultrastructure of the extracellular matrix of tendon tissue, this example uses a transmission electron microscope (TEM) for detection. The specific procedure is as follows:

[0095] (1)Sample pre - fixation: Take the mouse tendon tissue samples that have undergone the operations in Example 2 and pre - fix them in a 2% (w / v) glutaraldehyde solution at 4°C for 2 hours.

[0096] (2)Sample fixation: Fix the pre - fixed samples in a 1% (w / v) osmium tetroxide solution at 4°C for 2 hours.

[0097] (3)Dehydration treatment: Perform dehydration treatment with a gradient ethanol solution (30%, 50%, 70%, 90%, 100%), with each dehydration treatment lasting 10 minutes. Further dehydrate with acetone.

[0098] (4)Embedding: Embed the dehydrated samples and appropriately heat to accelerate the curing of the embedding agent.

[0099] (5)Ultra - thin sectioning: Cut the embedded samples into ultra - thin sections (70 - 90 nm).

[0100] (6)Staining: Stain with uranyl acetate and lead citrate to enhance the contrast.

[0101] (7)Observation and measurement: Observe the ultrastructure of the tissue samples using a transmission electron microscope and measure the diameter of collagen fibrils using IPP6.0 software.

[0102] In this example, at least 3 independent samples in each group were detected, and approximately 1200 collagen fibrils were analyzed to reveal the ultrastructural changes under different experimental conditions.

[0103] The TEM observation of the ultrastructure is as Figure 5A shown. It can be seen from the figure that the muscle collagen fibrils shown in the GelMA - c - rFGF7 group, whether in the longitudinal or transverse direction, exhibit the characteristics of regular shape, consistent direction, uniform distribution, and moderate size, which are significantly better than the other two groups.

[0104] The results of measuring the diameter of collagen fibrils using IPP 6.0 software are as Figure 5B shown. Only the GelMA - c - rFGF7 group shows two peaks, indicating that the GelMA - c - rFGF7 group maintains large - diameter collagen fibrils and has an obvious effect of preventing them from transforming into small - diameter fibrotic - like collagen fibrils.

[0105] Example 6: Experiment on the effect of FGF7 knockout (KO) on mouse movement

[0106] The normal function of tendon tissue profoundly affects motor behavior. To explore the effect of FGF7 on motor function, this example aims to compare the motor function of mice with FGF7 knockout and wild-type mice (non-experimental mice). In this example, FGF7 was knocked out in mice at birth time points of 21 days and 10 weeks, and the maximum exhaustion treadmill experiment was conducted with wild-type mice respectively, as Figure 6A shown, mice of different groups were respectively placed in a mouse exercise treadmill for exercise ability detection. The results are respectively as Figure 6B , Figure 6C and Figure 6D shown, among which, Figure 6B shows the comparison of the maximum exhaustion speed of wild-type mice with mice with FGF7 knockout at 21 days after birth and mice with FGF7 knockout at 10 weeks after birth, Figure 6C shows the comparison of the running distance at exhaustion of wild-type mice with mice with FGF7 knockout at 21 days after birth and mice with FGF7 knockout at 10 weeks after birth, Figure 6D shows the comparison of the exhaustion time of wild-type mice with mice with FGF7 knockout at 21 days after birth and mice with FGF7 knockout at 10 weeks after birth. It can be seen from the figure that at the time points of 21D or 10w respectively, the FGF7KO mice are weaker than the wild-type mice in terms of the exhaustion time, running distance at exhaustion, and maximum exhaustion speed.

[0107] Example 7: Microscopic effect of FGF7KO on mouse tendon tissue

[0108] To explore the effect of FGF7 on the microscopic tissue structure of tendons, in this example, the patellar tendons and Achilles tendon tissues of mice at 14 days, 21 days, and 10 weeks after birth were subjected to HE staining, and the microscopic tissue structure of the tendons was histologically scored according to the scoring criteria in Example 15. At the same time, wild-type healthy mice were set as controls. Among them, the staining results and histological scores of the Achilles tendon (AT) tissue are as Figure 7A shown, and the staining results and histological scores of the patellar tendon (PT) tissue are as Figure 7B shown, where NS represents the measured value of the normal side. Through this example, it was found that the histological score of the tendon tissue of FGF7KO mice increased significantly, indicating that the tendon tissue was abnormal, thus verifying the obvious importance of FGF7 to tendon tissue.

[0109] Example 8: Effect of FGF7 on inhibiting tendon tissue fibrosis

[0110] To further determine the effect of FGF7 on inhibiting tendon tissue fibrosis in animals. In this example, the effect of FGF7 on inhibiting tendon tissue fibrosis was studied. In this example, 10-week-old mice were selected. Through DAPI immunofluorescence staining of tendon tissues of wild-type mice and FGF7-deficient mice (FGF7KO), it was found that the expressions of tendon proteins Col1a1 and TNMD in the tendon tissues of FGF7-deficient mice were significantly decreased ( Figure 8A 、 Figure 8B ), while the expressions of fibrosis Marker proteins COL3 and ACTA2 were significantly increased ( Figure 8C 、 Figure 8D ).

[0111] Example 9: Effect of FGF7KO on SCX-GFP expression in tendon tissues of transgenic mice with tendon marker gene SCX

[0112] SCX protein is generally considered a marker protein of tendon cells. In this example, an FGF7KO knockout mouse model with SCX-GFP background was constructed ( Figure 9A ). Through immunofluorescence ( Figure 9B ) and flow analysis ( Figure 9C ), it was found that the expression level of GFP protein decreased in the Achilles tendon tissues of SCX-GFP mice and SCX-GFP mice of FGF7KO (8 weeks), proving that after FGF7KO, the tendon tissues underwent non-tendon lineage directional differentiation changes.

[0113] Example 10: Effect of FGF7 on inhibiting fibrotic differentiation of mouse tendon stem cells

[0114] To explore whether FGF7 can promote the expression of tendon lineage proteins in mouse tendon stem cells and inhibit the expression of fibrotic proteins in mouse tendon stem cells, DAPI protein immunostaining was performed on tendon stem cells of rFGF7-treated (added with rFGF7 recombinant protein), control, and FGF7KO groups of mice using protein immunofluorescence experiments. It was found that in the rFGF7-treated group, the expression levels of tendon lineage-related Marker proteins MKX and EGR1 were increased compared with the control group, and the tendon lineage protein expression levels were increased. While in the FGF7KO group, the expression levels of tendon lineage-related Marker proteins MKX and EGR1 were decreased compared with the control group, and the tendon lineage protein expression levels were decreased ( Figure 10A ). At the same time, in the rFGF7-treated group, the expression levels of fibrosis marker proteins ACTA2 and COL3 were significantly decreased compared with the control group, and the fibrotic protein expression levels were significantly decreased. While in the FGF7KO group, the expression levels of fibrosis marker proteins ACTA2 and COL3 were generally increased compared with the control group, and the fibrotic protein expression levels were generally increased ( Figure 10B ).

[0115] Example 11: Effect of FGF7 on Inhibiting Fibrotic Differentiation of Human Tendon Stem Cells

[0116] To investigate whether FGF7 can promote the expression of tendon-related proteins in human tendon stem cells and inhibit the expression of fibrotic proteins in human tendon stem cells, DAPI protein immunostaining was performed on three groups of human tendon stem cells, namely the rFGF7-treated (added with rFGF7 recombinant protein) group, the control group, and the FGF7KO group, using protein immunofluorescence experiments. It was found that compared with the control group, the expression levels of tendon-related protein Marker proteins MKX and EGR1 increased in the rFGF7-treated group. In contrast, compared with the control group, the expression levels of tendon-related Marker proteins MKX and EGR1 decreased in the AZD4547-treated hTSPCs group (AZD4547 is a small molecule inhibitor that inhibits downstream of FGF7). At the same time, compared with the control group, the expression levels of fibrotic marker proteins ACTA2 and COL3 in the rFGF7-treated group were significantly reduced, while in the AZD4547-treated TSPCs group, the expression levels of fibrotic marker proteins ACTA2 and COL3 were generally increased compared with the control group ( Figure 11 ).

[0117] Example 12: Discovery of Fibrotic Cell Subpopulations in Tendon Tissue after FGF7 Knockout by Single-Cell Sequencing

[0118] In this example, single-cell sequencing was performed on the tendon tissues of FGF7KO and WT mice. Then, according to the gene expression characteristics, the cell population was divided into 9 cell subpopulations using Seurat single-cell analysis (Subpopulation 0: Urotensin 2 receptor-positive, lysyl oxidase-positive fibroblast subpopulation; Subpopulation 1: tenocyte subpopulation; Subpopulation 2: tenoblast subpopulation; Subpopulation 3: cell surface glycoprotein CD44-positive, microtubule-associated protein-positive tendon stem / progenitor cell subpopulation; Subpopulation 4: tenifying cell subpopulation; Subpopulation 5: non-mineralized tendon-bone cell subpopulation; Subpopulation 6: lubricin 4-positive tendon stem / progenitor cell subpopulation; Subpopulation 7: muscle-tendon cell subpopulation; Subpopulation 8: mineralized tendon-bone cell subpopulation. And Umap was used for visual display ( Figure 12 ), and a key cell subpopulation promoting tendon fibrosis appeared in Fgf7 - / - (FGF7 knockout) mice. We found that a fibrotic subpopulation (lox + uts2r + ) that should not exist in normal tendon samples at 8 weeks was abundantly present in 8W FGF7KO tendon samples.

[0119] Example 13: Effect of GelMA-c-rFGF7 in releasing FGF7 to enhance the mechanical performance of repaired tendon tissue

[0120] To verify the effect of the GelMA-c-rFGF7 system on the mechanical function of repaired injured tendon tissue, in this example, the patellar tendon tissue of rats 4 weeks after the operation in Example 2 was subjected to mechanical tests. It was found that the indexes of breaking stress level, mechanical modulus, and breaking load-bearing capacity in the GelMA-c-rFGF7 group were significantly better than those of the group implanted with GelMA alone or the control group, and almost showed mechanical functions equivalent to those of the tendon tissue of healthy rats (wild WT group) ( Figure 13A 、 Figure 13B and Figure 13C ). This example proves that implanting FGF7 using the GelMA-c-rFGF7 sustained-release system can effectively inhibit the fibrotic repair phenomenon existing in the repair of tendon tissue injury and can effectively treat tendon diseases.

[0121] Example 14: Verification experiment of using rFGF7 to inhibit tendon explant fibrosis

[0122] In this example, first, the isolated tendon tissue of SCX-GFP mice that had undergone fibrotic changes after being separated from the normal in vivo environment was used and cultured in a medium with or without rFGF7 for 7 days and then analyzed. Then, tenascin THBS4 and EGR1 immunostaining were performed on the isolated tendon tissue with rFGF7 added and its control group. It was found that compared with the control group, the above protein expressions in the isolated tendon tissue cultured in the rFGF7 medium for 7 days were higher than those in the control group ( Figure 14A and Figure 14B ), while the expressions of the fibrotic marker proteins COL3 and ACTA2 were lower than those in the control group ( Figure 14C and Figure 14D ).

[0123] Example 15: Content related to histological scoring

[0124] This example provides the histological scoring criteria for tendon tissue that may be used in other examples of the present invention. This example can be established independently or cross / merged with other examples to achieve.

[0125] The specific criteria are as follows:

[0126] Gross criteria: Normal: 0 points; Slightly abnormal: 1 point; Moderately abnormal: 2 points; Severely abnormal: 3 points.

[0127] Scoring grades: 0 points; 0.5 points; 1 point; 1.5 points; 2 points; 2.5 points; 3 points.

[0128] Fiber structure: Whether there is, whether it is continuous and dense, whether it is in bundles, whether it is wavy; the presence of fat, blood vessels or cavities results in a score reduction.

[0129] Having a continuous and dense fiber structure, in bundles and wavy: 0 points; having a fiber structure and in bundles: 1 point; having a fiber structure but not in bundles: 2 points; having no fiber structure: 3 points.

[0130] Fiber arrangement: Whether there is directionality; whether it is parallel; the angle between parallel bundles is less than 20°: 0 points; the angle between parallel bundles is greater than 30° and less than 45°: 1 point; the angle between parallel bundles is greater than 45° and less than 90°: 2 points; the parallel bundles are perpendicularly arranged: 3 points.

[0131]

[0132] Although the specification has described the preferred embodiments of the present invention, once those skilled in the art learn the basic creative concept, they may make additional changes and modifications to these embodiments or combinations of embodiments without creative efforts. Therefore, the protection scope of the present invention should be understood as the scope intended to be interpreted and covered by the claims, rather than being limited to the parts described in detail in the specification and embodiments of the present invention. Moreover, the content described in the present invention, including the preferred embodiments, includes all changes and modifications that fall within the scope of the present invention.

Claims

1. Application of fibroblast growth factor 7 in the preparation of a drug for inhibiting tendon tissue fibrosis, the drug comprising FGF7 containing a sustained-release system, and the preparation method of FGF7 containing a sustained-release system is: Weigh 10 μg of FGF7 dry powder and add 1 mL of standard solution containing 0.25% (w / v) LAP photoinitiator to prepare a 10 μg / mL FGF7 solution; then incubate FGF7 dissolved in 0.25% LAP photoinitiator standard solution with AC-PEG-NHS PEGylation at 4° in the dark for 24 hours; Weigh 1 g of GelMA powder, add 10 ml of 0.25% (w / v) LAP initiator standard solution, heat in the dark for 20 minutes, and then filter the obtained GelMA solution to obtain a GelMA hydrogel precursor solution; 1 ml of GelMA hydrogel precursor solution and 1 ml of PEGylated FGF7 were mixed thoroughly and centrifuged to obtain a GelMA-c-rFGF7 hydrogel mixed solution. FGF7 can be released by implanting the sustained-release system GelMA-c-rFGF7. The mechanism of action of FGF7 is: FGF7 knocks out lox, a key cell subpopulation that promotes tendon fibrosis + uts2r + , then FGF7 can inhibit lox + uts2r + production, thereby inhibiting fibrosis.

2. A drug for inhibiting tendon tissue fibrosis, characterized in that: The drug includes FGF7 containing a sustained-release system, and the preparation method of the FGF7 containing a sustained-release system is: Weigh 10 μg of FGF7 dry powder and add 1 mL of standard solution containing 0.25% (w / v) LAP photoinitiator to prepare a 10 μg / mL FGF7 solution; then incubate FGF7 dissolved in 0.25% LAP photoinitiator standard solution with AC-PEG-NHS PEGylation at 4° in the dark for 24 hours; Weigh 1 g of GelMA powder, add 10 ml of 0.25% (w / v) LAP initiator standard solution, heat in the dark for 20 minutes, and then filter the obtained GelMA solution to obtain a GelMA hydrogel precursor solution; 1 ml of GelMA hydrogel precursor solution and 1 ml of PEGylated FGF7 were mixed thoroughly and centrifuged to obtain a GelMA-c-rFGF7 hydrogel mixed solution. FGF7 can be released by implanting the sustained-release system GelMA-c-rFGF7. The mechanism of action of FGF7 is: FGF7 knocks out lox, a key cell subpopulation that promotes tendon fibrosis + uts2r + , then FGF7 can inhibit lox + uts2r + production, thereby inhibiting fibrosis.

3. Use of fibroblast growth factor 7 in the preparation of a drug for treating tendon injury, the drug comprising FGF7 containing a sustained-release system, and the preparation method of FGF7 containing a sustained-release system is: Weigh 10 μg of FGF7 dry powder and add 1 mL of standard solution containing 0.25% (w / v) LAP photoinitiator to prepare a 10 μg / mL FGF7 solution; then incubate FGF7 dissolved in 0.25% LAP photoinitiator standard solution with AC-PEG-NHS PEGylation at 4° in the dark for 24 hours; Weigh 1 g of GelMA powder, add 10 ml of 0.25% (w / v) LAP initiator standard solution, heat in the dark for 20 minutes, and then filter the obtained GelMA solution to obtain a GelMA hydrogel precursor solution; 1 ml of GelMA hydrogel precursor solution and 1 ml of PEGylated FGF7 were mixed thoroughly and centrifuged to obtain a GelMA-c-rFGF7 hydrogel mixed solution. FGF7 can be released by implanting the sustained-release system GelMA-c-rFGF7. The mechanism of action of FGF7 is: FGF7 knocks out lox, a key cell subpopulation that promotes tendon fibrosis + uts2r + , then FGF7 can inhibit lox + uts2r + production, thereby inhibiting fibrosis.

Images