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

Abstract

The invention provides application of the fibroblast growth factor 7 in inhibition of tissue and / or cell fibrosis, verifies that the fibroblast growth factor 7 in inhibition of tendon and / or stem cell fibrosis, and further provides application of the fibroblast growth factor 7 in preparation of the medicine in the aspect, and the application of the fibroblast growth factor 7 in inhibition of muscle tendon and / or stem cell fibrosis in inhibition of muscle tendon and / or stem cell fibrosis in inhibition of muscle tendon and / or stem cell fibrosis in inhibition of muscle tendon and / or stem cell fibrosis. The invention also discloses application in preparation of a medicine for treating tendon injury. The invention verifies the effect of the fibroblast growth factor 7 (FGF7) in inhibiting fibrotic protein expression of tendon stem cells through experiments, and further verifies the application prospect of the fibroblast growth factor 7 (FGF7) as a medicine for treating tendon-related diseases.

Description

Technical Field

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

[0002] Tendon injury is a common musculoskeletal injury. Currently, there is no ideal treatment for tendon injury or rupture. Clinical treatment is usually symptomatic, including drug therapy, physical therapy and surgical treatment, such as direct suture, autologous or allogeneic transplantation, which has poor healing ability, high incidence of complications and unstable long-term efficacy. It is easy to have tendon adhesion, calcification, reduced strength, and even re-rupture.

[0003] At present, growth factors have been widely used to promote tissue repair and regeneration. Among them, the fibroblast growth factor (FGF) family is a highly complex and functionally diverse protein molecule that plays a key role in regulating cell growth, differentiation, migration, and tissue repair. The FGF family has many members, 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 sophisticated regulatory network in the body. Among them, FGF7 is composed of 194 amino acids and is a secreted extracellular protein. Its main functional domain is located in the 32-194 segment and contains an N-type glycosylation modification. It has been reported that FGF7 can act on keratinocytes to promote their proliferation and differentiation, which is essential for the healing and regeneration of skin wounds. In addition, Palifermin, a recombinant form of FGF7, has been used in clinical practice and can effectively reduce the duration and severity of oral mucositis in patients with hematological cancers receiving intensive chemotherapy and radiotherapy, and help regenerate the oral mucosa.

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

[0005] But in fact, the article only demonstrates the effect of FGF7 in promoting stem cell phenotype and maintenance in vitro, and there is no detailed verification on whether FGF7 can act as a drug in the body. In fact, when treating a certain disease, in vitro stem cell expansion therapy is still a relatively expensive and less universal treatment method in terms of current technological development and living standards. People hope to be able to act directly on the body and obtain results 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 purpose of the present invention is to provide an application and medicine of fibroblast growth factor 7 in inhibiting tissue and / or cell fibrosis in view of the problems existing in the background technology.

[0008] In order to achieve the above technical objectives,

[0009] The first aspect of the present invention is to provide a use of fibroblast growth factor 7 in inhibiting tissue and / or cell fibrosis.

[0010] On this basis, the present invention further provides an application of 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 is to provide an application of fibroblast growth factor 7 in inhibiting tendon tissue fibrosis.

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

[0013] The third aspect of the present invention is to provide an application of 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 is to provide an application of fibroblast growth factor 7 in inhibiting 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 tendon explant fibrosis.

[0017] The fifth aspect of the present invention is to provide a drug for inhibiting tissue and / or cell fibrosis, wherein the drug comprises fibroblast growth factor-7.

[0018] Furthermore, the drug also includes 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 sustain the release of 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 is to provide a use 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 medicament 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:

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

[0026] (2) The present invention experimentally verifies 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.

[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 than the ordinary GelMA system. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a diagram of the experimental results of cell immunofluorescence analysis in Example 1 of the present invention.

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

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

[0031] Figure 3B This is a chart of stride results obtained from the gait analysis experiment of Example 3 of the present invention.

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

[0033] Figure 3D This is a chart of stride length AI results obtained from the gait analysis experiment of Example 3 of the present invention.

[0034] Figure 3E This is a chart of toe span AI results obtained from the gait analysis experiment of Example 3 of the present invention.

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

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

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

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

[0039] Figure 5B This is the electron microscopic distribution diagram of collagen diameter of regenerated tissue in Example 5 of the present invention.

[0040] Fig. 6A Schematic diagram of the maximum exhaustion treadmill experiment of Example 6 of the present invention.

[0041] Figure 6BThis is a graph showing the comparison of the maximum speed after exhaustion of FGF7 knockout (KO) mice and wild-type mice in Example 6 of the present invention.

[0042] Figure 6C This is a graph showing the comparison of exhaustive running distances of FGF7 knockout (KO) mice and wild-type mice in Example 6 of the present invention.

[0043] Fig.6D This is a graph showing the comparison of exhaustion time between FGF7 knockout (KO) mice and wild-type mice in Example 6 of the present invention.

[0044] Fig. 7A This is a diagram showing the experimental results 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 This is a diagram showing the experimental results of the effect of FGF7 knockout (KO) on the tendon tissue level (patellar tendon PT) in Example 7 of the present invention.

[0046] Fig. 8A This is a graph showing the experimental results of immunofluorescence staining and statistical data of the COL1A1 marker gene in tendon tissues of wild-type mice and FGF7KO mice in Example 8 of the present invention.

[0047] Figure 8B This is a graph showing the experimental results of immunofluorescence staining and statistical data of TNMD marker genes in tendon tissues of wild-type mice and FGF7KO mice in Example 8 of the present invention.

[0048] Figure 8C This is a graph showing the experimental results of immunofluorescence staining and statistical data of the COL3 marker gene in tendon tissues of wild-type mice and FGF7KO mice in Example 8 of the present invention.

[0049] Fig.8D This is a graph showing the experimental results of immunofluorescence staining and statistical data of the ACTA2 marker gene in tendon tissues of wild-type mice and FGF7KO mice in Example 8 of the present invention.

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

[0051] Fig. 9B This is a fluorescence confocal image of the GFP protein expression of SCX-GFP in the tendon tissue of transgenic mice expressing the tendon marker gene SCX after FGF7 knockout (KO) in Example 9 of the present invention.

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

[0053] Fig. 10A These are the immunofluorescence images and semi-quantitative fluorescence statistical charts (tendon system-related marker proteins MKX and EGR1) of the experiment on the effect of FGF7 on the inhibition of mouse tendon stem cell fibrosis in Example 10 of the present invention.

[0054] Fig. 10B These are the immunofluorescence images and semi-quantitative fluorescence statistical charts (fibrosis marker proteins COL3 and 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] Fig.11 These are the immunofluorescence images and semi-quantitative fluorescence statistical charts 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] Fig.12 This is a graph showing the experimental results of Example 12 of the present invention, in which single-cell sequencing was used to find the presence of fibrotic cell subpopulations in tendon tissue after FGF7 knockout.

[0057] Fig.13A This is a graph showing the experimental results (failure stress) of the effect of GelMA-c-rFGF7 releasing FGF7 to enhance the mechanical properties of repaired tendon tissue in Example 13 of the present invention.

[0058] Fig. 13B This is a graph showing the experimental results (modulus) of the effect of GelMA-c-rFGF7 releasing FGF7 to enhance the mechanical properties of repaired tendon tissue in Example 13 of the present invention.

[0059] Fig. 13C This is a graph showing the experimental results (destructive force) of the effect of using GelMA-c-rFGF7 to release FGF7 to enhance the mechanical properties of repaired tendon tissue in Example 13 of the present invention.

[0060] Fig.14A This is an immunofluorescence image and a semi-quantitative fluorescence statistical chart (THBS4) showing the effect of rFGF7 in inhibiting tendon explant fibrosis in Example 14 of the present invention.

[0061] Fig. 14B This is an immunofluorescence image and a semi-quantitative fluorescence statistical chart (EGR1) showing the effect of rFGF7 in inhibiting tendon explant fibrosis in Example 14 of the present invention.

[0062] Fig. 14CThis is an immunofluorescence image and a semi-quantitative fluorescence statistical chart (COL3) showing the effect of rFGF7 in inhibiting tendon explant fibrosis in Example 14 of the present invention.

[0063] Fig.14D This is an immunofluorescence image and a semi-quantitative fluorescence statistical chart (ACTA2) showing the effect of rFGF7 in inhibiting tendon explant fibrosis in Example 14 of the present invention. DETAILED DESCRIPTION

[0064] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is specifically, clearly and completely described in the following embodiments in conjunction with the accompanying drawings. It should be noted that the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Moreover, based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work, any modifications, equivalent substitutions, improvements, etc., should be included in the scope of protection of the present invention.

[0065] In addition, in order to better interpret the technical solution of the present invention, some technical terms of the present invention are first explained. In the text of the present invention, these explanations are mainly to help those skilled in the art understand and implement the technical solution of the present invention, rather than to limit the present invention. Moreover, there may be other technical understandings of these technical terms in other disclosures outside the present invention. In order to avoid technical ambiguity, when understanding the present invention, these explanations of the present invention should be based on them.

[0066] Fibroblast Growth Factor 7 (FGF7)

[0067] Fibroblast growth factor 7 (FGF7) is a member of the fibroblast growth factor family, also known as keratinocyte growth factor (KGF), which plays an important role in the regulation of embryonic development, cell proliferation and cell differentiation, and has multiple biological functions such as regulating cell differentiation, promoting mitosis, and inhibiting cell 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 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, and provide a new strategy for the treatment of nervous system diseases. However, it is worth noting that overexpression or abnormal activation of certain subtypes may also lead to the proliferation and metastasis of tumor cells, so the risks need to be carefully assessed when using FGF family members for disease treatment.

[0069] Tissue and / or cellular fibrosis

[0070] Fibrosis is a medical concept. Tissue fibrosis can occur in muscle tissue and multiple organs. The main pathological changes are an increase in fibrous connective tissue and a decrease in parenchymal cells in organ tissues. Continuous progression can lead to organ structural destruction and functional impairment, or even failure. Cellular fibrosis refers to the abnormal proliferation of cells and the loss of normal regulation in cells, which leads to the formation of a large number of connective tissue cells. These cells generally secrete a large amount of collagen and other collagen analogs, resulting in a large aggregation of collagen, which ultimately causes the cells to lose their normal function. Cellular fibrosis usually leads to pathological remodeling and has irreversible effects on certain organs and tissues.

[0071] In some prior art disclosures, fibroblasts are believed to have a certain positive relationship with fibrosis. For example, it has been reported that in some pathological conditions, fibroblasts can be overactive, leading to abnormal fibrosis. Another example is that it has been disclosed in some literature that fibroblasts can be activated into myofibroblasts when tissue is damaged. Short-term activation helps repair acute tissue damage, while continuous activation leads to organ scarring (fibrosis).

[0072] In the embodiments of the present invention, a plurality of experiments for verifying the effects of the present invention are included. Before describing these experiments and effects, the sources of the equipment, instruments and reagents used are first explained to facilitate technicians in this field to reproduce the technical solutions of the present invention.

[0073]

Equipment

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

[0075] Fluorescence confocal instrument model: N-STORM / A1R, purchased from: Nikon.

[0076]

Reagents

[0077] HE and Masson staining reagents HE staining solution catalog number: C0105S Masson staining solution catalog number: C0189S Purchased from: Bio-Tech Biotech Co., Ltd.;

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

[0079] Example 1: Cell immunofluorescence experiment In order to verify the effect of FGF7 on promoting tendon expression and inhibiting fibrosis in human tendon cells, in this example, human tendon cell samples were divided into two groups, one of which 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 culturing them separately, the two groups of cell samples were fixed, cell membrane permeabilized, blocked, incubated with primary and secondary antibodies, cell nuclei stained, and sealed. Finally, fluorescence confocal microscopy observation was performed. The results are shown in the figure. Figure 1 shown.

[0080] pass Figure 1 It can be seen that compared with the control group without FGF7, the expression of normal tendon marker proteins such as COL1A1 and MKX in the tenocytes of the cell culture medium solution group (rFGF7 group) added with FGF7 protein growth factor was reduced, while the expression of fibrosis marker proteins such as ACTA2 and COL3 was more obvious. The above experiments prove that FGF7 and tenocytes can directly interact, promote the normal expression of tendon function of tenocytes, and inhibit the occurrence of fibrosis.

[0081] Example 2: Flow chart of the treatment of rat tendon injury model with FGF7.

[0082] In order to explore the effect of FGF7 on tendon-related diseases, this example used 8-10 week old female New Zealand rats purchased from the Animal Center of Hangzhou Normal University for tendon injury surgical modeling. After the tendon injury surgical model was established, a therapeutic experiment was performed using FGF7 containing a sustained release system.

[0083] The experimental process is as follows Figure 2 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 iodine, a lateral incision was made on the knee joint, the skin was pulled to the middle position to expose the patellar tendon, a middle window was opened to model the patellar tendon, and the full-thickness resection was performed at the modeling site.

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

[0086] Weigh 10 μg of FGF7 dry powder and add 1 mL of standard solution containing 0.25% (w / v) LAP initiator to prepare a 10 μg / mL FGF7 solution. Then, FGF7 dissolved in 0.25% LAP photoinitiator standard solution was incubated with AC-PEG-NHS PEGylation at 4° for 24 hours in the dark. Weigh 1g of GelMA powder and add 10ml of 0.25% (w / v) LAP initiator standard solution. After heating in the dark for 20 minutes, the obtained GelMA solution was filtered to obtain a GelMA hydrogel precursor solution. Take 1ml of GelMA hydrogel precursor solution and 1ml of PEGylated FGF7 and mix them thoroughly. Centrifuge to obtain a GelMA-c-rFGF7 hydrogel mixed solution. FGF7 can be released by implanting the sustained-release system GelMA-c-rFGF7, and finally suture the wound at the modeling site. All rats were then given quiet care, and other experiments were performed during the nursing process. The rats were sacrificed 24 weeks after surgery, and their patellar tendon tissues could be obtained during or after this period for further evaluation.

[0087] Example 3: Gait analysis experiment

[0088] A walking track system was designed using acrylic material, and a restricted road was set up on it. White paper was covered on the road, and then black ink and red ink were prepared to fully smear the soles of the left and right hind limbs of the rats. The rats treated with Example 2 (2 weeks and 4 weeks after surgery) were placed at the starting point of the restricted road and allowed to walk freely until they completed the whole journey. A sham operation group (control group) and a wild-type healthy group were set up for comparison. Black and red footprints were left on the white paper, and the white paper covering the footprints was collected. The surgical groups corresponded to different sustained-release systems, and a total of four groups of footprints were obtained, as shown in FIG. Figure 3A As shown (taking the 4-week footprint 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. Figure 3B-Figure 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 sample processed in Example 2 and pre-fix it with 2% (w / v) glutaraldehyde solution at 4°C for 2 hours.

[0096] (2) Sample fixation: The pre-fixed samples were fixed with 1% (w / v) osmium hydroxide solution at 4°C for 2 h.

[0097] (3) Dehydration: Dehydrate with gradient ethanol solution (30%, 50%, 70%, 90%, 100%) for 10 minutes each time. Further dehydration with acetone.

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

[0099] (5) Ultrathin sectioning: The embedded samples are subjected to ultrathin sectioning (70-90 nm).

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

[0101] (7) Observation and measurement: The ultrastructure of tissue samples was observed using a transmission electron microscope, and the diameter of collagen fibrils was measured using IPP6.0 software.

[0102] In this example, at least three independent samples in each group were tested, and approximately 1,200 collagen fibrils were analyzed to reveal the ultrastructural changes under different experimental conditions.

[0103] TEM observation of ultrastructure Figure 5A As shown in the figure, it can be seen that the muscle collagen fibers displayed by the GelMA-c-rFGF7 group, whether in the longitudinal or transverse direction, have the characteristics of regular shape, consistent direction, uniform distribution, and moderate size, which is significantly better than the other two groups.

[0104] The results of measuring collagen fibril diameter using IPP 6.0 software are shown in Figure 2. Figure 5B As shown, only the GelMA-c-rFGF7 group showed two peaks, indicating that the GelMA-c-rFGF7 group maintained large-diameter collagen fibrils and had a significant effect in preventing them from transforming into small-diameter fibrotic-like collagen fibrils.

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

[0106] The normal function of tendon tissue profoundly affects motor behavior. In order to explore the effect of FGF7 on motor function, this example aims to compare the motor function of FGF7 knockout mice and wild-type mice (non-experimental mice). In this example, FGF7 was knocked out of mice born at 21 days and 10 weeks, and the maximum exhaustion treadmill experiment was performed on wild-type mice. Fig. 6A As shown in Figure 2, mice in different groups were placed on a mouse treadmill for exercise capacity testing. The results were shown in Figure 2. Figure 6B , Figure 6C and Fig.6D As shown, Figure 6B The figure shows the comparison of the maximum speed of exhaustion of wild-type mice and mice knocked out of FGF7 at 21 days after birth and mice knocked out of FGF7 at 10 weeks after birth. Figure 6C The figure shows the comparison of exhaustive running distances of wild-type mice, 21-day-old FGF7 knockout mice, and 10-week-old FGF7 knockout mice. Fig.6D The figure shows a comparison of the exhaustion time of wild-type mice and 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 FGF7KO mice are weaker than wild-type mice in terms of exhaustion time, exhaustion running distance and maximum exhaustion speed at 21D or 10w respectively.

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

[0108] In order to explore the effect of FGF7 on the microstructure of tendon, in this example, HE staining was performed on the patellar tendon and Achilles tendon tissues of mice at 14 days, 21 days, and 10 weeks of age, and the histological score of the tendon microstructure was performed according to the scoring criteria in Example 15. Wild-type healthy mice were used as controls. The staining results and histological scores of Achilles tendon (AT) tissues were as follows: Fig. 7A The staining results and histological scores of patellar tendon (PT) tissues are shown in Figure 7B As shown, NS represents the measured value of the normal side. Through this example, it was found that the microscopic tissue structure score of the tendon tissue of FGF7KO mice increased significantly, indicating that the tendon tissue was abnormal, thereby verifying that FGF7 is significantly important to the tendon tissue.

[0109] Example 8: Effect of FGF7 on Inhibition of Tendon Tissue Fibrosis

[0110] In order to further determine the effect of FGF7 on inhibiting fibrosis of mouse tendon tissue in vivo. In this example, the effect of FGF7 on inhibiting fibrosis of tendon tissue was studied. In this example, 10-week-old mice were selected, and DAPI immunofluorescence staining of tendon tissues of wild-type mice and FGF7-deficient mice (FGF7KO) was performed to observe that the expression of tenascin Col1a1 and TNMD in the tendon tissues of FGF7-deficient mice was significantly reduced ( Fig. 8A , Figure 8B ), while the expression of fibrosis marker proteins COL3 and ACTA2 was significantly increased ( Figure 8C , Fig.8D ).

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

[0112] SCX protein is generally considered to be a marker protein of tenocytes. In this example, an FGF7KO knockout mouse model with SCX-GFP as the background was constructed ( Fig.9A ), by immunofluorescence ( Fig. 9B ) and flow cytometry ( Fig. 9C ) found that the expression of GFP protein in the Achilles tendon tissue of SCX-GFP mice and FGF7KO SCX-GFP mice (8 weeks) was decreased, proving that the tendon tissue underwent non-tendon differentiation after FGF7KO.

[0113] Example 10: Effect of FGF7 on Inhibition of Fibrotic Differentiation of Mouse Tendon Stem Cells

[0114] In order to explore whether FGF7 can promote the expression of tendon proteins in mouse tendon stem cells and inhibit the expression of fibrotic proteins in mouse tendon stem cells, protein immunofluorescence experiments were used to perform DAPI protein immunostaining on the rFGF7-treated (rFGF7 recombinant protein added) group, control group, and FGF7KO group of mouse tendon stem cells. It was found that the expression levels of tendon proteins in the rFGF7-treated group, including the marker proteins MKX and EGR1, were increased compared with those in the control group, while in the FGF7KO group, the expression levels of tendon proteins in the tendon-related marker proteins MKX and EGR1 were decreased compared with those in the control group ( Fig. 10A At the same time, the expression levels of fibrosis marker proteins ACTA2 and COL3 in the rFGF7-treated group were significantly lower than those in the control group, while in the FGF7KO group, the expression levels of fibrosis marker proteins ACTA2 and COL3 were generally higher than those in the control group ( Fig. 10B ).

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

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

[0117] Example 12: Single-cell sequencing revealed the presence of fibrotic cell subpopulations in tendon tissue after FGF7 knockout

[0118] In this example, single-cell sequencing was performed on the tendon tissues of FGF7KO and WT mice, and then the cell population was divided into 9 cell subpopulations according to gene expression characteristics using Seurat single-cell analysis (No. 0: urotensin 2 receptor-positive, lysyl oxidase-positive fibroblast subpopulation; No. 1: tenocyte subpopulation; No. 2: tenoblast subpopulation; No. 3: cell surface glycoprotein CD44-positive microtubule polymerization protein-positive tendon stem / progenitor cell subpopulation; No. 4: tendinization cell subpopulation; No. 5: non-mineralized tendon bone cell subpopulation; No. 6: lubricating protein 4-positive tendon stem / progenitor cell subpopulation; No. 7: muscle tendon cell subpopulation; No. 8: mineralized tendon bone cell subpopulation. And Umap was used for visualization ( Fig.12 ), Fgf7 - / - In the FGF7 knockout mice, key cell subpopulations that promote tendon fibrosis appeared, and we found that in the 8-week-old normal tendon samples, there was a fibrotic subpopulation (lox + uts2r + ) were abundant in 8WFGF7KO tendon samples.

[0119] Example 13: Effect of GelMA-c-rFGF7 release on the mechanical properties of repaired tendon tissue

[0120] In order to verify the effect of the GelMA-c-rFGF7 system on the mechanical function of repairing damaged tendon tissue, this example conducted mechanical tests on rat patellar tendon tissue 4 weeks after surgery in Example 2, and found that the fracture stress level, mechanical modulus, and fracture bearing capacity of the GelMA-c-rFGF7 group were significantly better than those of the simple GelMA implantation or control group, and almost showed the same mechanical function as the healthy rat tendon tissue (wild WT group) ( Fig.13A , Fig. 13B and Fig. 13C ). This example proves that implanting FGF7 using the GelMA-c-rFGF7 sustained-release system can effectively inhibit the fibrosis repair phenomenon in tendon tissue damage and repair, and can effectively treat tendon diseases.

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

[0122] In this example, the in vitro tendon tissue of SCX-GFP mice that had undergone fibrosis after being separated from the normal living environment was first used and cultured in a medium containing or not containing rFGF7 for 7 days for analysis. Then, the in vitro tendon tissue with rFGF7 added and the control group were immunostained for tendon proteins THBS4 and EGR1. It was found that compared with the control group, the in vitro tendon tissue cultured in rFGF7 medium for 7 days had higher expression of the above proteins than the control group ( Fig.14A and Fig. 14B ), while the expression of fibrosis marker proteins COL3 and ACTA2 was lower than that in the control group ( Fig. 14C and Fig.14D ).

[0123] Example 15: Histological scoring related content

[0124] This embodiment provides tendon histological scoring criteria that may be used in other embodiments of the present invention. This embodiment can be established independently or cross-linked / combined with other embodiments.

[0125] The specific standards are as follows:

[0126] Gross standard: normal: 0 points; mild abnormal: 1 point; moderate abnormal: 2 points; severe abnormal: 3 points.

[0127] Rating levels: 0 points; 0.5 points; 1 point; 1.5 points; 2 points; 2.5 points; 3 points.

[0128] Fibrous structure: whether it exists, whether it is continuous and dense, whether it is in bundles, whether it is wavy; if there is fat, blood vessels or cavities, the score will be reduced.

[0129] There is a continuous and dense fiber structure, which is bundled and wavy: 0 points; there is a fiber structure, which is bundled: 1 point; there is a fiber structure, but not bundled: 2 points; there is no fiber structure: 3 points.

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

[0131]

[0132] Although the preferred embodiments of the present invention have been described in the specification, once the basic creative concept is known to those skilled in the art, they may make additional changes and modifications to these embodiments or combinations of embodiments without creative work. Therefore, the protection scope of the present invention should be understood as the scope that the claims are intended to explain and cover, and should not be limited to the parts described in detail in the specification and embodiments of the present invention, and the contents described in the present invention, including the preferred embodiments, include all changes and modifications that fall within the scope of the present invention.

Claims

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

2. Application of fibroblast growth factor 7 in the preparation of drugs for inhibiting tissue and / or cell fibrosis.

3. Application of fibroblast growth factor 7 in inhibiting tendon tissue fibrosis.

4. Application of fibroblast growth factor 7 in the preparation of drugs for inhibiting tendon tissue fibrosis.

5. Application of fibroblast growth factor 7 in inhibiting the fibrotic differentiation of tendon stem cells.

6. Application of fibroblast growth factor 7 in the preparation of drugs for inhibiting the fibrotic differentiation of tendon stem cells.

7. Application of fibroblast growth factor 7 in inhibiting fibrosis of tendon explants.

8. Application of fibroblast growth factor 7 in the preparation of drugs for inhibiting fibrosis of tendon explants.

9. A drug for inhibiting tissue and / or cell fibrosis, characterized in that: The drug includes fibroblast growth factor 7.

10. The drug for inhibiting tissue and / or cell fibrosis according to claim 4, characterized in that: 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.

11. Use of fibroblast growth factor 7 in the preparation of drugs for treating tendinopathy.

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