Application of transcription factor CREB3L1 in preparation of medicine for promoting repair of damaged tendon in old people

Abstract

The application discloses a transcription factor CREB3L1 The application relates to the technical field of biological medicines, and discloses an application of a transcription factor in the preparation of a medicine for promoting the repair of damaged tendons of the elderly. ‌ COL1A1 And SPARC The application discloses a transcription factor CREB3L1 which can be combined with the promoter region of the gene through software prediction, ‌COL1A1 And SPARC Transcription and translation are promoted, so that the curative effect of tendon injury repair of the elderly is improved. CREB3L1 In-vivo experiments prove that overexpression can significantly improve tendon sliding function, reduce tendon adhesion, and greatly improve tendon healing strength and Young's modulus, thereby promoting the healing of damaged tendons. CREB3L1 In-vitro experiments further prove that overexpression can significantly enhance the proliferation activity of human tendon cells. The application provides a novel and effective scheme for the clinical treatment of tendon injury of the elderly, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to transcription factors. CREB3L1 Application in the preparation of drugs that promote the repair of damaged tendons in the elderly. Background Technology

[0002] Tendons, as dense connective tissues connecting muscles and bones, are an important component of the human musculoskeletal system. Relying on their excellent mechanical properties and structural stability, they effectively transmit muscle contraction forces, ensuring normal limb movement. Currently, tendon injuries are increasingly prominent among the elderly, becoming a significant health issue affecting their quality of life. Due to the decline in physiological function, the tendon tissue of the elderly undergoes a series of degenerative changes, including disordered collagen fiber arrangement, loss of matrix components, and reduced fibroblast activity, significantly reducing the tendon's resistance to injury and making them a high-risk group for tendon injuries. Epidemiological survey data shows a distinctly high incidence of tendon injuries in the elderly. The age distribution of Achilles tendon ruptures exhibits a bimodal pattern, with the first peak around age 50, and a second peak occurring in the elderly. Furthermore, compared to younger populations, the incidence of tendon injuries such as rotator cuff tears and biceps tendon ruptures is significantly higher in the elderly, and these injuries are often triggered by minor external forces, sometimes even resulting in spontaneous ruptures, fully demonstrating the vulnerability of tendon tissues in the elderly.

[0003] Compared to younger people, the repair process of tendon injuries in the elderly presents significant limitations, becoming a challenge in clinical treatment. In young people, after tendon injury, fibroblasts can rapidly proliferate and synthesize large amounts of matrix components such as collagen, allowing for orderly reconstruction of collagen fibers and a rapid recovery of the tendon's mechanical properties to normal levels. However, in the elderly, the repair response is slow, the proliferation and secretory functions of fibroblasts are significantly weakened, collagen synthesis is insufficient, and fiber assembly is disordered. This results in the injured area primarily undergoing granulation tissue repair, leading to poor mechanical strength and unstable structure. This not only significantly prolongs the repair period but also greatly increases the risk of complications such as tendon adhesions, poor healing, and re-rupture.

[0004] Currently, clinical treatment for tendon injuries primarily involves surgical repair combined with postoperative rehabilitation training, supplemented by symptomatic treatments such as nonsteroidal anti-inflammatory drugs (NSAIDs) and nutritional supplements. However, existing treatments cannot fundamentally address the core issues in tendon injury repair in the elderly. Surgical repair can only achieve anatomical connection of the tendon and cannot improve the self-repairing ability of the tendon tissue in the elderly; while rehabilitation training can promote tendon function recovery, its effectiveness is often unsatisfactory due to the inherent limitations of the repair tissue; existing drugs mostly focus on relieving inflammation and reducing pain, lacking specific targeting for tendon injury repair, and cannot effectively promote collagen synthesis or improve fibroblast activity, thus failing to improve the quality of tendon injury repair in the elderly. Therefore, developing treatments that can effectively promote tendon injury repair in the elderly has become a crucial technological need that urgently needs to be addressed in the biomedical field. Summary of the Invention

[0005] The purpose of this invention is to provide transcription factors. CREB3L1 This invention addresses the problems existing in the prior art by being applied in the preparation of drugs that promote the repair of injured tendons in the elderly. It provides a novel and effective solution for the clinical treatment of tendon injuries in the elderly and has promising clinical application prospects.

[0006] This invention, through single-cell sequencing of aged and young tendon tissues, discovered... COL1A1 and SPARC Both genes were significantly downregulated in fibroblasts of aged tendon tissue. COL1A1 The α1 chain encoding type I collagen, its expression product and COL1A2 The encoded α2 chain assembles to form type I collagen heterotrimers, which undergo propeptide cleavage and then self-assemble into well-organized collagen fibers, serving as the structural and mechanical basis of tendons. Downregulation of SPARC expression directly reduces collagen synthesis and disrupts fiber arrangement, a key molecular marker of age-related tendon degeneration. As a collagen-binding stromal cell protein, SPARC maintains tendon homeostasis by regulating type I collagen fiber assembly and promoting mechanotransduction in fibroblasts. Deletion or mutation of SPARC leads to impaired tendon development and maturation. Therefore, it is hypothesized that… COL1A1 and SPARC The significant decline in these two genes may be an important reason why tendons are more susceptible to injury and have poor repair outcomes in the elderly. Figure 1 Based on this, the present invention has discovered a transcription factor CREB3L1 that can simultaneously bind to (A). COL1A1 and SPARC The promoter region of a gene promotes COL1A1 and SPARC The transcription and translation of these substances can improve the efficacy of tendon injury repair in the elderly. Figure 1 (B)

[0007] Based on this, the present invention provides the following solution: This invention provides transcription factors CREB3L1 Application in the preparation of drugs that promote the repair of damaged tendons in the elderly.

[0008] This invention also provides overexpression transcription factors CREB3L1 The application of the reagent in the preparation of drugs that promote the repair of damaged tendons in the elderly.

[0009] Furthermore, the reagent is an overexpression vector.

[0010] Furthermore, the overexpression vector is a lentiviral overexpression vector.

[0011] Furthermore, the reagent is a lentivirus.

[0012] This invention also provides a drug for promoting the repair of damaged tendons in the elderly, the active ingredient of which includes an overexpressed transcription factor. CREB3L1 The reagent.

[0013] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0014] The present invention also provides a method for promoting tendon cell proliferation in vitro, comprising overexpressing transcription factors in tendon cells. CREB3L1 The steps.

[0015] Furthermore, by overexpressing transcription factors CREB3L1 The reagent was used to transfect tendon cells to achieve overexpression of transcription factors in tendon cells. CREB3L1 .

[0016] Furthermore, the reagent is a lentivirus.

[0017] The present invention discloses the following technical effects: This invention discovers a transcription factor CREB3L1 It has the function of promoting the repair of damaged tendons in elderly individuals. In vivo experiments have confirmed that... CREB3L1 Overexpression significantly improves tendon gliding function, reduces peritendinous adhesions, and substantially enhances tendon healing strength and Young's modulus, promoting the healing of injured tendons. This transcription factor can simultaneously bind to... COL1A1 and SPARC The promoter region of the gene significantly promotes the transcription and translation of both, upregulates the expression of related proteins, and compensates for the deficiencies of insufficient collagen synthesis and disordered fiber assembly in aging tendons at the molecular level. In vitro experiments further demonstrate that... CREB3L1Overexpression significantly enhances the proliferative activity of human tendon cells. This invention achieves targeted regulation of tendon repair in elderly individuals, fundamentally improving the self-repair ability of elderly tendons, providing a novel and effective solution for the clinical treatment of tendon injuries in the elderly, and possessing promising clinical application prospects. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 A schematic diagram illustrating the molecular mechanism by which CREB3L1 promotes tendon injury repair in the elderly; where A represents... COL1A1 and SPARC A schematic diagram illustrating the mechanism by which downregulation of expression levels affects the repair of injured tendons in the elderly; B represents CREB3L1 targeted binding. ‌ COL1A1 , SPARC A schematic diagram illustrating the mechanism by which gene promoter regions promote transcription and translation, thereby enhancing collagen fiber and extracellular matrix synthesis and tendon tensile strength; Figure 2 The images show the results of the glide function test of rat tendons. A shows the flexion angles of the toes in the Con, LV-NC, and LV-Creb3l1 groups under different weight loads; B shows a statistical graph of the flexion angles of the toes in the Con, LV-NC, and LV-Creb3l1 groups under different weight loads; and C shows the morphological observation of tendon adhesions in the three groups of rats. Figure 3 The graph shows the results of the mechanical properties test of rat tendons; where A is the gross morphology of the tendons in the Con group, LV-NC group, and LV-Creb3l1 group; B is a statistical graph of the tendon healing strength in the three groups of rats; and C is a statistical graph of the Young's modulus of the tendons in the three groups of rats. Figure 4 for CREB3L1 Fluorescence detection image of in vivo transfection efficiency of overexpressing virus; Figure 5 Immunohistochemical staining images of CREB3L1, COL1A1, and SPARC protein expression in tendon tissues of three groups of rats; Figure 6 This is a graph showing the results of detecting the cell viability of human tendon cells at 24h, 48h, 72h, and 96h. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Terminology Explanation: CREB3L1 refers to cAMP response element binding protein 3-like protein 1, also known as OASIS. It is a member of the CREB3 transcription factor family and a key transmembrane transcription factor in endoplasmic reticulum stress response.

[0026] COL1A1 refers to the α1 subunit of type I collagen, which is a core structural protein of the extracellular matrix of tissues such as bone, skin, and tendons. It mainly participates in the assembly of collagen fibers, provides structural support and mechanical strength for tissues, and plays an important role in the mineralization of bone matrix.

[0027] SPARC refers to osteonectin, an acidic secretory protein rich in cysteine, belonging to matrix regulatory proteins. Its main functions are to bind collagen and calcium ions, regulate collagen assembly and bone mineralization, and participate in physiological processes such as cell-matrix interactions, tissue remodeling, and wound repair.

[0028] The rat involved in this invention Creb3l1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; human... CREB3L1 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0029] SEQ ID NO.1:

[0030] SEQ ID NO.2:

[0031] Example 1 I. Materials and Methods 1. Creb3l1 Construction of overexpression lentiviral vector Creb3l1 Both the overexpressing lentivirus (LV-Creb3l1) and the control lentivirus (LV-NC) were purchased from Synbio Technologies (Suzhou, Jiangsu, China).

[0032] Construction method of LV-Creb3l1: using rats Creb3l1 Using the gene as a template, a gene fragment with restriction enzyme sites was amplified by PCR. The gene fragment was cloned into the pLVX-AcGFP1-N1 lentiviral vector (purchased from Synbio Technologies) using EcoRI and BamHI restriction endonucleases, and packaged using a lentiviral expression system to obtain the recombinant virus LV-Creb3l1.

[0033] A control lentivirus LV-NC was constructed using the pLVX-AcGFP1-N1 empty vector.

[0034] 2. Establishment of a model of flexor tendon injury in aged rats Eighteen 18-month-old male SD rats (36 tendons) were used and then randomly divided into three groups: a surgical repair group (control group), a surgical repair group injected with LV-NC after surgery, and a surgical repair group injected with LV-Creb3l1 after surgery. Each tendon was injected with 2×10- 9 Each viral particle was 10 μL in volume. Twelve tendon samples from each group were used to assess gliding function. Nine tendon samples were then subjected to anatomical and biomechanical testing, while the remaining three were used for histological analysis without dissection.

[0035] For the surgical procedure, anesthesia was first administered via intraperitoneal injection of 3 mL / kg 2% sodium pentobarbital. A 0.05 mL solution of 50% lidocaine hydrochloride containing 1% adrenaline was then injected into the sole of the foot to prevent severe bleeding. After completely severing the flexor tendons with a scalpel blade, each tendon was repaired using a 2-strand Kessler technique with 5-0 polypropylene sutures (Prolene, Ethicon, Somerville, NJ, USA), followed by a circumferential repair. For the LV-NC and LV-Creb3l1 injection groups following surgical repair, 2×10 mm sutures were inserted along the suture hole direction using a microinjector before knotting. 9 The virus particles were slowly injected into the interior of each tendon. Finally, the skin was sutured with 3-0 nylon sutures (HOLYCON, Nantong, Jiangsu, China).

[0036] 3. Testing of tendon gliding function Three weeks later, the rats were euthanized, and the hind limbs were subsequently amputated at the knee joint. The proximal flexor tendon along the tibia was dissected at the proximal tarsal bone. The proximal end of the tendon was secured with two injection needles (to avoid tendon penetration), and the proximal end of the tendon was connected to a weight using a single-strand suture. A tendon load was applied by freely suspending weights (0, 5, 10, 15, and 20 g). Passive flexion of the toes was promoted by proximal weight traction, starting with full toe extension. The flexion angle was defined as the angle between the longest toe and the horizontal plane, and was recorded by photographs and a protractor.

[0037] 4. Biomechanical testing The rat paw was dissected to observe the morphology and appearance of the adhesions. The repaired tendon was then gently detached from the surrounding tissue and completely excised along its entire length for strength and elastic modulus testing. The tendon was fixed at both ends in the lower and upper clamps of a biomechanical testing instrument (Model 3365; Instron Corp.), with the repair site centered between the clamps. The upper clamp was then pulled upwards at a constant speed of 25 mm / min until the tendon ruptured. Load-displacement curves were recorded using Instron Series IX testing software, with a sharp drop indicating rupture at the repair site.

[0038] 5. In vivo transfection efficiency and immunohistochemical staining Complete paws containing tendon injuries from three groups of rats were removed and fixed with 4% paraformaldehyde at room temperature for 24 hours. The samples were then dehydrated and embedded in paraffin blocks. 5 μm sections were cut using a paraffin microtome for in vivo transfection efficiency observation and immunohistochemical staining analysis. After dewaxing and washing, the tissue sections were stained with DAPI, mounted with anti-fluorescent mounting medium, and the green fluorescence expression around the wells was observed under a microscope. For immunohistochemical staining, the sections underwent antigen retrieval, followed by blocking with tissue blocking solution at room temperature for 2 hours. Slides were then incubated overnight at 4°C with rabbit anti-CREB3L1 antibody (1:200, Boster, Wuhan, China), rabbit anti-SPARC antibody (1:500, Abcam, Cambridge, UK), and rabbit anti-COL1A1 antibody (1:500, Abcam), respectively. The slides were then washed three times with PBS and incubated with secondary antibodies at 37°C for 2 hours. After washing, the slides were stained with 3,3-diaminobenzidine (DAB, Sigma, St. Louis, MO, USA) and hematoxylin. Finally, the slides were sealed and observed under a microscope (Leica DMR 3000; Leica, Bensheim, Germany).

[0039] II. Results and Analysis 1. In vivo application Creb3l1 Overexpression of the virus significantly improved the sliding function of injured tendons in aged rats. Before dissecting the tendon, this invention first tests the sliding function of the rat tendon, which can be obtained from... Figure 2 The study found that, starting with a weight of 10 g, the flexion angle of the longest toe in the LV-Creb3l1 group gradually increased compared to the blank control group (Con) and the negative control group (LV-NC). Statistical analysis revealed that, although there was no statistically significant difference between 5 g and 10 g weights, the average flexion angle in the LV-Creb3l1 group was greater than that in the Con and LV-NC groups. When the load increased to 15 g, the average flexion angle in the LV-Creb3l1 group was 6.33°±4.46°, while the flexion angles in the Con and LV-NC groups were 3.50°±2.24° and 2.42°±2.27°, respectively. When the load increased to 20 g, the average flexion angle in the LV-Creb3l1 group was 9.67°±5.10°, while the flexion angles in the Con and LV-NC groups were 5.67°±3.94° and 4.25°±2.96°, respectively. Under both loads, the mean flexion angle of rats in the LV-Creb3l1 group was significantly greater than that of rats in the LV-NC group ( Figure 2 (B) After dissection, compared with the Con group and LV-NC group rats, the adhesions around the tendons of the LV-Creb3l1 group rats were more sparse ( Figure 2 (C)

[0040] 2. In vivo application Creb3l1 Overexpression of the virus significantly improved the mechanical properties of damaged tendons in aged rats. After dissecting the tendon, the healing of the tendon after injury repair was first observed grossly. It was observed that the tendon ends of the LV-Creb3l1 group rats were almost connected by milky white tendinous tissue, while the tendons of the Con group and LV-NC group rats were mostly connected by translucent granulation tissue at the tendon ends. Figure 3 (A). This invention utilizes a biomechanical testing instrument to detect the tensile strength and elastic modulus of tendons. Regarding healing strength, the healing strength of the tendons in the Con group and LV-NC group rats were 22.48±4.32 N and 23.60±8.76 N, respectively, while the healing strength of the LV-Creb3l1 group rats was 32.62±8.77 N, significantly increasing by 145% and 138%, respectively, showing a statistically significant difference. Figure 3(B) Regarding elastic modulus, the elastic modulus of the LV-Creb3l1 group rats was 11.69±3.01 N / mm, which was significantly greater than that of the Con group (8.10±1.22 N / mm) and the LV-NC group rats (8.60±1.42 N / mm). Figure 3 (C)

[0041] 3. Creb3l1 In vivo transfection efficiency of overexpressing the virus Observation of sections showed no specific green fluorescence around the suture holes in the tendons of rats in the Con group, while a large number of green fluorescent cells were observed around the suture holes in the tendons of rats in the LV-NC and LV-Creb3l1 groups. Figure 4 ).

[0042] 4. In vivo application Creb3l1 Overexpression of the virus can promote the expression of CREB3L1, COL1A1, and SPARC proteins. This invention uses immunohistochemical staining to observe the expression of three proteins, CREB3L1, COL1A1, and SPARC, in the tendon tissue around the suture hole. It can be observed that compared to the Con group and the LV-NC group, the positive signals of CREB3L1, COL1A1, and SPARC in the tendon tissue of the LV-Creb3l1 group were all stronger than those in the other two control groups. Figure 5 ).

[0043] In summary, the present invention will Creb3l1 After overexpressing lentivirus and applying it to aged rats, the mechanical properties of the repaired tendons were evaluated, and the expression of CREB3L1, COL1A1, and SPARC proteins was analyzed. The results showed that it significantly improved the sliding function and enhanced the healing strength of aged rats. This is because the transcription factor CREB3L1 can simultaneously bind to... Col1a1 and Sparc The promoter region of a gene, through overexpression Creb3l1 It can promote Col1a1 and Sparc The transcription and translation of these transcription factors were investigated, thereby increasing the expression of COL1A1 and SPARC proteins in aged tendon tissue, thus clarifying the role of overexpression of transcription factors. Creb3l1 It can promote the repair of tendon injuries.

[0044] Example 2 I. Materials and Methods 1. CREB3L1 Construction of overexpression lentiviral vector CREB3L1Both the overexpressing lentivirus (LV-Creb3l1) and the control lentivirus (LV-NC) were purchased from Synbio Technologies (Suzhou, Jiangsu, China).

[0045] The construction method of LV-Creb3l1: based on human... CREB3L1 Using the gene as a template, a gene fragment with restriction enzyme sites was amplified by PCR. The gene fragment was cloned into the pLVX-AcGFP1-N1 lentiviral vector using EcoRI and BamHI restriction endonucleases, and packaged using a lentiviral expression system to obtain the recombinant virus LV-Creb3l1.

[0046] A control lentivirus LV-NC was constructed using the pLVX-AcGFP1-N1 empty vector.

[0047] 2. Culture of human tendon cells Discarded tendon tissue was obtained from elderly patients with tendon rupture and rinsed with sterile PBS. Tissue blocks approximately 2 mm × 2 mm × 2 mm were cut and placed in culture dishes containing complete culture medium (90% DMEM, 10% fetal bovine serum, 100 μg / mL streptomycin, 100 U / mL penicillin) and cultured in a cell culture incubator (37℃, 5% CO2). After 7 days of culture, tendon cells migrated from the tissue blocks and proliferated rapidly. Human tendon cell lines were obtained through passages for CCK8 assays.

[0048] 3. CCK8 Experiment Two experimental groups were set up: an LV-NC negative control group and an LV-Creb3l1 experimental group. Human tendon cells were seeded at a density of 10,000 cells per well in four 96-well plates (measured at 24h, 48h, 72h, and 96h post-transfection), with six wells per plate and three replicates per group. When cell confluence reached approximately 80%, cells were transfected according to the experimental groups, with approximately 1 × 103 cells per well. 5 Collect virus particles and, after 24 h of culture, discard the supernatant and replace with fresh culture medium. For the CCK8 plate to be tested, add 90 μL of fresh culture medium and 10 μL of 10×CCK-8 solution (Servicebio) to each well, and continue incubation in a cell culture incubator for 2 h. Finally, use a microplate reader to detect the absorbance value of each well at a wavelength of 450 nm.

[0049] II. Experimental Results CCK8 assays revealed that overexpression occurred at 24 and 48 hours post-transfection. CREB3L1 The tendon cells in the LV-Creb3l1 group did not show significant proliferative activity. However, at 72 and 96 hours, the cell viability in the LV-Creb3l1 group was significantly increased compared to the LV-NC negative control group. Figure 6 Therefore, CREB3L1 Overexpression can promote the proliferation of human tendon cells, thereby facilitating tendon injury repair.

[0050] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Transcription factors CREB3L1 Its application in the preparation of drugs that promote the repair of damaged tendons in the elderly is characterized by, The transcription factor CREB3L1 The nucleotide sequence is shown in SEQ ID NO.

2.

2. The application of a reagent that overexpresses the transcription factor Creb3l1 in the preparation of a drug that promotes the repair of injured tendons in the elderly, characterized in that... The transcription factor CREB3L1 The nucleotide sequence is shown in SEQ ID NO.

2.

3. The application according to claim 2, characterized in that, The reagent is an overexpression vector.

4. The application according to claim 3, characterized in that, The overexpression vector is a lentiviral overexpression vector.

5. The application according to claim 2, characterized in that, The reagent is a lentivirus.

6. A method for promoting tendon cell proliferation in vitro, characterized in that, Including overexpression of transcription factors in tendon cells CREB3L1 The steps.

7. The method according to claim 6, characterized in that, By overexpressing transcription factors CREB3L1 The reagent was used to transfect tendon cells to achieve overexpression of transcription factors in tendon cells. CREB3L1 .

8. The method according to claim 7, characterized in that, The reagent is a lentivirus.

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