CN117679493B - Medicine for treating or preventing tendinosis through immune regulation and application thereof - Google Patents
Medicine for treating or preventing tendinosis through immune regulation and application thereof Download PDFInfo
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Abstract
The invention discloses a medicament for treating or preventing tendinosis through immune regulation and application thereof, wherein an active ingredient of the medicament contains a midbrain astrocyte-derived neurotrophic factor or a virus capable of expressing the midbrain astrocyte-derived neurotrophic factor. Compared with the existing tendinosis treatment medicine, the medicine can relieve symptoms, can realize effective treatment aiming at etiology, and can effectively avoid systemic toxic and side effects.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a medicine for treating or preventing tendinosis through immune regulation and application thereof.
Background
Tendinosis (tendinopathy) is a disease which is a kind of disease mainly characterized clinically by pain, reduced function and reduced exercise tolerance, and is usually a degeneration of tendon tissue caused by excessive use and repeated strong traction of muscle, and good hair sites include rotator cuff tendon, medial and lateral epicondylitis of elbow joint, patellar tendon, gluteal tendon, achilles tendon, etc. In recent years, along with the continuous growth of sports population, the incidence rate of tendinosis is continuously increased, and the exercise capacity and the quality of life of patients are seriously affected. The current tendinosis treatment mode is mainly symptomatic treatment, and the effective treatment mode aiming at the etiology is lacking, so that the degeneration process of tendon tissues cannot be delayed.
Immune responses play an important role in the development of tendinopathy, while macrophages play a key role therein. Macrophages can infiltrate into damaged tendons, regulate local inflammatory and healing processes, and thus play an important role. Macrophages are classified into M1 and M2 phenotypes, where M1-type macrophages release various pro-inflammatory cytokines, whereas M2-type macrophages are able to participate in the later healing process, with the ability to clear damaged cells and tissue debris and promote tendon regeneration. Based on the unique properties of macrophages, modulating macrophage heterogeneity may be an effective way to ameliorate tendinosis.
Mesoastrocyte-derived neurotrophic factor (MESENCEPHALIC ASTROCYTE-derived neurotrophic factor, MANF) is an evolutionarily conserved neurotrophic factor that is found in vertebrate and invertebrate species and is expressed to varying degrees in tissues such as the brain, liver, heart, spleen, lung and skeletal muscle of humans and mice, both in the endoplasmic reticulum (endoplasmic reticulum, ER) and golgi apparatus, where brain tissue is found mainly in the cerebral cortex, hippocampus, cerebellum purkinje cells and thalamus and hypothalamus. MANF is a secreted protein, and has unique three-dimensional structure, can activate intracellular signal channels, regulate and control neuronal structures, functions and survival as well as the traditional neurotrophic factors (Neurotrophic Factors, NTF), plays a key role in the growth, survival and maturation of neurons, also participates in endoplasmic reticulum stress (endoplasmic reticulum stress, ERS) and the pathophysiological processes such as inflammatory reaction regulation, has antioxidant, anti-inflammatory and anti-apoptosis activities, and can play a role in protecting cells in animal models of non-neuronal diseases, including retinal injury, diabetes, liver injury, myocardial infarction, nephrotic syndrome and the like. Recent studies have also shown that MANF can play an important role in immune regulation by promoting the conversion of macrophages from M1 type to M2 type, but there has been no related study on whether MANF can improve tendon injury degeneration through immune regulation during tendinopathy.
Adeno-associated viruses (AAV) are increasingly powerful in the field of gene therapy, and more clinical trials are being used for the treatment of specific diseases. AAV is considered to have better safety due to its lower immunogenicity and site-specific integration capacity. Up to now, FDA approved AAV-based gene therapies include Luxturna of Sparktherapeutics, zolgensma of Avexis and ROCTAVIAN of eu approved PTC's Upstaza and BioMarin, respectively, for the treatment of hereditary retinal disease, spinal muscular atrophy, AADC deficiency, and hemophilia. However, there is no AAV-related application for a while in tendinosis treatment.
With the continuous advancement of construction of sports, sports population is growing, resulting in a year-by-year increase in the incidence of tendinopathy, and particularly in the athlete population. At present, the treatment modes aiming at tendinosis are mostly local treatment, and mainly focus on relieving pain and improving functions, and effective repair of damaged degenerated tendon tissues cannot be realized, so that the treatment effects are poor, the side effects are large and other limitations exist. The long-term use of commonly used drugs such as non-steroidal anti-inflammatory drugs can cause serious adverse gastrointestinal reactions and cardiovascular diseases; novel biological agents such as Platelet-rich plasma (PRP) currently lack high levels of research evidence to demonstrate their therapeutic effectiveness; stem cell therapy, which has a significant influence on proliferation and differentiation of stem cells due to donor specificity (such as age and sex), and cell culture techniques are also affected by unstable factors such as safety and quality control, and has a great limit in wide application; surgical suturing is the only effective treatment for tendon rupture, but surgery causes great trauma to the patient and has a high recurrence rate, causing serious physical and psychological distress to the patient. Thus, there is an urgent need to develop novel therapeutic drugs for tendinosis.
Disclosure of Invention
For the above reasons, the present invention aims to provide a medicament for treating tendinosis through immune regulation and application thereof. Specifically, in order to achieve the purpose of the present invention, the present invention adopts the following technical scheme:
In one aspect, the invention relates to a medicament for the treatment or prevention of tendinopathy by immunomodulation, the active ingredient of which comprises a mesogenic astrocyte-derived neurotrophic factor or a virus capable of expressing a mesogenic astrocyte-derived neurotrophic factor.
In a preferred embodiment of the present invention, the virus capable of expressing a mesogenic astrocyte-derived neurotrophic factor is an adenovirus. The invention uses the advantages of high AAV safety and strong specificity as a delivery carrier to realize the local high-efficiency expression of the neurotrophic factor MANF in damaged degenerated tendon tissues, and promotes the transformation of macrophages infiltrated in damaged parts from an M1 pro-inflammatory phenotype to an M2 repair phenotype, thereby remarkably reducing inflammatory response and promoting repair and regeneration of the tendon tissues.
In a preferred embodiment of the invention, the adenovirus has the nucleotide sequence shown in SEQ ID No. 5.
In a preferred embodiment of the invention, the medicament is an injection.
The invention also relates to application of the medicine in preparing a medicament for preventing or treating tendinosis.
In a preferred embodiment of the invention, the agent is used to relieve pain, improve balance, reduce inflammatory response, restore tendon tissue fiber alignment and/or inhibit collagen fiber alignment disorder.
Advantageous effects
The invention utilizes Adeno-associated virus (AAV) capable of over-expressing midbrain astrocyte-derived neurotrophic factor (MESENCEPHALIC ASTROCYTE-derived neurotrophic factor, MANF) to carry out local injection treatment on a rat model with tendinosis, and proves that the AAV-MANF has good treatment effect on tendinosis through multiple aspects of behavioral manifestations, general appearance of tendon tissues, histopathology and the like, thereby providing theoretical basis for developing new clinical treatment strategies of tendinosis. Compared with the existing tendinosis treatment medicine, the medicine can relieve symptoms, can realize effective treatment aiming at etiology, and can effectively avoid systemic toxic and side effects.
Drawings
Fig. 1: schematic of the structure map of vector GV 411;
Fig. 2: von frey test results for group 1-4 rats;
fig. 3: hot plate experimental results for group 1-4 rats;
fig. 4: balance bar test results for group 1-4 rats;
fig. 5: group 1-4 a general view of the achilles tendon site in rats;
fig. 6: group 1-4 rat achilles tendon tissue scanning electron microscope detection results;
Fig. 7: h & E staining of group 1-4 rat achilles tendon tissue;
Fig. 8: group 1-4 rats Achilles tendon tissue Masson staining results;
Fig. 9: the Western blot test results can further confirm that AAV-MANF viruses can successfully transfect cells and express MANF proteins.
Detailed Description
In order to further understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless otherwise specified, all reagents involved in the examples of the present invention are commercially available products and are commercially available.
Examples
1. Construction of the over-expressed adeno-associated viral vector AAV-MANF
1.1 Cloning of MANF-encoding Gene into viral vector GV411
① Gene information
Gene name: MANF (NM_ 006010)
Species: human body
② Carrier information
Carrier name: GV411, the structure map of which is shown in FIG. 1.
Element sequence: CMV-betaGlobin-MCS-3Flag-SV40 PolyA
Cloning site: bamHI/NheI
③ Acquisition of the Gene fragment of interest
Table 1: PCR amplified primer sequence (containing exchange pairing base, enzyme cutting site and 5' end part sequence of target gene for PCR fishing target gene):
PCR product size: 593bp
④ Recombinant plasmid construction
Product exchange into linearized expression vectors
Table 2: PCR identification primer sequence:
and (3) PCR identification: the size of the PCR product of the positive transformant is 730bp.
Comparing the positive clone sequencing results, wherein the thickened part in the sequencing results is consistent with the MANF sequence of the target gene, which shows that the transformation is successful:
aactggaggtggaggtagtggaatggatcccgccaccatgaggaggatgtgggccacgcaggggctggcggtggcgctggctctgagcgtgctgccgggcagccgggcgctgcggccgggcgactgcgaagtttgtatttcttatctgggaagattttaccaggacctcaaagacagagatgtcacattctcaccagccactattgaaaacgaacttataaagttctgccgggaagcaagaggcaaagagaatcggttgtgctactatatcggggccacagatgatgcagccaccaaaatcatcaatgaggtatcaaagcctctggcccaccacatccctgtggagaagatctgtgagaagcttaagaagaaggacagccagatatgtgagcttaagtatgacaagcagatcgacctgagcacagtggacctgaagaagctccgagttaaagagctgaagaagattctggatgactggggggagacatgcaaaggctgtgcagaaaagtctgactacatccggaagataaatgaactgatgcctaaatatgcccccaaggcagccagtgcacggaccgatttggctagcgactacaaggatgacgatgacaaggattacaaagacgacgatgataaggactataaggatgatgacgacaaaaagctttaaa
1.2 The recombinant expression plasmids described above were co-transfected into AAV-293 cells (providing the trans-acting factors required for AAV replication and packaging) with pHelper (carrying adenovirus-derived genes) and pAAV-RC (carrying AAV replication and capsid genes). Recombinant AAV-MANF was assembled in packaging cells 2 to 3 days post transfection.
1.3 AAV-MANF viral particles are collected from infected AAV-293 cells, typically the AAV particles are enriched in packaging cells, so that a substantial portion of the AAV particles can be recovered by harvesting the cells and then lysing the cells to release the AAV particles to the supernatant. The viral supernatant obtained in this step is then used in an infection experiment to infect various mammalian cell lines. At the same time, the virus in the supernatant can also be concentrated and reserved.
1.4 The virus supernatant from the previous step is concentrated and purified, and the primary supernatant contains a plurality of cellular protein molecules and fragments, and most of the cellular proteins and residual CsCl ions can be removed by 2 CsCl density gradient centrifugation and 1 ultrafiltration.
1.5 The titer of the resulting virus was measured by quantitative PCR, which gives the physical titer of the AAV genome packaged into particles. In the experiment, a standard curve is established by using plasmids as standard substances, and the titer of the sample to be detected is obtained after the AAV sample to be detected is compared with the standard curve.
TABLE 3 Table 3
Table 4: standard curve copy number
TABLE 5
Standard curve y= -3451LOG (X) +41.47, where r2=1.000, eff=94.9%
Titer calculation
Substituting Ct value of target sample into formula to obtain titer of sample to be measured, multiplying dilution multiple to obtain original titer of sample, averaging titer values of four groups to obtain average titer, namely final titer (7.62E+13 v.g./mL) of sample.
TABLE 6
The Western blot assay results shown in FIG. 9 further demonstrate that AAV-MANF viruses can successfully transfect cells and express MANF proteins.
Table 7: fig. 9 sample illustrates:
the results illustrate: the experimental group detected specific protein bands around 26KD, confirming successful overexpression of MANF in cells.
2. Tendinosis rat model establishment and AAV-MANF injection operation
Animal selection: male SD rats, 250g, were randomly divided into 4 groups of 8.
① AAV injection
Group 1 (blank): 30 mu L physiological saline is injected to hind limb achilles tendon
Group 2 (tendinosis modeling): 30 mu L physiological saline is injected to hind limb achilles tendon
Group 3 (control AAV treatment): hindlimb achilles tendon injection 1E+11 vg AAV control virus (30 [ mu ] L physiological saline solution)
Group 4 (AAV-MANF treatment): hindlimb achilles tendon injection 1E+11 vg AAV-MANF (dissolved in 30 [ mu ] L physiological saline)
Type I collagenase injection was performed 7 days later.
② Type I collagenase injection
Group 1 (blank): hindlimb achilles tendon injection 30 [ mu ] L PBS
Group 2 (tendinosis modeling): hindlimb achilles tendon injection of collagenase type 0.3 mg I (30 μl PBS)
Group 3 (control AAV treatment): hindlimb achilles tendon injection of collagenase type 0.3 mg I (30 μl PBS)
Group 4 (AAV-MANF treatment): hindlimb achilles tendon injection of collagenase type 0.3 mg I (30 μl PBS)
After 28 days, behavioural tests are performed and materials are obtained to complete the subsequent pathological detection.
3. Behavioural testing
The ciliated mechanical stimulation needle (von Frey) test, the hot plate test, and the balance bar test were performed on each group of rats, and the degree of pain in the hind limb and the balance of the limb were measured. Von frey and hot plate experiments showed that rats (Model) in the tendinosis Model group had significantly increased limb pain sensitivity compared to normal control group rats (NC), whereas AAV-MANF treatment significantly improved this symptom (fig. 2, 3). The balance bar results showed that the tendinosis model rats had significantly reduced limb balance compared to normal control rats, while AAV-MANF treatment significantly enhanced limb balance in rats (fig. 4).
4. General visual assessment
The hind limbs of each group of rats were dissected and the achilles tendon sites were photographed in general. The results showed that the rats in the tendinosis model group showed obvious bleeding, swelling and tissue degeneration at the achilles tendon site, the inflammatory response was obvious, and the AAV-MANF treatment significantly delayed this pathology compared to the normal control group rats (fig. 5).
5. Histopathological detection
The achilles tendon tissue of each group of rats was taken and examined by a scanning electron microscope, and the results showed that the arrangement of the achilles tendon tissue fibers of the rats in the tendinosis model group was significantly disturbed as compared with that of the normal control group of rats, and the AAV-MANF treatment allowed the arrangement of the achilles tendon tissue fibers of the rats to be restored to a certain extent (FIG. 6).
H & E and Masson staining results show that severe collagen fiber arrangement disorder and inflammatory infiltration exist in tendinous tissue of tendinous rats, whereas collagen arrangement is obviously more orderly in the tendinous tissue of rats in AAV-MANF treatment group, fiber volume is thickened, inflammatory response degree is obviously improved, and the whole is close to that of normal rats (figures 7 and 8).
The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations to the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.
Claims (4)
1. Use of a medicament for the treatment of tendinopathy, the active ingredient of which comprises a virus capable of expressing a astrocyte-derived neurotrophic factor in the middle brain, said virus capable of expressing a astrocyte-derived neurotrophic factor being an adenovirus, for the manufacture of a medicament for the treatment of tendinopathy.
2. The use according to claim 1, wherein the adenovirus has the nucleotide sequence shown in SEQ ID No. 5.
3. The use according to claim 1, wherein the medicament is an injection.
4. Use according to any one of claims 1-3, for the preparation of a medicament for alleviating pain, improving balance, alleviating inflammatory response, restoring tenosynovial fiber alignment and/or inhibiting collagen fiber alignment disorders.
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