Application of sJAG1 protein in improvement of SVF performance

Abstract

The invention relates to the field of regenerative medicine and tissue engineering, in particular to application of sJAG1 protein to improvement of SVF performance. When the sJAG1-improved SVF is used for treating osteoarthritis, the anti-inflammatory capacity, the tissue repair capacity and the cartilage regeneration capacity of the SVF can be enhanced, so that the treatment effect on osteoarthritis is improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the use of sJAG1 protein in improving the performance of SVF. Background Art

[0002] Osteoarthritis, also known as osteoarthrosis, degenerative arthritis, proliferative arthritis, senile arthritis, etc. It is a common disease in middle-aged and elderly people caused by the degeneration of articular cartilage, resulting in joint pain and joint dysfunction (including joint deformities). Commonly referred to as bone spurs, osteophytes, bowlegs, cervical spondylosis, patellar chondromalacia, lumbar diseases, such as disc herniation caused by the degeneration of the lumbar intervertebral disc, all belong to the category of osteoarthritis. The main pathological changes of osteoarthritis are the degeneration and disappearance of cartilage, as well as the reactive hyperplasia of bone spurs formed at the attachment of the marginal ligaments of the joint and subchondral bone, which causes joint pain, stiffness, deformity and dysfunction. Clinically, the disease can be divided into two categories: primary and secondary. Primary osteoarthritis refers to joint lesions that are not related to other diseases with age aging, while secondary osteoarthritis is caused by injuries, inflammation, genetics, metabolism, endocrine and other diseases. Current treatment methods include non-steroidal anti-inflammatory drug treatment, cell therapy, surgical therapy, etc., but there are still problems such as high treatment costs and strong side effects.

[0003] SVF, scientifically named "stromal vascular fraction", is an effective component extracted from adipose tissue autologous to the patient, and is a cell population formed by a mixture of various repair-functional cells and cytokines. Research shows that SVF contains mesenchymal stem cells, M2 macrophages and TGF-β1 that promote the regeneration of chondrocytes. Therefore, collecting the patient's own SVF to treat osteoarthritis is an effective method. However, the quality of SVF varies greatly, resulting in poor treatment effects on osteoarthritis. Summary of the Invention

[0004] Based on the above technical problems, the present invention provides a use of sJAG1 in improving the performance of SVF, and a use of SVF improved by sJAG1 in treating osteoarthritis. Experiments have proved that sJAG1 improves the heterogeneity of SVF, promotes the polarization of M1 macrophages into M2 macrophages and prevents neutrophil infiltration by inhibiting the Notch signal transduction pathway, thereby improving the treatment effect of osteoarthritis.

[0005] The specific technical solutions provided by the present invention are as follows: In the first aspect of the present invention, a use of sJAG1 in improving the performance of SVF is provided. The SVF is adipose tissue from the patient's own body, and is a multi-cell mixture rich in mesenchymal stem cells, macrophages, endothelial cells and various cytokines digested by collagenase.

[0006] As a preferred embodiment of the present invention, the sJAG1 is used to enhance the anti-inflammatory, tissue repair or cartilage regeneration promoting ability of SVF.

[0007] In a second aspect of the present invention, there is provided a pharmaceutical composition, which is formed by mixing sJAG1 and SVF, and the dosage ratio of sJAG1 to SVF in each 1 μL of the mixed solution is 0.08 ng - 0.5 ng: 10,000 cell amounts.

[0008] In a third aspect of the present invention, there is provided a drug, which comprises the pharmaceutical composition.

[0009] As a preferred embodiment of the present invention, the drug has any one of the following uses: (1) Anti-inflammatory; (2) Tissue repair; (3) Cartilage regeneration promotion.

[0010] As a preferred embodiment of the present invention, the drug takes the pharmaceutical composition as the sole active ingredient.

[0011] As a preferred embodiment of the present invention, the drug is compounded from the pharmaceutical composition and pharmaceutically acceptable excipients.

[0012] As a preferred embodiment of the present invention, the drug is an injection preparation. The injection preparation is used by intra-articular injection into the joint capsule.

[0013] In a fourth aspect of the present invention, there is provided an application of the pharmaceutical composition or the drug in the preparation of a drug for treating osteoarthritis.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention discovers that using sJAG1 during the treatment of osteoarthritis with SVF can improve the treatment performance of SVF. By regulating the SVF cell population with sJAG1, upregulating M2 macrophages, increasing the survival rate of cells such as ADSC, and preventing the infiltration of neutrophils, the anti-inflammatory, tissue repair and cartilage regeneration promoting abilities of SVF can be enhanced, thereby improving the treatment effect of osteoarthritis. Description of the Drawings

[0015] Figure 1 is the mRNA expression level of the MMP8 gene in SVF treated and untreated with sJAG1.

[0016] Figure 2 is the detection of the immune cell surface marker CD45 in SVF.

[0017] Figure 3 is the staining map of the sagittal section of the joint of a mouse untreated after DMM surgery.

[0018] Figure 4 Sagittal section staining map of the joints of mice treated with SVF 2 weeks after DMM surgery.

[0019] Figure 5 Sagittal section staining map of the joints of mice treated with sJAG1 after DMM surgery.

[0020] Figure 6 Sagittal section staining map of the joints of mice treated with sJAG1 + SVF combination after DMM surgery. Detailed implementation manners

[0021] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the accompanying drawings, where the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the accompanying drawings are exemplary and are intended to explain the present invention, and should not be construed as a limitation to the present invention.

[0022] Osteoarthritis is a chronic joint degenerative disease, and its final manifestation after onset is cartilage degeneration. Current treatment methods include non-steroidal anti-inflammatory drug treatment, cell therapy, surgical therapy, etc., but there are still problems such as high treatment costs and strong side effects.

[0023] SVF is to extract effective components from adipose tissue extracted from the patient's own body, and is a cell population formed by a mixture of various cells with repair functions and cytokines. Research shows that SVF contains mesenchymal stem cells, M2 macrophages, TGF-β1, etc. that promote chondrocyte regeneration. Therefore, collecting the patient's own SVF to treat osteoarthritis is an effective method. However, the quality of SVF varies, resulting in poor treatment effects on osteoarthritis.

[0024] The research team of the present invention found in the research that: SVF plays a good role in the treatment of osteoarthritis due to its rich variety of cells, growth factors, and immune regulatory factors, and has the ability to promote chondrocyte regeneration and repair. Among them, sJAG1, as a competitive inhibitory ligand of the Notch receptor, can further inhibit the reduction of M2 macrophages by inhibiting the activation of the Notch signal transduction pathway in the treatment of OA, prevent the infiltration of neutrophils at the joints, inhibit the production of inflammatory factors such as TNF-α and IL-1β to slow down inflammation, and can improve the arthritis condition by promoting chondrocyte synthesis and weakening extracellular matrix degradation. Therefore, by adding sJAG1 protein, the quality problem of SVF caused by heterogeneity can be improved, and thus osteoarthritis caused by cartilage degeneration and the imbalance of the intra-articular capsule environment can be treated.

[0025] In some embodiments of the present invention, SVF is a multi - cell mixture derived from the patient's own adipose tissue, rich in mesenchymal stem cells, macrophages, endothelial cells and various cytokines. It can promote chondrocyte regeneration and inhibit the exacerbation of inflammation through its own differentiation ability and anti - inflammatory components.

[0026] In the present invention, the "M1 macrophage" refers to a subtype of macrophage mainly involved in promoting inflammation, tissue damage and pathogen repair. The "M2 macrophage" refers to a subtype of macrophage mainly involved in anti - inflammation, tissue repair and immune regulation. The "neutrophil" refers to an important white blood cell in the immune system.

[0027] In some embodiments of the present invention, sJAG1 can inhibit inflammatory factors in SVF, promote the polarization of M1 macrophages to M2 macrophages, promote the secretion of hyaluronic acid, inhibit the degradation of chondrocyte extracellular matrix, improve the anti - inflammatory, immune regulation and cartilage repair abilities of SVF, thereby improving the therapeutic effect of osteoarthritis.

[0028] In some embodiments of the present invention, co - injecting sJAG1 protein and SVF into the joint capsule cavity of osteoarthritis patients can optimize the therapeutic performance by increasing the number of M2 macrophages and inhibiting the increase in the number of M1 macrophages.

[0029] 1. Materials and Methods 1.1 Preparation method of murine SVF Collect adipose tissue from 4 - week - old C57BL / 6 (purchased from Shanghai Jihui), digest it with collagenase I (Beyotime, ST2294) at 37°C for 30 min, add one - volume of DMEM, then centrifuge at 2000 rpm for 10 min at 4°C, and discard the supernatant; then resuspend with 5 mL of DMEM, filter through a 40 - μm sieve, centrifuge at 2000 rpm for 10 min at 4°C, discard the supernatant, resuspend with 1 mL of complete medium containing 10% FBS, spread it on a 6 - cm culture plate, and culture it at 37°C with 5% carbon dioxide for 4 h; after discarding the supernatant, wash it three times with 1×PBS and continue culturing to obtain SVF.

[0030] 1.2 RNA extraction and real - time fluorescence quantitative PCR Extract total RNA using TRIzol (Takara) according to the pre - set program. Both RNA reverse transcription and qRTPCR use Vazyme kits. The MMP8 primers are obtained through the primer bank and ncbi database, and β - actin is used as an internal reference for mRNA level.

[0031] 1.3 Flow cytometry analysis The marker of macrophages in SVF is CD45. After SVF was separated, it was co-cultured with sJAG1 for 3 days in a culture dish, collected in 100 μL 1×PBS, and CD45 antibody (Biolegend, 103107) was added and incubated at 4°C in the dark for 30 minutes. After centrifugation to remove the supernatant, it was washed once with 1×PBS and loaded on an instrument (LSRFortessa) to detect the CD45 positive cell rate.

[0032] 1.4 Construction of mouse medial meniscus instability model (DMM) Male mice aged 12 weeks or above (purchased from Shanghai Jihui) were selected, the knee joint cavity of the mice was opened, the patella was pushed aside, the medial meniscus was located, the menisco-tibial ligament was cut, and the opened joint capsule cavity was sutured. The mice were allowed to move freely in the cage for 1.5 to 2 months. During this period, a roller was added to the cage to increase the activity of the mice so that joint wear could better induce OA.

[0033] 1.5. Intra-articular injection therapy Two weeks after surgery, the mouse DMM model was divided into a control group, a SVF-only injection group, a sJAG1-only injection group, and a SVF+sJAG1 combination treatment group. The control group was injected with 5 μL of saline; the SVF group was injected with 5 μL of SVF with a total cell count of 50,000 cells; the sJAG1 treatment group was injected with 5 μL of 0.5 ng sJAG1 protein; and the combination treatment group was injected with a total volume of 5 μL of a mixture of 50,000 cells of SVF and 0.5 ng sJAG1. Samples were collected one week after treatment.

[0034] 1.6 OARSI score The mouse knee joints were fixed in 4% paraformaldehyde for one day and then changed to EDTA decalcification solution (Biosharp, BL616b) for decalcification on a shaker at room temperature for 10 days. The sections were then embedded in paraffin and stained with safranin fast green (Servicebio, G1053) after rehydration with xylene-transparent ethanol. The cartilage degeneration was observed under an optical microscope to give the OARSI score.

[0035] 2. Statistical analysis Statistical analysis was performed using GraphPad Prism 9.5 for Windows (GraphPad Software Inc., San Diego, CA, USA). All data were obtained from at least three independent experiments and are presented as mean ± standard error of the mean (S.EM.). Statistical analysis was performed using t-test for two-group samples or one-way ANOVA Dunnett's test for multiple groups of samples, and p < 0.05 was considered statistically significant.

[0036] 3. Results 3.1. sJAG1 inhibits the expression of MMP8 in SVF MMP8 is highly expressed in OA and is a matrix metalloenzyme with extracellular matrix degradation function. As Figure 1 shown, in the present invention, RNA extraction, reverse transcription and RT-qPCR were performed on SVF and SVF added with sJAG1, and it was verified that the expression of MMP8 in SVF added with sJAG1 decreased.

[0037] 3.2. sJAG1 increases the proportion of immune cells in SVF SVF is a mixed cell population of adipose stem cells, vascular endothelial cells, macrophages, T cells, etc., among which macrophages account for the highest proportion, and M2 macrophages have anti-inflammatory effects. Cyril Lucien Bouland et al. Frontiers in Cell and Developmental Biology recorded in the paper titled "Treatment of medication-related osteonecrosis of the jaw with cell therapy" that the proportion of CD45+ cells was 54.6±7.4% without treatment, while the present invention Figure 2 shows that the CD45+ cell rate in SVF treated with sJAG1 increases, and it can be further speculated that this SVF can optimize the performance of treating OA.

[0038] 3.3. The combined treatment of sJAG1 and SVF can alleviate the condition of OA in mice Figures 3 - 6 Saline, SVF, sJAG1 and sJAG1+SVF combination were injected into C57BL / 6 mice 2 weeks after DMM surgery. One week after treatment, the knee joints of the mice were sampled and stained with safranin-fast green. The red part is cartilage tissue and the green part is bone tissue. As Figures 3 - 6 shown, the experimental results after combined treatment are more complete in the red part than those in the control group and the single treatment group.

[0039] In summary, the present invention shows that sJAG1 can optimize the treatment process of osteoarthritis by promoting the polarization of M1 macrophages into M2 macrophages, inhibiting neutrophil infiltration and inhibiting the expression of MMP8 metalloproteinase in SVF.

[0040] The above has described an embodiment of the present invention in detail, but the content described is only a preferred embodiment of the present invention and cannot be considered as limiting the scope of implementation of the present invention. All equivalent changes and improvements made according to the scope of the application of the present invention should still fall within the scope covered by the patent of the present invention.

Claims

1. Use of sJAG1 protein in improving the performance of SVF.

2. The use according to claim 1, characterized in that, The sJAG1 protein is used to enhance the anti-inflammatory, tissue repair or cartilage regeneration promoting ability of SVF.

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition is formed by mixing the sJAG1 protein described in claim 1 with SVF, and the dosage ratio of sJAG1 to SVF in each 1 μL of the mixture is 0.08 ng - 0.5 ng: 10,000 cell amounts.

4. A drug, characterized in that, It includes the pharmaceutical composition described in claim 3.

5. The drug according to claim 4, characterized in that, The drug has any one of the following uses: (1) Anti-inflammatory; (2) Tissue repair; (3) Cartilage regeneration promotion.

6. The drug according to claim 1, characterized in that, The drug uses the pharmaceutical composition as the sole active ingredient.

7. The drug according to claim 1, characterized in that, The drug is compounded from the pharmaceutical composition and pharmaceutically acceptable excipients.

8. The medicament according to claim 7, characterized in that, The drug is an injection preparation.

9. Use of the pharmaceutical composition described in claim 3 or the drug described in claim 4 in the preparation of a drug for treating osteoarthritis.

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