Use of LAMP2A in ankylosing spondylitis

Abstract

The present invention belongs to the field of biomedical technology and discloses the use of LAMP2A in ankylosing spondylitis. By detecting the expression levels of LAMP2A in bone marrow mesenchymal stem cells of normal healthy individuals and ankylosing spondylitis patients, the present invention finds that the expression level of LAMP2A is significantly up-regulated in ankylosing spondylitis patients. LAMP2A has a positive regulatory effect on the osteogenic differentiation of human mesenchymal stem cells, and the increased expression of LAMP2A is an important cause of abnormal pathological osteogenesis in ankylosing spondylitis patients. The present invention takes LAMP2A as a new therapeutic target for ankylosing spondylitis, can relieve the pathological osteogenesis of ankylosing spondylitis patients, and has a good effect on controlling the abnormal formation of osteophytes and the occurrence of spinal deformities in ankylosing spondylitis patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the use of LAMP2A in ankylosing spondylitis. Background Art

[0002] Ankylosing spondylitis (AS) is a common bone immune-related disease and an autoimmune disease mainly characterized by chronic inflammation of joints and spine. Currently, there is still a lack of effective early diagnosis methods and effective early diagnosis and treatment means, nor are there measures to completely cure the disease. As the disease progresses, patients will gradually develop spinal and joint deformities, seriously affecting the patient's living and working abilities, causing great physical and mental pain to the patient, and also causing a great social and economic burden.

[0003] Chronic inflammation and pathological osteogenesis are the two major characteristics of ankylosing spondylitis. In recent years, the basic research on the chronic inflammation of ankylosing spondylitis has advanced by leaps and bounds, and various theories such as the unfolded protein response, bacterial infection, inflammatory cells and factors have been proposed, and some research results are gradually being translated into clinical practice. However, the other core feature of ankylosing spondylitis, pathological osteogenesis, has received less attention, and its specific molecular mechanism has not been clarified, which brings great difficulties to the early diagnosis and treatment of ankylosing spondylitis. Existing diagnostic techniques are difficult to predict the progression speed and severity of pathological osteogenesis, and existing treatment methods are also difficult to effectively reverse and delay pathological osteogenesis, ultimately resulting in severe joint stiffness and ankylosis in ankylosing spondylitis patients.

[0004] Therefore, it is particularly important to deeply study the occurrence and development mechanism of pathological osteogenesis in ankylosing spondylitis and explore new diagnosis and treatment targets, in order to provide a new theoretical basis for the early diagnosis and treatment and clinical treatment of ankylosing spondylitis. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide the use of LAMP2A in ankylosing spondylitis. It is hoped that by using LAMP2A as a treatment target, a new type of drug for treating ankylosing spondylitis can be developed and manufactured, providing a new diagnosis and treatment method for clinically controlling the progressive reduction of bone mass in ankylosing spondylitis patients and preventing the occurrence of pathological fractures.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] In the first aspect, the present invention applies a reagent for detecting the expression level of LAMP2A in bone marrow mesenchymal stem cells in the preparation for assisting in the diagnosis of ankylosing spondylitis.

[0008] By detecting the expression level of LAMP2A in bone marrow mesenchymal stem cells of normal healthy people and patients with ankylosing spondylitis, it was found that the expression level of LAMP2A was significantly up-regulated in patients with ankylosing spondylitis compared with normal healthy people. By intervening in the level of LAMP2A, it was found that LAMP2A has a positive regulatory effect on the osteogenic differentiation of human mesenchymal stem cells. Further studies have shown that the increased expression of LAMP2A is an important cause of abnormal pathological osteogenesis in patients with ankylosing spondylitis.

[0009] As a preferred embodiment of the application described in the present invention, the amino acid sequence of LAMP2A is as shown in SEQ ID No.1; compared with the healthy control, the expression level of LAMP2A is up-regulated.

[0010] In a second aspect, the present invention provides a pharmaceutical composition for treating ankylosing spondylitis, comprising an LAMP2A expression inhibitor.

[0011] The present invention constructed a disease model of ankylosing spondylitis, SKG mice, and intravenously injected shRNA adeno-associated virus knocked out of LAMP2A into the tail vein. Micro-CT further analyzed the osteophyte formation in the spines of the mice and found that obvious pathological osteogenesis occurred in the spines of the ankylosing spondylitis group, with a large number of osteophytes formed, indicating that the ankylosing spondylitis mouse model was successfully established. The osteophytes in the LAMP2A knockout group of ankylosing spondylitis mice were significantly reduced and were similar to those in the normal control group, indicating that knocking out LAMP2A can effectively relieve the pathological osteogenesis in the animal model of ankylosing spondylitis mice, suggesting that LAMP2A is an important target for treating ankylosing spondylitis.

[0012] As a preferred embodiment of the pharmaceutical composition for treating ankylosing spondylitis described in the present invention, the LAMP2A expression inhibitor is at least one of gRNA, shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular compounds, peptides, and antibodies.

[0013] Furthermore, the base sequences of the siRNA are as shown in SEQ ID No.2 and 3.

[0014] As a preferred embodiment of the pharmaceutical composition for treating ankylosing spondylitis described in the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0015] In a third aspect, the present invention applies an LAMP2A expression inhibitor in the preparation of a drug for treating and / or preventing abnormal pathological osteogenesis.

[0016] LAMP2A of the present invention can be used as a new therapeutic target for ankylosing spondylitis, which can alleviate the pathological osteogenesis in ankylosing spondylitis patients and has a good effect on controlling the abnormal formation of osteophytes and the occurrence of spinal deformities in ankylosing spondylitis patients.

[0017] In a fourth aspect, the present invention applies an LAMP2A expression inhibitor in the preparation of a drug for treating and / or preventing ankylosing spondylitis.

[0018] By using LAMP2A as a therapeutic target, a new therapeutic drug for ankylosing spondylitis can be developed, which can control the progressive reduction of bone mass and prevent the occurrence of pathological fractures.

[0019] As a preferred embodiment of the application of the present invention, the LAMP2A expression inhibitor is at least one of gRNA, shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular compounds, peptides, and antibodies.

[0020] Further, the base sequences of the siRNA are as shown in SEQ ID No.2 and 3.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] The present invention discovers that compared with normal healthy people, the expression level of LAMP2A is significantly up-regulated in ankylosing spondylitis patients, and LAMP2A has a positive regulatory effect on the osteogenic differentiation of human mesenchymal stem cells. Further research shows that the increase in LAMP2A expression is an important cause of abnormal pathological osteogenesis in ankylosing spondylitis patients. Knockout of LAMP2A can effectively alleviate the pathological osteogenesis in the animal model of ankylosing spondylitis mice. LAMP2A can be used as a new therapeutic target for ankylosing spondylitis, which can alleviate the pathological osteogenesis in ankylosing spondylitis patients and has a good effect on controlling the abnormal formation of osteophytes and the occurrence of spinal deformities in ankylosing spondylitis patients. By using LAMP2A as a therapeutic target, a new therapeutic drug for ankylosing spondylitis can be developed, which can control the progressive reduction of bone mass and prevent the occurrence of pathological fractures. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 For immunofluorescence detection of LAMP2A levels in hMSCs of normal people and AS patients.

[0024] Figure 2 For Western blot detection of protein levels in hMSCs of normal people and AS patients.

[0025] Figure 3 For alizarin red staining and quantitative analysis of osteogenic differentiation;

[0026] Figure 4 To detect the osteogenic differentiation level by alkaline phosphatase

[0027] Figure 3 and Figure 4 In both the si group, siRNA knockdown is indicated, and in the oe group, lentiviral overexpression is indicated

[0028] Figure 5 It is a schematic diagram of the grouping and treatment of SKG mice, an animal model of ankylosing spondylitis

[0029] Figure 6 It is a three-dimensional reconstruction map of the mouse spine Specific implementation mode

[0030] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention

[0031] The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all commercially available unless otherwise specified

[0032] Example 1:

[0033] Detect the expression level of LAMP2A in human bone marrow mesenchymal stem cells (hMSCs) of normal people and ankylosing spondylitis patients. Human bone marrow mesenchymal stem cells (hMSCs) of ankylosing spondylitis patients were obtained from the clinical department, and the human bone marrow mesenchymal stem cells (hMSCs) of healthy controls were from the outpatient healthy physical examination population. Both ankylosing spondylitis patients and healthy controls signed informed consent forms

[0034] The amino acid sequence of LAMP2A is:

[0035] MVCFRLFPVPGSGLVLVCLVLGAVRSYALELNLTDSENATCLYAKWQMNFTVRYETTNKTYKTVTISDHGTVTYNGSICGDDQNGPKIAVQFGPGFSWIANFTKAASTYSIDSVSFSYNTGDNTTFPDAEDKGILTVDELLAIRIPLNDLFRCNSLSTLEKNDVVQHYWDVLVQAFVQNGTVSTNEFLCDKDKTSTVAPTIHTTVPSPTTTPTPKEKPEAGTYSVNNGNDTCLLATMGLQLNITQDKVASVININPNTTHSTGSCRSHTALLRLNSSTIKYLDFVFAVKNENRFYLKEVNISMYLVNGSVFSIANNNLSYWDAPLGSSYMCNKEQTVSVSGAFQINTFDLRVQPFNVTQGKYSTAQDCSADDDNFLVPIAVGAALAGVLILVLLAYFIGLKHHHAGYEQF。

[0036] (1) Immunofluorescence detection

[0037] 1) Seed the cells into a cell culture plate, confocal dish or sterile glass coverslip.

[0038] 2) After different treatments, fix the cells with 4% paraformaldehyde for 25 minutes and permeabilize with 0.1% Triton X-100 for 15 minutes.

[0039] 3) Block the cells with 10% goat serum for 30 minutes and incubate with the corresponding primary antibody overnight at 4°C.

[0040] 4) Incubate the cells with a fluorescein-labeled secondary antibody at room temperature for 1 hour.

[0041] 5) Stain the cell nuclei with 4',6-diamidino-2-phenylindole (DAPI).

[0042] 6) Observe the fluorescence staining and take pictures using a fluorescence microscope or confocal microscope. The images are acquired by an immunofluorescence imaging and confocal imaging system and analyzed with ImageJ software.

[0043] (2) Western blot detection

[0044] 1) Treat hMSCs from ankylosing spondylitis patients and healthy controls with protein lysate respectively, and determine the protein concentration of each protein sample by BCA protein concentration assay; add 5×SDS-PAGE protein loading buffer to the collected protein samples, heat at 100°C for 10 min, and the protein loading amount is 20 μg.

[0045] 2) 10% SDS-PAGE electrophoresis: Use a constant voltage of 90 V for 30 min in the stacking gel, and use a constant voltage of 120 V for 100 min when the bromophenol blue enters the separating gel.

[0046] 3) Transfer to a PVDF membrane. Place the transfer tank in ice water, with a transfer current of 300 mA and a transfer time of 90 min.

[0047] 4) Immediately block with 5% skim milk for 60 min; wash the membrane 3 times with TBST, 5 min each time.

[0048] 5) According to the instructions of the primary antibody, dilute the primary antibody with 5% skim milk and incubate overnight in a refrigerator at 4°C. The next day, wash the membrane 3 times with TBST, 5 min each time; according to the instructions of the secondary antibody, dilute the secondary antibody with TBST and incubate at low speed on a shaker at room temperature for 1 h; wash the membrane 3 times with TBST, 5 min each time.

[0049] 6) Detect the protein expression level as Figure 2 shown.

[0050] It can be seen from Figure 1 and Figure 2 that immunofluorescence detection shows that the level of LMAP2A in hMSCs of ankylosing spondylitis patients is higher than that of normal people; after extracting the total protein from hMSCs of normal people and ankylosing spondylitis patients, Western blot detection shows that the level of LAMP2A in ankylosing spondylitis patients is higher than that of normal people; that is, the expression level of LAMP2A is significantly up-regulated in ankylosing spondylitis patients.

[0051] Example 2:

[0052] (1) Intervention of the LAMP2A level of hMSC

[0053] Take hMSCs from ankylosing spondylitis patients and healthy controls, and inoculate 0.6×10 5 hMSC cells into 12-well plates respectively. Discard the original medium the day after seeding, and perform overexpression of LAMP2A (LAMP2A was designed and constructed by Shanghai Heyuan Biotechnology Co., Ltd.) and knockdown siRNA transfection on hMSC. Replace the medium 24 h after overexpression lentivirus transfection, and replace the induction medium 6 h after siRNA transfection.

[0054] LAMP2A with overexpression was designed and constructed by Shanghai Heyuan Biotechnology Co., Ltd.

[0055] The LAMP2A siRNA sequences are as follows:

[0056] sense 5′-GGCAGGAGUACUUAUUCUATT-3′;

[0057] antisense 5'-UAGAAUAAGUACUCCUGCCTT-3′;

[0058] The control siRNA sequences are as follows:

[0059] sense 5′-UUCUCCGAACGUGUCACGUTT-3′;

[0060] antisense 5′-ACGUGACACGUUCGGAGAATT-3′.

[0061] (2) Induction of adipogenic / osteogenic differentiation of hMSCs

[0062] After knockdown or overexpression of the cells, osteogenic differentiation induction medium was added for corresponding osteogenic differentiation induction, and the osteogenic differentiation medium was replaced every 3 days.

[0063] The formulation of the osteogenic differentiation induction medium is as follows: 10% FBS DMEM, 0.1 μM dexamethasone, 10 mM glycerol phosphate, 50 μM vitamin C, and 100 IU / mL penicillin-streptomycin.

[0064] (3) Detection of the osteogenic differentiation level of hMSCs

[0065] The osteogenic differentiation level was detected on the 14th day of osteogenic differentiation induction. Alizarin red staining and alkaline phosphatase were used to detect the osteogenic differentiation of the cells.

[0066] As Figure 3 shown, alizarin red staining and quantitative analysis showed the osteogenic differentiation of LAMP2A knockdown and overexpression of hMSCs in normal individuals and patients with ankylosing spondylitis. After overexpression of LAMP2A, the osteogenic differentiation ability of hMSCs in both normal individuals and patients with ankylosing spondylitis increased, and the degree of osteogenic differentiation of hMSCs in normal individuals and patients with ankylosing spondylitis was almost the same. After knockdown of LAMP2A, the osteogenic differentiation ability of hMSCs in both patients with ankylosing spondylitis and normal individuals decreased, and the degree of osteogenic differentiation of hMSCs in normal individuals and patients with ankylosing spondylitis was similar, with no significant difference.

[0067] As Figure 4As shown, the osteogenic differentiation of hMSCs from normal subjects and ankylosing spondylitis patients after LAMP2A knockdown and overexpression was detected by alkaline phosphatase. After overexpressing LAMP2A, the osteogenic differentiation ability of hMSCs from both normal subjects and ankylosing spondylitis patients increased, and the degree of osteogenic differentiation of hMSCs from normal subjects and ankylosing spondylitis patients was almost the same. After knocking down LAMP2A, the osteogenic differentiation ability of hMSCs from both ankylosing spondylitis patients and normal subjects decreased, and the degree of osteogenic differentiation of hMSCs from normal subjects and ankylosing spondylitis patients was similar, with no significant difference.

[0068] The results of alizarin red staining and alkaline phosphatase detection of cell osteogenic differentiation indicate that the abnormal high expression of LAMP2A is an important cause of abnormal osteogenesis of hMSCs in ankylosing spondylitis patients. The above in vitro cell experiments show that by interfering with the level of LAMP2A, LAMP2A has a positive regulatory effect on the osteogenic differentiation of human mesenchymal stem cells (hMSCs).

[0069] Example 3:

[0070] (1) Establish a model

[0071] Female BALB / c mice at 24 weeks of age were selected and intraperitoneally injected with proteoglycan (once every 0, 3, and 6 weeks for a total of 3 injections, 100 μg each time). An animal model of ankylosing spondylitis, SKG mice, was established.

[0072] (2) shRNA adeno-associated virus for knocking out LAMP2A

[0073] The shRNA adeno-associated virus for knocking out LAMP2A was designed and constructed by Shanghai Heyuan Biotechnology Co., Ltd.

[0074] (3) Group administration

[0075] As Figure 5 shown, normal BABL / c mice and the constructed ankylosing spondylitis SKG mice were divided into three groups, including the BABL / c group, the SKG group, and the SKG + LAMP2A knockout group. Seven days after the establishment of the SKG mouse model, the three groups were respectively intravenously injected with 100 μL of normal saline, 5 × 10 11 empty vector and LAMP2A knockout adenovirus via the tail vein once every 2 weeks.

[0076] (4) Detection of mouse spinal osteophytes

[0077] The detection of mouse spinal osteophytes started from the 9th week after administration. The mice in the three groups were sacrificed by cervical dislocation. After sacrifice, the mouse spine was taken for Micro-CT scanning, and three-dimensional reconstruction of the mouse spine was performed to evaluate the formation of spinal osteophytes.

[0078] No abnormal deaths or side effects such as infections were observed in any of the three groups of treatments. By comparing the BABL / c group and the ankylosing spondylitis SKG group, it was determined that the number of vertebral bones in the ankylosing spondylitis SKG group was lower than that in the BABL / c group. In the ankylosing spondylitis model SKG group, obvious pathological osteogenesis occurred in the spine, with a large number of osteophytes formed, and the number of osteophytes was significantly higher than that in the BABL / c group, indicating that the establishment of the ankylosing spondylitis SKG mouse model was successful.

[0079] As Figure 6 shown, Micro-CT three-dimensional reconstruction could reveal the differences in the number of osteophytes in the spines of mice in different groups. By comparing the ankylosing spondylitis SKG group and the SKG+LAMP2A knockout group, it was found that the number of osteophytes in the ankylosing spondylitis SKG mice after LAMP2A knockout intervention was significantly restored, the osteophytes were significantly reduced, and the number of osteophytes was close to that of the control BABL / c mice, indicating that LAMP2A knockout could effectively inhibit the formation of osteophytes in ankylosing spondylitis. That is, it shows that LAMP2A knockout can effectively alleviate the pathological osteogenesis in the animal model of ankylosing spondylitis mice. Knocking out LAMP2A can significantly interfere with the progression of ankylosing spondylitis. The expression level of LAMP2A is negatively correlated with pathological osteogenesis, suggesting that LAMP2A is an important target for the treatment of ankylosing spondylitis.

[0080] In summary, in this invention, the expression levels of LAMP2A in bone marrow mesenchymal stem cells (hMSCs) of normal people and ankylosing spondylitis patients were detected, and it was found that the expression level of LAMP2A was significantly up-regulated in ankylosing spondylitis patients. By intervening in the level of LAMP2A, it was found that LAMP2A had a positive regulatory effect on the osteogenic differentiation of human mesenchymal stem cells (hMSC). Further research showed that the increase in LAMP2A expression was an important cause of abnormal pathological osteogenesis in ankylosing spondylitis patients. In the in vivo laboratory, an ankylosing spondylitis disease model SKG mouse was constructed, and an shRNA adeno-associated virus with LAMP2A knockout was injected into the tail vein. Further analysis of the formation of osteophytes in the mouse spine by Micro-CT found that obvious pathological osteogenesis occurred in the spine of the ankylosing spondylitis group, with a large number of osteophytes formed, indicating that the establishment of the ankylosing spondylitis mouse model was successful. The number of osteophytes in the LAMP2A knockout group of ankylosing spondylitis mice was significantly reduced and was similar to that of the normal control group, indicating that LAMP2A knockout can effectively alleviate the pathological osteogenesis in the animal model of ankylosing spondylitis mice, suggesting that LAMP2A is an important target for the treatment of ankylosing spondylitis.

[0081] The LAMP2A of the present invention can be used as a new therapeutic target for ankylosing spondylitis, which can relieve the pathological osteogenesis in patients with ankylosing spondylitis and has a good effect on controlling the abnormal formation of osteophytes and the occurrence of spinal deformities in patients with ankylosing spondylitis. By developing a new therapeutic drug for ankylosing spondylitis with LAMP2A as the therapeutic target, progressive bone loss can be controlled and pathological fractures can be prevented.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A pharmaceutical composition for treating ankylosing spondylitis, characterized in that, it comprises an inhibitor of LAMP2A expression; the inhibitor of LAMP2A expression is siRNA; the base sequences of the siRNA are shown in SEQ ID No.2 and 3.

2. The pharmaceutical composition according to claim 1, characterized in that, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

3. Use of an inhibitor of LAMP2A expression in the preparation of a drug for treating and / or preventing abnormal pathological osteogenesis, characterized in that, the inhibitor of LAMP2A expression is siRNA; the base sequences of the siRNA are shown in SEQ ID No.2 and 3.

4. Use of an inhibitor of LAMP2A expression in the preparation of a drug for treating and / or preventing ankylosing spondylitis, characterized in that, the inhibitor of LAMP2A expression is siRNA; the base sequences of the siRNA are shown in SEQ ID No.2 and 3.

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