Adipose mesenchymal stem cell preparation as well as preparation method and application thereof

Abstract

The invention discloses an adipose-derived mesenchymal stem cell preparation, the preparation comprises adipose-derived mesenchymal stem cells and a monoclonal antibody 1D4, the monoclonal antibody 1D4 is a TNF-alpha and IL-23 inhibitor, and amino acid sequences of a heavy chain variable region and a light chain variable region of the monoclonal antibody 1D4 are respectively shown as SEQ ID NO.3 and SEQ ID NO.4. The invention further discloses a preparation method of the adipose-derived mesenchymal stem cell preparation. On the basis of the adipose-derived mesenchymal stem cells, the adipose-derived mesenchymal stem cell preparation prepared by combining TNF-alpha protein and IL-23 protein inhibitors has a good treatment effect on psoriasis. And a new direction is provided for treatment of psoriasis.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an adipose-derived mesenchymal stem cell preparation and a preparation method and application thereof. Background Art

[0002] Adipose-derived Mesenchymal Stem Cells (ADSCs) are a type of adult stem cells derived from adipose tissue, which have multidirectional differentiation potential and strong biological characteristics. These stem cells have broad application prospects in clinical medicine and biomedical research.

[0003] Psoriasis is a chronic, relapsing skin disease characterized by erythema and scaling. In recent years, adipose-derived mesenchymal stem cells (AD-MSCs) have shown potential in the treatment of psoriasis.

[0004] Adipose-derived mesenchymal stem cells have unique immunomodulatory capabilities and can secrete a large number of growth factors and immunomodulatory factors, thereby inhibiting the body's excessive immune response. Such stem cells can be easily obtained from adipose tissue and have the ability to differentiate into multiple cell lineages to provide repair, maintenance or enhancement of various tissue functions.

[0005] In clinical studies, Lu Chuanjian's team at Guangdong Provincial Hospital of Traditional Chinese Medicine evaluated the feasibility, safety, and effectiveness of AD-MSCs infusion in the treatment of moderate to severe psoriasis through a single-arm trial. The study included 7 subjects with moderate to severe psoriasis, and the results showed that AD-MSCs treatment was safe and well tolerated. In addition, the latest research results released by Xiangya Hospital of Central South University also showed that autologous adipose-derived mesenchymal stem cells were used to treat patients with psoriasis vulgaris. After several MSC infusions, the patient's PASI (lesion activity index) decreased significantly.

[0006] In summary, adipose-derived mesenchymal stem cells have important application prospects in the treatment of psoriasis. Their immunomodulatory function and multidirectional differentiation ability make them a promising treatment. Current clinical trial results also support this view, indicating that AD-MSCs treatment is safe and effective, providing a new treatment option for patients with psoriasis.

[0007] Although adipose-derived mesenchymal stem cells have shown great potential in theoretical and experimental studies, they still face many challenges in terms of isolation, culture, differentiation control, safety, immune rejection, and clinical application. Future research needs to further optimize these issues to promote their widespread clinical application. Summary of the invention

[0008] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide an adipose-derived mesenchymal stem cell preparation and a preparation method and application thereof.

[0009] Therefore, the present invention discloses an adipose-derived mesenchymal stem cell preparation, comprising adipose-derived mesenchymal stem cells and monoclonal antibody 1D4, wherein the monoclonal antibody 1D4 is a TNF-α and IL-23 inhibitor, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 1D4 are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0010] Preferably, the monoclonal antibody 1D4 of the present invention specifically binds to TNF-α protein, and the amino acid sequence of the monoclonal antibody 1D4 specifically binding to TNF-α protein is STHVLLTHTISRIAVSYQT.

[0011] Preferably, the monoclonal antibody 1D4 of the present invention specifically binds to IL-23 protein, and the amino acid sequence of the monoclonal antibody 1D4 specifically binding to IL-23 protein is SAHPLVGHMDLREEGDEET.

[0012] Preferably, the content of adipose-derived mesenchymal stem cells in the preparation of the present invention is 5×10 5 cells / 0.2ml.

[0013] Preferably, the content of the monoclonal antibody 1D4 in the preparation of the present invention is 5 mg / kg.

[0014] Preferably, the adipose-derived mesenchymal stem cells and monoclonal antibody 1D4 described in the present invention are both diluted with PBS buffer.

[0015] In another aspect, the present invention also discloses a use of the monoclonal antibody 1D4 in preparing an adipose-derived mesenchymal stem cell preparation for treating psoriasis.

[0016] The adipose-derived mesenchymal stem cell preparation prepared by the present invention based on adipose-derived mesenchymal stem cells combined with an inhibitor of TNF-α protein and IL-23 protein (monoclonal antibody 1D4) has a good therapeutic effect on psoriasis, providing a new direction for the treatment of psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Adipose-derived mesenchymal stem cells.

[0018] Figure 2 Amino acid sequence alignment results of human TNF-α protein and human IL-23 protein.

[0019] Figure 3 Western blot detection results of monoclonal antibody 1D4 on recombinant human TNF-α protein and recombinant human IL-23 protein.

[0020] Figure 4Clinical treatment outcomes for psoriasis.

[0021] Figure 5 Results of TNF-α and IL-23 detection in serum. DETAILED DESCRIPTION

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0023] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0024] Example 1: Isolation and culture of adipose-derived mesenchymal stem cells

[0025] Adipose-Derived Mesenchymal Stem Cells (ADSCs) are a type of adult stem cell with multidirectional differentiation potential, which are widely used in regenerative medicine and tissue engineering. The preparation method mainly includes the following steps:

[0026] 1. Collection and pretreatment of fat tissue: Collect enough fat samples from subcutaneous fat tissue of human or animal. Usually 2-100ml of fat is collected. Cut and wash the collected fat to remove blood, fiber and other impurities, centrifuge at 1000r / min, 5min. Take the upper layer of fat tissue and the lower layer of cells.

[0027] 2. Digestion and separation: Add trypsin and collagenase with a final concentration of 0.1% to the adipose tissue. Digest in a 37°C water bath shaker for 30 minutes. Centrifuge at 700g / min for 5 minutes. Keep the lower layer of cells, mix the two cells, resuspend the cells in PBS buffer, and centrifuge again at 700g / min for 5 minutes. Resuspend the cells in the primary culture medium and inoculate them into a culture bottle, and culture them in a 37°C, 5% carbon dioxide incubator. Recorded as P0 generation.

[0028] 3. Culture and expansion: Observe cell growth. Change the medium by half on the third day, and change the medium every 3 days thereafter. When the cells reach 90% confluence, subculture them, digest them with 0.05% trypsin, and subculture them at a ratio of 1:4. Subculture them to the third generation for identification, transplantation or cryopreservation. Figure 1 shown.

[0029] 4. Cryopreservation and storage: The ADSCs that have passed the inspection are cryopreserved for long-term storage and clinical application. The formula of the cryopreservation solution is 60% complete culture medium, 30% fetal bovine serum (FBS), and 10% dimethyl sulfoxide (DMSO).

[0030] Example 2: Preparation of TNF-α and IL-23 inhibitors (monoclonal antibodies)

[0031] We first compared the amino acid sequences of human TNF-α protein (amino acid sequence as shown in SEQ ID NO.1) and human IL-23 protein (amino acid sequence as shown in SEQ ID NO.2) Figure 2 ), and the results showed that the two protein sequences had good similarity. We designed and synthesized two antigen epitope peptides, one for TNF-α protein (amino acid sequence is STHVLLTHTISRIAVSYQT, position in SEQ ID NO.1 is aa71~aa89), and the other for IL-23 protein (amino acid sequence is SAHPLVGHMDLREEGDEET, position in SEQ ID NO.2 is aa27~aa45).

[0032] We mixed the two synthetic polypeptides in equal mass ratios to prepare the vaccine. Female BALB / c mice aged 6 to 8 weeks were immunized for a total of three immunizations, all of which were subcutaneously injected at multiple points. The antigen immunization dosage for each mouse was 100 μg each time. The first immunization was emulsified with an equal amount of Freund's complete adjuvant, the second immunization was emulsified with an equal amount of Freund's incomplete adjuvant, and the third immunization was mixed with an equal amount of PBS buffer and injected intraperitoneally. Blood was collected 7 days after the third immunization, and the antibody titer was measured by an indirect method to determine whether antibodies were generated against both TNF-α protein and IL-23 protein. Through the test, a mouse with a titer of 1:10000 was screened.

[0033] Take the spleen of the mouse with higher titer, homogenize it and take the spleen cells, fuse it with myeloma cells, and then screen the hybridoma cells. After screening and ELISA detection, a good hybridoma cell is screened. Expand and freeze in liquid nitrogen for later use.

[0034] Healthy female mice aged 6 to 7 weeks were intraperitoneally injected with 0.5 ml of liquid paraffin. Seven days later, each mouse was intraperitoneally injected with 1×10 6The hybridoma cells were expanded and the mice were observed. After about 7 to 10 days, the abdominal cavity of the mice was obviously swollen and the ascites was collected. The ascites was centrifuged at 4000 rpm for 10 minutes at room temperature, and the supernatant was aspirated as the ascites of the mice. The ascites of the mice was taken, purified by commercial Protein G column, dialyzed and sterilized, and filtered to obtain a monoclonal antibody (labeled as monoclonal antibody 1D4). The concentration of monoclonal antibody 1D4 was measured using a BCA kit, and the monoclonal antibody was stored at -80°C for standby use after aliquoting. At the same time, the sequence of monoclonal antibody 1D4 was detected and analyzed. The amino acid sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 1D4 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0035] Monoclonal antibody 1D4 was used for western blot detection of recombinant human TNF-α protein (ab259410) and recombinant human IL-23 protein (ab106889). Monoclonal antibody 1D4 has good specificity and sensitivity to recombinant human TNF-α protein and recombinant human IL-23 protein. Figure 3 shown.

[0036] Example 3: Study on the treatment of psoriasis

[0037] Twenty-five healthy male Balb / c mice aged 7 to 8 weeks were selected and randomly divided into 5 groups (Groups 1 to 4 in Table 1), of which 4 groups were used for model construction and one group was used as a normal control (Group 5 in Table 1). All mice in the model construction group were shaved on the back, with a shaved area of ​​2.5 cm × 2.5 cm; Imiquimod (IMQ) ointment (Sichuan Mingxin Pharmaceutical Co., Ltd.) was applied to the back skin of the mice for 5 days (Day 1, Day 2, Day 3, Day 4 and Day 5 of model construction) to construct a psoriasis mouse model, and the control group was given the same dose of vaseline ointment. At the same time, the drugs listed in Table 1 were used for treatment on the day before, Day 2 and Day 4 of model construction. On the 7th day of model construction, the incidence of psoriasis in mice was observed and the mice were killed, and serum was taken for subsequent testing.

[0038] Clinical observation results show that ( Figure 4 ), the therapeutic effects were ranked as group 3>group 4>group 2>group 1, that is, the monoclonal antibody 1D4 prepared by the present invention had the best therapeutic effect in combination with adipose-derived mesenchymal stem cells.

[0039] The TNF-α and IL-23 in serum were detected using commercial kits (Shanghai ELISA Biologicals). The results showed that ( Figure 5 ), TNF-α and IL-23 showed the same trend in each group, and their contents were ranked as group 1>group 2>group 4>group 3>group 5. This further indicates that the monoclonal antibody 1D4 prepared by the present invention combined with adipose-derived mesenchymal stem cells has a better therapeutic effect on psoriasis.

[0040] Table 1 Mouse grouping and drug information

[0041]

[0042] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An adipose-derived mesenchymal stem cell preparation, characterized in that: The preparation comprises adipose-derived mesenchymal stem cells and monoclonal antibody 1D4, wherein the monoclonal antibody 1D4 is a TNF-α and IL-23 inhibitor, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 1D4 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

2. The preparation according to claim 1, characterized in that The monoclonal antibody 1D4 specifically binds to TNF-α protein, and the amino acid sequence of the monoclonal antibody 1D4 specifically binding to TNF-α protein is STHVLLTHTISRIAVSYQT.

3. The preparation according to claim 1, characterized in that The monoclonal antibody 1D4 specifically binds to the IL-23 protein, and the amino acid sequence of the monoclonal antibody 1D4 specifically binds to the IL-23 protein is SAHPLVGHMDLREEGDEET.

4. The preparation according to claim 1, characterized in that The content of the adipose-derived mesenchymal stem cells in the preparation is 5×10 5 cells / 0.2ml.

5. The preparation according to claim 1, characterized in that The content of the monoclonal antibody 1D4 in the preparation is 5 mg / kg.

6. The preparation according to claim 1, characterized in that The adipose-derived mesenchymal stem cells and monoclonal antibody 1D4 were both diluted with PBS buffer.

7. Use of the monoclonal antibody 1D4 according to claim 1 in the preparation of an adipose-derived mesenchymal stem cell preparation for the treatment of psoriasis.

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