Autologous stem cell preparation, preparation method thereof and application of autologous stem cell preparation in improvement of human immunity

Abstract

The invention provides an autologous stem cell preparation and a preparation method thereof, and aims to improve the immunocompetence of stem cells and enhance the application potential of the stem cells in treatment. The method comprises the following steps: firstly, collecting peripheral blood from healthy adult volunteers and separating mononuclear cells; then amplifying the cells under a specific culture condition, and performing immune activation through a TLR7 / 8 agonist and an anti-CXCR4 single-domain antibody; then, evaluating the phenotype and cytokine secretion level of the stem cells after immune activation through a flow cytometry and an ELISA (Enzyme-Linked Immunosorbent Assay) method; and finally, carrying out freeze-drying treatment for long-term storage, and ensuring that the freeze-dried stem cells can effectively recover the immunocompetence. The prepared autologous stem cell preparation can be used for improving the immunity of a human body and has a relatively good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cells, in particular to an autologous stem cell preparation and a preparation method thereof, and also to the application of the preparation in improving human immunity. Background Art

[0002] With the continuous development of stem cell technology, stem cells have shown great potential in regenerative medicine, immune regulation and the treatment of various diseases. Autologous stem cells, as a type of cell collected from an individual, have stronger immune tolerance and lower immune rejection than allogeneic stem cells, so they have unique advantages in clinical applications. Especially in the field of immune regulation, autologous stem cells can effectively enhance the body's immune function, regulate the balance of the immune system, and thus enhance the body's immune response to pathogens.

[0003] Traditional immune enhancement methods mostly rely on exogenous drugs, vaccines or immunopotentiators, but these methods have some limitations. For example, exogenous drugs may cause long-term side effects, and the effects are mostly short-term, making it difficult to maintain the long-term activity of the immune system. Vaccines may not be able to address all immune needs, especially for some individuals with weak immune function, and the effect is not good.

[0004] At present, the application of stem cells in immune system regulation is still in the exploratory stage. Studies have shown that stem cells can regulate immune responses, improve immune tolerance and enhance immune memory by secreting a variety of cytokines and directly interacting with immune cells. Therefore, the use of autologous stem cell preparations to regulate the immune system and improve immunity has become a new research direction. Especially in the face of problems such as immune weakness and aging-related immune system decline, autologous stem cell preparations show a wide range of application prospects. . Summary of the invention

[0005] The present invention provides an autologous stem cell preparation, which uses a specific preparation method to separate, culture and activate autologous stem cells from the human body, and then uses a specific treatment method to prepare a final preparation, and is used to improve human immunity. The preparation can not only effectively enhance the function of immune cells, but also improve the body's immune response to external pathogens.

[0006] Therefore, the present invention discloses an autologous stem cell preparation in one aspect, wherein the preparation is prepared by the following steps:

[0007] Step 1: Stem cell collection and isolation: Peripheral blood was collected from healthy adult volunteers and mononuclear cells were isolated by density gradient centrifugation;

[0008] Step 2: Stem cell expansion and immune activation: The isolated monocytes were cultured in a medium containing 10% fetal bovine serum, and TLR7 / 8 agonists and anti-CXCR4 single domain antibodies were added for immune activation;

[0009] Step 3: Freeze-drying and preparation storage: The immune-activated stem cells are collected by centrifugation, resuspended in a culture medium containing 10% glycerol, and then freeze-dried to obtain a stem cell preparation that can be stored for a long time.

[0010] Preferably, the TLR7 / 8 agonist of the present invention is CL264, and its concentration is 1 μg / mL.

[0011] Preferably, the VNAR sequence of the anti-CXCR4 single domain antibody of the present invention is as shown in SEQ ID NO.1, and its use concentration is 1 μg / mL.

[0012] Preferably, the sequencing of the freeze-drying treatment in step 3 of the present invention is: pre-freezing at -80°C for 2 hours, low-speed freeze-drying at -40°C for 12 hours, and finally keeping in a -80°C refrigerator.

[0013] In one aspect, the present invention also discloses a use of the anti-CXCR4 single domain antibody in stem cell activation.

[0014] The present invention treats autologous stem cells with immune activation factors (TLR7 / 8 agonists and anti-CXCR4 single domain antibodies) to enhance the immune function of stem cells and improve their immune response ability in vivo; this method not only enhances the immunoregulatory effect of autologous stem cells, but also avoids the immune rejection reaction that may be caused by allogeneic stem cells, and has strong safety. At the same time, the present invention uses freeze-drying technology to process autologous stem cells after immune activation, so that they can be stored for a long time in a low temperature environment and can maintain the immune activity of the cells; this freeze-dried preparation is easy to transport and store, and has significant practical application value. In addition, the present invention proposes a new direction of applying autologous stem cell preparations to enhance immunity, especially in the elderly, immunocompromised or immune system dysfunction patients, as a long-term immunoregulatory therapy; through the method of the present invention, the body's immune response can be significantly enhanced and the disease resistance can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 SDS-PAGE test results of anti-CXCR4 single domain antibodies, where 1 is an anti-CXCR4 single domain antibody.

[0016] Figure 2 Western blot test results of anti-CXCR4 single domain antibody, where 1 is the recombinant CXCR4 protein. DETAILED DESCRIPTION

[0017] 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.

[0018] 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.

[0019] Example 1: Preparation of autologous stem cell preparation

[0020] Step 1: Stem cell collection and isolation

[0021] (1) Sample collection: 5 mL of peripheral blood was collected from a healthy adult female volunteer (30 years old, weighing 60 kg). The volunteer did not take any medication before sampling to ensure the quality of the blood sample.

[0022] (2) Cell separation: using Ficoll-Paque TM PLUS density gradient centrifugation was used to separate peripheral blood mononuclear cells (PBMCs). The steps are as follows:

[0023] Add an equal volume of PBS to the collected blood and mix gently. TM PLUS (GE Healthcare) solution was centrifuged at 1200 g for 30 minutes at room temperature to separate the mononuclear cell layer. The mononuclear cell layer was removed and washed twice with PBS to obtain mononuclear cells (about 5 × 10 6 cell).

[0024] (3) Flow cytometric analysis: Surface markers of monocytes were detected by flow cytometry (BD FACSCalibur). The proportion of CD34+ cells was 10% and that of CD45+ cells was 80%.

[0025] Step 2: Stem cell expansion and immune activation

[0026] (1) Cell culture: The isolated monocytes were inoculated in RPMI 1640 medium containing 10% fetal bovine serum (FBS) at a rate of 2×10 cells per well. 6 The cells were cultured in an incubator at 37°C and 5% CO2. The culture medium was changed every 48 hours.

[0027] (2) Immune activation treatment: After the initial culture for 48 hours, immune activation factors were added: TLR7 / 8 agonist (CL264, 1 μg / mL) and anti-CXCR4 single domain antibody (1 μg / mL) (as shown in Example 3). The culture was continued for 48 hours to ensure that the cells grew under immune activation conditions.

[0028] (3) Evaluation of immune activity

[0029] 1) Cell phenotype analysis: Flow cytometry was used to evaluate stem cell surface markers after immune activation, and the expression of CD86 and MHC-II was observed. It was measured that the expression of CD86 on the cell surface of the immune activation group increased to 70%, and the expression of MHC-II increased to 65%. The expression of CD86 and MHC-II in the control group (unactivated cells) was 25% and 20%, respectively. This shows that immune activation significantly increases the expression of CD86 and MHC-II on the cell surface.

[0030] It should be noted that during immune activation, adding only TLR7 / 8 agonists or anti-CXCR4 single domain antibodies can increase the expression of CD86 and MHC-II on the cell surface (only 38-45% and 30-35%), but it is far less effective than adding both at the same time. This suggests that adding both at the same time can have a good synergistic stimulatory effect.

[0031] 2) Cytokine secretion analysis: The ELISA method was used to determine the cytokine secretion level of stem cells after immune activation. The test results are as follows:

[0032] IFN-γ: immune activation group (1000±50pg / mL), control group (unactivated cells) (150±30pg / mL);

[0033] IL-2: immune activation group (500±20pg / mL), control group (unactivated cells) (50±10pg / mL);

[0034] IL-6: immune activation group (200±30pg / mL), control group (unactivated cells) (20±5pg / mL);

[0035] TNF-α: immune activation group (300±40pg / mL), control group (unactivated cells) (50±15pg / mL).

[0036] This shows that immune activation significantly improves the secretion capacity of stem cells for immune factors. It should be noted that during immune activation, only adding TLR7 / 8 agonists or anti-CXCR4 single domain antibodies can improve the secretion capacity of stem cells for immune factors, but it is far less effective than adding both at the same time, which shows that adding both at the same time can have a good synergistic stimulatory effect.

[0037] Step 3: Freeze-drying and preparation storage

[0038] (1) Freeze-drying: The immune activated stem cells were collected by centrifugation and resuspended in a medium containing 10% glycerol. The freeze-drying process was performed according to the following procedure: pre-freeze at -80°C for 2 hours, freeze-dry at -40°C for 12 hours, and finally stored in a -80°C refrigerator.

[0039] (2) Evaluation of freeze-dried stem cell preparations: Freeze-dried stem cells were revived using PBS. Flow cytometry analysis showed that the survival rate of the revived cells was 85%, which was high and could effectively maintain their immune activity.

[0040] Step 4: Quality Control

[0041] (1) Cell proliferation detection: The proliferation of stem cells was determined by MTT method. The results showed that the proliferation rate (OD value) of the immune activation group was 2.5±0.2, which was significantly higher than that of the control group (unactivated cells) (1.2±0.1).

[0042] (2) Cell purity detection: The stem cell population after immune activation was analyzed by flow cytometry. The results showed that CD34+ cells accounted for 90% and CD45+ cells accounted for 85%.

[0043] Example 2: Application of autologous stem cell preparations in improving immunity

[0044] 1. Animal model selection: 20 healthy male mice aged 6 weeks were selected and randomly divided into an experimental group (10 mice) and a control group (10 mice). The mice in the experimental group were injected with 50 μL of immune activated autologous stem cell preparation, and the mice in the control group were injected with the same amount of PBS.

[0045] 2. Immune evaluation: The immune function of mice was evaluated by the following methods every week after the experiment:

[0046] (1) Serum IgG level: The IgG level in mouse serum was measured using ELISA.

[0047] (2) T cell activity: The proportion and activity of T cells in the peripheral blood of mice were analyzed by flow cytometry.

[0048] (3) NK cell activity: The cytotoxic activity of NK cells was detected using the LDH release method.

[0049] 3. Experimental results:

[0050] (1) Serum IgG level: experimental group: 1.5±0.1 mg / mL, control group: 1.2±0.1 mg / mL. Autologous stem cells after immune activation significantly increased the IgG level of mice.

[0051] (2) T cell activity: experimental group: 120±15 cell / μL, control group: 90±12 cell / μL. After injection of autologous stem cell preparations, the T cell activity of mice increased significantly, indicating that their immune system's response ability was enhanced.

[0052] (3) NK cell killing activity: experimental group: 45±5%, control group: 30±5%. The NK cell killing activity of mice in the experimental group was significantly enhanced, indicating that the cytotoxic activity of the immune system was improved.

[0053] The above results show that autologous stem cell preparations significantly enhance the immune function of mice, and the cells after immune activation show enhanced immune memory and immune response capabilities. The improvement in immunity is also reflected in the increase in serum immunoglobulin levels and the improvement in the activity of T cells and NK cells.

[0054] Example 3: Preparation of anti-CXCR4 single domain antibody

[0055] 1. Immunization

[0056] The recombinant human CXCR4 protein (ab155716) was emulsified with FCA and prepared into a vaccine (0.5 mg / mL). The vaccine was administered to sharks by subcutaneous injection at a dose of 0.5 mL. Two weeks after the first immunization, the vaccine was prepared again using the same dose of recombinant CXCR4 protein emulsified with FIA, and the second immunization was performed in the same manner and dose. Two weeks later, the third immunization was performed again in the same manner and dose. Seven days after the third immunization, peripheral blood was collected from sharks, lymphocytes were isolated, and total RNA of B cells was extracted using the TRIzol method, and reverse transcription was performed to synthesize cDNA according to the instructions of the kit.

[0057] 2. Cloning and sequence analysis

[0058] The shark single domain antibody (VNAR fragment) was amplified by designing specific primers. The primers used are as follows:

[0059] F: 5'-gtcctggctcagggagcggt-3', R: 5'-ggtacggttggcctgttgagg-3'.

[0060] PCR amplification conditions were as follows: initial denaturation at 94°C for 5 minutes; followed by 35 cycles (94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute); and finally extension at 72°C for 5 minutes.

[0061] The amplified VNAR fragment was cloned into the pGEM-T vector and transformed into DH5α E. coli competent cells. After selecting the positive clones, the plasmid was extracted and sequenced, and finally the VNAR sequence of the anti-CXCR4 single domain antibody (SEQ ID NO.1) was obtained.

[0062] 3. Expression and purification

[0063] The VNAR sequence of the anti-CXCR4 single domain antibody was cloned into the pET28a vector and transformed into Escherichia coli BL21 (DE3) competent cells. When the cells were cultured to an OD600 value of 0.6, 1mM IPTG was added to induce protein expression. After 4 hours of culture, the cells were collected and soluble proteins were extracted by ultrasonic disruption. The anti-CXCR4 single domain antibody was purified by nickel column affinity chromatography. The purified antibody was dialyzed and sterilized through a 220nm filter membrane, and finally frozen at -80°C for use.

[0064] 4. Purity analysis and functional verification

[0065] The purity of the purified anti-CXCR4 single domain antibody was analyzed by SDS-PAGE, and the results showed that ( Figure 1 ) Its purity is higher than 90%.

[0066] The prepared anti-CXCR4 single domain antibody was used to perform Western blot detection on the recombinant CXCR4 protein to verify whether the antibody can specifically bind to the recombinant CXCR4 protein. The results showed that ( Figure 2 ), which can specifically bind to the approximately 33kDa recombinant human CXCR4 protein (ab155716).

[0067] At the same time, by calculating the expressed and purified protein, the expression level of the antibody can reach more than 0.5g / L, proving that the antibody has a high expression yield and is suitable for large-scale production.

[0068] 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 autologous stem cell preparation, characterized in that: The preparation is prepared by the following steps: Step 1: Stem cell collection and isolation: Peripheral blood was collected from healthy adult volunteers and mononuclear cells were isolated by density gradient centrifugation; Step 2: Stem cell expansion and immune activation: The isolated monocytes were cultured in a medium containing 10% fetal bovine serum, and TLR7 / 8 agonists and anti-CXCR4 single domain antibodies were added for immune activation; Step 3: Freeze-drying and preparation storage: The immune-activated stem cells are collected by centrifugation, resuspended in a culture medium containing 10% glycerol, and then freeze-dried to obtain a stem cell preparation that can be stored for a long time.

2. The autologous stem cell preparation according to claim 1, characterized in that The TLR7 / 8 agonist is CL264, and its concentration is 1 μg / mL.

3. The autologous stem cell preparation according to claim 1, characterized in that The VNAR sequence of the anti-CXCR4 single domain antibody is shown in SEQ ID NO. 1, and its use concentration is 1 μg / mL.

4. The autologous stem cell preparation according to claim 1, characterized in that The sequencing of the freeze-drying treatment in step 3 is as follows: pre-freezing at -80°C for 2 hours, low-speed freeze-drying at -40°C for 12 hours, and finally keeping in a -80°C refrigerator.

5. Use of the anti-CXCR4 single domain antibody according to claim 3 in stem cell activation.

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