Method for inducing umbilical cord mesenchymal stem cells to secrete cell factors and application thereof

Abstract

The invention discloses a method for inducing umbilical cord mesenchymal stem cells to secrete cell factors. The method comprises the following steps: (1) separating UC-MSCs; (2) culture of the UC-MSCs; (3) multiple induction of UC-MSCs: when the confluence degree of the cells cultured in the step (2) reaches 80%, carrying out induction treatment; preparing an induction liquid from a proper amount of TGF-beta, EGF and FGF, adding the induction liquid into the culture medium, and lightly and uniformly mixing; adding the induction liquid into a cell culture dish, and inducing for 48 hours; the method comprises the following steps of: after induction, preparing enhanced induction liquid which comprises IL-1beta (interleukin-1beta), TNF (tumor necrosis factor)-alpha (alpha) and an Anti-CD105-CD44 monoclonal antibody; adding the enhanced induction liquid into the cell culture dish, and continuously inducing for 24 hours; and after 24 hours, collecting cell culture supernate for detecting cell factors. The method provided by the invention can efficiently induce UC-MSCs to secrete a plurality of cytokines, and has significant clinical application potential.

Description

Technical Field

[0001] The present invention relates to the fields of cell biology and regenerative medicine, and in particular to a method for inducing umbilical cord mesenchymal stem cells (UC-MSCs) to efficiently secrete cytokines based on multiple signal pathways and an application thereof in disease treatment. Background Art

[0002] Umbilical cord mesenchymal stem cells (UC-MSCs) have become a research hotspot in the field of regenerative medicine and cell therapy due to their low immunogenicity, strong proliferation ability, and multidirectional differentiation potential. Compared with other types of stem cells, UC-MSCs are relatively simple to obtain, do not require invasive surgery, and do not involve complex ethical issues, so their application prospects are very broad. However, existing technologies mainly focus on the basic isolation and culture of UC-MSCs, and there are relatively few systematic studies on their functional regulation and cytokine secretion in different microenvironments.

[0003] Traditional UC-MSCs induction methods usually rely on the stimulation of a single growth factor or compound, which often only induces cells to secrete certain specific types of cytokines and cannot fully stimulate the full potential of UC-MSCs. More importantly, these methods are insufficient in terms of stimulation efficiency and stability, resulting in low levels of cytokine secretion and a single type, which is difficult to meet the needs of clinical applications. In order to better exert the immunomodulatory, anti-inflammatory, and tissue repair effects of UC-MSCs, it is urgent to develop a novel and effective induction strategy that can simultaneously activate multiple cell signaling pathways and enhance the secretion capacity of UC-MSCs, thereby achieving efficient regulation and enrichment of multiple cytokines. This will lay the foundation for further promoting the application of UC-MSCs in the fields of tissue regeneration, immunomodulation, and anti-inflammatory therapy. Summary of the invention

[0004] The purpose of the present invention is to provide a method for inducing umbilical cord mesenchymal stem cells to secrete cytokines and application thereof.

[0005] Therefore, in one aspect, the present invention discloses an Anti-CD105-CD44 monoclonal antibody, the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0006] Preferably, the monoclonal antibody of the present invention specifically binds to the position 199-211 of the CD105 protein, and its amino acid sequence is LPGHSAGPRTVTV.

[0007] Preferably, the monoclonal antibody of the present invention specifically binds to the position 115-127 of the CD44 protein, and its amino acid sequence is LPNAFDGPITITI.

[0008] In another aspect, the present invention further discloses a method for inducing umbilical cord mesenchymal stem cells to secrete cytokines, the method comprising the following steps:

[0009] (1) Isolation of UC-MSCs;

[0010] (2) Cultivation of UC-MSCs;

[0011] (3) Multiple induction of UC-MSCs: When the cells cultured in step (2) reach 80% confluence, induction treatment is performed; appropriate amounts of TGF-β, EGF, and FGF are taken to prepare induction solution, which is added to the culture medium and gently mixed; the induction solution is added to the cell culture dish and induced for 48 hours; after induction, an enhanced induction solution is prepared, which contains IL-1β, TNF-α, and Anti-CD105-CD44 monoclonal antibodies; the enhanced induction solution is added to the cell culture dish and the induction is continued for 24 hours; after 24 hours, the cell culture supernatant is collected for cytokine detection.

[0012] Preferably, the concentrations of TGF-β, EGF and FGF in the induction solution of the present invention are 100 ng / mL, 50 ng / mL and 50 ng / mL respectively.

[0013] Preferably, the concentrations of IL-1β, TNF-α and Anti-CD105-CD44 monoclonal antibody in the enhanced induction solution of the present invention are 50 ng / mL, 100 ng / mL and 50 ng / mL respectively.

[0014] In another aspect, the present invention also discloses a use of the Anti-CD105-CD44 monoclonal antibody in inducing umbilical cord mesenchymal stem cells to secrete cytokines.

[0015] The present invention significantly enhances the cytokine secretion capacity of umbilical cord mesenchymal stem cells by regulating multiple signal pathways. Compared with the existing technology, it has higher innovation and practicality, and provides new ideas and methods for the application of UC-MSCs in regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Standard curves of various cytokines.

[0017] Figure 2 SDS-PAGE image of Anti-CD105-CD44 monoclonal antibody. DETAILED DESCRIPTION

[0018] 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.

[0019] 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.

[0020] Example 1: Isolation of UC-MSCs

[0021] 1. Take the umbilical cord of a healthy full-term newborn (with the consent of the volunteer and in compliance with ethical requirements) and place it under sterile conditions.

[0022] 2. Wash the umbilical cord with saline to remove blood and impurities.

[0023] 3. Cut the umbilical cord into small pieces of 3-5 cm, place them in a culture medium containing 0.1% collagenase, and digest them in a 37°C water bath for 30 minutes, gently shaking and mixing every 10 minutes.

[0024] 4. After digestion, remove the small segment of umbilical cord, filter it through a 200-mesh cell sieve, and collect the filtered liquid.

[0025] 5. Centrifuge (1000 rpm, 5 minutes), collect the precipitated cells and discard the supernatant.

[0026] 6. Wash the cell pellet twice with sterile PBS and centrifuge again to collect the cells.

[0027] Example 2: Cultivation of UC-MSCs

[0028] 1. Resuspend the cell suspension isolated in Example 1 in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and adjust the cell concentration to 1×10 6 cells / mL.

[0029] 2. Inoculate cells in T25 culture flasks at 37°C and 5% CO 2 Cultivated under conditions.

[0030] 3. Replace the culture medium every 2-3 days to remove dead cells and metabolites and observe the cell growth status.

[0031] Example 3: Multiple induction of UC-MSCs

[0032] 1. When the cells of Example 2 reached 80% confluence, induction treatment was performed.

[0033] 2. Take appropriate amount of TGF-β, EGF, and FGF, prepare induction solution (100ng / mL TGF-β, 50ng / mL EGF, 50ng / mL FGF), add to the culture medium, and mix gently.

[0034] 3. Add the induction solution to the cell culture dish and induce for 48 hours.

[0035] 4. After induction, an enhanced induction solution was prepared, comprising IL-1β and TNF-α (50 ng / mL and 100 ng / mL), and 50 ng / mL of Anti-CD105-CD44 monoclonal antibody (as specifically described in Example 5).

[0036] 5. Add the enhanced induction solution to the cell culture dish and continue induction for 24 hours.

[0037] 6. After 24 hours, the cell culture supernatant was collected for cytokine detection.

[0038] Example 4: Cytokine Detection

[0039] The commercial kit was used to detect the concentration of cytokines in the cell culture supernatant, including IL-6, IL-10, TNF-α, VEGF, TGF-β, etc. The test results showed that the cytokine secretion of the UC-MSCs supernatant prepared by the present invention was significantly increased, and the concentration of cytokines was higher after adding Anti-CD105-CD44 monoclonal antibodies. The details are shown in Table 1.

[0040] Table 1 Detection results of various cytokines (pg / mL)

[0041]

[0042] The test process of the kit is briefly described as follows: Collect the cell culture supernatant after induction and operate using sterile techniques. According to the instructions of the ELISA kit, dilute the culture supernatant to the recommended concentration and add it to a 96-well plate. Perform the reaction according to the instructions of the kit, and the incubation time and temperature are based on the specific requirements of the kit. Use an enzyme-labeled instrument to measure the light absorption value of each well, and according to the standard curve (such as Figure 1 The concentration of each cytokine was calculated.

[0043] Example 5: Preparation and testing of Anti-CD105-CD44 monoclonal antibodies

[0044] 1. Preparation of Anti-CD105-CD44 Monoclonal Antibody

[0045] 1. By comparing the amino acid sequences of CD105 protein and CD44 protein, two polypeptides were designed and synthesized, one of which was derived from CD105 protein, the position of the polypeptide in the CD105 protein (amino acid sequence as shown in SEQ ID NO.1) was 199-211, and its amino acid sequence was LPGHSAGPRTVTV; the other polypeptide was derived from CD44 protein, the position of the polypeptide in the CD44 protein (amino acid sequence as shown in SEQ ID NO.2) was 115-127, and its amino acid sequence was LPNAFDGPITITI.

[0046] 2. Subsequently, the two polypeptide segments were coupled to the carrier protein BSA using a classic chemical coupling method. The two polypeptides coupled to BSA were mixed in an equal mass ratio and emulsified with complete Freund's adjuvant, and 100 μg of the polypeptide mixture was injected subcutaneously into each mouse. The two polypeptides coupled to BSA were then emulsified with incomplete Freund's adjuvant and injected three times in the same way. The immunization was repeated every 2 weeks for a total of 4 immunizations. Serum was collected two weeks after the end of the immunization, and the specific antibody titers of CD105 and CD44 proteins were detected by ELISA. Mice with high titers of both proteins were selected for cell fusion.

[0047] 3. On the third day after the last immunization, collect the spleen of the mouse, prepare the spleen cell suspension and mix it with myeloma cells at a ratio of 10:1, and use 40% PEG solution at 37°C for cell fusion. After fusion, dilute the cell suspension and add it to HAT selection medium, place at 37°C, 5% CO 2 Incubate in an incubator and protect from light. After 7 days, select the cultured hybridoma cells, and screen the cell lines that can produce specific antibodies against the two proteins by ELISA. Subsequently, the screened positive cell lines were verified by ELISA to detect their binding activity to the two proteins, and the positive cell lines were cloned by limited dilution until a stable monoclonal cell line was obtained. Finally, the monoclonal antibody produced was purified using a protein G column to obtain a monoclonal antibody against CD105-CD44. The monoclonal antibody was sterilized by filtration with a 220nm filter membrane and stored at -80°C for later use.

[0048] 2. Testing of Anti-CD105-CD44 Monoclonal Antibodies

[0049] 1. The concentration of the purified monoclonal antibody was measured using a BCA kit. The result showed that the concentration of the monoclonal antibody was 6.15 mg / ml.

[0050] 2. The purity of monoclonal antibodies was analyzed by SDS-PAGE. The results are shown in Figure 2 , showing that its purity is over 95%.

[0051] 3. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody were analyzed. The results showed that the amino acid sequences of the heavy chain and the light chain variable region corresponded to SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0052] 4. The binding activity of the monoclonal antibody and the commercially available monoclonal antibody was detected by ELISA method. The results are shown in Table 2. The binding activity of the monoclonal antibody of the present invention is better than that of the commercially available monoclonal antibody.

[0053] Table 2 Monoclonal antibody ELISA test results (OD450nm)

[0054]

[0055] The steps of the above ELISA detection method are briefly described as follows:

[0056] (1) Antigen coating: Dilute CD105 protein (ab54338) and CD44 protein (ab173996) to a final concentration of 1 μg / mL using carbonate buffer. Add 100 μL of the corresponding antigen solution to each well of a 96-well ELISA plate and shake gently to ensure that the antigen is evenly distributed. Incubate overnight at 4°C to allow the antigen to be fully coated on the ELISA plate.

[0057] (2) Washing step: The next day, discard the liquid in the wells and add 200 μL PBST (PBS containing 0.05% Tween-20) to each well for washing. Repeat the washing 3-5 times, leaving it for 1-2 minutes each time to ensure the removal of unbound antigens. Between each wash, be sure to pour out the PBST and pat the bottom of the ELISA plate dry on absorbent paper to avoid residual liquid.

[0058] (3) Blocking: Add 200 μL of PBST blocking solution containing 1% BSA to each well and incubate at 37°C for 2 hours to block the uncoated well surface to prevent nonspecific binding in subsequent steps. After blocking, discard the blocking solution and no additional washing is required.

[0059] (4) Antibody incubation: The monoclonal antibody to be tested was first diluted to 1 mg / mL, and then further diluted to final concentrations of 1:10000, 1:20000, and 1:30000, respectively. 100 μL of the diluted antibody solution was added to each well, ensuring that it was mixed evenly, and incubated at 37°C for 1 hour to allow the antibody to fully bind to the antigen.

[0060] (5) Washing: Repeat the washing procedure in step (2) to ensure that unbound antibodies are removed.

[0061] (6) Add secondary antibody for incubation: Dilute HRP-labeled secondary antibody (anti-mouse IgG-HRP) at a ratio of 1:5000, and add 100 μL of the diluted secondary antibody solution to each well. Incubate at 37°C for 30 minutes to ensure that the secondary antibody is fully bound to the primary antibody.

[0062] (7) Wash again: Wash the plate 3-5 times according to the washing procedure in step (2) to ensure that unbound secondary antibody is completely removed.

[0063] (8) Color reaction: Add 100 μL of TMB color substrate to each well, incubate at 37°C in the dark for 10 min, and observe the color change from colorless to blue.

[0064] (9) Termination of reaction: Add 50 μL of 2 M sulfuric acid to each well to terminate the color development reaction. The color will then change from blue to yellow.

[0065] (10) OD value determination: Set the wavelength to 450 nm on the microplate reader, read the absorbance (OD value) of each well, and record the experimental results.

[0066] Example 6: Therapeutic study of UC-MSCs

[0067] 1. Establishment of animal model: 30 healthy BALB / c mice of 8-10 weeks old and weighing 18-22g were selected, regardless of gender, and adaptively raised for one week. Each mouse was injected with a mixed solution of D-GalN 800mg / kg and LPS 50μg / kg according to body weight, ensuring uniform injection to avoid damage to organs. After injection, the mice were closely observed for 12 hours, and typical symptoms of acute liver injury model such as mental depression, decreased appetite, and unkempt hair were successfully induced.

[0068] 2. Experimental grouping and treatment: 30 mice were randomly divided into 3 groups, 10 mice in each group. Within 2 to 6 hours of model establishment, cells were injected into the tail vein. The grouping and treatment were as follows:

[0069] Control group: injected with an equal volume of PBS;

[0070] Uninduced UC-MSCs group: 1×10 6 Uninduced UC-MSCs;

[0071] Induced UC-MSCs group: 1×10 6 UC-MSCs induced in Example 3 above.

[0072] 3. Result detection: Mouse serum was collected 24 hours, 48 ​​hours, and 72 hours after injection to detect the levels of liver injury-related indicators (ALT, AST) and inflammatory factors (IL-6, TNF-α, IL-10).

[0073] The results showed (as shown in Table 3) that compared with the control group, the ALT and AST levels in the uninduced UC-MSCs group were significantly reduced at each time point (P<0.01), while the decrease in the induced UC-MSCs group was greater, indicating that induced UC-MSCs have a stronger protective effect against liver injury.

[0074] The results showed (as shown in Table 4) that the secretion of pro-inflammatory factors IL-6 and TNF-α in the induced UC-MSCs group at each time point was significantly lower than that in the other groups (P<0.01), while the secretion of anti-inflammatory factor IL-10 was significantly increased, indicating that the induced UC-MSCs have more advantages in regulating inflammatory response.

[0075] Table 3 ALT and AST test results of each group (U / L)

[0076]

[0077] Table 4 Detection results of inflammatory factors in each group

[0078]

[0079] This experiment shows that the induced umbilical cord mesenchymal stem cells (UC-MSCs) exhibit significant therapeutic effects in a mouse acute liver injury model. It should be noted that the UC-MSCs prepared by the present invention also have good therapeutic effects on other diseases.

[0080] 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 Anti-CD105-CD44 monoclonal antibody, characterized in that: The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

2. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody specifically binds to the position 199-211 of the CD105 protein, and its amino acid sequence is LPGHSAGPRTVTV.

3. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody specifically binds to the position 115-127 of the CD44 protein, and its amino acid sequence is LPNAFDGPITITI.

4. A method for inducing umbilical cord mesenchymal stem cells to secrete cytokines, characterized in that: The method comprises the following steps: (1) Isolation of UC-MSCs; (2) Cultivation of UC-MSCs; (3) Multiple induction of UC-MSCs: When the cells cultured in step (2) reach 80% confluence, induction treatment is performed; appropriate amounts of TGF-β, EGF, and FGF are taken to prepare an induction solution, which is added to the culture medium and gently mixed; the induction solution is added to the cell culture dish and induced for 48 hours; after induction, an enhanced induction solution is prepared, which contains IL-1β, TNF-α, and the Anti-CD105-CD44 monoclonal antibody described in claim 1; the enhanced induction solution is added to the cell culture dish and the induction is continued for 24 hours; after 24 hours, the cell culture supernatant is collected for cytokine detection.

5. The method according to claim 4, characterized in that The concentrations of TGF-β, EGF and FGF in the induction solution are 100 ng / mL, 50 ng / mL and 50 ng / mL respectively.

6. The method according to claim 4, characterized in that The concentrations of IL-1β, TNF-α and Anti-CD105-CD44 monoclonal antibody in the enhanced induction solution are 50 ng / mL, 100 ng / mL and 50 ng / mL respectively.

7. Use of the Anti-CD105-CD44 monoclonal antibody according to claim 1 in inducing umbilical cord mesenchymal stem cells to secrete cytokines.

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