CN101514333B - Immunologic tolerance dendritic cell, preparation method thereof and special culture medium - Google Patents

Abstract

The invention discloses an immune tolerance dendritic cell, a preparation method and a special culture medium thereof. The medium is adding mesenchymal stem cells to the medium of dendritic cells. The preparation method is to add precursor cells of dendritic cells and induction factors into the culture medium. The co-culture ratio of the mesenchymal stem cells and the precursor cells of the dendritic cells is (1-2):(1-10), preferably 1:1. The precursor cells of dendritic cells are CD34 ⁺ cells. The inducers include GM-CSF, TNF-α, IL-4 and LPS. The invention provides the application of immune tolerance type dendritic cells in the preparation of body immune tolerance medicine. The immune tolerance dendritic cells of the invention can lower the expression of co-stimulatory molecules, reduce inflammatory factors, increase the secretion of inhibitory cytokines, have angiogenic ability and inhibit the proliferation of allogeneic T cells. The operation of the invention is simple, convenient and practical, and a stable culture technology system for selectively activating dendritic cells is established.

Description

A kind of immunotolerant dendritic cell and its preparation method and special medium

technical field

The invention relates to a dendritic cell, a preparation method thereof and a special culture medium, in particular to an immune tolerance dendritic cell, a preparation method thereof and a special culture medium.

Background technique

Dendritic cell (DC) is the most important antigen-presenting cell, named for its many dendritic or pseudopodia-like processes when mature. It participates in the recognition, processing and presentation of antigens, and is the initiator of the body's immunity. It plays a unique and important role in the induction of immune responses, can activate T cells and antigen-specific killer T cells, and plays an important role in anti-tumor and important role in the immune response to microorganisms. In recent years, DC-related research has gradually become a hot spot in immunology, and more and more attention has been paid to the basic research and clinical application of DC. In the early study of DC, due to the lack of understanding of its origin, differentiation, development, and maturation, DCs could only be isolated from different tissues. The number of cells obtained in this way was extremely small, and the study of its functional characteristics was greatly limited. Currently, DCs obtained by in vitro induction mainly include two sources, namely peripheral blood mononuclear cells and hematopoietic stem cells (including bone marrow and umbilical cord blood). Umbilical cord blood is the best candidate for induction of DC because of its convenient source, weak immunogenicity, and a large number of hematopoietic stem cells. Studies have shown that DCs induced from cord blood CD34 ⁺ hematopoietic stem cells have higher purity and more powerful functions. Different DC cell populations play different or even opposite roles in the immune response. DCs induced by conventional induction methods can stimulate the body's immune response and kill pathogens and tumor cells. Recent studies have found that DC (Alternatively Activated Dendritic Cells, AADC) induced by the inhibitory cytokine IL-10 is a dendritic cell with selective immunomodulatory activity, which can reduce inflammatory factors by secreting inhibitory cytokines. The secretion and angiogenesis of DC can induce immune tolerance and reduce inflammation. It has completely opposite functions to traditional DC, and has a very broad application prospect in the treatment of autoimmune diseases.

Mesenchymal stem cells were first isolated from bone marrow. They are a type of tissue stem cells derived from mesoderm with multi-directional differentiation potential and self-renewal ability. The ability of muscle cells, liver cells and other adult cells to differentiate is the precursor of various stromal cells in the bone marrow hematopoietic microenvironment. The latest research shows that mesenchymal stem cells can secrete IL-6, IL-12, PGE2, IL-10 and other immunosuppressive cytokines, which can significantly reduce immune responses in both basic and clinical applications.

Contents of the invention

The purpose of the present invention is to provide an immune tolerance dendritic cell, its preparation method and special culture medium.

The present invention provides a special culture medium for immune-tolerant dendritic cells, which is to add mesenchymal stem cells as a trophoblast to the culture medium for dendritic cells.

The culture medium of the dendritic cells is: DMEM-LG medium containing 5%-15% (volume percentage) of fetal bovine serum or DMEM-LG medium with a concentration of 5%-15% (volume percentage) of fetal bovine serum Complete 1640 medium.

Wherein, the mesenchymal stem cells are derived from human bone marrow or commercialized human mesenchymal stem cell lines.

Another object of the present invention is to provide a method for preparing immune-tolerant dendritic cells, adding precursor cells of dendritic cells and inducing factors to produce immune-tolerant dendritic cells.

Wherein, the co-culture ratio of the mesenchymal stem cells and the precursor cells of the dendritic cells is (1-2):(1-10); preferably 1:1.

The precursor cells of the dendritic cells are from human umbilical cord blood or commercial dendritic cell precursor cell lines; the precursor cells of the dendritic cells are CD34 ⁺ cells.

The aforementioned inducers are GM-CSF, TNF-α, IL-4 and LPS.

Wherein, the concentration of the GM-CSF is 10ng-100ng/ml, preferably 10-30ng/ml; the concentration of the TNF-α is 10ng-100ng/ml, preferably 10-30ng/ml; the IL- The concentration of 4 is 10ng-100ng/ml, preferably 10-30ng/ml; the concentration of the LPS is 10ng-1000ng/ml, preferably 100-1000ng/ml; wherein, GM-CSF and TNF-α are added to the tree The precursor cells of dendritic cells were added to the medium on day 1, IL-4 was added to the medium on day 6, and LPS was added to the medium on day 12.

The temperature of the above cultivation is 35-38°C, the _CO concentration is 5-8% (volume percentage), the humidity is saturated humidity, and the cultivation time is 14 days; the temperature is preferably 37°C, and _the CO concentration is preferably 5%.

The medium was replaced with fresh medium every 3 days from the start of the culture.

The culturing also includes subculturing.

Another object of the present invention is to provide immune tolerant dendritic cells obtained according to the above preparation method.

Another object of the present invention is to provide the application of immune tolerance dendritic cells in the preparation of immune tolerance drugs.

The immunotolerant dendritic cells of the present invention have low expression of co-stimulatory molecules CD80, CD86, HLA-DR, etc., reduce inflammatory factors, increase the secretion of inhibitory cytokines IL-10, THF-β, etc., and have angiogenic ability And inhibiting the proliferation of allogeneic T cells can cause body tolerance and reduce inflammation. Therefore, the immune tolerance dendritic cells of the present invention are of great significance in the preparation of drugs for autoimmune diseases, transplant rejection and allergic diseases.

The present invention is simple to operate, convenient and practical. Compared with the existing methods: less cytokines are used, the cost is low, and the expanded cells simulate the characteristics of cells in the body to the greatest extent. Therefore, the method of the present invention is useful in basic research and It will have broad prospects in clinical application.

Description of drawings

Figure 1 is a morphological picture of cell growth.

Fig. 2 is the result of identifying DC surface markers by flow cytometry.

Figure 3 shows the results of MSC-DC inhibiting the proliferation of allogeneic T cells.

Figure 4 is the RT-PCR electrophoresis diagram.

Figure 5 shows the results of MSC-DC tube forming experiments.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The raw materials used in this experiment are as follows: Percoll (1.073g/ml) is a product of Sigma; Ficoll (1.077g/ml) is purchased from Institute of Hematology, Chinese Academy of Medical Sciences; Percoll is purchased from Sigma; fetal bovine serum (FBS) It is the product of Hyclone Company; mouse anti-human monoclonal antibodies related to flow cytometry: CD14-PE, CD34-FITC, HLA-DR-FITC,, CD1a-FITC, CD83-PE, CD86-PE, CD80-PE are BectonDickinson Company product; PBS (8.0g NaCl, 2.9g Na ₂ HPO ₄ , 12H ₂ O, 0.2g KCl, 0.24g KH ₂ PO ₄ dissolved in 1000ml deionized water, adjust the pH value to 7.4, unless otherwise specified, used in this article PBS is all this formula); cytokines TNF-α, GM-CSF, and IL-4 are all products of Peprotech Company; LPS is a product of Sigma Company; RT-PCR kit is a product of Takara Company.

The primers used in the following examples are products of Beijing Bomaide Technology Development Co., Ltd., and the specific primer sequences are shown in Table 1

Table 1. Primer sequences for each target factor

Example 1. Obtaining mesenchymal stem cells can be purchased commercially (Nanjing Kaiji Biological Co., Ltd.), or isolated and cultured.

1. Take 2ml of healthy adult bone marrow, dilute it with an equal amount of PBS, add it to the lymphocyte separation medium (percoll), centrifuge at 900g for 20 minutes, absorb the mononuclear cell layer, wash twice with PBS, discard the supernatant, and wash with 10% FBS, 100U/ml penicillin and 100U/ml streptomycin were resuspended in DMEM-LG (Gibco Company), and inoculated into a 75cm ² culture flask (Costa Company) at a high density of 1-2×10 ⁶ cells/cm ² , at 37°C, cultured in a 5% CO ₂ incubator. Change the medium after 48 hours, remove the suspended cells, and change the medium every 3 to 4 days. When the cells grow close to confluence, they are digested with 0.25% trypsin (Sigma Company) and frozen for use in experiments.

Example 2, Induction of immune tolerant dendritic cells (MSC-DC) and DC, IL-10-DC

1. Recover mesenchymal stem cells and inoculate 24-well plates at 5×10 ⁴ /well.

2. Obtain CD34 ⁺ cells, which can be purchased from commercial channels (Nanjing Kaiji Biological Co., Ltd.), or can be separated from cord blood CD34 ⁺ cells by MACS method. The discarded umbilical cord blood obtained from the hospital was added to an equal volume of PBS, and then a quarter of 5% methylcellulose was added, after mixing, centrifuged at 1000rpm for 10min. Discard the supernatant, add PBS (pH value 7.4) to resuspend, then slowly add to the test tube containing 5ml Ficoll, 1500rpm, 20min. Take the middle mononuclear cell layer, wash twice with PBS, 1000rpm, 10min. Buffer A (containing 0.5% BSA; 2mM EDTA-2Na ⁺ in PBS solution) was resuspended and counted. After centrifugation, add 300 μl buffer A/1×10 ⁸ cells, 100 μl blocking antibody (FcRBlocking Reagent)/1×10 ⁸ cells, and 100 μl CD34 magnetic bead antibody (CD34 Microbeads)/1×10 ⁸ cells. Incubate at 4°C for 30 min. After washing once with buffer A, discard the supernatant. Add 2ml of buffer A, slowly flow through the magnetic column, and add 2ml of buffer A to rinse the column after all the flow is complete. The column was taken out from the magnetic rack, washed with 1ml of buffer A, and the obtained cells were CD34 ⁺ hematopoietic stem cells.

3. Use DMEM-LG (Gibco Company) medium containing 10% FBS, 100U/ml penicillin, and 100U/ml streptomycin to induce and culture dendritic cells; 10 ⁴ /cm ² were added to CD34 ⁺ cells on MSC; at the same time, GM-CSF (20ng/ml) and TNF-α (20ng/ml) were added on the first day, and half of the medium was changed every 3 days. On the 6th day, IL-420ng/ml was added.

The normal DC group and IL-10 group of the control group directly inoculated CD34 ⁺ cells on a 24-well plate, and its culture medium was DMEM-LG (Gibco Inc. ) culture medium. In the normal DC group, GM-CSF (20ng/ml) and TNF-α (20ng/ml) were added on the first day, and half of the medium was changed every 3 days. IL-4 (20ng/ml) was added on the 6th day; and GM-CSF (20ng/ml) and TNF-α (20ng/ml) were added to the IL-10 group on the 1st day, and the medium was changed every 3 days. On day 6, IL-4 (20 ng/ml) and IL-10 (20 ng/ml) were added.

4. Cultivate to the 12th day, add LPS 1μg/ml, stimulate for 48 hours, gently pipette the upper layer of cells, collect them after centrifugation, and freeze them in a liquid nitrogen tank for later use.

Example 3, Identification of immune tolerant dendritic cells (MSC-DC)

1. Observation of cell morphology

In the process of the induction culture in Example 1, as shown in Figure 1 (A, B, and C of Figure 1 are cell morphology photos of MSC-DC at 0, 7, and 14 days, wherein, B is after adding IL-4 and culturing It was taken on the 1st day after adding LPS, and C was taken on the 2nd day after adding LPS; D, E, and F in Figure 1 are photos of the cell morphology of routinely induced DCs at 0, 7, and 14 days), and it can be found that, MSC-DCs began to proliferate and protrude a small amount at about 7 days after induction, while conventionally induced DCs had larger cell bodies, significantly increased number of processes, and cells aggregated together. At 14 days, the cells in the two groups proliferated to varying degrees, but the cells in the MSC-DC group still maintained a round shape, while the cells in the control group had protruded a large number of protrusions.

2. Detection of surface markers by flow cytometry

Count the cells, adjust the cell MSC-DC concentration to 1×10 ⁶ /ml, add to EP tube, wash once with PBS, centrifuge at 1500rpm for 10min; discard the supernatant, leave 100-200μl left, blow and mix the cells; add monoclonal antibodies CD14, CD1a, CD83, CD86, CD80 and HLA-DR, and set a tube as a blank control; 4 ℃, dark reaction 30min; Fix with 1% paraformaldehyde, place at 4°C for testing, and use flow cytometry to detect cell surface markers within 3 days.

IL-10-induced IL-10-DCs and routinely induced DCs were subjected to the same procedure and then flow cytometer was used to detect cell surface markers.

The test results are shown in Table 2 and Figure 2. It can be seen that only the expression of CD1a and CD83 on the surface of IL-10-induced IL-10-DC was lower than that of conventionally induced DC; while MSC-DC had a total of Stimulatory molecules such as CD86, CD80, and HLA-DR were all lowly expressed.

Table 2. The expression of co-stimulatory molecules on the surface of DCs induced by three methods (%)

CD1a CD14 CD83 HLA-DR CD86 CD80 DC 81.8 7.5 23.3 90.0 91.3 13.3 MSC-DC 14.4 4.7 7.7 45.7 18.8 1.7 IL-10-DC 28.7 89.9 4.8 91.4 90.0 77.4

3. ³ H incorporation experiment to detect the function of MSC-DC

Count the DC, IL-10-DC and MSC-DC cells collected in Example 1, and adjust the concentration to 2×10 ⁶ /ml; separate CD3 ⁺ T cells from the blood of the blood donor, and adjust the concentration to 1×10 ⁷ /ml. Inoculate cells into 96-well plates according to T: DC ratio gradient 1:2, 1:5, 1:10, 1:20, 1:40, 1:80, and the culture medium is 1640 medium (Gibco) containing 20% FBS . After 84 hours of co-cultivation, 1 μci/ml of ³ H was added, the cells were collected after 12 hours of continuous culture, and the cpm value was counted with a liquid scintillation instrument. Each group has 3 repetitions. The proliferation level of T cells is expressed by stimulation index (SI): SI=(measurement well cpm-control well cpm)/control well cpm (the control group is ^{the 3} H absorption value cpm of CD3 ⁺ T cells cultured alone)

Figure 3 shows: the ability of each dendritic cell to inhibit the proliferation of allogeneic T cells is MSC-DC>IL-10-DC>DC, and MSC-DC has a strong inhibition when T:MSC-DC is 1:2 The ability of allogeneic T cells to proliferate.

4. RT-PCR detection of expression of cytokines and chemokines

Total RNA was extracted with TRizol reagent, and cDNA was synthesized by reverse transcription in strict accordance with the instructions of the reverse transcription kit (TaKaRa Company) using it as a template, and PCR amplification was performed using primers according to the following conditions: pre-denaturation at 94°C for 2 minutes, 94°C for 30 seconds , 56°C for 30 seconds, 72°C for 60 seconds, a total of 35 cycles, and finally 72°C for 5 minutes. After the reaction was completed, 10 μl of PCR products were taken for electrophoresis in 1.5% agarose gel. After electrophoresis, observe and image under the gel analyzer (Figure 4).

The results showed that compared with conventionally induced DCs, MSC-DCs can secrete inhibitory cytokines such as IL-10 and TGF-β, reduce the secretion of inflammatory cytokines such as IL-12, and at the same time lower the expression of chemokines such as CCR5 and CCR6 .

5. Tube forming experiment to detect the tube forming ability of MSC-DC

(1) Prepare a 48-well cell culture plate (costa) and 20-200 μl Tips and freeze at -20°C; Matrigel (Sigma) thaw at 4°C.

(2) Take one well of the 48-well plate and use 30 μl Matrigel to spread the glue, and use a pre-cooled Tip to move quickly to prevent the Matrigel from solidifying too quickly at room temperature and causing the glue surface to be uneven. Place at 37°C for 30 minutes to allow the gel to solidify.

(3) For cell counting, take 2×10 ⁴ MSC-DC and routinely induced DC cells and add them to wells covered with Matrigel gel respectively, add cell culture medium DMEM-LG to 200 μl, 37°C, 5% CO ₂ saturated humidity The results of tube formation were observed with an inverted microscope, as shown in Figure 5. in,

A and B of Figure 5 are the results of tube formation of MSC-DC and conventionally induced DC cells on Matrigel gel respectively; Figure 5C is the electrophoresis graph of MSC-DC and conventionally induced DC cells respectively obtained by RT-PCR.

From A and B of Figure 5, it can be seen that MSC-DC has the ability to form tubes on Matrigel glue, and has the characteristics of endothelial cell vascular network; while conventionally induced DC does not have the ability to form tubes on Matrigel glue, indicating that MSC-DC can Secretion of tubule-associated cytokines.

Figure 5C shows that conventionally induced DCs can only secrete a small amount of VGEF, while MSC-DCs can secrete large amounts of vascular induction factors such as b-FGF and VEGF, which induce MSC-DCs to differentiate into mature endothelial cells, participate in angiogenesis, and induce immunity tolerance.

Claims (8)

1. A culture medium for immune-tolerant dendritic cells is to add mesenchymal stem cells as a trophoblast in the culture medium for dendritic cells; The culture medium of the dendritic cells is: DMEM-LG medium containing 5%-15% (volume percentage) fetal bovine serum. 2. The culture medium of immune-tolerant dendritic cells according to claim 1, wherein the mesenchymal stem cells are derived from commercial human mesenchymal stem cell lines. 3. A method for preparing immune-tolerant dendritic cells, characterized in that: to the culture medium of immune-tolerant dendritic cells according to claim 1 or 2, adding the precursor of dendritic cells Somatic cells and inducing factors to generate immune-tolerant dendritic cells; The co-culture ratio of the precursor cells of the mesenchymal stem cells and dendritic cells is (1-2): (1-10); The inducing factors are GM-CSF, TNF-α, IL-4 and LPS; The concentration of the GM-CSF is 10ng-100ng/ml; the concentration of the TNF-α is 10ng-100ng/ml; the concentration of the IL-4 is 10ng-100ng/ml; the concentration of the LPS is 10ng- 1000ng/ml; Among them, GM-CSF and TNF-α were added to the medium on the first day when the precursor cells of dendritic cells were added, IL-4 was added to the medium on the sixth day, and LPS was added to the culture on the 12th day base. 4. The preparation method according to claim 3, characterized in that: the co-culture ratio of the mesenchymal stem cells and the precursor cells of the dendritic cells is 1:1. 5. The preparation method according to claim 3 or 4, characterized in that: the precursor cells of the dendritic cells are from human umbilical cord blood or commercial dendritic cell precursor cell lines; The precursor cells of dendritic cells are CD34 ⁺ cells. 6. The preparation method according to claim 3, characterized in that: the concentration of the GM-CSF is 10-30ng/ml; the concentration of the TNF-α is 10-30ng/ml; the concentration of the IL-4 The concentration is 10-30ng/ml; the concentration of the LPS is 100-1000ng/ml. 7. The immunotolerant dendritic cells obtained by the preparation method according to any one of claims 3-6. 8. The application of the immunotolerant dendritic cells according to claim 7 in the preparation of immune tolerance medicines.

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