CN114480277B - A kind of method and medium for stimulating dendritic cell maturation - Google Patents

Abstract

The invention discloses a method for stimulating the maturation of dendritic cells. In the process of stimulating and maturing immature dendritic cells, anhydrous ethanol is added to stimulate the maturation culture. The above-mentioned method for stimulating the maturation of dendritic cells specifically includes the following steps: (1) spreading immature dendritic cells in a culture dish, adding a medium for culture, and changing the medium after they adhere to the wall; (2) placing the immature dendritic cells in a culture dish A medium containing absolute ethanol is added to the cells obtained in step (1) to stimulate culture for at least 24 hours, and the cells and supernatant are collected. The invention has been proved by experiments that it can effectively promote the maturation of DC cells, and the effect of stimulating maturation is better than that of the LPS and TNF-α groups in the prior art, and can effectively upregulate the expression of cell surface antigens CD54, CD80, CD86 and antigen presentation molecule MHC II .

Description

A kind of method and medium for stimulating dendritic cell maturation

technical field

The invention relates to a method for stimulating maturation of dendritic cells and a culture medium for stimulating maturation, belonging to the field of biotechnology.

Background technique

Dendritic cells (DCs) are the most powerful antigen-presenting cells (APCs) that can activate naive T lymphocytes. DC2.4 cells are immortalized cell lines established by transfecting C57BL/6 mouse bone marrow cells with oncogenes v-myc and v-raf. They have strong phagocytic function and are a standard for DC research. immature cell lines. Stimulation by chemical agents can change the expression of cell surface antigens and promote their maturation.

Dendritic cell vaccine (DC vaccine) refers to mature DCs loaded with relevant antigens, and is one of the most promising new immunotherapy methods developed in recent years. The clinical treatment of certain tumors with DC vaccines has achieved significant efficacy, and DC vaccines loaded with HBV surface antigen (HBsAg) can induce anti-HBV-specific immune responses in healthy volunteers and chronic hepatitis B (CHB) patients. Therefore, the development of HBV-specific DC vaccine is of great significance in the clinical application of the treatment of CHB, and the establishment of a quality control and evaluation system for the preparation of DC vaccine is the premise of HBV-specific DC vaccine clinical trials.

There is no uniform standard for the method of stimulating the maturation of DC cells in vitro. At present, the existing technology mainly uses TNF-α or LPS (lipopolysaccharide) to stimulate the maturation of DCs. LPS and TNF-α stimulated the maturation of DCs poorly. The main technical problem is that they can stimulate DC maturation to a certain extent, but still cannot obtain highly mature DCs that match the clinically desired efficacy. Studies have shown that TNF-α-matured DC cells are functionally defective and can hardly secrete IL-12 after cell maturation. The premise of DC-induced immune activation is that a sufficient number of highly mature DCs, expressing a sufficient amount of antigenic peptides and secreting a large number of cytokines are required to effectively induce an immune response and exert clinical efficacy. LPS stimulation of DC maturation also has an insurmountable operational problem, that is, different LPS manufacturers result in different LPS purity, so the LPS concentration used to stimulate DC maturation is also different, resulting in cumbersome operations and uncertain results. In addition, the storage conditions of LPS are relatively strict. It can only be stored at 4 °C for one month. If you want to store it for a long time, it needs to be stored at -20 °C, but repeated freezing and thawing should be avoided, which increases the complexity of the operation.

SUMMARY OF THE INVENTION

The primary technical problem to be solved by the present invention is to provide a method for stimulating the maturation of dendritic cells.

Another technical problem to be solved by the present invention is to provide a medium for stimulating the maturation of dendritic cells.

In order to achieve the above object, the present invention adopts the following technical scheme:

According to a first aspect of the embodiments of the present invention, a method for stimulating the maturation of dendritic cells is provided. In the process of stimulating and maturing immature dendritic cells, a ripening agent absolute ethanol is added to stimulate the maturation culture. The method steps are as follows :

(1) Spread the immature dendritic cells in a petri dish, add the medium for culture, and change the medium after they adhere to the wall;

(2) The cells obtained in step (1) are stimulated and cultured in a medium containing a ripening agent absolute ethanol for at least 24 hours, and the cells and supernatant are collected.

Preferably, the added concentration of the absolute ethanol is 400 mmol/L to 800 mmol/L.

Preferably, the medium is 1640 complete medium.

According to a second aspect of the embodiments of the present invention, there is provided a method for stimulating the maturation of dendritic cells, comprising the following steps:

(1) Obtain dendritic cell precursor mononuclear cell PBMC, and obtain immature dendritic cells in the first medium;

(2) promoting the maturation of the cells obtained in step (1) in the second medium for at least 24 hours to obtain dendritic cells;

The first medium is a medium containing GM-CSF and IL-4, and the second medium is a ripening agent absolute ethanol added to the first medium.

Preferably, the added concentration of the absolute ethanol is 400 mmol/L to 800 mmol/L.

Preferably, the first medium is serum-free AIM-V medium.

Preferably, the final concentration of GM-CSF is 800-1000 U/ml, and the final concentration of IL-4 is 800-1000 U/ml.

According to a third aspect of the embodiments of the present invention, there is provided a culture medium for stimulating dendritic cell maturation, the culture medium is adding absolute ethanol to the dendritic cell culture medium, and the concentration of the absolute ethanol added is: 400 mmol/L～800 mmol/L.

Preferably, the working concentration of the absolute ethanol is 400 mmol/L～800 mmol/L.

The beneficial effects of the present invention are as follows:

The invention solves the problems in the prior art that the ripening effect of the ripening agent is not good, the concentration of the ripening agent is not uniform, and the operation is difficult. The invention proposes a method for stimulating the maturation of dendritic cells by using anhydrous ethanol as a ripening agent, which is proved by experiments to effectively promote the maturation of DC cells, and the effect of stimulating maturation is better than that of the LPS and TNF-α groups in the prior art, and It can effectively up-regulate the expression of cell surface antigens CD54, CD80, CD86 and antigen-presenting molecule MHC II. On the other hand, those skilled in the art routinely use absolute ethanol for cell damage modeling, and the present invention overcomes the habitual thinking of those skilled in the art. Another advantage of anhydrous ethanol as a ripening agent is that the standard of anhydrous ethanol sold by various manufacturers is consistent, which is very convenient for dendritic cell maturation-promoting culture operations, convenient to purchase and low cost.

Description of drawings

Fig. 1A is 200mmol/L absolute ethanol ripening effect figure;

Fig. 1B is 400mmol/L absolute ethanol ripening effect diagram;

Fig. 1C is 600mmol/L absolute ethanol ripening effect figure;

Fig. 1D is the effect diagram of 800mmol/L anhydrous ethanol ripening;

Figure 1E is the effect of 20ug/ml LPS ripening;

Figure 1F 1000U/ml TNF-α ripening effect diagram;

Figure 2A is a diagram of normal DC2.4 cells;

Figure 2B is a cell diagram after 24h stimulation with 600mmol/L absolute ethanol;

Figure 2C is a cell diagram after 24h stimulation with 800mmol/L absolute ethanol;

Figure 2D is a cell diagram after 1200mmol/L absolute ethanol stimulation for 24h;

Figure 3A is a graph of CD86 expression in DC2.4 cells not stimulated by absolute ethanol;

Figure 3B shows the expression of CD86 in DC2.4 cells stimulated by absolute ethanol (600mmol/l);

Figure 3C is a picture of DC2.4 cell exosomes without anhydrous ethanol stimulation;

Figure 3D Exosomes of DC2.4 cells stimulated by absolute ethanol (600mmol/l);

Figure 4A is a graph of MoDC-CD54 expression;

Figure 4B is a graph of MoDC-CD80 expression;

Figure 4C is a graph of MoDC-CD86 expression;

Figure 5A is a cell diagram of the unstimulated group on the 8th day;

Figure 5B is a cell diagram of the LPS stimulation group on the 8th day;

Figure 5C is the cell diagram of the TNF-α stimulation group on the 8th day;

Fig. 5D is the cell chart on the 8th day in the absolute ethanol group.

Detailed ways

In order for those skilled in the art to have a clear understanding of the present invention, specific embodiments are used for detailed description below. The methods used in each embodiment are conventional methods unless otherwise specified.

The materials and sources used in the following examples are as follows:

1640 medium (Gibco); fetal bovine serum (Gibco); serum-free AIM-V medium (Gibco); absolute ethanol (Beijing Oriental Shibo); DC2.4 (Fenghui Bio); M-MHC II (BD ); M-CD86 (BD); CD63 (BD); H-CD54 (BD); H-CD80 (BD); H-CD86 (BD); granulocyte-macrophage colony stimulating factor (GM-CSF ) ( PeproTech) tumor necrosis factor-a (TNF-a) (PeproTech, USA); interleukin-4 (IL-4) (PeproTech).

Example 1 Comparison of maturation-promoting effects of different concentrations of anhydrous ethanol on DC2.4 cells

As the initiator of adaptive immune response, DCs are the most powerful professional antigen-presenting cells, which can recognize antigens on the surface of tumor cells, enhance the immunogenicity of tumor cells and effectively avoid immune escape. It exhibits efficient antigen presentation function and the function of inducing naive T cell activation to participate in immune response. Anhydrous ethanol is generally used to make liver injury models in this field. Anhydrous ethanol has a certain negative effect on cells. We set up a concentration gradient comparison and found that anhydrous ethanol can play a good role in DC cells at a specific concentration. Ripening effect, and the concentration of anhydrous ethanol is easy to achieve the same, easy to buy, low cost, easy to store.

Source of material:

1. Experimental method and grouping

(1) Experimental method: Spread DC2.4 cells into a 10cm petri dish, add 1640 complete medium (RP-MI 1640 medium containing 10% fetal bovine serum), and change the medium after they adhere to the wall;

(2) The cultured cells were divided into 6 groups, and different concentrations of absolute ethanol, LPS and TNF-α were added to the medium of the 6 groups; the concentrations of absolute ethanol were 200mmol/L, 400mmol/L, 600mmol/L, respectively. L and 800mmol/L, LPS is 20ug/ml, TNF-α is 1000U/ml;

(3) After stimulation for 24h, cells were collected separately.

(4) Centrifuge the 6 groups of cells at 800 r/min for 5 min, resuspend the cells in 100 ul, add 5 ul of MHC II antibody, place on ice, incubate in the dark for 30 min, add 1 ml of PBS to wash off excess antibodies, and adjust the cell concentration to 1 ×10 ⁶ /ml-1 × 10 ⁷ /ml, take 500ul to EP tube, label, use quantitative imaging analysis flow cytometer on the machine.

2. Experimental results

As shown in Figure 1A to Figure 1F, the results of flow cytometry showed that the expression level of MHC II molecules on the surface of DC2.4 cells in the 200 mmol/L absolute ethanol group was 1.52%, and that on the surface of DC2.4 cells in the 400 mmol/L absolute ethanol group was 1.52%. The expression level of MHC II molecules was 2.38%, the expression level of MHC II molecules on the surface of DC2.4 cells in 600mmol/L absolute ethanol group was 25.02%, and the expression level of MHC II molecules on the surface of DC2.4 cells in 800 mmol/L absolute ethanol group The expression level of MHC II molecules on the surface of DC2.4 cells in 20ug/ml LPS group was 2.36%, and the expression level of MHC II molecules on the surface of DC2.4 cells in 1000U/ml TNF-α group was 2.11%.

The above data indicated that LPS and TNF-α had poor effect on stimulating DC2.4 maturation. The expression levels of MHC II molecules in the 400-800 mmol/L absolute ethanol group were higher, and the expression level of MHC II molecules in the 600 mmol/L absolute ethanol group was the highest, indicating that the optimal concentration of DC2.4 stimulated by absolute ethanol was 400-800 mmol /l.

As shown in Figure 2A to Figure 2D, Figure 2A shows normal DC2.4 cells, Figure 2B shows 600 mmol/L absolute ethanol after stimulation for 24 hours, Figure 2C shows 800 mmol/L absolute ethanol stimulation for 24 hours, and Figure 2D shows 1200 mmol/L After 24h stimulation with absolute ethanol, it can be seen from the figure that normal DC2.4 cells grow adherently and are epithelial-like, and there is no difference between cells and normal cells after stimulation with 800 mmol/L absolute ethanol. It was observed by optical microscope that when the concentration of absolute ethanol was 1200mmol/L, a large number of cells died and retracted and became suspended, indicating that this concentration has caused great damage to cells and should not be used as a concentration range for stimulating DC2.4 maturation. . Therefore, the concentration of absolute ethanol below 800mmol/L is a safe and reliable stimulation concentration for DC cells.

Example 2 Comparative experiment of CD86 expression in DC2.4 cells stimulated with absolute ethanol and not stimulated with absolute ethanol

1. Experimental method

(1) Divide DC2.4 cells into two groups (stimulated group and unstimulated group), spread them into 10cm petri dishes, and add 1640 complete medium (RP-MI 1640 medium containing 10% fetal bovine serum).

(2) After the stimulation group adhered to the wall, the medium was changed, 1640 complete medium + 600 mmol/L absolute ethanol (10 ml: 0.35 ml) was added, and the cells were stimulated for 24 hours, and the cells and supernatant were collected respectively.

(3) Collect DC2.4 cells and supernatant in the unstimulated group.

(4) Centrifuge the two types of cells at 800 r/min for 5 min, resuspend the cells in 100 ul, add 5 ul of CD86 antibody, place on ice, incubate in the dark for 30 min, add 1 ml of PBS to wash off excess antibody, and adjust the cell concentration to 1× 106/ml-1×107/ml, take 500ul to EP tube, label, and use quantitative imaging analysis flow cytometer on the machine.

(5) Centrifuge the collected two cell supernatants (40ml) at 2000r/min for 10min, remove large cell debris, take the supernatant, 10000r/min, 30min, remove the residual cell debris and organelles, etc., take the supernatant . Use an ultracentrifuge to continue centrifuging the supernatant at 110,000 r/min for 70 min. Discard the supernatant and resuspend it with the backflow liquid from the tube wall to obtain exosomes. Add 10 ul of CD63 antibody and 5 ul of CD86 antibody, and place on ice to protect from light. Incubate for 45min. Add PBS to wash off excess antibodies, centrifuge again with an ultracentrifuge at 110,000 r/min, 70 min, discard the supernatant, resuspend with the backflow liquid from the tube wall, pipet to an EP tube, label, and use quantitative imaging to analyze the flow cytometer machine.

2. Experimental results

As shown in Figures 3A-3D and Table 1.

Table 1

DC2.4 DC2.4 exosomes Not stimulated 7.69% 0.09% after stimulation 46.74% 3.43%

3. Conclusion

As shown in Figure 3A to Figure 3D, Figure 3A is the flow chart of unstimulated DC2.4 cells, the CD86 expression level is 7.69%, Figure 3B is the flow chart of DC2.4 cells stimulated by absolute ethanol, CD86 The expression level is 46.74%; Figure 3C shows the flow cytometry results of the supernatant exosomes of unstimulated DC2.4 cells, the CD86 expression level is 0.09%, and Figure 3D shows the supernatant exosomes of DC2.4 cells stimulated with absolute ethanol The flow chart of the results showed that the CD86 expression level was 3.43%. (CD63 is an exosome marker, so the expression level of exosome CD86 depends on the double-positive ratio of CD63 and CD86.) After DC2.4 cells were stimulated by absolute ethanol, the expression of cell surface antigen CD86 increased significantly, and DC2.4 secreted The expression of CD86 in exosomes also increased significantly, indicating that the method of the present invention can up-regulate the expression of DC2.4 cell surface antigen and promote the maturation of DC2.4.

Example 3 DC ripening experiment in patients with chronic hepatitis B

1. Experimental method

(1) Cell culture:

0d: Separation of PBMCs: The peripheral blood of patients with artificial hepatitis B in Beijing You'an Hospital was collected, PBMCs were separated, and cell debris and platelets were removed by washing with PBS. Suspend the cells in serum-free AIM-V medium, count, adjust the cell concentration to 5×10 ⁶ /ml, and plate them in a 6-well plate with 2-3ml of AIM-V medium per well, 37°C, 5% CO ₂ Incubate for 1-2h in the incubator to make monocytes adherent. When the incubation is over, aspirate the liquid and non-adherent cells in each culture well, and add 1 ml of AIM-V medium to each well, which contains GM-CSF at a final concentration of 800 U/ml and IL-1000 U/ml. 4. Incubate for 2 days in a 37°C, 5% CO ₂ incubator.

2d: Observe the cells under an inverted microscope, change the medium in full, and continue culturing in an incubator.

5d: Observe the cells under an inverted microscope, change half of the medium, and continue to culture in an incubator.

6d: Observe the cells under an inverted microscope and group them into blank control (unstim); TNF-a group: add TNF-a (1000U/ml); absolute ethanol group: add anhydrous ethanol (600mmol/l) as a booster. Mature factor, continue to culture for 1 d. (all final concentrations)

8d: Observe the cells of each group under an inverted microscope, collect the suspended cells and loose adherent cells in each well, take 10ul of the cells to determine the survival ratio of MoDC cells, take the cell suspensions of each group, and centrifuge them to detect the phenotype of MoDC.

(2) Flow cytometry to detect the expression of MoDC surface molecules

The immature and mature MoDC were washed with CD54, CD80, and CD86 monoclonal antibodies for 20 min at room temperature in the dark, and 1 ml of PBS was added to wash off excess antibodies, labeled, harvested and analyzed by FACS under Cellquest.

2. Experimental results

As shown in Figure 4A to Figure 4C, the mean fluorescence intensities of CD54 in the MoDC blank group, TNF-a group, and absolute ethanol stimulation group were 407, 418, and 851, respectively; the CD80 in the MoDC blank group, TNF-a group, and absolute ethanol stimulation group The mean fluorescence intensities were 26, 24.9, and 33.6, respectively; the mean fluorescence intensities of CD86 in the MoDC blank group, TNF-a group, and absolute ethanol stimulation group were 5.19, 9.53, and 28.8, respectively.

3. Conclusion

Flow cytometry results showed that after MoDC was stimulated with absolute ethanol, the expression levels of cell surface molecules CD54, CD80 and CD86 were significantly higher than those in blank control group and TNF-a group. It shows that absolute ethanol can effectively stimulate the maturation of MoDC and promote the expression of its surface molecules.

Example 4 Observation of ripening cell state under electron microscope

1. Experimental method: PBMCs were cultured to the 6th day for grouping and divided into unstimulated group, LPS group, TNF-a group and absolute ethanol group (the PBMC culture method was the same as in Example 3 above). Photographs were taken with a light microscope on day 1, day 3, and day 8.

2. Experimental conclusion: As shown in Fig. 5A to Fig. 5D, in order to stimulate the cells on the 8th day, through optical microscope observation, it is found that the mature dendritic cells are larger in size and have multiple protrusions compared with the immature cells. Compared with the other control groups, the number of mature cells in the absolute ethanol group increased significantly, and the cell shape and size were uniform, indicating that the ripening function of absolute ethanol was better than other groups.

Claims (6)

1. A method for stimulating dendritic cell maturation is characterized in that absolute ethyl alcohol is added in the process of stimulating the maturation of immature dendritic cells to stimulate maturation culture, and the method comprises the following steps:

(1) laying immature dendritic cells in a culture dish, adding a culture medium for culturing, and changing the culture medium after the cells adhere to the wall;

(2) adding a culture medium containing absolute ethyl alcohol into the cells obtained in the step (1) to stimulate and culture for at least 24 hours, and collecting the cells and supernatant;

the addition concentration of the absolute ethyl alcohol is 400 mmol/L-800 mmol/L.

2. The method for stimulating the maturation of dendritic cells according to claim 1, wherein said culture medium is 1640 complete medium.

3. A method of stimulating the maturation of dendritic cells comprising the steps of:

(1) obtaining dendritic cell precursor mononuclear cells (PBMC), and obtaining immature dendritic cells under the culture of a first culture medium;

(2) subjecting the cells obtained in step (1) to mature culture in a second culture medium for at least 24 hours to obtain dendritic cells;

the first culture medium is a culture medium containing GM-CSF and IL-4, and the second culture medium is absolute ethyl alcohol obtained by adding a ripener into the first culture medium;

the addition concentration of the absolute ethyl alcohol is 400 mmol/L-800 mmol/L.

4. The method for stimulating dendritic cell maturation according to claim 3, wherein the first medium is serum-free AIM-V medium.

5. The method for stimulating dendritic cell maturation according to claim 3, wherein the final concentration of GM-CSF is 800-1000U/ml and the final concentration of IL-4 is 800-1000U/ml.

6. The application of the absolute ethyl alcohol in preparing the ripener for stimulating the maturation of the dendritic cells is characterized in that the addition concentration of the absolute ethyl alcohol is 400 mmol/L-800 mmol/L.

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