Application and method of shikonin in inducing activated dendritic cells in vitro

Abstract

The present invention discloses an application and method of shikonin in inducing activated dendritic cells in vitro, and belongs to the technical field of dendritic cell culture. The present invention finds that shikonin can activate DC. Activated DC induced by shikonin highly expresses CD80 and CD86; compared with activated DC induced by LPS, it can upregulate the Th1 / Th17 ratio, enhance the killing ability of T cells on gastric cancer cells, and has a strong anti-tumor effect; it can effectively activate T cells and has the effect of killing tumor cells; it also has good stability and activity for DC itself. The present invention uses shikonin to induce and culture activated DC in vitro, and obtains highly active and highly immune-functional activated DC from peripheral blood, and this DC can be stored in liquid nitrogen. The activated DC induced and cultured or revived after low-temperature storage by the method of the present invention has good activity and can treat gastric cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of dendritic cell culture, and specifically relates to an application and method of shikonin in inducing activated dendritic cells in vitro. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Gastric cancer (GC) is the fifth most common malignant tumor in the world, and its mortality rate ranks fourth. In the past 10 years, the rapid development of immunotherapy has brought new hope to gastric cancer patients, especially tumor vaccine therapy, which has become a rising star in this field. Vaccine therapy can stimulate the increase of specific lymphocytes and induce a strong immune response to tumor antigens. Compared with traditional treatments, immunotherapy has low toxicity, long-lasting efficacy, and helps long-term survival. Dendritic cell (DC) tumor vaccine is one of the most promising types at present, and it has achieved relatively ideal results in preliminary trials of various tumors.

[0004] DC is the key to activating adaptive immunity. It not only has an immune coordination function and clears foreign invading viruses, bacteria and other microbial infections, but also can regulate the immune system and induce immune tolerance in a stable state, thereby maintaining the balance of the immune system. Inducing changes in its functional state has become an important strategy for immunotherapy. Isolated DCs usually need to be co-cultured with cytokines to reach a mature state. Studies have shown that using agonists to activate DCs in vitro can more effectively activate effector cells to enter the tumor microenvironment and kill tumors. The lack of an effective DC activation adjuvant may cause DCs to remain in an unactivated state or induce tolerance, resulting in poor DC vaccine effects. Related antibodies or agonist inducers can induce DC maturation and activation, which is more conducive to the treatment of tumors. However, some activation inducers can form an immunosuppressive tumor microenvironment, produce immunosuppressive effects, and are not conducive to the subsequent immune effects of the vaccine.

[0005] Shikonin (SK) is one of the active ingredients of traditional Chinese medicine Lithospermum officinale. Its main active ingredient is akanin, with molecular formula C 16 H 16 O5, has multiple effects such as anti-tumor, anti-inflammatory, anti-diabetic, immunomodulatory, etc. At present, there is no report on the method of inducing activated DCs in vitro with shikonin. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide an application and method of shikonin in inducing activated dendritic cells in vitro. The present invention utilizes shikonin to induce and culture activated DCs in vitro, thereby obtaining highly active activated DCs with strong immune function for the treatment of gastric cancer.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] The first aspect of the present invention provides the use of shikonin in inducing activated dendritic cells in vitro.

[0009] In some embodiments of the present invention, the activated dendritic cells have one or more of the following functions:

[0010] (1) High expression of co-stimulatory molecules CD80 and CD86;

[0011] (2) Co-culture with T cells to promote the secretion of IL-12p70, IFN-γ, and TNF-α;

[0012] (3) Co-culture with T cells to promote the transformation of T cells to Th1 and Th17;

[0013] (4) Co-culture with T cells to enhance the ability of T cells to kill gastric cancer cells.

[0014] The second aspect of the present invention provides a method for inducing activated dendritic cells in vitro with shikonin, comprising the following steps:

[0015] Obtain peripheral blood mononuclear cells and culture them to obtain dendritic cell precursor cells;

[0016] Cytokines are added and cultured until the dendritic cell precursor cells differentiate into dendritic cells;

[0017] Adding shikonin induced dendritic cells to differentiate into activated dendritic cells.

[0018] In some embodiments of the present invention, obtaining peripheral blood mononuclear cells comprises the following steps:

[0019] Peripheral blood was collected, diluted, and separated to obtain peripheral blood mononuclear cells.

[0020] In some embodiments of the present invention, the peripheral blood mononuclear cells are cultured in 1640 medium containing 10-15% autologous inactivated plasma. It can be understood that the autologous inactivated plasma is plasma homologous to the cells induced by the culture.

[0021] In some embodiments of the present invention, the cytokines are IL-4 and GM-CSF;

[0022] After adding cytokines, the concentration of IL-4 was 500 U / mL and the concentration of GM-CSF was 1000 U / mL.

[0023] In some embodiments of the present invention, after adding shikonin, the concentration of shikonin is 1.4-1.5 μg / mL.

[0024] In some embodiments of the present invention, the method further comprises: collecting activated dendritic cells, treating them with a freezing solution, and then cryopreserving them.

[0025] The freezing solution includes 10% DMSO and 90% FBS by mass percentage. The concentration of activated dendritic cells in the freezing solution is 2×10 6 / mL. The collected activated dendritic cells were treated with freezing solution and then divided into cryopreservation tubes or freezing bags and stored at -196°C in liquid nitrogen.

[0026] The third aspect of the present invention provides an activated dendritic cell, wherein the activated dendritic cell is prepared by the method of inducing activated dendritic cells with shikonin in vitro;

[0027] The activated dendritic cells have one or more of the following functions:

[0028] (1) High expression of co-stimulatory molecules CD80 and CD86;

[0029] (2) Co-culture with T cells to promote the secretion of IL-12p70, IFN-γ, and TNF-α;

[0030] (3) Co-culture with T cells to promote the transformation of T cells to Th1 and Th17;

[0031] (4) Co-culture with T cells to enhance the ability of T cells to kill gastric cancer cells.

[0032] The fourth aspect of the present invention provides a use of the activated dendritic cells in the preparation of a drug for treating gastric cancer.

[0033] The activated dendritic cells prepared by the present invention have strong immune function, can be used as an activated dendritic cell product, and can be used to treat gastric cancer.

[0034] The beneficial effects of the present invention are:

[0035] The present invention found that shikonin can activate DC. The activated DC induced by shikonin highly expresses CD80 and CD86; compared with the activated DC induced by LPS, it can upregulate the Th1 / Th17 ratio, enhance the killing ability of T cells on gastric cancer cells, and has a strong anti-tumor effect; it can effectively activate T cells and has the effect of killing tumor cells; it also has better stability and activity for DC itself.

[0036] The present invention provides an in vitro culture method for inducing activation of dendritic cells derived from peripheral blood mononuclear cells. By inducing and culturing activated DCs in vitro with shikonin, activated DCs with high activity and strong immune function can be obtained from peripheral blood, and such DCs can be stored in liquid nitrogen. The activated DCs induced and cultured or revived after cryopreservation by the method of the present invention have good activity and can treat gastric cancer, which will be beneficial to the treatment of gastric cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0038] Figure 1 The expression of CD80 and CD86 on activated DCs detected by flow cytometry after 7 days of induction culture in Example 1, Comparative Example 1 and Comparative Example 2 of the present invention, A is the percentage diagram, and B is the mean fluorescence intensity diagram; compared with the PBS+DC group, ** P <0.01; compared with LPS+DC, △ P <0.05, △△ P <0.01;

[0039] Figure 2 Figure 1 shows the content of IL-12p70 (A), IFN-γ (B), and TNF-α (C) in the culture supernatant detected by Elisa after 7 days of induction culture in Example 1, Comparative Example 1, and Comparative Example 2 of the present invention, and co-cultured with T lymphocytes for 1 day; compared with the PBS+DC group, ** P <0.01; compared with LPS+DC, △ P <0.05, △△ P <0.01;

[0040] Figure 3 After 7 days of induction culture in Example 1, Comparative Example 1 and Comparative Example 2 of the present invention, DC cells were co-cultured with T lymphocytes for 1 day, and the expression of T-bet (A), RORγt (B), Tbet / CD4 + T ratio (C), RORγt / CD4+ Compared with the PBS+DC group, * p <0.05,** P <0.01; compared with LPS+DC, △ P <0.05, △△ P <0.01;

[0041] Figure 4 The killing ability of spleen effector T cells on gastric cancer cells was detected by LDH method after 7 days of induction culture in Example 1, Comparative Example 1 and Comparative Example 2 of the present invention, and co-cultured with T lymphocytes for 1 day; compared with the PBS+DC group, * P <0.01; compared with LPS+DC, △△ P <0.01;

[0042] Figure 5 This is a graph showing the activity of activated dendritic cells induced in vitro by shikonin after freezing and thawing in Example 1 of the present invention.

[0043] Figure 1-Figure 4 In the table, PBS+DC refers to DC cells induced and cultured using PBS in Comparative Example 1, SK+DC refers to DC cells induced and cultured using SK in Example 1, and LPS+DC refers to DC cells induced and cultured using LPS in Comparative Example 2. DETAILED DESCRIPTION

[0044] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0045] Example 1

[0046] A method for inducing activated dendritic cells in vitro with shikonin comprises the following steps:

[0047] Peripheral blood was collected from healthy individuals and passed the transfusion-transmitted infectious disease test. After dilution and resuspending with PBS + 2% FBS (1:1), mononuclear cells were separated using lymphocyte separation fluid. Mononuclear cells were washed with PBS + 2% FBS buffer and cultured with 1640 medium containing 10% autologous inactivated plasma at a concentration of 1×10 7 / mL per dish were inoculated in 60 mm culture dishes, and the DC precursor cells were obtained by washing the suspended cells after adhering to the wall for 1 hour in an incubator at 37°C, >5% CO2 and saturated humidity. 3mL / dish of clinical grade 1640 medium was added to the culture dish again and cultured overnight at 37°C, 5% CO2 and saturated humidity.

[0048] On the next day (i.e., day 0), cytokines IL-4 (500 U / mL) and GM-CSF (1000 U / mL) were added to induce DC cells.

[0049] During the culture process, shikonin (1.44 μg / mL) was added on day 7 to induce differentiation into activated DCs, and the cells were collected on day 8.

[0050] The activated DCs induced by shikonin collected on the 8th day were treated with the freezing solution and then packaged into cryopreservation tubes or freezing bags for storage in liquid nitrogen.

[0051] Activated DCs after induction culture or cryopreservation are used as dendritic cells for the treatment of gastric cancer.

[0052] All the above processes are carried out in a sterile environment.

[0053] Comparative Example 1

[0054] A method for inducing activated dendritic cells in vitro, which differs from Example 1 in that PBS (1.44 μg / mL) is added on day 7 to induce differentiation. The remaining steps are consistent with those of Example 1.

[0055] Comparative Example 2

[0056] A method for inducing activated dendritic cells in vitro, which differs from Example 1 in that lipopolysaccharide (LPS) (1.44 μg / mL) is added on day 7 to induce differentiation. The remaining steps are consistent with those of Example 1.

[0057] Performance testing:

[0058] (1) Activated DC immunophenotyping was performed by flow cytometry. CD11c antibody, CD80 antibody, CD86 antibody, and MHCII antibody were used for cell surface markers.

[0059] Test results such as Figure 1 shown. Figure 1 The expression of CD80 and CD86 on activated DCs was detected by flow cytometry after 7 days of induction with PBS, SK, and LPS. Figure 1 It was found that activated DCs cultured by shikonin induced expression of costimulatory molecules CD80 and CD86 at higher levels than mature DCs.

[0060] (2) Peripheral blood mononuclear cells were isolated by density gradient centrifugation from peripheral blood and CD4 + T cell immunomagnetic bead sorting kit (Miltenyi Company) was used for sorting, CD4 +T cells were used as effector T cells. Effector T cells were stimulated and activated with activated DC or allogeneic mature DC treated with mitomycin to form a mixed lymphocyte reaction system at a ratio of 1:10 and cultured at 37°C, >5% CO2 and saturated humidity, and the culture supernatant was collected.

[0061] like Figure 2 As shown, the results were analyzed by Elisa method to detect the contents of IL-12p70, IFN-γ, and TNF-α. Figure 2 The following is a graph showing the levels of IL-12p70, IFN-γ, and TNF-α in the supernatant of the culture medium of the cells induced by PBS, SK, and LPS for 7 days and then co-incubated with the sorted T cells for 1 day. Figure 2 It can be seen that the co-culture of activated DCs induced by shikonin and T cells can promote the secretion of IL-12p70, IFN-γ and TNF-α factors.

[0062] like Figure 3 As shown, activated DCs cultured with shikonin induced upregulation of the expression of T-bet and RORγt on T cells. Figure 3 The cells were induced with PBS, SK, and LPS for 7 days, and then co-cultured with sorted T cells for 1 day. The effects of DC and T lymphocyte co-culture on T-bet, RORγt, and Tbet / CD4 were detected by flow cytometry. + T、RORγt / CD4 + Effect diagram of T and Th1 / Th17 ratio. Figure 3 It can be seen that the co-culture of activated DCs induced by shikonin and T cells can promote the transformation of T cells into Th1 and Th17.

[0063] like Figure 4 As shown, activated DCs cultured with shikonin increased the cytotoxicity of T cells against gastric cancer cells. Figure 4 The figure shows the killing ability of spleen effector T cells on gastric cancer cells detected by LDH method after induction culture with PBS, SK, and LPS for 7 days and co-incubation with T cells for 1 day. Figure 4 It can be seen that shikonin-induced activated DCs enhance the killing ability of T cells against gastric cancer cells.

[0064] In summary, the activated DCs cultured by shikonin induction in the present invention highly express the co-stimulatory molecules CD80 and CD86; can promote the secretion of IL-12p70, IFN-γ and TNF-α factors when co-cultured with T cells; can promote the transformation of T cells to Th1 and Th17 when co-cultured with T cells, and mainly activate Th1 cells; can enhance the killing ability of T cells against gastric cancer cells. The activated DCs cultured by shikonin induction were pre-treated with cryopreservation solution, and then divided into cryovials or freezing bags and stored at -196°C with liquid nitrogen. After recovery, the activity can reach 95.8% ( Figure 5 ), which can be used to treat gastric cancer.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of shikonin in inducing activated dendritic cells in vitro; The concentration of shikonin is 1.4-1.5 μg / mL.

2. The use according to claim 1, characterized in that The activated dendritic cells have one or more of the following functions: (1) High expression of co-stimulatory molecules CD80 and CD86; (2) Co-culture with T cells to promote the secretion of IL-12p70, IFN-γ, and TNF-α; (3) Co-culture with T cells to promote the transformation of T cells to Th1 and Th17; (4) Co-culture with T cells to enhance the ability of T cells to kill gastric cancer cells.

3. A method for preparing activated dendritic cells by induction of shikonin in vitro, characterized in that: The steps include: Obtain peripheral blood mononuclear cells and culture them to obtain dendritic cell precursor cells; Cytokines are added and cultured until the dendritic cell precursor cells differentiate into dendritic cells; The addition of shikonin induced the differentiation of dendritic cells into activated dendritic cells; After adding shikonin, the concentration of shikonin was 1.4-1.5 μg / mL.

4. The method for inducing activated dendritic cells in vitro using shikonin according to claim 3, characterized in that: The method of obtaining peripheral blood mononuclear cells comprises the following steps: Peripheral blood was collected, diluted, and separated to obtain peripheral blood mononuclear cells.

5. The method for inducing activated dendritic cells in vitro using shikonin according to claim 3, characterized in that: The peripheral blood mononuclear cells were cultured with 1640 medium containing 10-15% autologous inactivated plasma.

6. The method for inducing activated dendritic cells in vitro using shikonin according to claim 3, characterized in that: The cytokines are IL-4 and GM-CSF; After adding cytokines, the concentration of IL-4 was 500 U / mL and the concentration of GM-CSF was 1000 U / mL.

7. The method for inducing activated dendritic cells in vitro using shikonin according to claim 3, characterized in that: The method further comprises: collecting activated dendritic cells, treating them with freezing liquid, and then freezing and preserving them.

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