Application of atorvastatin calcium in preparing an anti-tumor preparation for enhancing dendritic cell vaccine

Abstract

The present invention discloses the application of atorvastatin calcium in the preparation of an anti-tumor preparation for enhancing dendritic cell vaccines, belonging to the technical field of immune adjuvants. The application of atorvastatin calcium disclosed by the present invention in the preparation of an anti-tumor preparation for enhancing dendritic cell vaccines. Atorvastatin calcium can be used in the preparation of dendritic cell vaccines, significantly up-regulating the expression of pro-inflammatory factors Tnfa, Il6, Il12p40, and Il12p35 and the expression of surface co-stimulatory molecules CD80 and CD86 in dendritic cells, promoting the ability of dendritic cells to mediate the activation of CD4+ T cells and CD8+ T cells and their differentiation into IFNγ-secreting effector cells, and effectively enhancing the anti-tumor effect of dendritic cell vaccines. The present invention provides a method for using atorvastatin calcium in the preparation of dendritic cell vaccines to promote the efficacy of dendritic cell vaccines, providing a new direction for tumor prevention and treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of immune adjuvants, and more particularly to the application of atorvastatin calcium in the preparation of an anti-tumor preparation for enhancing dendritic cell vaccines. Background Art

[0002] Dendritic cell vaccines are prepared by modern high-tech biotechnology. Peripheral blood collected from tumor patients is induced and cultured in vitro into a large number of dendritic cells and then transfused back into the patients. By activating the patients' adaptive immune response, cancer cells can be precisely killed, and an immune memory can also be induced in the body, enabling the patients to obtain a long-term anti-cancer effect. Since Professor Ralph Steinman, the discoverer of dendritic cells, extended his expected lifespan of several months after being diagnosed with pancreatic cancer to four and a half years using dendritic cell vaccines in 2011, preclinical studies and clinical trials in the past decade or so have shown the superior effects of dendritic cell vaccines in enhancing immune responses and prolonging the overall survival of patients. This has made dendritic cell vaccines the focus of global immunotherapy research and development. Researchers have found that dendritic cell vaccines have advantages such as a multiplier effect where one cell can activate hundreds or thousands of T cells and mobilize other immune cells, almost no toxic side effects, and the ability to reconstruct the patients' immune protection system and continuously exert an anti-cancer effect. However, there are still many dilemmas and challenges in the current clinical application of dendritic cell vaccines, such as vaccine sources, vaccine antigen and adjuvant selection, and migratory limitations. Among them, the selection of adjuvants is crucial for inducing highly mature and efficient dendritic vaccines during in vitro culture.

[0003] Therefore, providing the application of atorvastatin calcium in the preparation of an anti-tumor preparation for enhancing dendritic cell vaccines is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0004] In view of this, the present invention provides the application of atorvastatin calcium in the preparation of an anti-tumor preparation for enhancing dendritic cell vaccines.

[0005] Atorvastatin calcium is an organic compound with the chemical formula 2(C 33 H 34 FN2O5).Ca and a molecular weight of 1155.34. Currently, as a prescription drug, it is mainly used for: (1) treating hypercholesterolemia; (2) reducing the risks of myocardial infarction, stroke, and revascularization in patients with coronary heart disease (such as acute coronary syndrome, stable coronary heart disease, after coronary revascularization, etc.) or other atherosclerotic cardiovascular diseases (such as ischemic cardiomyopathy, ischemic stroke, transient ischemic attack, peripheral atherosclerotic disease, etc.).

[0006] The present invention "repurposes an old drug" by using atorvastatin calcium as an immune adjuvant in the preparation of dendritic cell vaccines. Treating dendritic cells with 4 μM atorvastatin calcium for 20 hours can significantly enhance the expression of inflammatory factors and co-stimulatory molecules in dendritic cells, and improve their ability to promote the activation of CD4 + T cells and CD8 + T cells and the secretion of the effector factor IFNγ, and enhance the anti-tumor effect of dendritic cell vaccines.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] An immune adjuvant, wherein the immune adjuvant is atorvastatin calcium.

[0009] Furthermore, the application of atorvastatin calcium as an immune adjuvant in the preparation of dendritic cell vaccines.

[0010] During the preparation of dendritic cell vaccines, dendritic cells are treated with atorvastatin calcium, and the specific steps are as follows:

[0011] (1) Bone marrow cells without red blood cells are plated at a concentration of 1×10 6 / ml, 1 ml / well in a 24-well culture plate, and cultured in an incubator at 37°C with 5% carbon dioxide for 7 days. The cells are replaced with fresh medium on the 3rd, 5th, and 6th days of culture, and immature dendritic cells with low immunogenicity are obtained on the 7th day.

[0012] (2) The immature dendritic cells obtained in the above step are resuspended in fresh complete medium, treated with atorvastatin calcium for 20 hours before LPS-induced dendritic cell maturation, and then LPS is added for stimulation for 5 hours after 20 hours of atorvastatin calcium treatment to obtain mature dendritic cells with strong immunogenicity.

[0013] The complete medium is RPMI1640 medium containing GM-CSF, IL-4, fetal bovine serum, penicillin, streptomycin, and glutamine; the final concentrations of GM-CSF, IL-4, fetal bovine serum, penicillin, streptomycin, and glutamine in the complete medium are 10 ng / ml, 5 ng / ml, 10%, 100 U / ml, 100 μg / ml, and 2 mM in sequence.

[0014] The specific operation for replacing the medium on the 3rd day in step (1) is: carefully aspirate 800 μl of the original medium from each well of the 24-well culture plate, and then add an equal amount of freshly prepared complete medium preheated to 37°C.

[0015] The specific operation of changing the culture medium at 5 days and 6 days in step (1) is as follows: Carefully aspirate 500 μl of the original culture medium from each well of the 24-well culture plate, and then add an equal amount of freshly prepared complete culture medium preheated to 37 °C.

[0016] In step (2), the final concentration of atorvastatin calcium is 0.5 - 4 μM.

[0017] In step (2), the final concentration of LPS is 100 ng / ml.

[0018] Furthermore, the application of atorvastatin calcium in the preparation of an anti-tumor preparation for enhancing dendritic cell vaccine.

[0019] Furthermore, the application of atorvastatin calcium in the preparation of a preparation for enhancing the expression of pro-inflammatory factors Tnfa, Il6, Il12p40, and Il12p35 in dendritic cells.

[0020] Furthermore, the application of atorvastatin calcium in the preparation of a preparation for enhancing the expression of co-stimulatory molecules CD80 and CD86 in dendritic cells.

[0021] Furthermore, atorvastatin calcium is used in the preparation of a preparation for enhancing dendritic cells to promote CD4 + T cells and CD8 + T cell activation preparation.

[0022] Furthermore, atorvastatin calcium is used in the preparation of a preparation for enhancing dendritic cells to promote CD4 + T cells and CD8 + T cell differentiation preparation towards IFNγ-secreting effector cells.

[0023] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses the application of atorvastatin calcium in the preparation of an anti-tumor preparation for enhancing dendritic cell vaccine. Dendritic cells are professional antigen-presenting cells, which can present the antigen taken up and processed to T cells through the major histocompatibility complex to activate them, and promote the differentiation of T cells into effector T cells secreting effector factors such as IFNγ by upregulating the expression of co-stimulatory molecules and cytokines, and thus play a key role in anti-tumor and other immune responses. Atorvastatin calcium is currently mainly used as a prescription drug for the treatment of hypercholesterolemia and coronary heart disease, etc. Experiments have confirmed that atorvastatin calcium can be used in the preparation of dendritic cell vaccine, significantly upregulating the expression of pro-inflammatory factors Tnfa, Il6, Il12p40, and Il12p35 and the expression of surface co-stimulatory molecules CD80 and CD86 in dendritic cells, and promoting dendritic cell-mediated CD4 + T cells and CD8 +The ability of T cell activation and differentiation into IFNγ-secreting effector cells, and effectively enhance the anti-tumor effect of dendritic cell vaccines. The present invention provides a method for using atorvastatin calcium to promote the efficacy of dendritic cell vaccines in the preparation of dendritic cell vaccines, providing a new direction for tumor prevention and treatment. Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0025] Figure 1 The drawings are the detection results of the cell viability after treating dendritic cells with 0.5, 1, 2, 4, 6, 8, 10 μM atorvastatin calcium for 24 hours in the control group and the experimental group in Example 1 of the present invention.

[0026] Figure 2 It is the relative expression levels of inflammatory factors Tnfa, Il6, Il12p40, and Il12p35 after stimulating dendritic cells treated with control reagent and 4 μM atorvastatin calcium for different durations for 4 hours in Example 2 of the present invention; where a is dendritic cells treated with control reagent and 4 μM atorvastatin calcium for 2 hours, b is dendritic cells treated with control reagent and 4 μM atorvastatin calcium for 8 hours, c is dendritic cells treated with control reagent and 4 μM atorvastatin calcium for 14 hours, and d is dendritic cells treated with control reagent and 4 μM atorvastatin calcium for 20 hours.

[0027] Figure 3 It is the relative expression levels of inflammatory factors after stimulating dendritic cells treated with 0.5, 1, 2, 4 μM atorvastatin calcium for 20 hours in the control group and the experimental group for 4 hours in Example 3 of the present invention; where a is the relative expression level of Tnfa, b is the relative expression level of Il6, c is the relative expression level of Il12p40, and d is the relative expression level of Il12p35.

[0028] Figure 4 It is the flow cytometry detection results of the surface co-stimulatory molecule CD80 after stimulating dendritic cells treated with 0.5, 1, 2, 4 μM atorvastatin calcium for 20 hours in the control group and the experimental group for 16 hours in Example 4 of the present invention.

[0029] Figure 5Flow cytometry detection results of co-stimulatory molecule CD86 on the surface of dendritic cells treated with 0.5, 1, 2, and 4 μM atorvastatin calcium for 20 hours in the control group and experimental group in Example 4 of the present invention after 16 hours of LPS stimulation.

[0030] Figure 6 For the detection results of the promotion of CD4 + T cell activation by dendritic cells in the control group and those treated with 4 μM atorvastatin calcium for 20 hours in Example 5 of the present invention; where a is the flow cytometry detection result and b is the statistical chart of the percentage of activated cells.

[0031] Figure 7 For the detection results of the promotion of CD4 + T cell secretion of effector factor IFNγ by dendritic cells in the control group and those treated with 4 μM atorvastatin calcium for 20 hours in Example 5 of the present invention; where a is the flow cytometry detection result and b is the statistical chart of the percentage of cells expressing IFNγ.

[0032] Figure 8 For the detection results of the promotion of CD8 + T cell activation by dendritic cells in the control group and those treated with 4 μM atorvastatin calcium for 20 hours in Example 6 of the present invention; where a is the flow cytometry detection result and b is the statistical chart of the percentage of activated cells;

[0033] Figure 9 For the detection results of the promotion of CD8 + T cell secretion of effector factor IFNγ by dendritic cells in the control group and those treated with 4 μM atorvastatin calcium for 20 hours in Example 6 of the present invention; where a is the flow cytometry detection result and b is the statistical chart of the percentage of cells expressing IFNγ.

[0034] Figure 10 For the experimental results of immunizing mice with lung metastatic tumors with dendritic cell vaccines treated with PBS, the control group, and 4 μM atorvastatin calcium for 20 hours in Example 7 of the present invention; where a is the growth condition diagram of lung tumors and b is the statistical chart of the number of pulmonary metastatic tumor nodules. Detailed implementation manners

[0035] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0036] Example 1

[0037] Unless otherwise specified, the methods used in each embodiment are conventional methods.

[0038] The materials and their sources used in the following examples are as follows: atorvastatin calcium (Selleck); DMSO (Sigma); RPMI 1640 medium (Gibco); fetal bovine serum (Gibco); penicillin-streptomycin (Gibco); glutamine (Gibco); GM-CSF (R&D); IL-4 (Novus); LPS (Sigma); flow antibodies such as anti-CD11c, anti-CD11b, anti-CD80, anti-CD86, anti-CD4, anti-CD8, anti-TCRb, anti-CD25, anti-CD69, anti-IFNγ, and Fixable Viability Dye (FVD) (Invitrogen); CCK-8 kit (Beyotime); Ficoll (GE); naive CD4 + T cell sorting kit (StemCell); naive CD8 + T cell sorting kit (StemCell); Click's medium (Irvinesci).

[0039] The complete medium used in the following examples is RPMI 1640 medium containing GM-CSF, IL-4, fetal bovine serum, penicillin, streptomycin, and glutamine, and the final concentrations of GM-CSF, IL-4, fetal bovine serum, penicillin, streptomycin, and glutamine are 10 ng / ml, 5 ng / ml, 10%, 100 U / ml, 100 μg / ml, and 2 mM in sequence.

[0040] The T cell medium used in the following examples is Click's medium supplemented with fetal bovine serum, penicillin, streptomycin, and glutamine, and the final concentrations of fetal bovine serum, penicillin, streptomycin, and glutamine are 10%, 100 U / ml, 100 μg / ml, and 2 mM in sequence.

[0041] The polypeptide OVA used in the following examples 323-339 has the sequence ISQAVHAAHAEINEAGR (SEQ ID NO.1), and the polypeptide OVA 257-264 has the sequence SIINFEKL (SEQ ID NO.2), both of which are synthesized by Gil Biochemical (Shanghai) Co., Ltd.

[0042] The method for preparing bone marrow cells without red blood cells is as follows:

[0043] (1) Take the femurs and tibias of mice, remove the muscles, disinfect and wash, and flush the bone marrow cells into a 50 ml centrifuge tube with PBS that has been autoclaved and pre-cooled at 4°C. Centrifuge at 400 g for 5 minutes, and remove the supernatant;

[0044] (2) Resuspend the cells with PBS. Filter the resuspended cells through a 40-μm filter membrane to remove impurities such as broken bones, and centrifuge at 400 g for 5 minutes to remove the supernatant.

[0045] (3) Resuspend the cells with 1 ml of erythrocyte lysate to lyse the red blood cells in the bone marrow cells. Add PBS to a volume of 50 ml to terminate the lysis, and centrifuge at 400 g for 5 minutes to remove the supernatant.

[0046] (4) Resuspend the cells with 50 ml of PBS, centrifuge at 400 g for 5 minutes, and remove the supernatant to obtain bone marrow cells without red blood cells.

[0047] (5) Resuspend the cells with complete medium and count them for standby.

[0048] In Examples 1-7, the specific operation of changing the medium on the 3rd day of step (1) is as follows: Carefully aspirate 800 μl of the original medium from each well of the 24-well culture plate, and then add an equal amount of freshly prepared complete medium preheated to 37°C.

[0049] The specific operation of changing the medium on the 5th and 6th days of step (1) is as follows: Carefully aspirate 500 μl of the original medium from each well of the 24-well culture plate, and then add an equal amount of freshly prepared complete medium preheated to 37°C.

[0050] Experiment on the effect of atorvastatin calcium at different concentrations on dendritic cell activity after 24-hour treatment in Example 1

[0051] Experimental method and grouping: (1) Resuspend the bone marrow cells without red blood cells with complete medium at a concentration of 1×10 6 / ml, plate them at 1 ml / well in a 24-well culture plate, and culture them in an incubator at 37°C with 5% carbon dioxide for 7 days. Change the medium for the cells on the 3rd, 5th, and 6th days, and obtain immature dendritic cells with low immunogenicity on the 7th day. (2) Resuspend the above-mentioned immature dendritic cells in fresh complete medium at a concentration of 1×10 6 / ml, and add 0.5, 1, 2, 4, 6, 8, 10 μM atorvastatin calcium to treat the dendritic cells for 24 hours. The control group is treated with DMSO, the dissolution reagent of atorvastatin calcium, to treat the dendritic cells for 24 hours. (3) Detect the cell activity through a CCK-8 kit.

[0052] The experimental results are as Figure 1As shown, compared with the control group cells, the survival rates of dendritic cells treated with 0.5, 1, 2, 4, 6, and 8 μM atorvastatin calcium for 24 hours did not change significantly. Among them, the average survival rates of cells in the groups treated with 0.5, 1, 2, and 4 μM atorvastatin calcium were all greater than 95%, but the average survival rates of cells in the groups treated with 6 μM and 8 μM atorvastatin calcium were lower than 90% (88.47% and 81.95% respectively). In addition, compared with the control group cells, the survival rate of dendritic cells treated with 10 μM atorvastatin calcium for 24 hours decreased significantly (P < 0.001), and the average survival rate of cells was only 63.51%.

[0053] The above experimental results show that treating dendritic cells with 0.5, 1, 2, and 4 μM atorvastatin calcium for 24 hours does not affect cell viability. Treating dendritic cells with 6 μM and 8 μM atorvastatin calcium for 24 hours has a small impact on cell viability. Treating dendritic cells with 10 μM atorvastatin calcium for 24 hours will significantly reduce cell viability. Therefore, in subsequent experiments, dendritic cells were treated with 0.5 - 4 μM atorvastatin calcium.

[0054] Example 2 Experiment on the effect of treating dendritic cells with atorvastatin calcium for different durations on the expression of inflammatory factors in dendritic cells

[0055] Experimental method and grouping: (1) Resuspend bone marrow cells without red blood cells in complete medium at a concentration of 1×10 6 / ml, and plate 1 ml per well in a 24-well culture plate. Incubate in an incubator at 37°C with 5% carbon dioxide for 7 days. Replace the medium on the 3rd, 5th, and 6th days of culture. On the 7th day, obtain immature dendritic cells with low immunogenicity. (2) Resuspend the above-mentioned immature dendritic cells in fresh complete medium at a concentration of 1×10 6 / ml and divide them into four groups: For the first group of dendritic cells, add the control reagent DMSO or treat with 4 μM atorvastatin calcium for 2 hours, and then stimulate with 100 ng / ml LPS for 4 hours; for the second group of dendritic cells, add the control reagent DMSO or treat with 4 μM atorvastatin calcium for 8 hours, and then stimulate with 100 ng / ml LPS for 4 hours; for the third group of dendritic cells, add the control reagent DMSO or treat with 4 μM atorvastatin calcium for 14 hours, and then stimulate with 100 ng / ml LPS for 4 hours; for the fourth group of dendritic cells, add the control reagent DMSO or treat with 4 μM atorvastatin calcium for 20 hours, and then stimulate with 100 ng / ml LPS for 4 hours. (3) After harvesting the cells, extract RNA and reverse-transcribe it into the first strand of cDNA. Detect the relative expression levels of Tnfa, Il6, Il12p40, and Il12p35 by real-time fluorescence quantitative PCR, with the internal reference gene Actin as a reference. The primer sequences used are shown in Table 1.

[0056] Table 1

[0057]

[0058]

[0059] The experimental results of the effect of 4 μM atorvastatin calcium treatment for 2 hours followed by LPS stimulation for 4 hours on the expression of inflammatory factors in dendritic cells are as follows Figure 2 shown in a. The relative expression levels of Tnfa, Il6, Il12p40, and Il12p35 in dendritic cells treated with 4 μM atorvastatin calcium for 2 hours followed by LPS stimulation for 4 hours showed no significant difference compared with the control group

[0060] The experimental results of the effect of 4 μM atorvastatin calcium treatment for 8 hours followed by LPS stimulation for 4 hours on the expression of inflammatory factors in dendritic cells are as follows Figure 2 shown in b. The relative expression levels of Il6 and Il12p35 in dendritic cells treated with 4 μM atorvastatin calcium for 8 hours followed by LPS stimulation for 4 hours showed no significant difference compared with the control group, but the relative expression levels of Tnfa and Il12p40 were significantly upregulated compared with the control group. The relative expression level of Tnfa was 1.57 times that of the control group, and the relative expression level of Il12p40 was 1.86 times that of the control group

[0061] The experimental results of the effect of 4 μM atorvastatin calcium treatment for 14 hours followed by LPS stimulation for 4 hours on the expression of inflammatory factors in dendritic cells are as follows Figure 2 shown in c. The relative expression levels of Tnfa, Il6, Il12p40, and Il12p35 in dendritic cells treated with 4 μM atorvastatin calcium for 14 hours followed by LPS stimulation for 4 hours were significantly upregulated compared with the control group. The relative expression level of Tnfa was 1.76 times that of the control group, the relative expression level of Il6 was 1.89 times that of the control group, the relative expression level of Il12p40 was 2.24 times that of the control group, and the relative expression level of Il12p35 was 1.37 times that of the control group

[0062] The experimental results of the effect of 4 μM atorvastatin calcium treatment for 20 hours followed by LPS stimulation for 4 hours on the expression of inflammatory factors in dendritic cells are as follows Figure 2 shown in d. The relative expression levels of Tnfa, Il6, Il12p40, and Il12p35 in dendritic cells treated with 4 μM atorvastatin calcium for 20 hours followed by LPS stimulation for 4 hours were significantly upregulated compared with the control group. The relative expression level of Tnfa was 2.19 times that of the control group, the relative expression level of Il6 was 2.67 times that of the control group, the relative expression level of Il12p40 was 2.94 times that of the control group, and the relative expression level of Il12p35 was 1.67 times that of the control group

[0063] The above experimental results showed that the expression of inflammatory factors Tnfa, Il6, Il12p40, and Il12p35 in dendritic cells was not affected by LPS stimulation for 4 hours after treatment with 4 μM atorvastatin calcium for 2 hours. However, prolonging the treatment duration of atorvastatin calcium upregulated the expression of these inflammatory factors to varying degrees. Among them, the effect of upregulating the expression of inflammatory factors Tnfa, Il6, Il12p40, and Il12p35 in dendritic cells was the most significant after treatment with 4 μM atorvastatin calcium for 20 hours followed by LPS stimulation for 4 hours.

[0064] Experiment on the effect of treating dendritic cells with different concentrations of atorvastatin calcium for 20 hours on the expression of inflammatory factors in dendritic cells

[0065] Experimental method and grouping: (1) Resuspend bone marrow cells without red blood cells in complete medium at a concentration of 1×10 6 / ml, and plate 1 ml per well in a 24-well culture plate. Incubate in an incubator at 37°C with 5% carbon dioxide for 7 days. Replace the culture medium on the 3rd, 5th, and 6th days of culture. On the 7th day, obtain immature dendritic cells with low immunogenicity. (2) Resuspend the above-mentioned immature dendritic cells in fresh complete medium at a concentration of 1×10 6 / ml. Add control reagent DMSO and 0.5, 1, 2, 4 μM atorvastatin calcium respectively to treat dendritic cells for 20 hours, and then stimulate with 100 ng / ml LPS for 4 hours; (3) After harvesting the cells, extract RNA and reverse transcribe it into the first strand of cDNA. Detect the relative expression levels of Tnfa, Il6, Il12p40, and Il12p35 by real-time fluorescence quantitative PCR, using the internal reference gene Actin as a reference. The primer sequences used are the same as those in Table 1.

[0066] The experimental results are as Figure 3 shown. Compared with the control group, the relative expression levels of inflammatory factors Tnfa, Il6, Il12p40, and Il12p35 in dendritic cells treated with 0.5, 1, 2, 4 μM atorvastatin calcium were increased to varying degrees. Moreover, the effect of atorvastatin calcium in promoting the expression of inflammatory factors in dendritic cells was dose-dependent. Within the range of 0.5 - 4 μM, the relative expression levels of inflammatory factors Tnfa, Il6, Il12p40, and Il12p35 in dendritic cells treated with 4 μM atorvastatin calcium were the highest.

[0067] Experiment on the effect of treating dendritic cells with different concentrations of atorvastatin calcium for 20 hours on the expression of co-stimulatory molecule CD80 in dendritic cells

[0068] Experimental method and grouping: (1) Resuspend bone marrow cells without red blood cells in complete medium at a concentration of 1×10 6Resuspend at a concentration of / ml and plate at 1 ml / well in a 24-well culture plate. Incubate in an incubator at 37°C with 5% carbon dioxide for 7 days. Replace the medium on days 3, 5, and 6 of the culture. On day 7, obtain immature dendritic cells with low immunogenicity. (2) Resuspend the above-mentioned immature dendritic cells in fresh complete medium at a concentration of 1×10 6 / ml. Add the control reagent DMSO and atorvastatin calcium at 0.5, 1, 2, and 4 μM respectively to treat the dendritic cells for 20 hours, and then stimulate with 100 ng / ml LPS for 16 hours. (3) After harvesting the cells, detect the expression levels of co-stimulatory molecules CD80 and CD86 on the surface of dendritic cells by flow cytometry surface staining. FVD - The cells are live cells, and CD11c in live cells + CD11b + The cells are dendritic cells, and the mean fluorescence intensities of CD80 and CD86 in dendritic cells can respectively reflect the expression levels of CD80 and CD86.

[0069] The results of flow cytometry detection of co-stimulatory molecule CD80 on the surface of dendritic cells are as Figure 4 shown. The mean fluorescence intensity of CD80 in dendritic cells of the control group is 1141, and the mean fluorescence intensities of CD80 in dendritic cells treated with 0.5, 1, 2, and 4 μM atorvastatin calcium are 1179, 1398, 1397, and 1432 in sequence.

[0070] The results of flow cytometry detection of co-stimulatory molecule CD86 on the surface of dendritic cells are as Figure 5 shown. The mean fluorescence intensity of CD86 in dendritic cells of the control group is 554, and the mean fluorescence intensities of CD86 in dendritic cells treated with 0.5, 1, 2, and 4 μM atorvastatin calcium are 612, 672, 790, and 903 in sequence.

[0071] The above experimental results show that atorvastatin calcium can up-regulate the expression of co-stimulatory molecules CD80 and CD86 on the surface of dendritic cells, and this up-regulation effect is dose-dependent. In the range of 0.5 - 4 μM, the expression levels of co-stimulatory molecules CD80 and CD86 on the surface of dendritic cells treated with 4 μM atorvastatin calcium are the highest.

[0072] Example 5 Experimental study on the effect of treating dendritic cells with atorvastatin calcium for 20 hours on dendritic cell-mediated CD4 + T cell activation and secretion of effector factor IFNγ

[0073] Experimental method and grouping: (1) Use complete medium to resuspend bone marrow cells without red blood cells at 1×10 6Resuspend at a concentration of / ml and plate at 1 ml / well in a 24-well culture plate. Incubate in an incubator at 37°C with 5% carbon dioxide for 7 days. Replace the medium on days 3, 5, and 6 of the culture. On day 7, obtain immature dendritic cells with low immunogenicity. (2) Resuspend the above-mentioned immature dendritic cells in fresh complete medium at a concentration of 1×10 6 / ml. Add the control reagent DMSO and 4 μM atorvastatin calcium to treat the dendritic cells for 20 hours, and then stimulate with 100 ng / ml LPS for 4 hours. (3) After the stimulation of the two groups of cells, collect the live cells by Ficoll centrifugation, resuspend them in T cell medium, and count for standby. (4) Purify the naive CD4 + T cells of OT-II mice using a naive CD4 + T cell sorting kit, resuspend them in T cell medium, and count for standby. (5) Co-culture the two groups of dendritic cells obtained from the above experimental steps with naive CD4 + T cells respectively. Each co-culture system is 500 μl and contains 2.5×10 4 dendritic cells, 2.5×10 5 naive CD4 + T cells, and 5 μg / ml OVA 323-339 . (6) After 36 hours of co-culture, detect the proliferation of CD4 + T cells by flow cytometry surface staining. After 6 days of co-culture, detect the level of IFNγ secreted by CD4 + T cells by flow cytometry intracellular staining. FVD - cells are live cells. Among the live cells, TCRβ + CD4 + cells are CD4 + T cells. Among the CD4 + T cells, CD25 + CD69 + cells are activated cells. The proportion of IFNγ + cells among the CD4 + T cells can reflect the level of IFNγ secretion.

[0074] CD4 + The results of flow cytometry detection of the activation status of T cells are shown in Figure 6 a. The proportions of activated cells, namely CD25 + CD69 + cells, among the CD4 + T cells co-cultured with control dendritic cells and dendritic cells treated with 4 μM atorvastatin calcium for 20 hours are 40.4% and 53.5% respectively.

[0075] CD4+ The statistical analysis results of T cell activation are as follows Figure 6 shown in b. The proportion of activated cells, namely CD25 + CD69 + in CD4 + T cells co - cultured with dendritic cells treated with 4 μM atorvastatin calcium for 20 hours was significantly higher than that of CD4 + T cells co - cultured with control dendritic cells (P < 0.01).

[0076] The above experimental results indicate that treatment with 4 μM atorvastatin calcium for 20 hours can significantly enhance the ability of dendritic cells to promote CD4 + T cell proliferation.

[0077] CD4 + The flow cytometry detection results of the effector factor IFNγ secreted by CD4 Figure 7 T cells are shown in a. The proportions of IFNγ + cells in CD4 + T cells co - cultured with control dendritic cells and dendritic cells treated with 4 μM atorvastatin calcium for 20 hours were 16.7% and 25.4% respectively.

[0078] CD4 + The statistical analysis results of the effector factor IFNγ secreted by CD4 Figure 7 T cells are shown in b. The proportion of IFNγ + cells in CD4 + T cells co - cultured with dendritic cells treated with 4 μM atorvastatin calcium for 20 hours was significantly higher than that of CD4 + T cells co - cultured with control dendritic cells (P < 0.001).

[0079] The above experimental results indicate that treatment with 4 μM atorvastatin calcium for 20 hours can significantly enhance the ability of dendritic cells to promote CD4 + T cells to secrete the effector factor IFNγ.

[0080] Example 6. Experiment on the effect of treating dendritic cells with atorvastatin calcium for 20 hours on dendritic cell - mediated CD8 + T cell activation and secretion of the effector factor IFNγ

[0081] Experimental method and grouping: (1) Use complete medium to resuspend bone marrow cells without red blood cells at 1×10 6Resuspend at a concentration of / ml, plate at 1 ml / well in a 24-well culture plate, and culture in an incubator at 37°C with 5% carbon dioxide for 7 days. Replace the medium on days 3, 5, and 6 of culture, and obtain immature dendritic cells with low immunogenicity on day 7. (2) Resuspend the above immature dendritic cells in fresh complete medium at a concentration of 1×10 6 / ml, add the control reagent DMSO and 4 μM atorvastatin calcium respectively to treat the dendritic cells for 20 hours, and then stimulate with 100 ng / ml LPS for 4 hours. (3) After the stimulation of the two groups of cells, take the live cells by Ficoll centrifugation, resuspend with T cell medium, and count for standby. (4) Purify naive CD8 + T cells of OT-I mice using a naive CD8 + T cell sorting kit, resuspend with T cell medium, and count for standby. (5) Co-culture the two groups of dendritic cells obtained from the above experimental steps with naive CD8 + T cells respectively. Each co-culture system is 500 μl and contains 2.5×10 4 dendritic cells, 2.5×10 5 naive CD8 + T cells, and 10 ng / ml OVA 257-264 . (6) After co-culture for 18 hours, detect the proliferation of CD8 + T cells by flow cytometry surface staining. After co-culture for 4 days, detect the level of IFNγ secreted by CD8 + T cells by flow cytometry intracellular staining. FVD - cells are live cells. Among the live cells, TCRβ + CD8 + cells are CD8 + T cells. Among the CD8 + T cells, CD25 + CD69 + cells are activated cells. The proportion of IFNγ + cells among CD8 + T cells can reflect the level of IFNγ secretion.

[0082] CD8 + The results of flow cytometry detection of the activation status of T cells are shown as Figure 8 a. The proportions of activated cells, namely CD25 + CD69 + cells, among the CD8 + T cells co-cultured with dendritic cells in the control group and dendritic cells treated with 4 μM atorvastatin calcium for 20 hours are 43.9% and 57.3% respectively.

[0083] CD8+ The statistical analysis results of T cell activation are as follows Figure 8 shown in b. The proportion of activated cells, namely CD25 + and CD69 + cells, in CD8 + T cells co-cultured with dendritic cells treated with 4 μM atorvastatin calcium for 20 hours was significantly higher than that in CD8 + T cells co-cultured with control dendritic cells (P < 0.001).

[0084] The above experimental results indicate that treatment with 4 μM atorvastatin calcium for 20 hours can significantly enhance the ability of dendritic cells to promote the proliferation of CD8 + T cells.

[0085] CD8 + The results of flow cytometry detection of the secretion of effector factor IFNγ by CD8 Figure 9 T cells are shown in a. The proportions of IFNγ + cells in CD8 + T cells co-cultured with control dendritic cells and dendritic cells treated with 4 μM atorvastatin calcium for 20 hours were 26.7% and 44.7% respectively.

[0086] CD8 + The statistical analysis results of the secretion of effector factor IFNγ by CD8 Figure 9 T cells are shown in b. The proportion of IFNγ + cells in CD8 + T cells co-cultured with dendritic cells treated with 4 μM atorvastatin calcium for 20 hours was significantly higher than that in CD8 + T cells co-cultured with control dendritic cells (P < 0.001).

[0087] The above experimental results indicate that treatment with 4 μM atorvastatin calcium for 20 hours can significantly enhance the ability of dendritic cells to promote the secretion of effector factor IFNγ by CD8 + T cells.

[0088] Example 7 Effect of Atorvastatin Calcium on the Antitumor Effect of Dendritic Cell Vaccine

[0089] 1) Experimental methods and grouping for preparing dendritic cell vaccine: (1) Resuspend bone marrow cells without red blood cells in complete medium at a concentration of 1×10 6 / ml, plate 1 ml / well in a 24-well culture plate, and culture in an incubator at 37°C with 5% carbon dioxide for 7 days. Replace the medium on the 3rd, 5th, and 6th days of culture. On the 7th day, obtain immature dendritic cells with low immunogenicity. (2) Resuspend the above-mentioned immature dendritic cells in fresh complete medium at a concentration of 1×106 / ml, respectively add the control reagent DMSO and 4 μM atorvastatin calcium to treat dendritic cells for 20 hours, and then add 100 ng / ml LPS and 10 ng / ml OVA 257-264 . (3) After 4 hours, collect the cells of the two groups respectively, take the live cells by Ficoll centrifugation, resuspend them with PBS after washing once with PBS, and count for standby.

[0090] 2) Experimental methods for establishing a mouse lung metastasis tumor model and in vivo immunization with dendritic cell vaccine: (1) Collect cells when melanoma cells B16-OVA are in the logarithmic growth phase, wash once with PBS, resuspend melanoma cells B16-OVA with PBS, and count. 5 . (2) Inject melanoma cells B16-OVA into the veins of wild-type mice, and inject 2×10 5 B16-OVA melanoma cells per mouse. (3) Divide the above mice into three groups: The first group injects PBS into the veins of mice 1 day, 4 days, and 7 days after intravenous injection of tumor cells; the second group injects the dendritic cell vaccine of the control group into the veins of mice 1 day, 4 days, and 7 days after intravenous injection of tumor cells, and injects 5×10 5 dendritic cells per mouse each time; the third group injects the dendritic cell vaccine treated with 4 μM atorvastatin calcium into the veins of mice 1 day, 4 days, and 7 days after intravenous injection of tumor cells, and injects 5×10 5 dendritic cells per mouse each time. (4) Observe the status of mice every day, euthanize the mice 16 days after intravenous injection of tumor cells, take the lung tissues of the mice to observe the growth of lung tumors and statistically analyze the number of tumor nodules metastasized to the lungs.

[0091] The growth of lung tumors in mice is as shown in Figure 10 a. The mice in the PBS treatment group have the most lung tumors. Both the dendritic cell vaccine of the control group and the dendritic cell vaccine treated with 4 μM atorvastatin calcium can significantly reduce the tumors in the lungs of mice, and the effect of the dendritic cell vaccine treated with 4 μM atorvastatin calcium in reducing lung tumors is stronger than that of the dendritic cell vaccine of the control group.

[0092] The statistical analysis results of the number of tumor nodules metastasized to the lungs of mice are as shown in Figure 10 b. Compared with the mice in the PBS treatment group, the number of metastatic tumor nodules in the lungs of mice immunized with the dendritic cell vaccine of the control group and the dendritic cell vaccine treated with 4 μM atorvastatin calcium both decreased significantly, and the number of metastatic tumor nodules in the lungs of mice immunized with 4 μM atorvastatin calcium was significantly less than that of mice immunized with the dendritic cell vaccine of the control group.

[0093] The above experimental results show that the immune adjuvant atorvastatin calcium in the present invention can significantly enhance the anti-tumor effect of dendritic cell vaccines.

[0094] The foregoing description of the disclosed embodiments enables those skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of atorvastatin calcium in the preparation of an anti-tumor preparation for enhancing dendritic cell vaccine; The tumor is a pulmonary metastatic tumor of melanoma.

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