A culture medium for improving DC cell activity in multiple myeloma patients and a culture method thereof

Abstract

The present invention discloses a culture medium for improving the activity of DC cells in patients with multiple myeloma and a culture method thereof, belonging to the field of biomedicine technology. The culture medium comprises RPMI 1640 culture medium and leonurine. When DC cells are cultured in vitro, the method significantly improves the activity and maturity of DC cells in healthy individuals and patients with multiple myeloma by adding an appropriate amount of leonurine, demonstrating its potential as a DC adjuvant and can be used in DC-based treatment strategies, such as DC vaccines and DC cell therapies.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a cell culture medium and a culture method for improving the activity of DC cells in multiple myeloma patients. Background Art

[0002] Multiple myeloma (MM) is a malignant tumor of plasma cells, currently the second most common hematologic malignancy. It remains incurable, and patients will eventually face relapse. MM is closely associated with chromosomal deletions, abnormal gene expression, and altered immune microenvironment, with immune disturbances present in the early stages of the disease. Therefore, immunotherapy strategies designed to enhance a patient's own anti-myeloma immunity can effectively eliminate cancer cells, even achieving a complete cure, by enhancing immune surveillance, immune activation, and immune killing.

[0003] Dendritic cells (DCs) play a key role in initiating the body's anti-tumor immune response. As the most important antigen-presenting cells, DCs present MHC molecules and tumor antigens to initial T cells for recognition, provide co-stimulatory signals required for T cell activation, and promote the differentiation and proliferation of Th cells and cytotoxic T lymphocytes, thus being the most critical first step in initiating the body's specific anti-tumor immune response.

[0004] Studies have shown that MM patients exhibit significant immunodeficiency, with severely immunodeficient DCs in these patients being one of the primary reasons for the inability to effectively and normally activate specific anti-tumor immunity, leading to the development and progression of the disease. Therefore, reversing the severe immunodeficiency of DCs in these patients and restoring or enhancing their activity and maturity are key challenges that need to be addressed.

[0005] Therefore, the immunotherapy strategy of culturing and inducing the formation of autologous DC cells from MM patients in vitro and significantly enhancing their activity and maturity, and then returning the DC cells to the patient's body has been a cutting-edge research direction at home and abroad in recent years, with good research and translational application prospects.

[0006] Conventional methods for in vitro culture and induction of DC formation generally include isolating peripheral blood mononuclear cells (PBMCs) from peripheral blood, culturing the PBMCs in a medium consisting of 1640 medium, 5 vol% human serum, 100 U / mL penicillin, and 0.1 mg / mL streptomycin at 37°C in a 5 vol% CO2 environment for 24 hours to allow DC precursor cells (monocytes) to adhere and attach to the wall. Non-adherent cells are then washed and discarded, and the DC precursor cells are cultured in a medium consisting of 1640 medium, 5 vol% human serum, 800 U / mL GM-CSF, 500 U / mL IL-4, 100 U / mL penicillin, and 0.1 mg / mL streptomycin at 37°C in a 5 vol% CO2 environment for 7 days to induce DC formation, and then the DC cells are harvested.

[0007] Current immunotherapy strategies that involve inducing the formation of autologous DCs from MM patients in vitro and then infusing them back into the patient's body have achieved some progress. However, while existing conventional DC induction and culture methods can improve the activity and maturity of patients' DCs to a certain extent, they are still far from achieving the highly activated and mature DCs required for clinical efficacy, which can stimulate significant anti-tumor immune responses and achieve significant clinical efficacy. Therefore, how to innovate and develop in vitro DC culture technologies that significantly enhance DC activity and maturity, and restore or significantly improve the activity and maturity of DCs in MM patients with immune deficiencies, is a key issue that needs to be addressed in current DC therapy strategies. Summary of the Invention

[0008] One of the purposes of the present invention is to provide a cell culture medium for improving the activity of DC cells to solve the above problems.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows: a cell culture medium for improving the activity of DC cells, including RPMI 1640 culture medium and leonurine.

[0010] Leonurine is a specific alkaloid contained in the traditional Chinese medicinal material Leonurine. It is the main effective monomer component of Leonurine and is expected to become one of the few successful original drugs originating from China. Leonurine has been reported to regulate sugar metabolism through methylglyoxal (MGO), thereby showing possible potential for treating diabetes. At the same time, previous studies by the inventor's research group have confirmed that in mammalian experimental models of mice and rhesus monkeys, Leonurine significantly improved the lipid profile in plasma and significantly reduced the levels of cholesterol, triglycerides and low-density lipoprotein in plasma. Studies have also reported that Leonurine can regulate lipid metabolism and thus improve atherosclerosis. Previous studies by the inventor's research group have reported that administration of Leonurine for 3 weeks can significantly reduce fasting blood sugar levels and increase plasma insulin levels in the db / db mouse model, while also reducing plasma triglyceride concentrations and increasing high-density lipoprotein concentrations.

[0011] In summary, the mechanism of action of leonurine is closely related to the regulation of sugar and lipid metabolism, but the immunoregulatory effect and mechanism of leonurine on human DCs have not been reported.

[0012] The research in this application shows that motherwort alkaloids can significantly increase the expression of MHC molecules (HLA-DR), co-stimulatory molecules (CD80\CD86\CD40), and maturation-related marker molecules (CD83) in in vitro cultured and induced DC cells, significantly improving the activity and maturity of DCs. Therefore, the inventors have pioneered the application of motherwort alkaloids to the in vitro culture and induction of DC cell formation. The acquisition of highly activated and mature DCs is a technological innovation with great potential and translational application prospects. The DC culture technology scheme for applying motherwort alkaloids to DC culture technology will be described in detail in the following section.

[0013] As a preferred technical solution: the concentration of the motherwort is 0.9-1.1 μM. The inventors have confirmed through a large number of experiments that when the concentration of motherwort is above 1.1 μM, the toxicity is very strong, the DC cell activity is poor and the effect cannot be achieved; while the concentration of motherwort is too low, the DC cell activity is not improved, the target efficacy is not good, and the effect cannot be achieved; the expression levels of DC activity / maturation-related molecules at different concentrations of motherwort are as follows Figure 10 shown.

[0014] According to the inventor's experiments, it is proved that the optimal concentration of leonurine is 1 μM.

[0015] As a further preferred technical solution: the culture medium also includes human serum, GM-CSF, IL-4, penicillin and streptomycin.

[0016] As a further preferred technical solution: the concentrations of the components are: 5 vol% human serum, 800 U / mL GM-CSF, 500 U / mL IL-4, 100 U / mL penicillin, and 0.1 mg / mL streptomycin.

[0017] The second object of the present invention is to provide a cell culture method for improving the activity of DC cells, and the technical solution adopted is: adding leonurine to the culture medium.

[0018] As a preferred technical solution, the following steps are included:

[0019] Peripheral blood mononuclear cells, i.e., PBMCs, were isolated from peripheral blood and cultured in 1640 culture medium at 37°C, 5 vol% CO2 for 24 hours. Non-adherent cells were then washed and discarded to obtain DC precursor cells. The DC precursor cells were then cultured in a culture medium consisting of 1640 culture medium, 5 vol% human serum, 800 U / mL GM-CSF, 500 U / mL IL-4, 100 U / mL penicillin, 0.1 mg / mL streptomycin, and 1 μM leonurine at 37°C, 5 vol% CO2 for 7 days to induce the formation of DC cells, and finally the DC cells were harvested.

[0020] Compared with the existing technology, the advantages of the present invention are: this method found that in vitro, motherwort ine at an appropriate concentration showed a significant effect of improving the activity and maturity of DCs in healthy people and multiple myeloma patients, showing the potential of motherwort ine as a DC adjuvant and can be used in DC-based therapeutic strategies, such as DC vaccines and DC cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 CD80 + CD86 + Comparison of the proportion of cells (DCs) in the total harvested cells between the HD group and the MM patient group;

[0022] Figure 2 CD80 between the leonurine group and the control group in healthy subjects + CD86 + Comparison of the proportion of cells (DCs) in the total harvested cells;

[0023] Figure 3 is the mean fluorescence intensity (MFI) of CD40 expression on DCs of healthy individuals (n=14);

[0024] Figure 4 is the mean fluorescence intensity (MFI) of CD83 expression on DCs of healthy subjects (n=14);

[0025] Figure 5 is the mean fluorescence intensity (MFI) of HLA-DR expression on DCs of healthy subjects (n=14);

[0026] Figure 6 CD80 expression between the leonurine group and the control group in MM patients + CD86 + Comparison of the proportion of cells in the total harvested cells;

[0027] Figure 7 is the mean fluorescence intensity (MFI) of CD40 expression on DCs of MM patients (n=11);

[0028] Figure 8 is the mean fluorescence intensity (MFI) of CD83 expression on DCs of MM patients (n=11);

[0029] Figure 9 is the mean fluorescence intensity (MFI) of HLA-DR expression on DCs of MM patients (n=11);

[0030] Figure 10 This is a graph showing the expression levels of DC activity / maturation-related molecules under different concentrations of leonurine. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the accompanying drawings.

[0032] Example 1:

[0033] A cell culture medium for improving DC cell activity, comprising the following components:

[0034] RPMI1640 medium, 5 vol% human serum, 800 U / mL GM-CSF, 500 U / mL IL-4, 100 U / mL penicillin, 0.1 mg / mL streptomycin and 1 μM leonurine.

[0035] Example 2

[0036] The method for improving DC cell activity using the culture medium of Example 1 comprises the following steps:

[0037] (1) Obtaining human peripheral blood mononuclear cells:

[0038] 20 ml of peripheral venous blood (anticoagulated with heparin) was drawn from healthy donors (HD; n = 14) or patients with multiple myeloma (MM) (n = 11), diluted 1:1 with 1× PBS, and slowly added to the surface of the lymphocyte separation medium. The cells were centrifuged at 400 × g for 30 minutes using a slow ramp-up / slow ramp-down pattern. The intermediate white cell layer was then extracted and collected to obtain PBMCs. PBMCs were then washed with 1× PBS and centrifuged at 400 × g for 10 minutes, and the supernatant was discarded.

[0039] (2) Induction of DC formation in vitro:

[0040] PBMCs were resuspended in 1640 medium containing 5 vol% human serum, 100 U / mL penicillin and 0.1 mg / mL streptomycin and divided into two groups. 6 The cells were cultured at a concentration of 100 cells / ml at 37°C and 5 vol% CO2 for 24 hours to allow DC precursor cells (monocytes) to fully adhere and attach to the wall, and then the non-adherent cells were washed and discarded. The DC precursor cells were cultured in a culture medium consisting of 1640 medium, 5 vol% human serum, 800 U / mL GM-CSF, 500 U / mL IL-4, 100 U / mL penicillin, 0.1 mg / mL streptomycin and 1 μM leonurine (or an equivalent amount of drug solvent DMSO) at 37°C and 5 vol% CO2 for 7 days to culture and induce the formation of DC cells, and then the DC cells were harvested.

[0041] Example 3

[0042] Assessment of DC cell maturity and activity

[0043] This example selected five molecules as markers of DC maturity: the DC cell MHC molecule HLA-DR (essential for the first signal to activate T cells), the costimulatory molecules CD86, CD80, and CD40 (essential for the second signal to activate T cells), and the maturation-related marker molecule (CD83);

[0044] The cells collected in Example 2 were washed and resuspended in pre-chilled 1× PBS, and then APC-labeled CD40 monoclonal antibody (mAb), APC-A750-labeled CD83 mAb, PE-labeled CD86 mAb, FITC-labeled CD80 mAb and Pacific Blue-labeled HLA-DR mAb or isotype-matched negative control mAb at the same concentration were added and incubated at 4°C for 30 minutes; Subsequently, the cells were washed twice and resuspended in pre-chilled 1× PBS for immunofluorescence analysis by flow cytometry. In the FACS analysis system, the CD80 in the total cells was circled. + CD86 +The cell population was used as DCs, and the mean fluorescence intensity (MFI) of CD40, CD83, and HLA-DR expression on DCs was analyzed;

[0045] Statistical analysis was performed using GraphPad Prism 5.0 software. Paired t-test was used to compare the expression of CD40, CD83, and HLA-DR between the leonurine group and the control group, and independent sample t-test was used to compare the expression of CD80 between the leonurine group and the control group. + CD86 + The proportion of cell populations, P < 0.05 was considered statistically significant, and the results are as follows:

[0046] 1) Comparison between HD-DC and MM-DC (without leonurine treatment)

[0047] This example compares the CD80 between HD group and MM patient group. + CD86 + The difference in the proportion of CD80 cells in the total harvested cells was found in the HD group. + CD86 + The cell proportion was significantly higher than that in the MM patient group (75.55%±9.69% vs 61.39%±4.86%, P=0.000). Figure 1 shown.

[0048] 2) Effects of Leonurine on Healthy Human DC Cells

[0049] This example analyzed the differences in DC cells between the leonurine group and the control group in 14 healthy subjects and found that CD80 + CD86 + There was no statistical difference in the proportion of cells in the total harvested cells between the leonurine group and the control group (75.45% ± 8.79% vs 75.55% ± 9.69%, P = 0.933). Figure 2 As shown;

[0050] However, the mean fluorescence intensity of CD40 expression on DCs was significantly higher in the leonurine group than in the control group (2.11×10 5 ±0.92×10 5 vs 1.81×10 5 ±0.72×10 5 , P = 0.049) (Table 1, Figure 3 ); the mean fluorescence intensity of CD83 expression on DCs was significantly higher in the leonurine group than in the control group (2.41×10 5 ±2.06×10 5 vs 2.14×10 5 ±1.71×10 5, P = 0.042) (Table 2, Figure 4 ); the mean fluorescence intensity of HLA-DR expression on DCs was significantly higher in the leonurine group than in the control group (12.21×10 5 ±4.20×10 5 vs11.11×10 5 ±3.94×10 5 , P = 0.013) (Table 3, Figure 5 ).

[0051] Table 1. Mean fluorescence intensity (MFI) of CD40 expression on DCs of healthy individuals (n=14) (10 5 )

[0052]

[0053] Figure 3 It can be seen that the mean fluorescence intensity of CD40 expression on DCs was compared between the leonurine group and the control group (HD, n=14). Figure 3 a shows the mean fluorescence intensity of CD40 expression on DCs in each HD leonurine group and control group; Figure 3 b shows that the mean fluorescence intensity of CD40 expression on DCs was significantly higher in the leonurine group than in the control group (2.11×10 5 ±0.92×10 5 vs 1.81×10 5 ±0.72×10 5 , P = 0.049).

[0054] Table 2. Mean fluorescence intensity (MFI) of CD83 expression on DCs of healthy individuals (n=14) (10 5 )

[0055]

[0056] Figure 4 It can be seen that the mean fluorescence intensity of CD83 expression on DCs was compared between the leonurine group and the control group (HD, n=14). Figure 4 a shows the mean fluorescence intensity of CD83 expression on DCs in each HD leonurine group and control group; Figure 4 b shows that the mean fluorescence intensity of CD83 expression on DCs was significantly higher in the leonurine group than in the control group (2.41×10 5 ±2.06×10 5 vs 2.14×10 5 ±1.71×10 5 , P = 0.042).

[0057] Table 3. Mean fluorescence intensity (MFI) of HLA-DR expression on DCs of healthy individuals (n=14) (10 5 )

[0058]

[0059] Figure 5 It can be seen that the mean fluorescence intensity of HLA-DR expression on DCs was compared between the leonurine group and the control group (HD, n=14). Figure 5 a shows the mean fluorescence intensity of HLA-DR expression on DCs in each HD leonurine group and control group; Figure 5 b shows that the mean fluorescence intensity of HLA-DR expression on DCs was significantly higher in the leonurine group than in the control group (12.21×10 5 ±4.20×10 5 vs 11.11×10 5 ±3.94×10 5 , P = 0.013).

[0060] 3) Effects of Leonurine on DCs in MM Patients

[0061] This example analyzed the differences in DC cells between the leonurine group and the control group in 11 MM patients and found that CD80 + CD86 + The proportion of cells in the total harvested cells was significantly higher in the leonurine group than in the control group (64.86% ± 5.41% vs 61.39% ± 4.86%, P = 0.029) ( Figure 6 The mean fluorescence intensity of CD40 expression on DCs was significantly higher in the leonurine group than in the control group (1.99×10 5 ±0.98×10 5 vs 1.72×10 5 ±0.78×10 5 , P = 0.025) (Table 4, Figure 7 ); the mean fluorescence intensity of CD83 expression on DCs was significantly higher in the leonurine group than in the control group (0.30×10 5 ±0.18×10 5 vs 0.27×10 5 ±0.16×10 5 , P = 0.020) (Table 5, Figure 8 ); the mean fluorescence intensity of HLA-DR expression on DCs was significantly higher in the leonurine group than in the control group (7.49×10 5 ±2.58×10 5 vs 6.80×10 5 ±2.18×10 5, P = 0.006) (Table 6, Figure 9 ).

[0062] Table 4. Mean fluorescence intensity (MFI) of CD40 expression on DCs of MM patients (n=11) (10 5 )

[0063]

[0064] Figure 7 It can be seen that the mean fluorescence intensity of CD40 expression on DCs was compared between the leonurine group and the control group (MM, n=11). Figure 7 a shows the mean fluorescence intensity of CD40 expression on DCs in the leonurine group and control group of each MM patient; Figure 7 b shows that the mean fluorescence intensity of CD40 expression on DCs was significantly higher in the leonurine group than in the control group (1.99×10 5 ±0.98×10 5 vs 1.72×10 5 ±0.78×10 5 , P = 0.025).

[0065] Table 5. Mean fluorescence intensity (MFI) of CD83 expression on DCs of MM patients (n=11) (10 5 )

[0066]

[0067] Figure 8 It can be seen that: the comparison of the mean fluorescence intensity of CD83 expression on DCs between the leonurine group and the control group (MM, n = 11); Figure 8 a shows the mean fluorescence intensity of CD83 expression on DCs in the leonurine group and control group of each MM patient; Figure 8 b shows that the mean fluorescence intensity of CD83 expression on DCs was significantly higher in the leonurine group than in the control group (0.30×10 5 ±0.18×10 5 vs 0.27×10 5 ±0.16×10 5 , P = 0.020).

[0068] Table 6. Mean fluorescence intensity (MFI) of HLA-DR expression on DCs of MM patients (n=11) (10 5 )

[0069]

[0070] Figure 9It can be seen that: the comparison of the mean fluorescence intensity of HLA-DR expression on DCs between the leonurine group and the control group (MM, n = 11); Figure 9 a shows the mean fluorescence intensity of HLA-DR expression on DCs in the leonurine group and control group of each MM patient; Figure 9 b shows that the mean fluorescence intensity of HLA-DR expression on DCs was significantly higher in the leonurine group than in the control group (7.49×10 5 ±2.58×10 5 vs 6.80×10 5 ±2.18×10 5 , P = 0.006).

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A cell culture medium for improving the activity of DC cells in patients with multiple myeloma, characterized in that: The culture medium consists of 1640 culture medium, leonurine, human serum, GM-CSF, IL-4, penicillin and streptomycin, wherein the concentration of leonurine is 0.9-1.1 μM, the concentration of human serum is 5 vol%, the concentration of GM-CSF is 800 U / mL, the concentration of IL-4 is 500 U / mL, the concentration of penicillin is 100 U / mL, and the concentration of streptomycin is 0.1 mg / mL.

2. A cell culture method for improving the activity of DC cells in multiple myeloma patients, characterized in that: The steps include: Peripheral blood mononuclear cells, i.e., PBMCs, were isolated from peripheral blood and cultured in 1640 culture medium at 37°C, 5 vol% CO2 for 24 hours. Non-adherent cells were then washed and discarded to obtain DC precursor cells. The DC precursor cells were then cultured in a culture medium consisting of 1640 culture medium, 5 vol% human serum, 800 U / mL GM-CSF, 500 U / mL IL-4, 100 U / mL penicillin, 0.1 mg / mL streptomycin, and 0.9-1.1 μM leonurine at 37°C, 5 vol% CO2 for 7 days to induce the formation of DC cells, and finally the DC cells were harvested.

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