CN115678848A

CN115678848A - Culture medium for improving activity of DC cells of multiple myeloma patients and culture method thereof

Info

Publication number: CN115678848A
Application number: CN202211364879.9A
Authority: CN
Inventors: 代景莹; 何林; 陈成; 叶子琛
Original assignee: Sichuan Academy Of Medical Sciences Sichuan Provincial People's Hospital
Current assignee: Sichuan Academy Of Medical Sciences Sichuan Provincial People's Hospital
Priority date: 2022-11-02
Filing date: 2022-11-02
Publication date: 2023-02-03

Abstract

The invention discloses a culture medium for improving the activity of DC cells of patients with multiple myeloma and a culture method thereof, belonging to the technical field of biological medicines, wherein the culture medium comprises an RPMI1640 culture medium and leonurine; when the method is used for culturing DC cells in vitro, the addition of a proper amount of leonurine shows the effect of remarkably improving the activity and maturity of the DC cells of healthy people and patients with multiple myeloma, shows the potential of being used as a DC adjuvant, and can be used for DC-based treatment strategies, such as DC vaccine and DC cell therapy.

Description

Culture medium for improving activity of DC (dendritic cell) of multiple myeloma patient and culture method thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to a cell culture medium and a culture method for improving the activity of DC cells of patients with multiple myeloma.

Background

Multiple Myeloma (MM) is a malignant tumor of plasma cell origin, which is the second most serious malignancy of the blood system with high incidence and is not curable until now, and patients will face disease recurrence. The onset of MM is closely related to chromosome deletion, gene abnormal expression, immune microenvironment change, etc., and immune disorders have already been developed in early stages of the disease. Therefore, the immunotherapy strategy for improving the autologous anti-myeloma immunity of the patient can effectively eliminate cancer cells in vivo by improving the immune monitoring, immune activation and immune killing effects of the organism, and even achieve the aim of thorough cure.

Dendritic Cells (DCs) play a key role in initiating an anti-tumor immune response of a body, and the DC cells serve as the most important antigen presenting cells, recognize initial T cells by presenting MHC molecules and tumor antigens, provide co-stimulation signals required for T cell activation, and promote differentiation and proliferation of Th cells and cytotoxic T lymphocytes, thereby being the most key first step in initiating a specific anti-tumor immune response of the body.

Studies have shown that MM patients exhibit significant immunodeficiency, in which severely immunodeficient DC cells in patients are one of the major causes of failure of normal, effective activation of specific anti-tumor immunity, leading to disease development. Therefore, reversing the severe immunodeficiency state of the DC cells in the body of the patient and recovering or improving the activity and the maturity of the DC cells of the patient are one of the key problems to be solved urgently.

Therefore, the immunotherapy strategy of culturing and inducing in vitro to form the DC cells of the MM patient from the self source and obviously enhancing the activity and maturity of the DC cells and then returning the DC cells to the patient is the leading hot spot direction of the domestic and foreign research in recent years, and has good research and transformation application prospects.

The conventional method for culturing and inducing DC cells in vitro generally comprises the following steps: separating Peripheral Blood Mononuclear Cells (PBMCs) from peripheral blood, adding PBMCs to a medium consisting of 1640 medium, 5vol% human serum, 100U/mL penicillin and 0.1mg/mL streptomycin, at 37 deg.C, 5vol% CO ₂ Culturing in the environment for 24 hours to allow DC precursor cells (monocytes) to adhere and adhere. Thereafter, the non-adherent cells were washed off and discarded, and the DC precursor cells were treated with CO at 37 ℃ and 5vol% in a medium consisting of 1640 medium, 5vol% human serum, 800U/mL GM-CSF, 500U/mL IL-4, 100U/mL penicillin, and 0.1mg/mL streptomycin ₂ The cells were cultured in the environment for 7 days to induce formation of DC cells, and thereafter the DC cells were harvested.

Currently, immunotherapy strategies for forming MM patient autologous DCs through in vitro culture and induction and returning the MM patient autologous DCs to patients have been developed to a certain extent, but although the activity and maturity of the patient DCs can be improved to a certain extent by the existing conventional DC in vitro induction culture methods, the highly activated and mature DCs required by clinical curative effects stimulate significant anti-tumor immune effects of organisms, and the significant clinical curative effects are still different. Therefore, how to innovate the technology and research and develop an in vitro DC culture technology for remarkably improving the DC activity and the maturity, the DC activity and the maturity of the immune deficiency of the MM patient are recovered to be normal or remarkably improved, and the method is a key problem to be solved urgently in the current DC treatment strategy.

Disclosure of Invention

It is an object of the present invention to provide a cell culture medium for increasing DC cell activity, so as to solve the above problems.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a cell culture medium for increasing DC cell activity comprises RPMI1640 medium, and leonurine.

Leonurine (Leonurine) is a specific alkaloid contained in traditional Chinese medicinal material leonurus, is a main effective monomer component in leonurus, and is expected to become one of a few successful original medicaments originated from China. Leonurine has been reported to modulate carbohydrate metabolism by Methylglyoxal (MGO) and thus to display potential for the treatment of diabetes. Meanwhile, the preliminary research of the subject group of the inventor proves that leonurine remarkably improves the lipid profile in plasma and obviously reduces the levels of cholesterol, triglyceride and low-density lipoprotein in the plasma in the mammal experimental models of mice and rhesus monkeys. Leonurine has also been reported to regulate lipid metabolism and thereby ameliorate atherosclerosis. Previous studies by the inventors' group have reported that leonurine administration for 3 weeks significantly reduced fasting blood glucose levels and increased insulin levels in the plasma in a db/db mouse model, while lowering plasma triglyceride concentrations and increasing high density lipoprotein concentrations.

In conclusion, the action mechanism of leonurine is closely related to the regulation of sugar and lipid metabolism, but the immunoregulation action and mechanism of leonurine on human DC are not reported yet.

The research shows that the leonurine can obviously improve the expressions of MHC molecules (HLA-DR), co-stimulatory molecules (CD 80\ CD86\ CD 40) and maturation related marker molecules (CD 83) of DC cells cultured and induced in vitro, and obviously improve the activity and the maturity of the DC. Therefore, the inventor creatively applies the leonurine to the DC culture technology for culturing and inducing DC cells to form in vitro, so as to obtain highly activated and mature DC as a technical innovation with good potential and transformation application prospect, and the DC culture technical scheme for applying the leonurine to the DC culture technology is elaborated in the following part.

As a preferred technical scheme: the concentration of the leonurine is 0.9-1.1 mu M. A large number of experiments prove that the toxicity is very strong when the concentration of the leonurine is more than 1.1 mu M, and the DC cell activity is poor, so that a good effect cannot be achieved; the concentration of leonurine is too low, the activity of DC cells is not well improved, the target effect is not good, and the effect cannot be achieved; the expression levels of DC activity/maturation associated molecules at different concentrations of leonurine are shown in figure 10.

According to the test of the inventor, the method has the following steps: the concentration of leonurine is preferably 1. Mu.M.

As a further preferred technical scheme: the culture medium also comprises human serum, GM-CSF, IL-4, penicillin and streptomycin.

As a further preferable technical proposal: the concentration of the components is as follows: 5vol% human serum, 800U/mL GM-CSF, 500U/mL IL-4, 100U/mL penicillin, 0.1mg/mL streptomycin.

The second purpose of the invention is to provide a cell culture method for improving DC cell activity, which adopts the technical scheme that: adding leonurine into the culture medium.

The preferable technical scheme comprises the following steps:

separating peripheral blood mononuclear cells, i.e., PBMCs, from peripheral blood, said PBMCs being treated in 1640 medium at 37 ℃, 5vol% CO ₂ Culturing in environment for 24 hr, washing, removing nonadherent adherent cells to obtain DC precursor cells, and culturing the DC precursor cellsThe precursor cells were cultured in 1640 medium, 5vol% human serum, 800U/mL GM-CSF, 500U/mL IL-4, 100U/mL penicillin, 0.1mg/mL streptomycin and 1. Mu.M leonurine at 37 ℃ in a medium of 5vol% CO ₂ The cells were cultured for 7 days to induce DC cell formation, and finally the DC cells were harvested.

Compared with the prior art, the invention has the advantages that: the method finds that the leonurine in vitro shows a remarkable effect of improving the DC activity and maturity of healthy people and patients with multiple myeloma at a proper concentration, shows the potential of the leonurine as a DC adjuvant, and can be used for DC-based treatment strategies, such as DC vaccine and DC cell therapy.

Drawings

FIG. 1 shows a CD80 ⁺ CD86 ⁺ Comparison of cells (DCs) in total harvested cells between the HD and MM patient groups;

FIG. 2 is a graph showing CD80 between leonurine group and control group of healthy persons ⁺ CD86 ⁺ Comparison of the proportion of cells (DCs) in the total harvested cells;

fig. 3 is Mean Fluorescence Intensity (MFI) of CD40 expression on healthy human (n = 14) DCs;

figure 4 is the Mean Fluorescence Intensity (MFI) of CD83 expression on healthy human (n = 14) DCs;

FIG. 5 is Mean Fluorescence Intensity (MFI) of HLA-DR expression on healthy human (n = 14) DCs;

FIG. 6 shows CD80 between leonurine group and control group of MM patients ⁺ CD86 ⁺ Comparison of the proportion of cells in the total harvested cells;

figure 7 is Mean Fluorescence Intensity (MFI) of CD40 expression on DCs of MM patients (n = 11);

fig. 8 is Mean Fluorescence Intensity (MFI) of CD83 expression on MM patients (n = 11) DCs;

fig. 9 is Mean Fluorescence Intensity (MFI) of HLA-DR expression on MM patients (n = 11) DCs;

FIG. 10 is a graph of DC activity/maturation associated molecule expression levels at different concentrations of leonurine.

Detailed Description

The invention will be further explained with reference to the drawings.

Example 1:

a cell culture medium for increasing DC cell activity, consisting of:

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

Example 2

A method of increasing DC cell activity using the medium of example 1, comprising the steps of:

(1) Obtaining human peripheral blood mononuclear cells:

20ml of peripheral venous blood (heparin anticoagulation) from Healthy donors (HD; n = 14) or MM patients (n = 11) were drawn and treated with 1 XPBS 1: diluting 1, slowly adding into the upper part of the liquid level of the lymphocyte separation liquid, centrifuging for 30min in a slow-rising and slow-reducing mode at 400 Xg, and then extracting and collecting a middle white cell layer to obtain the PBMCs. Thereafter, PBMCs were washed with 1 XPBS followed by centrifugation at 400 Xg for 10min and the supernatant discarded;

(2) In vitro induction to form DC:

the PBMCs were resuspended in 1640 medium containing 5vol% human serum, 100U/mL penicillin and 0.1mg/mL streptomycin, divided into two groups at 2X 10 ⁶ Cell concentration per ml at 37 ℃ 5vol% CO ₂ Culturing in environment for 24 hr to make DC precursor cell (monocyte) adhere sufficiently, washing off and discarding cell; the DC precursor cells were then incubated in 1640 medium, 5vol% human serum, 800U/mL GM-CSF, 500U/mL IL-4, 100U/mL penicillin, 0.1mg/mL streptomycin, and 1. Mu.M leonurine (or equivalent pharmaceutical vehicle DMSO) at 37 ℃ with CO 5vol% ₂ The cells were cultured for 7 days to culture, induce the formation of DC cells, and thereafter the DC cells were harvested.

Example 3

Evaluation of DC cell maturation and Activity

Five molecules of the DC cell MHC molecule HLA-DR (necessary for activating a first signal of a T cell), costimulatory molecules CD86, CD80 and CD40 (necessary for activating a second signal of the T cell) and a maturation associated marker molecule (CD 83) are selected as markers of the DC maturation degree;

the cells collected in example 2 were washed, resuspended in precooled 1 × PBS, then added with 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 and incubated at 4 ℃ for 30 minutes; subsequently, cells were washed twice and resuspended in pre-cooled 1 × PBS for immunofluorescence analysis by flow cytometry. In FACS analysis System, CD80 in Total cells was circled ⁺ CD86 ⁺ The cell population was analyzed as DCs for Mean Fluorescence Intensity (MFI) of CD40, CD83 and HLA-DR expression on DCs, respectively;

statistical analysis was performed using GraphPad Prism 5.0 software. Paired t-test comparing the expression of leonurine groups with control groups for CD40, CD83 and HLA-DR, independent sample t-test for comparing CD80 between leonurine and control groups ⁺ CD86 ⁺ The proportion of the cell population, P < 0.05, was considered statistically significant and the results were as follows:

1) Comparison of HD-DC with MM-DC (without leonurine treatment)

This example compares CD80 between the HD and MM patient groups ⁺ CD86 ⁺ The difference of the cell ratio in the total harvested cells was found to be CD80 in the cells harvested in the HD group ⁺ CD86 ⁺ The cell proportion was significantly higher than in the MM patient group (75.55% ± 9.69%/vs 61.39% ± 4.86%, P = 0.000), as shown in fig. 1.

2) Effect of leonurine on healthy human DC cells

This example analyzes the DC cell difference between the leonurine group and the control group of 14 healthy persons in a whole group and finds CD80 ⁺ CD86 ⁺ The proportion of cells in the total harvested cells was not statistically different between the leonurine group and the control group (75.45% ± 8.79% vs 75.55% ± 9.69%, P = 0.933), as shown in fig. 2;

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

TABLE 1 Mean Fluorescence Intensity (MFI) of CD40 expression on healthy human (n = 14) DCs (10) ⁵ )

As can be seen in fig. 3: comparison of mean fluorescence intensity of CD40 expression on DCs between leonurine and control groups (HD, n = 14). FIG. 3a shows the mean fluorescence intensity of CD40 expression on DCs of each of the HD leonurine groups and the control group; FIG. 3b shows that the mean fluorescence intensity of CD40 expression on DCs was significantly higher in the leonurine group than in the control group (2.11X 10) ⁵ ±0.92×10 ⁵ vs 1.81×10 ⁵ ±0.72×10 ⁵ ，P＝0.049)。

TABLE 2 Mean Fluorescence Intensity (MFI) of CD83 expression on DCs of healthy humans (n = 14) (10) ⁵ )

As can be seen in fig. 4: comparison of mean fluorescence intensity of CD83 expression on DCs between leonurine and control groups (HD, n = 14). FIG. 4a shows the mean fluorescence intensity of CD83 expression on DCs of each of the HD leonurine groups and the control group; FIG. 4b shows that the mean fluorescence intensity of CD83 expression on DCs was significantly higher in the leonurine group than in the control group (2.41X 10) ⁵ ±2.06×10 ⁵ vs 2.14×10 ⁵ ±1.71×10 ⁵ ，P＝0.042)。

TABLE 3 Mean Fluorescence Intensity (MFI) of HLA-DR expression on healthy human (n = 14) DCs (10) ⁵ )

FIG. 5 shows a comparison of the mean fluorescence intensity of HLA-DR expression on DCs between the leonurine group and the control group (HD, n = 14). FIG. 5a shows the mean fluorescence intensity of HLA-DR expression on DCs of each of the HD leonurine groups and the control group; FIG. 5b shows that the mean fluorescence intensity of HLA-DR expression on DCs is significantly higher in the leonurine group than in the control group (12.21X 10) ⁵ ±4.20×10 ⁵ vs 11.11×10 ⁵ ±3.94×10 ⁵ ，P＝0.013)。

3) Effect of leonurine on DC cells of MM patients

This example analyzes the DC cell difference between leonurine group and control group of 11 MM patients as a whole, and finds 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) (fig. 6). The mean fluorescence intensity of CD40 expression on DCs was significantly higher in the leonurine group than in the control group (1.99X 10) ⁵ ±0.98×10 ⁵ vs 1.72×10 ⁵ ±0.78×10 ⁵ P = 0.025) (table 4, fig. 7); the mean fluorescence intensity of CD83 expression on DCs was significantly higher in the leonurine group than in the control group (0.30X 10) ⁵ ±0.18×10 ⁵ vs 0.27×10 ⁵ ±0.16×10 ⁵ P = 0.020) (table 5, fig. 8); the mean fluorescence intensity of HLA-DR expression on DCs was also significantly higher in the leonurine group than in the control group (7.49X 10) ⁵ ±2.58×10 ⁵ vs 6.80×10 ⁵ ±2.18×10 ⁵ P = 0.006) (table 6, fig. 9).

Table 4 Mean Fluorescence Intensity (MFI) of CD40 expression on mm patients (n = 11) DCs (10) ⁵ )

As can be seen in fig. 7: comparison of mean fluorescence intensity of CD40 expression on DCs between leonurine and control groups (MM, n = 11). FIG. 7a shows mean fluorescence intensity of CD40 expression on DCs of leonurine and control groups of each MM patients; FIG. 7b shows CD40 expression on DCsThe average fluorescence intensity of the compound is obviously higher in the leonurine group than in the control group (1.99 multiplied by 10) ⁵ ±0.98×10 ⁵ vs 1.72×10 ⁵ ±0.78×10 ⁵ ，P＝0.025)。

Mean Fluorescence Intensity (MFI) of CD83 expression on MM patients (n = 11) DCs (10) ⁵ )

As can be seen in fig. 8: comparison of mean fluorescence intensity of CD83 expression on DCs between leonurine and control groups (MM, n = 11); FIG. 8a shows the mean fluorescence intensity of CD83 expression on the leonurine group and control DCs of each MM patients; FIG. 8b shows that the mean fluorescence intensity of CD83 expression on DCs was significantly higher in the leonurine group than in the control group (0.30X 10) ⁵ ±0.18×10 ⁵ vs 0.27×10 ⁵ ±0.16×10 ⁵ ，P＝0.020)。

TABLE 6 Mean Fluorescence Intensity (MFI) of HLA-DR expression on MM patients (n = 11) DCs (10) ⁵ )

As can be seen in fig. 9: comparison of mean fluorescence intensity of HLA-DR expression on DCs between leonurine and control groups (MM, n = 11); FIG. 9a shows the mean fluorescence intensity of HLA-DR expression on DCs of leonurine groups and control groups of each MM patients; FIG. 9b shows that the mean fluorescence intensity of HLA-DR expression on DCs is significantly higher in the leonurine group than in the control group (7.49X 10) ⁵ ±2.58×10 ⁵ vs 6.80×10 ⁵ ±2.18×10 ⁵ ，P＝0.006)。

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. A cell culture medium for increasing DC cell activity in a patient with multiple myeloma comprising 1640 medium, wherein: also comprises leonurine.

2. The cell culture medium for increasing DC cell activity in multiple myeloma patients according to claim 1, wherein: the concentration of the leonurine is 0.9-1.1 mu M.

3. The cell culture medium for increasing DC cell activity in multiple myeloma patients according to claim 1, wherein: the culture medium also comprises human serum, GM-CSF, IL-4, penicillin and streptomycin.

4. The cell culture medium of claim 3 for increasing DC cell activity in multiple myeloma patients, wherein: the concentration of the components is as follows: 5vol% human serum, 800U/mL GM-CSF, 500U/mL IL-4, 100U/mL penicillin, 0.1mg/mL streptomycin.

5. A cell culture method for increasing DC cell activity in a patient with multiple myeloma, comprising: adding leonurine into the culture medium.

6. The cell culture method of claim 5 for increasing DC cell activity in multiple myeloma patients, comprising the steps of:

separation of peripheral blood mononuclear cells, PBMCs, from peripheral blood, said PBMCs being purified in 1640 medium at 37 ℃, 5vol% CO ₂ Culturing for 24 hours in the environment, washing off and discarding the cells not adhered to the wall to obtain DC precursor cells, and then adding the DC precursor cells into a culture medium consisting of 1640 culture medium, 5vol% human serum, 800U/mL GM-CSF, 500U/mL IL-4, 100U/mL penicillin, 0.1mg/mL streptomycin and 1 mu M leonurine to perform the reaction at 37 ℃ and 5vol% of CO ₂ Culturing for 7 days to induce DC cells, and finally harvesting the DC cells.