Application of traditional Chinese medicine polysaccharide in induction of peripheral blood mononuclear cells

Abstract

The invention provides application of traditional Chinese medicine polysaccharide in induction of peripheral blood mononuclear cells, and belongs to the technical field of immunotherapy. Eucommia polysaccharide, leonurus polysaccharide and cinnamon polysaccharide are preferably selected to induce peripheral blood mononuclear cells to prepare the CIK cells with efficient killing activity on tumors, the proportion of CD3 + CD56 + double positive cells and the proportion of CD3 + CD8 + double positive cells in the obtained CIK cells are greatly increased, and the killing rates of the CIK cells on various tumor cells are all 60% or above.

Description

Technical Field

[0001] The present invention relates to the technical field of immunotherapy, and particularly to the application of Chinese herbal polysaccharides in inducing peripheral blood mononuclear cells. Background Art

[0002] PBMC (Peripheral Blood Mononuclear Cells) refers to the cells with a single nucleus in peripheral blood, mainly including lymphocytes (such as T cells, B cells and natural killer cells) and monocytes. T cells generally account for 60% - 80% of lymphocytes, B cells account for about 10% - 20% of lymphocytes, natural killer cells (NK cells) account for about 5% - 15% of lymphocytes, and monocytes account for 10% - 20%. These cells play an indispensable role in the immune response process of the human body. In scientific research experiments, PBMC is widely used in fields such as cell therapy and drug screening.

[0003] With the rapid development of precision medicine, scientists have paid more and more attention to the role of PBMC in cell therapy. Among them, CIK cells are a type of immune cells with powerful anti-tumor and anti-viral abilities. They are a mixed immune cell population generated by in vitro culture and activation of peripheral blood mononuclear cells (PBMC). CIK immune cells have various biological characteristics, such as broad anti-tumor activity, the ability to rapidly kill tumor cells, the killing effect on various tumor types, and being unrestricted by specific antigens. Clinically, CIK immune cells have been widely used in tumor treatment.

[0004] The effector cells CD3+CD56+ cells in CIK cells are extremely rare in normal human peripheral blood, only 1% - 5%, but after in vitro culture with multiple factors for 28 - 30 days, the CD3+CD56+ cells increase rapidly and the anti-tumor activity is enhanced. Therefore, the method of stimulating and culturing CIK cells in vitro can further enhance their killing activity against tumor cells.

[0005] The commonly used cytokines in the in vitro culture of peripheral blood mononuclear cells mainly include anti-CD3 monoclonal antibody (CD3McAb), interleukin-2 (IL-2), interferon-γ (IFN-γ), and IL-1α, etc. Although these cytokines have improved the killing activity of CIK cells to a certain extent, the effect is still limited. For example, the killing rates of CIK cells induced and cultured with IFN-γ, IL-2, and CD3McAb were 67.28% against the human leukemia cell line K562, 63.79% against the human leukemia cell line KL-60, 65.06% against the human cervical cancer cell line Hela, 33.05% against the human hepatoma cell line SMMC7721, and 29.72% against the human melanoma cell line A375 at an effector-to-target ratio of 25:1. ([1] Duan Xiumei, Tan Yan, Song Yan, et al. Detection of the immunophenotype and in vitro killing activity of CIK by flow cytometry [J]. Journal of Jilin University (Medicine Edition), 2006(4): 639-642. DOI: A key scientific research project funded by the Science and Technology Department of Jilin Province (200504.).

[0006] In recent years, scientists have also paid attention to the role of Chinese herbal polysaccharides in inducing immune cells in vitro. For example, after inducing CIK cells with Angelica sinensis polysaccharide, the killing rate against K562 cells can reach 84.19% at an effector-to-target ratio of 40:1, but only 72.32% at an effector-to-target ratio of 10:1, showing no significant difference from the control group. Therefore, it is very necessary to develop Chinese herbal polysaccharides that are more efficient in activating CIK cells. Summary of the Invention

[0007] The purpose of the present invention is to provide Chinese herbal polysaccharides that can efficiently activate CIK cells and a method for culturing CIK cells, so that the cultured CIK cells have higher and more extensive tumor cell killing activity.

[0008] To achieve the above invention purpose, the present invention provides the following technical solutions:

[0009] The present invention provides the application of Chinese herbal polysaccharides in inducing peripheral blood mononuclear cells, and the Chinese herbal polysaccharides include Eucommia ulmoides polysaccharide, Leonurus japonicus polysaccharide, and Cinnamomum cassia polysaccharide.

[0010] The present invention also provides a method for inducing peripheral blood mononuclear cells to culture CIK cells, which includes the following steps:

[0011] (1) After culturing the isolated peripheral blood mononuclear cells in a medium containing IFN-γ, add IL-2 and anti-CD3 monoclonal antibody and continue culturing;

[0012] (2) After the culture in step (1) is completed, add the above-mentioned Chinese herbal polysaccharides and continue culturing to obtain CIK cells.

[0013] Preferably, the added concentration of IFN-γ is 800 - 1200 U / ml, and the culture time in the medium containing IFN-γ is 20 - 24 h.

[0014] Preferably, the added concentration of IL-2 is 200 - 400 U / ml, and the added concentration of anti-CD3 monoclonal antibody is 20 - 30 ng / ml.

[0015] Preferably, the time for continued culture after adding IL-2 and anti-CD3 monoclonal antibody is 3 - 4 d.

[0016] Preferably, the Chinese herbal polysaccharides are eucommia ulmoides polysaccharide, leonurus japonicus polysaccharide and cinnamomum cassia polysaccharide. The added concentration of eucommia ulmoides polysaccharide is 30 - 50 μg / ml, the added concentration of leonurus japonicus polysaccharide is 10 - 15 μg / ml, and the added concentration of cinnamomum cassia polysaccharide is 10 - 15 μg / ml.

[0017] Preferably, the time for continued culture after adding the Chinese herbal polysaccharides is 8 - 16 d.

[0018] The present invention also provides CIK cells cultured by the above-mentioned culture method.

[0019] The present invention also provides the application of the above-mentioned CIK cells in the preparation of drugs for killing tumor cells.

[0020] Preferably, the tumor cells include erythroleukemia, leukemia, cervical cancer, liver cancer and melanoma.

[0021] The present invention preferably uses eucommia ulmoides polysaccharide, leonurus japonicus polysaccharide and cinnamomum cassia polysaccharide to induce peripheral blood mononuclear cells to prepare CIK cells with high killing activity against tumors. The proportion of CD3+CD56+ double-positive cells and CD3+CD8+ double-positive cells in the obtained CIK cells increases significantly, and the killing rate against various tumor cells is above 60%. Detailed implementation manners

[0022] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0023] Example 1

[0024] 1. Isolation of peripheral blood mononuclear cells (PBMC)

[0025] Collect fresh peripheral blood from healthy individuals. High-quality PBMC can be isolated by density gradient centrifugation. The key to isolating PBMC by density gradient centrifugation lies in the mononuclear cell separation solution (Ficoll solution). The density of the separation solution is between that of red blood cells and PBMC. Its density characteristics enable lymphocytes and monocytes to be enriched in the upper layer of the separation solution after density gradient centrifugation, while red blood cells and granulocytes, due to their relatively high density, will be in the lower layer of the separation solution. We obtain purified PBMC by collecting the buffy coat cells in the upper layer of the separation solution.

[0026] Specific steps: Dilute the whole blood by adding an equal volume of PBS. Take a 50 ml centrifuge tube and add the separation solution. The ratio of the separation solution to the sample volume is 1:1. Slightly tilt the separation solution tube, and then use a Pasteur pipette to slowly add the diluted blood sample to the tube wall 1 cm above the liquid surface of the separation solution. Centrifuge (650×g, 20 min). After centrifugation, four separation layers can be seen. From top to bottom, they are plasma, buffy coat, separation solution, and red blood cells. PBMC is enriched in the buffy coat. Use a Pasteur pipette to aspirate the buffy coat as clean as possible and transfer it to a 15 ml centrifuge tube. When aspirating, note that the pipette tip should be 3 mm above the buffy coat. Then add PBS to resuspend to 10 ml and take a sample for counting.

[0027] 2. Preparation of Chinese herbal polysaccharides

[0028] (1) Eucommia ulmoides polysaccharide: Select commercially available Eucommia ulmoides bark as the sample. Weigh 10 g, crush it through a 20-mesh sieve, take the material passing through the sieve, place it in a round-bottom flask, add 10 times the weight of distilled water, heat to 100 °C, and extract for 3 h. Filter, take the residue and repeat the extraction 2 more times. Combine the three filtrates, concentrate to 1 / 10 of the original volume, then add absolute ethanol to make the final concentration 95% in it, let it stand for precipitation, take the precipitate, wash it 3 times with absolute ethanol, and dry it to obtain Eucommia ulmoides polysaccharide.

[0029] (2) Leonurus japonicus polysaccharide: Commercially available Leonurus japonicus. Weigh 10 g of Leonurus japonicus powder (60 mesh), add it to a conical flask, add 10 times the weight of distilled water, heat to 45 °C, extract under ultrasonic conditions of 220 W for 10 min, filter by suction, collect the filtrate and concentrate to 1 / 10 of the original volume, remove protein by the Savage method, then add a mixture of chloroform and n-butanol (volume ratio 4:1) to the concentrated solution for extraction. Take the aqueous phase and add 3 times the volume of 80% ethanol solution, let it stand overnight, collect the precipitate, wash it with absolute ethanol and acetone until colorless, and dry it to obtain Leonurus japonicus polysaccharide.

[0030] (3) Cinnamon polysaccharide: Commercially available cinnamon was pulverized to 40 mesh with a pulverizer. 10 g was weighed and added to a Soxhlet extractor for defatting. After evaporating the solvent, an 80% (v / v) ethanol solution was added for extraction. The extraction temperature was 80 °C, the solid-liquid ratio was 1:15, and the time was 3 h. The extract was collected, filtered, concentrated to 1 / 5 of the original volume, 3 volumes of 95% ethanol were added, allowed to stand for precipitation for 4 h, centrifuged, the precipitate was collected and dried to obtain cinnamon polysaccharide.

[0031] 3. Induction of peripheral blood mononuclear cells (cultivation of CIK cells)

[0032] The isolated peripheral blood mononuclear cells were suspended in RPMI-1640 complete culture medium (containing 10% fetal bovine serum), and the cell density was 1×10 6 cells / ml. They were inoculated into 15 mm culture dishes, IFN-γ was added, and the final concentration was 1000 U / ml. They were cultured in an incubator at 37 °C and 5% CO2 for 24 h; then IL-2 and anti-CD3 monoclonal antibody were added, and the final concentrations were 300 U / ml and 25 ng / ml respectively, and the culture continued for 4 d. Then Chinese herbal polysaccharides (eucommia ulmoides polysaccharide 40 μg / ml, leonurus heterophyllus polysaccharide 12 μg / ml, cinnamon polysaccharide 12 μg / ml) were added and the culture continued for 12 d. The medium was changed every 4 days, and IL-2, anti-CD3 monoclonal antibody and Chinese herbal polysaccharides were re-added after changing the medium.

[0033] 4. Detection of CIK cell immunophenotype

[0034] The cultured CIK cells were harvested, centrifuged at 680×g for 5 min, the supernatant was discarded, washed once with PBS, and the cell density was adjusted to 2×10 6 cells / ml. Flow cytometry was used to analyze the immunophenotype on the surface of CIK cells. The results showed that the proportion of CD3+CD56+ double-positive cells was greater than 70%, and the proportion of CD3+CD8+ double-positive cells was greater than 80%.

[0035] 5. Detection of CIK cell tumor killing ability

[0036] Human erythroleukemia cell line K562, human leukemia cell line KL-60, human cervical cancer cell line Hela, human liver cancer cell line SMMC7721, and human melanoma cell line A375 were used as target cells. Each target cell was cultured to the logarithmic growth phase, and the cell concentration was adjusted to 1×10 5 cells / ml. The concentration of CIK cells was also adjusted to 1×10 5cells / ml, as effector cells, experiments were conducted at effector-to-target ratios of 10:1 and 5:1. Specifically: 100 μl of target cells and effector cells were added to a 96-well plate, and the plate was placed in an incubator at 37°C and 5% CO2. After culturing for 24 h, detection was performed using the CCK-8 method. At the same time, target cell culture wells and effector cell culture wells were set as blank controls. CIK cells not induced with Chinese herbal polysaccharides were set as the control group, with an effector-to-target ratio of 10:1. The OD value at 450 nm was measured, and the killing rate was calculated.

[0037] Killing rate (%) = 1 - [(OD value of experimental well - OD value of effector cell well) / OD value of target cell well].

[0038] From the detection results (Table 1), it can be seen that when the effector-to-target ratio of the CIK cells cultured in the present invention was 10:1, the killing rate against K562 cells was 90.65%, the killing rate against KL-60 cells was 89.82%, the killing rate against Hela cells was 86.44%, the killing rate against SMMC7721 cells was 69.46%, and the killing rate against A375 cells was 61.85%.

[0039] Table 1 Detection results of the tumor killing ability of CIK cells (%)

[0040]

[0041] From the above examples, it can be seen that the present invention provides a kind of CIK cells with significant killing effect on tumor cells, and the killing rates against various tumor cells such as human erythroleukemia cell line K562, human leukemia cell line KL-60, human cervical cancer cell line Hela, human liver cancer cell line SMMC7721, and human melanoma cell line A375 are all above 60%.

[0042] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of Chinese herbal polysaccharides in inducing peripheral blood mononuclear cells, characterized in that, The traditional Chinese medicine polysaccharides include eucommia ulmoides polysaccharides, leonurus japonicus polysaccharides and cinnamomum cassia polysaccharides.

2. A method for inducing the culture of CIK cells from peripheral blood mononuclear cells, characterized in that, It includes the following steps: (1) After culturing the isolated peripheral blood mononuclear cells in a medium containing IFN-γ, add IL-2 and anti-CD3 monoclonal antibody and continue culturing; (2) After the culturing in step (1) is completed, add the traditional Chinese medicine polysaccharides described in claim 1 and continue culturing to obtain CIK cells.

3. The cultivation method according to claim 2, characterized in that, The added concentration of the IFN-γ is 800-1200 U / ml, and the culturing time in the medium containing IFN-γ is 20-24 h.

4. The culturing method according to claim 3, wherein The added concentration of the IL-2 is 200-400 U / ml, and the added concentration of the anti-CD3 monoclonal antibody is 20-30 ng / ml.

5. The cultivation method according to claim 4, characterized in that The time for continuing culturing after adding IL-2 and anti-CD3 monoclonal antibody is 3-4 d.

6. The cultivation method according to claim 5, characterized in that, The traditional Chinese medicine polysaccharides are eucommia ulmoides polysaccharides, leonurus japonicus polysaccharides and cinnamomum cassia polysaccharides. The added concentration of the eucommia ulmoides polysaccharides is 30-50 μg / ml, the added concentration of the leonurus japonicus polysaccharides is 10-15 μg / ml, and the added concentration of the cinnamomum cassia polysaccharides is 10-15 μg / ml.

7. The culturing method according to claim 6, wherein The time for continuing culturing after adding the traditional Chinese medicine polysaccharides is 8-16 d.

8. CIK cells obtained by the culturing method according to any one of claims 1 to 7.

9. Use of the CIK cells according to claim 8 in the preparation of a drug for killing tumor cells.

10. The application according to claim 9, characterized in that, The tumor cells include erythroleukemia, leukemia, cervical cancer, liver cancer and melanoma.

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