Application of Chinese medicine polysaccharide in inducing peripheral blood mononuclear cells

Abstract

The application provides application of traditional Chinese medicine polysaccharide in induction of peripheral blood mononuclear cells, and belongs to the technical field of immunotherapy. The application preferably uses eucommia ulmoides polysaccharide, motherwort polysaccharide and cinnamomum cassia polysaccharide to induce peripheral blood mononuclear cells to prepare CIK cells with high 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 rate on various tumor cells is all above 60%.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunotherapy, and particularly relates to application of traditional Chinese medicine polysaccharide in inducing peripheral blood mononuclear cells. BACKGROUND

[0002] PBMC (Peripheral Blood Mononuclear Cells) refers to cells with 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% to 80% of lymphocytes, B cells account for about 10% to 20% of lymphocytes, natural killer cells (NK cells) account for about 5% to 15% of lymphocytes, and monocytes account for about 10% to 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 the fields of 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 kind of immune cells with strong anti-tumor and anti-viral ability. They are a mixed immune cell population produced by in vitro culture and activation of peripheral blood mononuclear cells (PBMC). CIK immune cells have multiple biological properties, such as extensive anti-tumor activity, the ability to rapidly kill tumor cells, killing effect on multiple tumor types, and not being limited by specific antigens. In clinical practice, CIK immune cells have been widely used in tumor treatment.

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

[0005] The cytokines commonly used 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 can improve the killing activity of CIK cells to a certain extent, the effect is still limited. For example, the killing rate of CIK cells induced by IFN-γ, IL-2 and CD3McAb to human erythroleukemia cell line K562 is 67.28% at an effector-target ratio of 25:1, to human leukemia cell line KL-60 is 63.79%, to human cervical cancer cell line Hela is 65.06%, to human hepatocarcinoma cell line SMMC7721 is 33.05%, and to human melanoma cell line A375 is 29.72%. ([1] Duan XM, Tan Y, Song Y, et al. Flow cytometry detection of CIK immune phenotype and in vitro killing activity [J]. Journal of Jilin University (Medical Sciences), 2006(4):639-642. DOI: Jilin Province Science and Technology Department Key Scientific Research Project (200504.).

[0006] In recent years, scientists have also paid attention to the role of Chinese medicine polysaccharides in inducing immune cells in vitro. For example, after angelica polysaccharides induce CIK cells, the killing rate to K562 cells can reach 84.19% at an effector-target ratio of 40:1, but only 72.32% at an effector-target ratio of 10:1, which has no significant difference with the control group. Therefore, it is necessary to develop Chinese medicine polysaccharides that can more efficiently activate CIK cells. SUMMARY

[0007] The purpose of the present application is to provide Chinese medicine polysaccharides for efficiently activating CIK cells and a method for culturing CIK cells, so that the obtained CIK cells have higher and more extensive tumor cell killing activity.

[0008] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0009] The present application provides the application of Chinese medicine polysaccharides in inducing peripheral blood mononuclear cells, wherein the Chinese medicine polysaccharides include eucommia polysaccharides, motherwort polysaccharides and cassia polysaccharides.

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

[0011] (1) After the isolated peripheral blood mononuclear cells are cultured in the culture medium containing IFN-γ, IL-2 and anti-CD3 monoclonal antibody are added for further culture;

[0012] (2) After the culture in step (1) is completed, the Chinese medicine polysaccharides are added for further culture, and CIK cells are obtained.

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

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

[0015] Preferably, the culture time after the addition of the IL-2 and the anti-CD3 single antibody is 3-4 d.

[0016] Preferably, the Chinese medicinal polysaccharide is Eucommia ulmoides polysaccharide, Leonurus heterophyllus polysaccharide and Cinnamomum cassia polysaccharide, the added concentration of the Eucommia ulmoides polysaccharide is 30-50 μg / ml, the added concentration of the Leonurus heterophyllus polysaccharide is 10-15 μg / ml, and the added concentration of the Cinnamomum cassia polysaccharide is 10-15 μg / ml.

[0017] Preferably, the culture time after the addition of the Chinese medicinal polysaccharide is 8-16 d.

[0018] The application further provides a CIK cell obtained by the culture method.

[0019] The application further provides an application of the CIK cell in the preparation of a medicine for killing tumor cells.

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

[0021] The application preferably uses Eucommia ulmoides polysaccharide, Leonurus heterophyllus polysaccharide and Cinnamomum cassia polysaccharide to induce peripheral blood mononuclear cells to prepare CIK cells with high killing activity to tumor cells. 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 rate to various tumor cells is all above 60%. DETAILED DESCRIPTION

[0022] The technical solutions provided by the application will be described in detail below with reference to the examples, but they should not be understood as limiting the protection scope of the application.

[0023] Example 1

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

[0025] Fresh peripheral blood of healthy people was collected, and high-quality PBMCs were isolated by density gradient centrifugation. The key to the density gradient centrifugation method for separating PBMCs is the single nuclear cell separation solution (Ficoll solution). The density of the separation solution is between red blood cells and PBMCs. The density characteristics make lymphocytes and mononuclear cells enriched in the upper layer of the separation solution after density gradient centrifugation, while red blood cells and granulocytes are in the lower layer of the separation solution due to their higher density. We obtained purified PBMCs by collecting the white membrane layer cells in the upper layer of the separation solution.

[0026] Specific steps: Add the same volume of PBS to the whole blood for dilution, take a 50ml centrifuge tube, add the separation solution, and the volume ratio of the separation solution to the sample is 1:1. Slightly tilt the separation solution tube, then use a Pasteur pipette to slowly add the diluted blood sample to the tube wall 1cm above the separation solution surface. Centrifuge (650xg, 20min), after centrifugation, four layers of separation can be seen, from top to bottom, plasma, white membrane layer, separation solution and red blood cells, and PBMCs are enriched in the white membrane layer. Use a Pasteur pipette to remove the white membrane layer as completely as possible and transfer it to a 15ml centrifuge tube. When sucking, the pipette tip should be 3mm above the white membrane layer. Then resuspend to 10ml with PBS, take a sample for counting.

[0027] 2. Preparation of traditional Chinese medicine polysaccharides

[0028] (1) Eucommia ulmoides polysaccharides: Select commercially available Eucommia ulmoides bark as sample, weigh 10g, crush through 20 mesh sieve, take the undersize, put in round bottom flask, add 10 times weight of distilled water, heat to 100℃, extract for 3h. Filter, take the residue and repeat extraction for 2 times, collect the three times of filtrate, concentrate to 1 / 10 of the original volume, then add anhydrous ethanol to make the final concentration 95%, stand for precipitation, take the precipitate, wash with anhydrous ethanol for 3 times, dry to obtain Eucommia ulmoides polysaccharides.

[0029] (2) Motherwort polysaccharides: commercially available motherwort, weigh 10g of motherwort powder (60 mesh), add to a conical flask, add 10 times weight of distilled water, heat to 45℃, extract under the condition of 220W ultrasonic for 10min, filter, concentrate the filtrate to 1 / 10 of the original volume, remove protein by Savage method, then add a mixture of chloroform and n-butanol (volume ratio 4:1) to the concentrated solution, take 3 times volume of 80% ethanol solution, stand overnight, collect the precipitate, wash with anhydrous ethanol and acetone until colorless, dry to obtain motherwort polysaccharides.

[0030] (3) Cinnamon polysaccharide: commercially available cinnamon was ground to 40 mesh, 10 g was weighed and defatted in a Soxhlet extractor, and then 80% (v / v) ethanol solution was added for extraction after the solvent was evaporated. The extraction temperature was 80°C, the solid-liquid ratio was 1:15, the time was 3 h, the extraction liquid was collected and filtered, concentrated to 1 / 5 of the original volume, 3 volumes of 95% ethanol were added, and precipitated for 4 h. After centrifugation, the precipitate was collected and dried to obtain cinnamon polysaccharide.

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

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

[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, and the cells were washed once with PBS and adjusted to a cell density of 2×10 6 6 / 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 hepatocellular carcinoma cell line SMMC7721, and human melanoma cell line A375 were used as target cells. The target cells were cultured to the logarithmic growth phase, and the cell concentration was adjusted to 1×10 5 6 / ml, and the concentration of CIK cells was also adjusted to 1×10 5The target cells and the effector cells were added in 96-well plates at 100 μl each, and were placed in a 37℃, 5% CO2 incubator for 24 hours, and then were detected by CCK-8 method. Meanwhile, the target cell culture wells and the effector cell culture wells were set as blank controls. The CIK cells induced by the Chinese medicine polysaccharide were set as a control group, and the effector-target ratio was 10:1. The OD value at 450 nm was measured, and the killing rate was calculated.

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

[0038] According to the detection results (Table 1), the killing rate of the CIK cells cultured according to the present application on K562 cells was 90.65% when the effector-target ratio was 10:1, the killing rate on KL-60 cells was 89.82%, the killing rate on Hela was 86.44%, the killing rate on SMMC7721 was 69.46%, and the killing rate on A375 cells was 61.85%.

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

[0040]

[0041] According to the above examples, the present application provides CIK cells with a significant tumor cell killing effect, and the killing rate on human erythroleukemia cell line K562, human leukemia cell line KL-60, human cervical cancer cell line Hela, human hepatoma cell line SMMC7721, and human melanoma cell line A375 is all above 60%.

[0042] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for inducing peripheral blood mononuclear cells to culture CIK cells, characterized in that, It comprises the following steps: (1) after the isolated peripheral blood mononuclear cells are cultured in the culture medium containing IFN-γ for 20-24 hours, IL-2 and anti-CD3 monoclonal antibody are added to continue culturing for 3-4 days; The adding concentration of the IFN-γ is 800-1200 U / ml; The adding concentration of the IL-2 is 200-400 U / ml, and the adding concentration of the anti-CD3 monoclonal antibody is 20-30 ng / ml; (2) after the culturing in step (1) is finished, traditional Chinese medicine polysaccharide is added to continue culturing for 8-16 days to obtain CIK cells; The traditional Chinese medicine polysaccharide is eucommia ulmoides polysaccharide, motherwort polysaccharide and cinnamon polysaccharide, the adding concentration of the eucommia ulmoides polysaccharide is 30-50 μg / ml, the adding concentration of the motherwort polysaccharide is 10-15 μg / ml, and the adding concentration of the cinnamon polysaccharide is 10-15 μg / ml; The preparation method of the eucommia ulmoides polysaccharide is that eucommia ulmoides bark is crushed, 20-mesh screen is used, the undersize material is taken, is placed in a round bottom flask, 10 times weight of distilled water is added, heating to 100 DEG C is carried out, and extraction is carried out for 3 hours, filtration is carried out, the filter residue is taken and is repeatedly extracted for 2 times, the three times filter liquor is collected and concentrated to 1 / 10 of the original volume, then anhydrous ethanol is added, the final concentration is 95%, and the precipitation is taken, the precipitate is washed with anhydrous ethanol for 3 times, and drying is carried out to obtain eucommia ulmoides polysaccharide; The preparation method of the motherwort polysaccharide is that motherwort is crushed to 60 mesh, is added into a conical flask, 10 times weight of distilled water is added, heating to 45 DEG C is carried out, and extraction is carried out under the condition of 220 W ultrasonic for 10 min, suction filtration is carried out, the filter liquor is collected and concentrated to 1 / 10 of the original volume, the protein is removed by Savage method, the mixed solution of chloroform and n-butyl alcohol is added to the concentrated liquor for extraction, the volume ratio is 4:1, 3 times volume of 80% ethanol solution is added, and the precipitation is collected after standing overnight, the precipitation is washed with anhydrous ethanol and acetone until colorless, and drying is carried out to obtain motherwort polysaccharide; The preparation method of the cinnamon polysaccharide is that cinnamon is crushed to 40 mesh, is added into a soxhlet extractor for defatting, after the solvent is dried, 80% (v / v) ethanol solution is added for extraction, the extraction temperature is 80 DEG C, the solid-liquid ratio is 1:15, the time is 3 hours, the extraction liquid is collected and filtered, and concentrated to 1 / 5 of the original volume, 3 times volume of 95% ethanol is added, the precipitation is collected after standing for 4 hours, and drying is carried out to obtain cinnamon polysaccharide.

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