Method for inhibiting growth of tumor or cancer cells by using NK cells

Abstract

The invention provides a method for inhibiting tumor or cancer cell growth through NK cells, and belongs to the technical field of biological medicine. According to the technical scheme, a method for separating and amplifying the NK cells is firstly constructed, the method comprises the following steps: mixing human peripheral blood from a donor with a lymphocyte separating medium, centrifuging, collecting a plasma layer, collecting a second layer of annular milk white mononuclear cells, cleaning and resuspending; and amplifying the NK cells obtained by separation in an X-VIVO 15 culture medium added with IL-2, IL-15 and K562 cells at the initial concentration of (0.5-1.5) * 10 < 6 > cells / mL for 14-15 days to obtain an NK cell finished product. On the basis, the NK cells are infused to a donor patient in an intravenous infusion mode, and a pharmaceutical composition containing rhizoma polygonati, angelica sinensis, flatstem milkvetch seeds, radix dipsaci, white hyacinth beans and other components is administered in the course of treatment, so that the inhibiting effect on tumors and cancer cells can be remarkably improved. The oral medicine disclosed by the invention is matched with an NK cell therapy, has an exact synergistic effect, and is good in safety and remarkable in technical advantage.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a method for inhibiting tumor or cancer cell growth by NK cells. BACKGROUND

[0002] Natural killer cells (NK) are important immune cells of the body, which are not only related to anti-tumor, anti-viral infection and immune regulation, but also participate in the occurrence of hypersensitivity and autoimmune diseases in some cases, and can recognize target cells and kill mediators. NK cells are derived from bone marrow lymphoid stem cells, and their differentiation and development depend on the bone marrow and thymus microenvironment, and are mainly distributed in bone marrow, peripheral blood, liver, spleen, lung and lymph nodes. Unlike T and B cells, NK cells are a type of lymphocyte that can non-specifically kill tumor cells and virus-infected cells without prior sensitization.

[0003] Because the killing activity of NK cells is not MHC restricted and does not depend on antibodies, it is called natural killing activity. NK cells are rich in cytoplasm and contain large azureophilic granules, and the content of the granules is positively correlated with the killing activity of NK cells. The killing effect appears early after NK cells act on target cells, and the killing effect can be seen in 1 hour in vitro and 4 hours in vivo. The target cells of NK cells are mainly some tumor cells (including some cell lines), virus-infected cells, some self-tissue cells (such as blood cells), parasites, etc., so NK cells are important immune factors for the body to resist tumors and infections, and also participate in type II hypersensitivity and graft-versus-host reaction.

[0004] NK cell immunotherapy is a new type of immunotherapy based on NK cells, which extracts immature immune cells (blood sampling) from the body, then activates and cultures them in the laboratory to make them have the ability to recognize and kill tumor cells efficiently, and then infuses them into the body to activate and enhance the body's immunity, thereby achieving the dual effect of treatment and prevention of tumors. In the prior art, the inhibitory effect of conventional NK cell reinfusion therapy on tumor and cancer cell growth still needs to be improved. In view of this problem, some researches believe that the use of other drugs in the process of giving NK cells is expected to improve the overall efficacy, but the research on such drugs is still not perfect, and the ideal combination drug has not been found. SUMMARY

[0005] The technical problem to be solved by the present application is how to provide a synergist for inhibiting tumor and cancer cells by NK cells.

[0006] To achieve the above technical purpose, the present application adopts the following technical scheme: A method for inhibiting tumor or cancer cell growth by NK cells, comprising: 1) Add human peripheral blood lymphocyte separation medium into a centrifuge tube, then add the same volume of the donor blood sample onto the surface of the separation medium, centrifuge, take the plasma layer into a new centrifuge tube, keep at 55-60℃ for 20-40 min, then centrifuge to take the supernatant, and suck the mononuclear cell layer into another centrifuge tube, add deionized water, centrifuge to discard the supernatant, resuspend the cells with deionized water, centrifuge to discard the supernatant, and obtain the NK cells; 2) Resuspend the NK cells obtained in step 1) into X-VIVO 15 medium containing 5% serum, adjust the cell density to 0.5-1.5×10 6 cells / mL, and culture at 37℃ in a 5% CO2 environment, and harvest the NK cells; 3) Intravenously return the NK cells harvested in step 2) to the donor at a single dose of 0.2-0.4×10 9 cells, and orally take a pharmaceutical composition during the period from 3 days before the first intravenous return to 30 days after the last intravenous return; the daily dose of the oral administration is 15-20 g; the pharmaceutical composition comprises the following ingredients by weight: 8-10 parts of Rhizoma Polygonati, 4-6 parts of Angelica sinensis, 6-8 parts of Hedysarum, 3-5 parts of Dipsacus, 2-4 parts of Lablab, 4-5 parts of Albizia, 3-4 parts of Raphanus, 2-3 parts of Gigeria, 1-2 parts of Amomum, 2-3 parts of Platycladus, and 2-3 parts of Fritillaria.

[0007] Preferably, in step 1), after the first centrifugation, the system is divided into four layers from top to bottom, the first layer is the plasma layer, the second layer is the annular milky white mononuclear cell layer, the third layer is the transparent separation medium layer, and the fourth layer is the red blood cell layer.

[0008] Preferably, in step 2), 200 U / mL IL-2, 10 ng / mL IL-15 and 10 6 cells / mL K562 cells are added to the culture system.

[0009] Preferably, in step 2), the NK cells are passaged every 2-3 days, and the cytokines and new K562 cells are supplemented.

[0010] Preferably, in step 2), the NK cells are harvested on the 14th-15th day after the start of the culture.

[0011] Preferably, in step 3), the number of times of the intravenous return is 2-3, and the interval between the adjacent two intravenous returns is 4-6 weeks.

[0012] Preferably, the pharmaceutical composition further comprises 3-4 parts by weight of Acanthopanax.

[0013] Preferably, the pharmaceutical composition further comprises 1-2 parts by weight of Drynaria.

[0014] As preferred, the pharmaceutical composition further comprises 1-3 parts by weight of Euphorbia humifusa.

[0015] As preferred, the pharmaceutical composition is composed of the following ingredients by weight: 9 parts of Polygonatum sibiricum, 5 parts of Angelica sinensis, 7 parts of Hedysarum polybotrys, 4 parts of Dipsacus asper, 3 parts of Laburnum, 4.5 parts of Semen Raphani, 2.5 parts of Galla Rhois, 1.5 parts of Amomum villosum, 2.5 parts of Platycladus orientalis, 2.5 parts of Fritillaria walujewii, 3.5 parts of Eleutherococcus senticosus, 1.5 parts of Drynaria fortunei, and 2 parts of Euphorbia humifusa.

[0016] The present application provides a method for inhibiting tumor or cancer cell growth by NK cells. The technical scheme firstly constructs a method for separating and expanding NK cells. The method mixes human peripheral blood from a donor with lymphocyte separation medium, collects the plasma layer after centrifugation, collects the second layer of circular milky white mononuclear cells, and resuspends after washing. For the separated NK cells, the initial concentration is 0.5-1.5×10 6 cells / mL, and the NK cells are expanded in X-VIVO 15 culture medium added with IL-2, IL-15 and K562 cells for 14-15 days to obtain NK cell products. On this basis, the NK cells are transfused to the donor patient by intravenous transfusion, and a pharmaceutical composition containing Polygonatum sibiricum, Angelica sinensis, Hedysarum polybotrys, Dipsacus asper, Laburnum and the like is given during the treatment course, which can significantly improve the inhibition effect on tumor and cancer cells. The oral drug of the present application is combined with the NK cell therapy, has an exact synergistic effect, is good in safety, and has significant technical advantages. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is a statistical result graph of tumor inhibition rate of patients in the embodiment of the present application. DETAILED DESCRIPTION

[0018] The specific embodiments of the present application will be described in detail below. In order to avoid too many unnecessary details, the structures or functions that are well known will not be described in detail in the following examples. The approximate language used in the following examples can be used for quantitative description, indicating that the quantity can be allowed to have certain changes without changing the basic functions. Unless defined, the technical and scientific terms used in the following examples have the same meaning as generally understood by those skilled in the art to which the present application belongs. Example 1

[0019] A method for inhibiting tumor or cancer cell growth by NK cells, comprising: 1) Add human peripheral blood lymphocyte separation medium into a centrifuge tube, then add the same volume of blood sample of the ovarian cancer patient donor onto the liquid surface of the separation medium, centrifuge, take the plasma layer into a new centrifuge tube, keep at 56℃ for 30 min, then centrifuge to take the supernatant, suck the mononuclear cell layer into another centrifuge tube, add deionized water, centrifuge to discard the supernatant, resuspend the cells with deionized water, centrifuge to discard the supernatant, and obtain NK cells; 2) Resuspend the NK cells obtained in step 1) into X-VIVO 15 medium containing 5% serum, adjust the cell density to 1×10 6 cells / mL, and culture at 37℃ in a 5% CO2 environment, and harvest the NK cells; 3) Intravenously return the NK cells harvested in step 2) to the ovarian cancer patient donor at a single dose of 0.3×10 9 cells, from 3 days before the first intravenous return to 30 days after the last intravenous return, and orally take a pharmaceutical composition during the period; the daily dose of the oral administration is 18 g; the pharmaceutical composition comprises the following components in parts by weight: Rhizoma Polygonati 9 parts, Angelica sinensis 5 parts, Astragalus complanatus 7 parts, Dipsacus asper 4 parts, Laburnum 3 parts, Alstonia scholaris 4.5 parts, Raphanus sativus 3.5 parts, Gigeria sinensis 2.5 parts, Amomum villosum 1.5 parts, Platycladus orientalis 2.5 parts, Fritillaria walujewii 2.5 parts. Example 2

[0020] A method for inhibiting the growth of tumor or cancer cells by NK cells, comprising: 1) Add human peripheral blood lymphocyte separation medium into a centrifuge tube, then add the same volume of blood sample of the ovarian cancer patient donor onto the liquid surface of the separation medium, centrifuge, take the plasma layer into a new centrifuge tube, keep at 56℃ for 30 min, then centrifuge to take the supernatant, suck the mononuclear cell layer into another centrifuge tube, add deionized water, centrifuge to discard the supernatant, resuspend the cells with deionized water, centrifuge to discard the supernatant, and obtain NK cells; 2) Resuspend the NK cells obtained in step 1) into X-VIVO 15 medium containing 5% serum, adjust the cell density to 1×10 6 cells / mL, and culture at 37℃ in a 5% CO2 environment, and harvest the NK cells; 3) Intravenously return the NK cells harvested in step 2) to the ovarian cancer patient donor at a single dose of 0.3×10 9A single dose of 0.3 x 10 cells / mL of the NK cells harvested in step 2) is intravenously transfused to the ovarian cancer patient donor, and an oral drug composition is taken from 3 days before the first intravenous transfusion to 30 days after the last intravenous transfusion, with a daily dose of 18 g; the drug composition comprises the following ingredients by weight: Rhizoma Polygonati 9 parts, Angelica 5 parts, Fructus Amori 7 parts, Dipsacus 4 parts, Lablab 3 parts, Cortex Albiziae 4.5 parts, Semen Raphani 3.5 parts, Gigeriae 2.5 parts, Piperis Longi 1.5 parts, Platycladi 2.5 parts, Fritillariae 2.5 parts, Acanthopanax 3.5 parts, Sedi 1.5 parts, Herba Hyperici 2 parts.

[0021] In step 1), after the first centrifugation, the system is divided into four layers from top to bottom, the first layer is the plasma layer, the second layer is the annular milky white mononuclear cell layer, the third layer is the transparent separation liquid layer, and the fourth layer is the red blood cell layer. In step 2), 200 U / mL IL-2, 10 ng / mL IL-15 and 10 6 cells / mL K562 cells are added to the culture system. In step 2), the NK cells are subcultured every 2-3 days to supplement the cytokines and new K562 cells. In step 2), the NK cells are harvested on the 15th day after the start of culture. In step 3), the number of intravenous transfusions is 3, and the interval between adjacent two intravenous transfusions is 5 weeks. Example 3

[0022] A method for inhibiting the growth of tumor or cancer cells by NK cells, comprising: 1) Add human peripheral blood lymphocyte separation medium to a centrifuge tube, then add the same volume of blood sample of the ovarian cancer patient donor to the liquid surface of the separation medium, centrifuge, take the plasma layer to a new centrifuge tube, keep at 56℃ for 30 min, then centrifuge to take the supernatant, suck the mononuclear cell layer to another centrifuge tube, add deionized water, centrifuge to discard the supernatant, resuspend the cells with deionized water, centrifuge to discard the supernatant, and obtain NK cells; 2) Resuspend the NK cells obtained in step 1) in X-VIVO 15 medium containing 5% serum, adjust the cell density to 1 x 10 6 cells / mL, and culture at 37℃ in a 5% CO2 environment, and harvest the NK cells; 3) A single dose of 0.3 x 10 9 cells / mL of the NK cells harvested in step 2) is intravenously transfused to the ovarian cancer patient donor.

[0023] In step 1), after the first centrifugation, the system is divided into four layers from top to bottom, the first layer is the plasma layer, the second layer is the annular milky white mononuclear cell layer, the third layer is the transparent separation liquid layer, and the fourth layer is the red blood cell layer. In step 2), 200 U / mL IL-2, 10 ng / mL IL-15 and 10 6 cells / mL K562 cells are added to the culture system. In step 2), the NK cells are passaged every 2-3 days, and the cytokines and new K562 cells are supplemented. In step 2), the NK cells are harvested on the 15th day after the start of culture. In step 3), the number of times of intravenous infusion is 3, and the interval between adjacent two times of intravenous infusion is 5 weeks. Experimental example

[0024] Human ovarian cancer A2780 cells were cultured to the logarithmic phase, trypsinized, centrifuged at 800 rpm for 5 min, and then added with complete culture medium to blow and mix the cells to prepare a single cell suspension with a cell density of 1.5×10000 cells / 100 μL. 100 μL of cell solution was added to each well of a 96-well plate, and 3 replicate wells were set for each group. The plate was placed in a constant temperature incubator and cultured at 37°C and 5% CO2 overnight.

[0025] The pharmaceutical compositions used in Example 1 and Example 2 were respectively prepared into different concentrations (0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4, 8, 10, 20 mg / mL), 200 μL of DMEM culture medium was added to each well, and 3 replicate wells were set for each group. After 48 h of culture, CCK8 solution was replaced, and after 2 h of incubation, the plate was placed in an enzyme marker at 450 nm to measure the absorbance value, and the cell viability IC50 value was calculated. The experimental results showed that the IC50 values of Example 1 and Example 2 were 1.2 mg / mL and 1.0 mg / mL, respectively, proving that the pharmaceutical compositions of Example 1 and Example 2 had a definite inhibitory effect on ovarian cancer cells.

[0026] For the patients in Example 1-Example 3, abdominal enhanced CT detection was performed before and after treatment, respectively, to obtain the long diameter and short diameter data of the tumor, and calculate the tumor inhibition rate.

[0027] Tumor volume = 1 / 2 × (tumor long diameter × tumor short diameter 2 ); Tumor inhibition rate = (average tumor volume before treatment - average tumor volume after treatment) / average tumor volume before treatment × 100%.

[0028] The experimental results show that the tumor inhibition rates of Example 1 and Example 2 are 66.4% and 79.1% respectively, and the tumor inhibition rate of Example 3 is 37.3%. The tumor inhibition rate of Example 3 reaches the target level of previous studies, but is significantly lower than that of Example 1 and Example 2, thus it can be seen that the oral administration of the pharmaceutical composition provided by the present application during the execution of the NK cell therapy can significantly improve the tumor inhibition effect.

[0029] After the treatment, the venous blood of the patients was taken, and the percentages of lymphocyte subgroups (CD3 + , CD4 + , CD8 + ) and CD4 + / CD8 + in the peripheral blood were examined by flow cytometry. The experimental results show that the percentage of CD3 + of the patient in Example 1 is 54.33%, the percentage of CD4 + is 50.56%, the percentage of CD8 + is 32.27%, and CD4 + / CD8 + is 1.57; the percentage of CD3 + of the patient in Example 2 is 52.15%, the percentage of CD4 + is 54.21%, the percentage of CD8 + is 34.03%, and CD4 + / CD8 + is 1.59; the percentage of CD3 + of the patient in Example 3 is 50.40%, the percentage of CD4 + is 43.72%, the percentage of CD8 + is 29.58%, and CD4 + / CD8 + is 1.48. Thus it can be seen that the present application significantly improves the immune level of the patients.

[0030] The above detailed description of the embodiments of the present application, but the content described is only the preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement and improvement within the scope of the present application should be included in the protection scope of the present application.

Claims

1. A method for inhibiting tumor or cancer cell growth using NK cells, characterized in that, include: 1) Add human peripheral blood lymphocyte separation medium to a centrifuge tube, then add the same volume of donor blood sample to the surface of the separation medium, centrifuge, transfer the plasma layer to a new centrifuge tube, keep at 55~60℃ for 20~40 min, then centrifuge to collect the supernatant, aspirate the mononuclear cell layer to another centrifuge tube, add deionized water, centrifuge and discard the supernatant, resuspend the cells in deionized water, centrifuge again and discard the supernatant to obtain NK cells; 2) Resuspend the NK cells obtained in step 1) in X-VIVO 15 medium containing 5% serum, and adjust the cell density to 0.5~1.5×10⁶ cells / day. 6 NK cells were harvested by culturing at 37°C and 5% CO2 at a density of cells / mL. 3) With 0.2~0.4×10 9 The single dose of NK cells is obtained by intravenously reinfusing the NK cells harvested in step 2) into the donor, starting 3 days before the first intravenous infusion and ending 30 days after the last intravenous infusion. During this period, the drug composition is taken orally at a daily dose of 15-20g. The drug composition comprises the following components in parts by weight: 8-10 parts of Polygonatum sibiricum, 4-6 parts of Angelica sinensis, 6-8 parts of Astragalus complanatus, 3-5 parts of Dipsacus asper, 2-4 parts of Dolichos lablab, 4-5 parts of Albizia julibrissin, 3-4 parts of Raphanus sativus, 2-3 parts of Gallus gallus domesticus gizzard lining, 1-2 parts of Piper longum, 2-3 parts of Platycladus orientalis seed, and 2-3 parts of Fritillaria cirrhosa.

2. The method for inhibiting tumor or cancer cell growth using NK cells according to claim 1, characterized in that, In step 1), after the first centrifugation, the system is divided into four layers from top to bottom: the first layer is the plasma layer, the second layer is the ring-shaped milky white mononuclear cell layer, the third layer is the transparent separation liquid layer, and the fourth layer is the red blood cell layer.

3. The method for inhibiting tumor or cancer cell growth using NK cells according to claim 1, characterized in that, In step 2), the culture system contained 200 U / mL IL-2, 10 ng / mL IL-15, and 10 6 K562 cells / mL.

4. The method for inhibiting tumor or cancer cell growth using NK cells according to claim 3, characterized in that, In step 2), NK cells are passaged every 2-3 days to replenish cytokines and new K562 cells.

5. The method for inhibiting tumor or cancer cell growth using NK cells according to claim 4, characterized in that, In step 2), NK cells are harvested on day 14-15 after the start of culture.

6. The method for inhibiting tumor or cancer cell growth using NK cells according to claim 1, characterized in that, In step 3), the intravenous infusion is performed 2 to 3 times, with an interval of 4 to 6 weeks between two adjacent intravenous infusions.

7. The method for inhibiting tumor or cancer cell growth using NK cells according to claim 1, characterized in that, The pharmaceutical composition also includes 3-4 parts by weight of Acanthopanax senticosus.

8. The method for inhibiting tumor or cancer cell growth using NK cells according to claim 1, characterized in that, The pharmaceutical composition also includes 1 to 2 parts by weight of Drynaria fortunei.

9. The method for inhibiting tumor or cancer cell growth using NK cells according to claim 1, characterized in that, The pharmaceutical composition also includes 1 to 3 parts by weight of Eclipta prostrata.

10. A method for inhibiting tumor or cancer cell growth using NK cells according to claim 1, characterized in that, The pharmaceutical composition comprises the following components in parts by weight: 9 parts Polygonatum sibiricum, 5 parts Angelica sinensis, 7 parts Astragalus complanatus, 4 parts Dipsacus asper, 3 parts Dolichos lablab, 4.5 parts Albizia julibrissin bark, 3.5 parts Raphanus sativus seed, 2.5 parts Gallus gallus domesticus gizzard lining, 1.5 parts Piper longum, 2.5 parts Platycladus orientalis seed, 2.5 parts Fritillaria cirrhosa, 3.5 parts Acanthopanax senticosus, 1.5 parts Drynaria fortunei, and 2 parts Eclipta prostrata.

Images