CN111733129A - A method for the induction of high-efficiency DC-CIK cells by polymyocytes - Google Patents

Abstract

The invention relates to the technical field of immunity, in particular to the use of a copolymer of polyinosinic acid and polycytidylic acid-polyinosinic acid (Poly I:C), which is a ligand for type 3 Toll-like receptors in animals. After activation of TLR-3, it can mediate a series of immune responses in the body, and has a good promotion effect on the specific and non-specific immunity of the body. Compared with the control group, the DC-CIK induced by polymyocytes in the present invention has higher proliferation, higher differentiation (especially CD3+CD4-CD8+, CTL) and higher tumoricidal activity. Autologous high-efficiency DC-CIK and other immune cells induced by polymyocytes are used in the treatment of clinical tumor patients. In order to restore or improve the immune function of tumor patients, improve the body's own immune system to kill tumor cells and inhibit their proliferation. Reduce tumor cell load, remove tiny residual lesions or significantly inhibit the proliferation of residual tumor/cancer cells, eliminate recurrence and metastasis factors, increase the possibility of cure, prolong survival time, and improve quality of life. So as to achieve the purpose of treating tumor/cancer.

Description

A method for the induction of high-efficiency DC-CIK cells by polymyocytes

technical field

The invention relates to the technical field of immunity, in particular to the use of a copolymer of polyinosinic acid and polycytidylic acid-polyinosinic acid (polyinosinic: polycytidylic acid copolymer, Poly I:C), and polyinosinic acid is a kind of 3 type in animal body. The ligand of Toll-like receptor, after activation of TLR-3, can mediate a series of immune responses in the body, such as inducing the secretion of cytokines such as interferon, interleukin, tumor necrosis factor, etc., promoting monocytes, macrophages, lymphocytes, etc. And the proliferation and maturation of dendritic cells, etc., to the body's specific immunity and non-specific immunity have a good role in promoting.

The proliferation and differentiation of DC-CIK (macrophage-cytokine-activated tumor killer cells) cells are synergistically induced by adding polymyocytes to the induction medium, which greatly improves the tumoricidal activity of DC-CIK. In order to obtain a polymyocyte-induced autologous high-efficiency DC-CIK cells, which can be used in the treatment of clinical tumor patients. It can better kill residual tumor cells, reduce the factors of tumor recurrence and metastasis, increase the possibility of cure, prolong the survival time of patients, and improve the quality of life of patients.

Background technique

The application of autologous immune cells (DC-CIK) in tumor biological therapy is an emerging, safe and effective tumor treatment mode, and it is a new therapeutic method for tumor treatment. The method uses biological technology and biological preparations to induce in vitro culture and expansion of immune cells-mononuclear cells (including dendritic cells, T lymphocytes, B lymphocytes) collected and isolated from the peripheral blood of tumor patients. , back into the patient's body to stimulate and enhance the body's own immune function, so as to achieve the purpose of tumor treatment. Tumor biotherapeutics is the fourth most important method of tumor treatment after surgery, radiotherapy and chemotherapy.

Polyinosin is a copolymer of polyinosinic acid and polycytidylic acid. It is an efficient interferon (IFN) inducer with broad-spectrum anti-virus, stimulation of phagocytosis, regulation of immunity, anti-tumor, and anti-cerebral properties. Bacterial and protozoal infections, etc. At present, it is used in human medicine to treat hepatitis, herpes virus infection and some tumor diseases, etc., and it has shown its real clinical effect.

At the same time, polymyocytes are ligands of animal cell type 3 Toll-like receptors (TLR-3). After activation of TLR-3, polymyocytes can mediate a series of immune responses in the body, such as induction of interferon, interleukin, tumor necrosis The secretion of cytokines such as factors promotes the proliferation and maturation of immune cells (including: monocytes, macrophages, lymphocytes and dendritic cells), and promotes the production of antibodies in vivo. Polymyocytes can promote both specific and non-specific immunity of the body.

Immunomodulatory effect of polymyocytes: Immunity is a physiological protective function of the body. It is the body's ability to recognize and eliminate any foreign intrusion (viruses, bacteria, etc.) Ability to handle mutant cells and virus-infected cells in vivo. Modern immunology believes that immunity is the physiological response of the human body to identify and exclude "others". The immune capacity of the body can be divided into specific immunity and non-specific immunity, and the two are closely related. Non-specific immunity is an immune function formed by organisms in the process of germline development in the continuous struggle with pathogenic microorganisms, and can be inherited to offspring. It is closely related to the tissue characteristics and physiological functions of the human body. Specific immunity is the immune function acquired by the body after being stimulated by internal and external environmental factors. It can recognize the same antigen when it is contacted again, and the corresponding immunological response occurs, which requires the participation of highly differentiated tissues and cells to complete.

(1) Polymyocytes enhance the activity of immune cells;

(2) Polymyocytes promote the secretion of various cytokines;

(3) Induction effect of polymyocytes on Mx protein;

(4) Polymyocytes promote the production of antibodies in vivo.

Studies on the anti-tumor effect of polymyocytes have proved that polymyocytes can not only inhibit tumors caused by oncoviruses and oncoviruses such as polyoma virus and herpes simplex virus. The transplanted tumors, such as a variety of reticulum cell sarcoma, ascites tumor, S91 melanoma, breast tumor, fibrosarcoma, etc. in mice, as well as mouse sarcoma in hamsters, are inhibited to varying degrees.

Tumor biological therapy is an emerging tumor treatment mode with remarkable curative effect, and a new treatment method for autoimmune anti-cancer. It is a method of using biotechnology and biological preparations to culture and expand immune cells collected from patients in vitro and then infuse them into the patient's body to stimulate and enhance the body's autoimmune function, so as to achieve the purpose of treating tumors. Tumor biological therapy is the fourth largest tumor treatment technology after surgery, radiotherapy and chemotherapy.

The immune system is the body's defense system. On the one hand, it plays the function of removing bacteria, viruses, and foreign bodies. On the other hand, it eliminates senescent cells and mutated cells in the body (some mutated cells will become tumor cells). The outcome of the interaction between the body's immune system and tumor cells determines the final evolution of the tumor. In a healthy person, the immune system is strong enough to clear mutated cancer cells in time. However, for tumor patients, the immune system is generally low, which cannot effectively identify and kill tumor/cancer cells; on the other hand, the massive proliferation of tumor/cancer cells will further suppress the patient's immune function. There are various mechanisms to escape the recognition and killing of immune cells. The immunotherapy of tumor/cancer is to improve the immunogenicity of tumor/cancer by means of molecular biology technology and cell engineering technology, and to supplement the body with a sufficient number of normal immune cells. and related molecules, stimulate and enhance the body's anti-tumor immune response, improve the sensitivity of tumors/cancers to the body's anti-tumor/cancer immune effects, induce tumor/cancer specific and non-specific effector cells and molecules in vivo and in vitro, and achieve final clearance Tumor/Cancer Purpose.

Tumor/cancer biological therapy, its effect is not to kill all tumor/cancer cells, but because when the tumor/cancer cell load is significantly reduced, after the body's immune function is restored, by removing tiny residual lesions or significantly inhibiting residual tumor/cancer The way cancer cells proliferate for the purpose of treating tumors/cancers. Tumor/cancer immunotherapy is precisely through human intervention to mobilize the body's own immune system to kill cancer cells and inhibit their proliferation.

Both experiments and clinical studies have shown that the body's immune system has the effect of removing tumors/cancers. After the primary tumor/cancer is surgically removed or the local tumor/cancer is ablated by minimally invasive surgery such as argon fluoride knife, immunotherapy can kill the remaining tumor/cancer. It can eliminate the factors of recurrence and metastasis, increase the possibility of cure, prolong the survival time, and improve the quality of life.

As of 2013, tumor/cancer biological therapy has been regarded as the fourth treatment method after surgery, radiotherapy and chemotherapy. Biological therapy includes cytokine therapy, immune cell therapy, gene therapy, molecular targeted therapy and antibody therapy.

Biological cellular immunotherapy is the fourth largest tumor treatment technology after surgery, radiotherapy and chemotherapy. Biological cell immunotherapy for the treatment of tumors has the following advantages:

1. The effect is exact and the efficiency is high. For some tumors/cancers, the effective rate is as high as 70%.

2. No toxic and side effects of radiotherapy and chemotherapy, no pain for patients, good tolerance, and strong tumor killing specificity.

3. It can stimulate the systemic anti-cancer effect, and is also effective for multiple foci or metastatic malignant tumors.

4. It can help the body quickly restore the anti-cancer immune system destroyed by radiotherapy and chemotherapy, and improve the long-term anti-cancer ability.

5. It has remarkable anti-recurrence effect on tumor/cancer after operation, and has good long-term anti-cancer effect.

6. It can be used alone or in combination with other treatment methods. Single effective, multiple use, the effect is better.

Autologous DC-CIK cells are one of many immune cell therapies. DC-CIK cells can recognize tumor cells by different mechanisms, lyse tumor cells by penetrating the closed tumor cell membrane through direct cytoplasmic particles; kill tumor cells by inducing tumor cell apoptosis; DC-CIK cells can secrete IL- 2. Various anti-tumor cytokines such as IL-6 and IFN-γ; DC-CIK cells can activate the body's own immune system and improve the body's immune function after infusion.

The therapy can be used alone or as a powerful adjunct after surgery, chemotherapy and radiotherapy, with remarkable results. Combined with surgical resection, intervention, radiofrequency, argon-helium knife and other treatments, it can remove extremely small tumor foci that cannot be surgically removed or scattered tumor cells in the body, and plays an important role in delaying or preventing tumor metastasis or recurrence; for some Cancer patients who are temporarily unsuitable for surgery, intervention or other treatments can also undergo DC-CIK cell therapy first to improve their physical function, improve their quality of life, and strive for other treatment opportunities.

Since the biological immunotherapy technology uses the body's own immune cells instead of traditional chemicals to kill tumor cells, this technology is safe and has no toxic side effects, and is suitable for the treatment of tumors/cancers at all stages. Since then, it has relieved the pain of countless tumor/cancer patients and brought good health to tumor patients!

Tumor biological therapy is the latest tumor treatment technology superior to surgery, radiotherapy and chemotherapy. It is a treatment method in which autologous immune cells that can kill tumors are cultivated in a high-standard laboratory through biotechnology, and then reinfused into the body to directly kill cancer cells. Different from traditional treatment methods, tumor biotherapy can directly identify and eliminate cancer cells existing in the blood and lymph of the human body without damaging the body's immune system and function, and restore and enhance the body's natural anti-cancer immune system and function. . Tumor biological therapy has the advantages of no harm to the body, no pain, and no need to be hospitalized. It can improve the patient's own immunity and quality of life, and is applicable to all tumor diseases. This biological therapy technology has been formally used in clinical practice.

DC stands for dendritic cells and was discovered in 1973. It is the most functional professional antigen-presenting cell in the body, which can efficiently ingest, process and present antigens. DC cells are closely related to the occurrence and development of tumors, and are one of the most effective ways to activate the body's immune system and stimulate resistance to tumor/cancer invasion. By activating and culturing DC cells loaded with tumor antigens in a large amount in vitro, and returning to the patient after the number of cells reaches a large scale, a strong anti-tumor immune response can be induced in the body. According to clinical verification, a large number of infiltrating DCs in most solid tumors results in a better prognosis for patients.

In tumor immunity, DCs cannot directly kill tumor cells, but can monitor and kill tumors by recognizing tumor cell-specific antigens and presenting their signals to T cells with killing effect. Cells are used in combination.

DCs are the most powerful antigen-presenting cells discovered so far (that is, provide tumor-related information to cells that normally exist in the body with tumor-killing activity), and play a role similar to "radar" in the human immune system; CIK is peripheral blood mononuclear cells. After being co-induced and cultured by a variety of cytokines in vitro, it produces a heterogeneous cell population with CD3+CD56+ T cells as the main effector cells. Therefore, CIK has both T cells and NK cells. These two types of human body mainly have the effect of anti-tumor active cells, and their anti-tumor activity in vitro is 100-1000 times stronger than that of LAK, CTL and TIL cultured in previous biological therapy. Medical scientists describe it more vividly: if T cells act as "cannonballs" in the body's immune system, then DC-CIK is equivalent to a more powerful "missile".

SUMMARY OF THE INVENTION

Polymyocytes are ligands of mammalian cell type 3 Toll-like receptors (TLR-3). After activation of TLR-3, polymyocytes can mediate a series of immune responses in the body, such as induction of interferon, interleukin, and tumor necrosis. The secretion of cytokines such as factors promotes the proliferation and maturation of immune cells (including: monocytes, macrophages, lymphocytes and dendritic cells), and promotes the production of antibodies in vivo. It has a good promoting effect on the specific immunity and non-specific immunity of the body.

Therefore, polymyocytes are selected as DC-CIK synergistic inducers, which can efficiently induce the proliferation, differentiation and maturation of autologous mononuclear cells, and then infuse them into patients for the treatment of tumor patients. In order to restore or improve the immune function of tumor patients, improve the body's own immune system to kill tumor cells and inhibit their proliferation. Reduce tumor cell load, remove tiny residual lesions or significantly inhibit the proliferation of residual tumor/cancer cells, eliminate recurrence and metastasis factors, increase the possibility of cure, prolong survival time, and improve quality of life. So as to achieve the purpose of treating tumor/cancer.

First, the peripheral blood of normal healthy voluntary blood donors was selected, and mononuclear cells were routinely isolated. DC-CIK cells were induced synergistically with different concentrations of polymyocytes, and the optimal effective concentration was established by measuring the number of cells proliferating, detecting their CD markers and killing activity on tumor cells by flow cytometry.

Description of drawings

Fig. 1 is the OD ₄₅₀ value according to the detection, with Graphpad Prism5 software, with the polymyocyte concentration as the abscissa, and the corresponding OD value as the ordinate to make the cell proliferation curve;

Figure 2 is a histogram made according to the counting results, using Graphpad Prism5 software, with the days of cell culture as the abscissa and the number of cells as the ordinate;

Figure 3 is a partition map made by selecting CD3+CD4-CD8+ cells by flow-through, with CD4 as the abscissa and CD8 as the ordinate;

Figure 4 is a histogram of the tumoricidal activity of the percentage of each group's tumoricidal activity, which was produced by Graphpad Prism5 software.

Detailed ways

Embodiment 1. Selection of the optimal concentration of polymyocytes

Material:

1. Poly muscle cells (Origin: Shanghai Yuanye Biotechnology Co., Ltd.; Cas: 24939-03-5)

2. Hyclone RPMI 1640

3. Peripheral anticoagulation in healthy volunteers

4. Conditioned medium: IL-2 100000IU/ml, anti-human CD3 humanized chimeric antibody 1000μg/ml, IL-1α

10000ng/ml, GMCSF10000ng/ml, gamma interferon 10000ng/ml.

5. CCK8 detection kit (Beijing Chemical Technology (Beijing) Co., Ltd., model: CK04)

6. Others: plate microplate reader (Shenzhen Redu Life Science Co., Ltd., model: RT-6100), carbon dioxide incubator (Thermo SCIENTIFIC, model: 3111), clean workbench (Thermo SCIENTIFIC, model: 1379), etc.

Prepare:

1. Preparation of DC-CIK medium: respectively take the plasma-containing medium of "Method Step 2", and add 500 μl DC-CIK conditioned medium according to the ratio of DC-CIK conditioned medium: basal medium = 1:100. .

method:

1. Aseptically collect 5ml of peripheral blood from healthy volunteers, anticoagulate with 250IU heparin, and mix gently and thoroughly. Transfer to a 15ml centrifuge tube and centrifuge at 2000rpm for 10 minutes;

2. Collect about 2.5ml of upper plasma and add it to 50ml of RPMI-1640 for later use;

3. Blood cell sediment, add normal saline to suspend to 10ml;

4. Slowly add the "step 3" sample along the tube wall into a 15ml disposable sterile plastic centrifuge tube that has been pre-added with 3ml of lymphocyte stratified fluid, so that the added blood overlaps the stratified fluid to form a clear interface (be careful not to shake);

5. Centrifuge at 2000rpm for 20min in a horizontal centrifuge and take it out carefully;

6. Gently insert a disposable sterile plastic Pasteur pipette into the centrifuge tube, suck out the white cloudy mononuclear cell layer, transfer it to a 15ml centrifuge tube, add normal saline to 15ml, mix the cells thoroughly, and centrifuge at 2000rpm for 5min. Repeat the washing twice in this way; trypan blue staining and counting, centrifuge again at 2000rpm for 5min, and discard the supernatant;

7. According to the cell count results, suspend the cell pellet in DC-CIK-Hyclone RPMI 1640 medium to make the final cell concentration 2×10 ⁵ cells/ml, for later use;

8. In 96-well cell culture plates, use DC-CIK-Hyclone RPMI 1640 medium to dilute the polymyocytes by 4-fold gradient, so that the final concentrations of polymyocytes are 1, 0.25, 0.0625... until 9.54E-07mg/ml . At the same time, a blank control without polymyocytes was set. 2 duplicate wells for each concentration, 100 μl per well;

9. Add the spare cells from "Step 7" separately, 100 μl per well;

10. Incubate in a saturated humidity, 5% carbon dioxide, 37°C carbon dioxide incubator for 72 hours;

11. 4 hours before the end of the culture, add 10 μl of CCK8 detection reagent to each well;

12. At the end of the culture, use a plate reader to detect the OD ₄₅₀ value.

result:

According to the detected OD ₄₅₀ value, use the Graphpad Prism5 software to make the cell proliferation curve with the polymyocyte concentration as the abscissa and the corresponding OD value as the ordinate.

in conclusion:

According to the OD ₄₅₀ value, the cell proliferation curve (bar graph) was made with Graphpad Prism5 software.

The final concentrations of polymyocytes were 1, 0.25, and 0.0625 mg/ml, respectively, which significantly stimulated the proliferation of DC-CIK cells.

Example 2 The effect of polymyocytes on the proliferation of DC-CIK

Material:

1. Polymuscular (Shanghai Yuanye Biotechnology Co., Ltd.; Cas: 24939-03-5)

2. Hyclone RPMI 1640

3. Peripheral anticoagulation in healthy volunteers

4. Conditioned medium: 100000 IU/ml IL-2, 1000 μg/ml anti-human CD3 humanized chimeric antibody, 10000 ng/ml IL-1α, 10000 ng/ml GMCSF, 10000 ng/ml gamma interferon.

5. Plate microplate reader (Shenzhen Redu Life Science Co., Ltd., model: RT-6100), carbon dioxide incubator (Thermo SCIENTIFIC, model: 3111), clean workbench (Thermo SCIENTIFIC, model: 1379), etc.

Prepare:

1. Preparation of control DC-CIK medium: take 250ml of HycloneRPMI 1640 medium containing 2% autologous plasma from "Method Step 2", add 2.5ml of DC-CIK conditioned medium to form Control DC-CIK- HycloneRPMI 1640 medium.

2. Preparation of DC-CIK medium in experimental group: 250ml of HycloneRPMI 1640 medium containing 2% autologous plasma of "method step 2" was taken, and 2.5ml of DC-CIK conditioned medium was added. Then polymyocytes were added to make the final concentration 1 mg/ml to form DC-CIK-Hyclone RPMI 1640 medium.

method:

1. Aseptically collect 20ml of peripheral blood from healthy volunteers, anticoagulate with 1000IU heparin, and mix gently and thoroughly. Transfer to a 50ml centrifuge tube and centrifuge at 2000rpm for 10 minutes;

2. Collect about 10ml of upper plasma and add it to 500ml of Hyclone RPMI-1640, and divide it into 2 bottles, ie each bottle is 250ml. spare;

3. Blood cell sediment, add normal saline to suspend to 20ml;

4. Slowly add the "step 3" sample along the tube wall to two 15ml disposable sterile plastic centrifuge tubes that have been pre-added with 2.5ml of lymphocyte stratification fluid, so that the added blood overlaps the stratified fluid. , forming a clear interface (be careful not to shake);

5. Centrifuge at 2000rpm for 20min in a horizontal centrifuge and take it out carefully;

6. Gently insert a disposable sterile plastic Pasteur pipette into the centrifuge tube, suck out the white cloudy mononuclear cell layer, transfer it to a 50ml centrifuge tube, add normal saline to 50ml, mix the cells thoroughly, and centrifuge at 2000rpm for 5min. Repeat the washing twice; for the third time, suspend the cells with normal saline, count with trypan blue, and divide them into two parts, so that each part of the cells contains 2.5×10 ⁶ mononuclear cells, centrifuge again at 2000 rpm for 5 min, and discard the supernatant. ;

7. According to the cell count results, use 5ml of Control DC-CIK-Hyclone RPMI 1640 and DC-CIK-Hyclone RPMI 1640 medium to suspend the cells respectively;

8. Transfer to 25cm ² cell culture flasks and mark them;

9. Incubate in a saturated humidity, 5% carbon dioxide, 37°C carbon dioxide incubator for 0, 72, 120, 168 hours;

10. Samples were taken at the end of the culture and counted by trypan blue staining.

result:

According to the counting results, use Graphpad Prism5 software to make a histogram with the days of cell culture as the abscissa and the number of cells as the ordinate; Figure 2 is the graph of "the effect of polymyocytes on the proliferation of DC-CIK".

in conclusion:

According to the above results, it can be seen that with the increase of culture time, the number of cells in the polymyocyte group and the non-polymyocyte group both increased. There was no significant difference in the number of cells on the third day between the two groups, but the number of cells in the polymyocytes group was significantly different on the fifth and seventh days compared with the non-aggregate myoblast group. .

Example 3 Phenotypic identification of high-efficiency DC-CIK induced by polymyocytes

Material:

1. Polymuscular (Shanghai Yuanye Biotechnology Co., Ltd.; Cas: 24939-03-5)

2. Hyclone RPMI 1640

3. Peripheral anticoagulation in healthy volunteers

4. Conditioned medium: 100000 IU/ml IL-2, 1000 μg/ml anti-human CD3 humanized chimeric antibody, 10000 ng/ml IL-1α, 10000 ng/ml GMCSF, 10000 ng/ml gamma interferon.

5. Flow labeled antibodies CD3:BV510, CD4:BUV496, CD8:BV786

6. Others: flow cytometer (BD, model: FACSymphony), carbon dioxide incubator (Thermo SCIENTIFIC, model: 3111), clean bench (Thermo SCIENTIFIC, model: 1379), etc.

Prepare:

1. Preparation of DC-CIK medium for control group: take 250ml of Hyclone RPMI1640 medium containing 2% autologous plasma from "Method Step 2", add 2.5ml of DC-CIK conditioned medium to obtain DC-CIK conditioned medium, namely into ControlDC-CIK-Hyclone RPMI 1640 medium.

2. Preparation of DC-CIK medium for experimental group: take 250ml of Hyclone RPMI1640 medium containing 2% autologous plasma from "Method step 2", and add 2.5ml of DC-CIK conditioned medium. Then polymyocytes were added to make the final concentration 1 mg/ml to form DC-CIK-Hyclone RPMI 1640 medium.

method:

1. Aseptically collect 20ml of peripheral blood from healthy volunteers, anticoagulate with 1000IU heparin, and mix gently and thoroughly. Transfer to a 50ml centrifuge tube and centrifuge at 2000rpm for 10min;

2. Collect about 10ml of upper plasma, respectively take 12.5ml of plasma and add it to 500ml of YQ-1640/RPMI-1640, and divide into 2 bottles, ie, each bottle is 250ml. spare;

3. Add the blood cell sediment to 20ml of normal saline; slowly add the "step 3" sample to the two 15ml disposable sterile plastic centrifuge tubes with 3ml of lymphocyte layering solution added in advance along the tube wall with a pipette. , so that the added blood overlaps the layered liquid to form a clear interface (be careful not to shake);

4. Centrifuge at 2000rpm for 20min in a horizontal centrifuge and take it out carefully;

5. Gently insert a disposable sterile plastic Pasteur pipette into the centrifuge tube, suck out the white cloudy mononuclear cell layer, transfer it to a 50ml centrifuge tube, add normal saline to 50ml, mix the cells thoroughly, and centrifuge at 2000rpm for 5min. Repeat the washing twice; for the third time, suspend the cells with normal saline, count with trypan blue, and divide them into two parts, so that each part of the cells contains 2.5×10 ⁶ mononuclear cells, centrifuge again at 2000 rpm for 5 min, and discard the supernatant. ;

6. According to the cell count results, use Control DC-CIK-Hyclone RPMI 1640DC-CIK-Hyclone RPMI 1640 medium to suspend the cell pellet in 5ml;

7. Transfer to 25cm ² cell culture flasks and mark them;

8. Incubate in a saturated humidity, 5% carbon dioxide, 37°C, carbon dioxide incubator for 0, 72, 120, 168 hours;

9. At the end of the culture, samples were taken for routine labeling with CD3, CD4, and CD8 fluorescent antibodies;

10. Detect with flow cytometer.

result:

Select CD3+CD4-CD8+ cells by flow cytometry, and make a partition map with CD4 as the abscissa and CD8 as the ordinate. Figure 3 is a graph of "Polymyocyte-induced high-efficiency DC-CIK phenotype identification".

in conclusion:

With the increase of cell culture time, the number of CD3+CD4-CD8+ cells increased to varying degrees. The number of CD3+CD4-CD8+ cells in the polymyocytes group was significantly higher than that without polymyocytes, and there was a significant difference.

Example 4 High-efficiency DC-CIK tumoricidal activity induced by polymyocytes

Material:

1. Poly muscle cells: (Shanghai Yuanye Biotechnology Co., Ltd.; Cas: 24939-03-5)

2. Hyclone RPMI 1640

3. Peripheral anticoagulation in healthy volunteers

4. Conditioned medium: 100000 IU/ml IL-2, 1000 μg/ml anti-human CD3 humanized chimeric antibody, 10000 ng/ml IL-1α, 10000 ng/ml GMCSF, 10000 ng/ml gamma interferon.

5. Human myeloid leukemia cell line K562

6. CCK8 detection kit (Beijing Chemical Technology (Beijing) Co., Ltd., model: CK04)

7. Plate microplate reader (Shenzhen Redu Life Science Co., Ltd., model: RT-6100), carbon dioxide incubator (Thermo SCIENTIFIC, model: 3111), clean workbench (Thermo SCIENTIFIC, model: 1379), etc.

Prepare:

1. Preparation of control DC-CIK medium: Take 250ml of Hyclone RPMI1640 medium containing 2% autologous plasma from "Method step 2" and add 2.5ml of DC-CIK conditioned medium to form Control DC-CIK-Hyclone RPMI1640 culture base.

2. Preparation of DC-CIK medium for experimental group: take 250ml of Hyclone RPMI1640 medium containing 2% autologous plasma from "Method step 2", and add 2.5ml of DC-CIK conditioned medium. Then polymyocytes were added to make the final concentration 1 mg/ml to form DC-CIK-Hyclone RPMI 1640 medium.

3. Target cells: K562 cells in logarithmic growth phase were taken, counted by trypan blue staining, and washed by conventional centrifugation. Then, the cell concentration was adjusted to 4×10 ⁴ cells/ml with Hyclone RPMI 1640 medium containing 5% newborn calf serum, for use.

method:

1. Aseptically collect 20ml of peripheral blood from healthy volunteers, anticoagulate with 1000IU heparin, and mix gently and thoroughly. Transfer to a 50ml centrifuge tube and centrifuge at 2000rpm for 10 minutes;

2. Collect about 10ml of upper plasma, add it to 500ml of RPMI-1640, and divide it into 2 bottles, ie each bottle is 250ml. spare;

3. Blood cell sediment, add normal saline to suspend to 20ml;

4. Slowly add the "step 3" sample along the tube wall to two 15ml disposable sterile plastic centrifuge tubes with 2.5ml lymphocyte layering solution added in advance, so that the added blood overlaps the layering solution. , forming a clear interface (be careful not to shake);

5. Centrifuge at 2000rpm for 20min in a horizontal centrifuge and take it out carefully;

6. Gently insert a disposable sterile plastic Pasteur pipette into the centrifuge tube, suck out the white cloudy mononuclear cell layer, transfer it to a 50ml centrifuge tube, add normal saline to 50ml, mix the cells thoroughly, and centrifuge at 2000rpm for 5min. Repeat the washing twice; for the third time, suspend the cells with normal saline, count with trypan blue, and divide them into two parts, so that each part of the cells contains 2.5×10 ⁶ mononuclear cells, centrifuge again at 2000 rpm for 5 min, and discard the supernatant. ;

7. According to the cell count results, use 5ml of Control DC-CIK-Hyclone RPMI 1640 and DC-CIK-Hyclone RPMI 1640 medium to suspend the cells respectively;

8. Transfer to 25cm ² cell culture flasks and mark them;

9. Incubate in a saturated humidity, 5% carbon dioxide, 37°C carbon dioxide incubator for 0, 72, 120, 168 hours;

10. Before the end of the culture, samples were taken and washed 3 times with normal saline, and were counted by trypan blue staining. 5×10 ⁶ cells were taken respectively, and the cells were suspended in Hyclone RPMI 1640. 2000rpm, centrifugation for 5min. The pellet was resuspended with 2.5ml of Hyclone RPMI 1640 medium containing 5% newborn calf serum to make the final concentration 2×10 ⁶ cells/ml;

11. Take a round-bottomed disposable sterile 96-well cell culture plate and add samples to it in Table 1 below. That is: for the control DC-CIK-Hyclone RPMI 1640 group cell tumoricidal activity test, add the sample as follows: in the experimental group, add 100 μl of Control DC-CIK-Hyclone RPMI 1640 group cells at a concentration of 2×10 ⁶ cells/ml to each well, and at the same time add the concentration of 2×10 6 cells/ml. 100 μl of K562 cells at 4×10 ⁴ cells/ml; 100 μl of Control DC-CIK-HycloneRPMI 1640 cells at a concentration of 2×10 ⁶ cells/ml were added to each well of the effect control group, and at the same time, 5% newborn calf serum was added. Hyclone RPMI 1640 100 μl; for the target cell group, only 100 μl of K562 cells with a concentration of 4×10 ⁴ cells/ml and 100 μl of HycloneRPMI 1640 containing 5% newborn calf serum can be added to each well.

12. Incubate in a saturated humidity, 5% carbon dioxide, 37°C carbon dioxide incubator for 16 hours; add CCK8 to each well, 10 μl per well. After 4 hours, the OD ₄₅₀ value was determined with a plate reader.

Table 1 Hyclone 1640 medium loading table

Remarks: Four duplicate wells were made in each group; unit: μl.

result:

1. Calculation of results: tumor killing percentage=[1-(OD ₄₅₀ of experimental group-OD ₄₅₀ of effector control group)/OD ₄₅₀ of target cell control group]×100%;

2. Substitute the detection data of each group into the above formula to obtain the tumoricidal activity of each group;

3. Using the Graphpad Prism5 software to make a histogram of the tumoricidal activity of the percentages of each group's tumoricidal activity; Fig. 4 is a graph of "highly effective DC-CIK tumoricidal activity induced by polymyocytes".

in conclusion:

According to the above results, it can be seen that with the increase of cell culture time, the tumoricidal activity of the two groups of cells increased to different degrees, and the tumoricidal activity of the polymyocytes group increased more significantly than that of the non-polymyocyte group, and there was a significant difference.

Claims (2)

1. A method for inducing DC-CIK cells efficiently by polyinosinic cells.

2. The polyinosinic-polycytidylic acid is used as a synergistic inducer of other immune cells (such as NK, CIK, DC, gamma T and the like) to efficiently induce the proliferation, differentiation and maturation of autologous mononuclear cells.

Images