CN1990044A - Preparation and use of human body immunocompetent cell DCCIK anti-tumor cell preparation - Google Patents
Preparation and use of human body immunocompetent cell DCCIK anti-tumor cell preparation Download PDFInfo
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Abstract
The invention discloses the human body immunocompetent cell (DCCIK) anti-tumor cell agent and preparation methods. The method induces patients peripheral blood to DC and CIK using relevant cytokine, mixes the DC and CIK cells according proportion and cultures together after using alpha-Galcer or CI repeatedly impact DC, then gets DCCIK cells. Compared with homologous CIK, its proliferative activity and cytotoxicity are improved. The DCCIK has specific targeting and highly effective wide spectrum kill tumor effect without relevant tumor antigen impact. It can prevent tumour transition and recurrence after tumour surgery, radiotheraphy and chemotherapy. It can extend patient survival time and improve life quality with the assistance of other therapies.
Description
Technical field:
The invention belongs to biological technical field, be specifically related to the preparation method and the application of human immunity competent cell (DCCIK) preparation.
Background technology:
It is of paramount importance means outside tumor operation, the chemotherapy and radiation that immunization therapy is generally acknowledged, to removing the intravital micrometastasis tumor cell of tumor patient, prevent the transfer and the recurrence of tumor and improve patient's survival rate and quality of life the tool important function.The specificity active immunity treatment of tumor and the research of passive adoptive immunotherapy.Over nearly 20 years, obtained huge advance made: (1) identify and purification cytokine (Miller DL, the et al.Science.1982 of an immune activation function; 215:689-390), it can enhancing body to the immunne response of cancerous cell.(2) develop can selectivity efficient the method for specific amplification immune effector cell, promptly obtain T and NK cell (GrimmEA, the et al.J Exp Med.1982 of a large amount of specificity tool killing tumor cells; 155:1823-1941), be used for immunization therapy.(3) obtain and understood the molecular characterization of various tumor specific antigens, for example MUC (Ioannides CG, et al.J Immunol.1993; 151:3693-3703) and PSA (Vesey SG, et al.Urology.1990; 35:483-486) etc.(4) confirm that (Dendritic Cell DC) is antigen presenting cell (AntigenPresenting Cells, APC) (the Steinman RM.Annu Rev Immunol.1991 of a key to dendritic cell; 9:271-296) play the regulation and control immunization.(5) recognize that tumor cell has the development variety of way to escape immune ability (Khong HT, et al.Nat Immunol.2002; 3:999-1005).(6) in rejecting has the tumor cell of immunity-stimulator antigen, play an important role (Shan Karan V, et al.Nature, 2001 of conclusive evidence immune system; 410:1107-1111).
It is truly feasible that above-mentioned these immunologic progress recognize researcher to manage the immunne response that starts body self to eliminate the malignant tumor growth in vivo.
In the adoptive immunotherapy of tumor, 5 kinds of immunocompetence effector lymphocytes that are used for the treatment of tumor have appearred in front and back.(1) the activated killer cell of lymphokine (Lymphokine activated killer cell, LAK) (Rosenberg SA, et al.N.Engl.J.Med.1985; 313:1485-1492).(2) tumor infiltrating lymphocyte (Tumorinfiltrating lymphocyte, TIL) (Itok, et al.J.Exp.Med.1988; 168:1419-1441).(3) the activated killer cell of anti-leukocyte differentiation antigen-3 monoclonal antibody (Anti-CD3McAb activated killer cell, CD3-AK) (Uberti JP, et al.Clin.Immunol.Immunother.1994; 70:234-240).(4) cytotoxic T cell (Cytotoxic T lymphocyte, CTL) (Aruga A, et al.Int.J.Cancer.1991; 49:19-24).(5) cytokine induced kill cell (Cytokine induced killer cell, CIK) (Schmidt-Wolf IGH, et al.J.Exp.Med.1991; 174:139-149).
Above-mentioned 5 kinds of immunocompetence preparations are used for finding behind the clinical treatment tumour:
(1) although LAK obtains immense success in the mouse tumor experiment, in clinical practice, because of its serious dependency to IL-2, can produce general toxicity, this limits its extensive use on human body.
(2) CTL and TIL, particularly CTL, speaking from theory is the immunologically competent cell that is expected most, but because of still can't obtaining enough clinical required quantity at present, and other limiting factors and use less.
(3) CD3-AK and CIK, the both is to be that stimulating factor stimulates the T lymphopoiesis with anti-CD3 monoclonal antibody, almost has identical proliferation activity and to the kill capability of tumor cell, but the CIK cell also depends on the participation of other cytokines, so CIK has stronger propagation and cytotoxic activity than CD3-AK.Contain a large amount of granules in the CIK cell cytoplasm, include esterase, perforin, cytolysin etc., can make target cell that dissolving and dead takes place.The expression that the CIK cell is also secreted the various kinds of cell factor and raised adhesion molecule can increase effector lymphocyte's antitumor action.
Dendritic cell (Dendritic cell, DC) be of paramount importance immunoregulation and the accessory cell of recently being found, it is topmost antigen presenting cell, has and catches, processed antigen is also offered the function of antigen molecule to the T lymphocyte, and express costimulatory molecules and adhesion molecule; And can secrete the Th1 cytokines IL-12 that in antitumor immunity of organism, plays a significant role, induce body to produce antigen specific T lymphocyte, recognition and killing tumor cell.
People find to add tumor patient again behind the DC of body when at the CIK cell culture, can interact each other, promote the maturation of both sides' cell, and induce than the stronger proliferation activity of homology CIK cell and the active cell mass of Geng Gao tumor cytotoxicity (Marten A, et al.J Immunother.2001; 24 (6): 502-510).
The inventor carries out common cultivation to DC and CIK on the basis to T lymphopoiesis characteristic research, discovery can produce the rate of increase and the stronger cell mass that presses down oncocyte cytotoxic activity more higher than former CIK cell.And this novel cell ordered surely into DCCIK (immunologically competent cell), the inventor confirms the DCCIK experimental group with this DCCIK at human body adenocarcinoma of lung nude mice animal model, and there were significant differences than CIK and normal saline (NC) two matched groups, and be respectively 60 ± 78 its life cycle; 40.5 ± 5.6 and 19.8 ± 1.2 (P＜0.01), and confirm that the treatment of DCCIK cellular immunization can improve the effect of being with tumor (pulmonary carcinoma, hepatocarcinoma, melanoma) nude mice chemotherapy, both have collaborative tumor killing effect, the tumor body is dwindled and disappear and simple chemotherapy group is only dwindled no extinction tests.This shows that DCCIK has the treatment meaning to kinds of tumors, and prompting selects for use DCCIK will have more advantage than CIK cell as the immunity therapeutic preparation of tumour patient clinically.
Summary of the invention:
Technical problem to be solved by this invention is that research design prepares the DCCIK cell preparation.
The invention provides a kind of human body immunocompetent cell DCCIK anti-tumor cell preparation, this cell preparation uses the PBMC in patient's autologous peripheral blood source, obtain to adhere to and non-adhesion two class cells through culture of isolated, induce through relevant cell factor again and produce DC and CIK cell respectively.With carrying out common cultivation in proportion with CIK again behind α-Galcer or the CI impact DC, promptly obtaining CD3+CD56+ is a main immunologically competent cell group (DCCIK).
The innovation of DCCIK of the present invention has been to use the DC before α-Galcer glycolipid matter and CI Calcium ionophore repeatedly impact common training, make DC to its catch, processed offers this antigen molecule to the T lymphocyte and carries out signal transduction when training altogether with CIK, polygenes activation is expressed and discharged more cytokine and raise CD3+CD56+ ratio in the T lymphocyte being in contact with one another of DC and CIK, this will greatly strengthen the cell toxicant killing activity of DCCIK to the tumor target cell.
The invention provides the preparation method of human body immunocompetent cell DCCIK anti-tumor cell preparation, this method comprises the following steps:
(1) preparation of PERIPHERAL BLOOD MONONUCLEAR CELL PBMC
Aseptic condition is got the tumor patient anticoagulation cirumferential blood with the blood system device down, behind the normal saline two-fold dilution, use lymphocyte separation medium Ficoll, 1.077, centrifugal 2000rpm * 25 ', separate PBMC, reuse Hanks liquid washing 2 times, mirror is the meter cell number down, regulates cell density with serum-free medium AIM-V at last and makes into 4 * 10
6/ ml cell suspension.
(2) non-adhesion separates with adherent cell
Cell suspension is moved into 6 orifice plate Nuclon, 37 ℃, 5%CO2, cultivate 2h, dash gently with pipet then and inhale cell, the non-adherent cell suspension is collected in induces the CIK cell standby in the centrifuge tube, adherent cell is just stayed on 6 orifice plates, and adding DC culture fluid 3ml/ hole is stand-by;
(3) non-adhesion CIK cell inducing and increasing
The non-adherent cell suspension inoculation in the centrifuge tube to the 25cm that contains IFN-γ 1000U/ml AIM-V culture fluid
2Culture bottle 100ml/ bottle is put 37 ℃, behind the 24-36h, the anti-CD3 monoclonal antibody 5 μ g/ml with the preparation of Hanks liquid is wrapped by 25cm in the 5%CO2 incubator
2Culture bottle adds final concentration IL-1 β 100U/ml AIM-V simultaneously, and IL-2 300U/mlAIM-V continues to cultivate 48h, and mirror is counting down, and transferring cell density with CIK training liquid is 4 * 10
5/ ml by above-mentioned the same terms amplification 1 time, is cultured to 5-7 days and promptly collects the CIK cell standby every 48h;
Anti-CD3 monoclonal antibody method for coating: in AIM-V training liquid, add anti-CD3 monoclonal antibody, make that to become anti-CD3 monoclonal antibody concentration be the coating buffer of 5～10 μ g/ml, at 25cm
2Add the 5ml coating buffer in the culture bottle, 4 ℃ spend the night or 37 ℃ of incubation 2h after, discard whole coating buffers and get final product;
(4) adhere to inducing and increasing of DC cell
Leaving on 6 orifice plates of adherent cell, every hole is added DC training liquid 3ml and is contained GM-CSF 800U/ml and IL-4500U/ml, in 37 ℃, 5%CO2 cultivated after 2 days, add α-Galcer 100ng/ml or CI (200ng/ml) in each hole, add totally 3 times every day 1 time, after the continued stimulus 3 days, standby in 5-7 days collecting cells;
(5) DC and CIK mix to cultivate altogether and produce the DCCIK cell at 1: 5
Get the DC and the CIK cell that are cultured to the 5th day, counting, centrifugal, collect DC and CIK cell respectively, transfer bicelluar density to be respectively 2 * 10 with the AIM-V serum-free medium
5With 1 * 10
6, press DC: CIK=1: 5 equal-volumes mix the back and move into 25cm
2Culture bottle 10ml/ bottle, in 37 ℃, 5%CO2 cultivates, and divides the bottle amplification culture 1 time every 48-72h by above cell density, can obtain 5 * 10 after 5 amplification culture
9～1 * 10
10The DCCIK cell;
(6) DCCIK cell preparation
Centrifugal collection 1～5 * 10
9Cell, centrifugal with 0.9% sodium chloride injection washing 2 times, abandon supernatant, cell is moved in the 250ml infusion bottle, add 2g human serum albumin and 250ml 0.9% sodium chloride injection, make the cell suspension preparation, keep sample, carry out the calibrating of every index, all eligibles, cryopreservation, stand-by.
The present invention uses the DCCIK cell and LAK, the same heterogeneous body cell mass that is of CIK cell that common cultivation obtains, and its main biological characteristics is as follows:
(1) cell is formed: the T lymphocyte is greater than 90% or more and a small amount of DC in the DCCIK cell.Each subgroup proportion has individual variation and certain excursion is arranged because of In vitro culture time length in the T lymphocyte, being generally CD3-CD56+T cell (NK cell) is 20～40%, the CD3+CD8+ cell is 20～50%, and CD3+CD56+ (NKT) cell is 50～70%.
(2) proliferation activity height: the proliferation activity of DCCIK cell is more powerful than CIK cell, and amplification in vitro is after 3～4 weeks, and the DCCIK cell is bigger 2～4 times and 20～40 times respectively than homology CIK cell and LAK cell.
(3) release cytokine amount is big: the amount of DCCIK cell release IFN-γ is 2～5 times of homology CIK cell.
(4) cytotoxicity height: compare with homology CIK cell, the DCCIK cells in vitro exceeds 10～20% to the lethal effect of specificity target cell, performance is to the specific killing of homology malignant cell, people's carcinoma animal model treatment experiment shows that the mean survival time (MST) of DCCIK treated animal is 1～2 times (seeing the animal experiment data statistic) of CIK treated animal approximately.
Experimental group: No.1 inoculation normal saline (N.S) matched group
No.2 CIK experimental group
No.3 DCCIK experimental group
Method: every animal inoculation A549 3 * 10
5Individual cell, inoculation back 24h, 72h, 144h tail vein respectively give normal saline, CIK cell (5 * 10
6), DCCIK (5 * 10
6), investigate 3 groups of tumorigenesis situation and curative effects of testing with the existence natural law as observation index.
CIK and DCCIK are to the influence of animal survival phase of A549 tumor
| Group (No) | Number of animals (only) | Life cycle (my god) | The P value |
| 1(N.S) | 6 | 20.2±17 | / |
| 2(CIK) | 6 | 44.7±7.9 | ＜0.001 |
| 3(DCCIK) | 6 | 59.8±7.8 | ＜0.001 |
As seen, the CIK group is compared its P value respectively all less than 0.001 with the DCCIK group with matched group, have the difference of highly significant between experimental group and the matched group, and also there were significant differences (P＜0.01) between CIK group and the DCCIK group.The result shows: CIK and DCCIK have shown the strong inhibition effect to the A549 tumor, its life cycle CIK group than having prolonged more than 1 times the life cycle of matched group, and the DCCIK group has prolonged than matched group and has been close to 2 times.Illustrate that DCCIK group shows the stronger tumor power that presses down than the CIK group, the immunity therapeutic preparation that prompting selects for use DCCIK to do tumour patient clinically will more have superiority than CIK.
The DCCIK cell preparation is mainly used in the clinical treatment of autologous tumor, can cooperate postoperative and put, the effectively transfer and the recurrence of anti-rotation tumor of auxiliary treatment after the chemotherapy.In addition, the DCCIK cell also can be used for some infectious disease and toxic chemical damage, and good clinical application prospect is arranged.
The specific embodiment
Below by specific embodiment, the invention will be further described:
1. the preparation of PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC)
Aseptic condition is got the tumor patient anticoagulation cirumferential blood of making a definite diagnosis with the blood system device down, after times dilutions such as normal saline (0.9%NaCl), with lymphocyte separation medium (Ficoll, 1.077), centrifugal (2000rpm * 25 '), separate PBMC, reuse Hanks liquid washing 2 times, mirror is the meter cell number down, uses serum-free medium (AIM-V) to regulate cell density at last and makes into 4 * 10
6/ ml cell suspension.
2. non-adhesion separates with adherent cell
Cell suspension is moved into 6 orifice plates (Nuclon), put 37 ℃, 5%CO2 cultivates 2-6h, dashes gently with pipet then and inhales cell, the non-adherent cell suspension is collected in induces the CIK cell standby in the centrifuge tube; And adherent cell is just stayed on 6 orifice plates, adds DC culture fluid (3ml/ hole).
3. the inducing and increasing of non-adhesion (CIK) cell
The non-adherent cell suspension inoculation in the centrifuge tube to the 25cm that contains IFN-γ (1000U/ml) AIM-V culture fluid
2Culture bottle (100ml/ bottle) is put 37 ℃, behind the 24-36h, the anti-CD3 monoclonal antibody (5 μ g/ml) with the preparation of Hanks liquid is wrapped by * 25cm in the 5%CO2 incubator
2Culture bottle adds final concentration IL-1 β (100U/ml AIM-V) simultaneously, and IL-2 (300U/ml AIM-V) continues to cultivate 48-72h, and mirror is counting down.Transferring cell density with CIK training liquid is 4 * 10
5/ ml by above-mentioned the same terms amplification 1 time, is cultured to 5-7 days and promptly collects the CIK cell standby every 48h.
4. adhere to inducing and increasing of (DC) cell
Leaving on 6 orifice plates of adherent cell, DC training liquid 3ml is added in every hole, contain GM-CSF (800U/ml) and IL-4 (500U/ml), in 37 ℃, 5%CO2 cultivated after 2 days, added Galcer (100ng/ml) or CI (200ng/ml) in each hole, add every day 1 time, totally 3 times, continued stimulus is after 3 days, and is standby in 5-7 days collecting cells.
Get DC and the CIK cell that is cultured to 5-7 days 5.DC produce the DCCIK cell with the common cultivation of CIK (mixing at 1: 5), counting, centrifugal, collect DC and CIK cell respectively, transfer bicelluar density to be respectively 2 * 10 with the AIM-V serum-free medium
5With 1 * 10
6(DC: CIK=1: 5) equal-volume mixes back immigration 25cm
2Culture bottle (10ml/ bottle), in 37 ℃, 5%CO2 cultivates, and divides the bottle amplification culture 1 time every 48-72h by above cell density, after 5 amplification culture, can obtain 5 * 10
9～1 * 10
10The DCCIK cell.
6.DCCIK cell preparation
Centrifugal collection 1～2 * 10
9Cell, centrifugal with 0.9% sodium chloride injection washing 2 times, abandon supernatant, cell is moved in the 250ml infusion bottle, add 2g human serum albumin and 250ml 0.9% sodium chloride injection, make cell suspension, keep sample, carry out the calibrating of every index, all eligibles, cryopreservation, stand-by.
Claims (5)
1, a kind of human body immunocompetent cell DCCIK anti-tumor cell preparation is characterized in that said preparation makes by following method,
(1) preparation of PERIPHERAL BLOOD MONONUCLEAR CELL PBMC
Aseptic condition is got the tumor patient anticoagulation cirumferential blood with the blood system device down, behind the normal saline two-fold dilution, use lymphocyte separation medium Ficoll, 1.077, centrifugal 2000rpm * 25 ', separate PBMC, reuse Hanks liquid washing 2 times, mirror is the meter cell number down, regulates cell density with serum-free medium AIM-V at last and makes into 4 * 10
6/ ml cell suspension;
(2) non-adhesion separates with adherent cell
Cell suspension is moved into 6 orifice plate Nuclon, 37 ℃, 5%CO2, cultivate 2-6h, dash gently with pipet then and inhale cell, the non-adherent cell suspension is collected in induces the CIK cell standby in the centrifuge tube, adherent cell is just stayed on 6 orifice plates, and adding DC culture fluid 3ml/ hole is stand-by;
(3) non-adhesion CIK cell inducing and increasing
The non-adherent cell suspension inoculation in the centrifuge tube to the 25cm that contains IFN-γ 1000U/ml AIM-V culture fluid
2Culture bottle, the 100ml/ bottle is put 37 ℃, behind the 24-36h, the anti-CD3 monoclonal antibody 5 μ g/ml with the preparation of Hanks liquid is wrapped by 25cm in the 5%CO2 training case
2Culture bottle adds final concentration IL-1 β 100U/ml AIM-V simultaneously, and IL-2300U/mlAIM-V continues to cultivate 48-72h, and mirror is counting down, and transferring cell density with the CIK culture fluid is 4 * 10
5/ ml by above-mentioned the same terms amplification 1 time, is cultured to 5-7 days and promptly collects the CIK cell standby every 48h;
Anti-CD3 monoclonal antibody method for coating: in AIM-V training liquid, add anti-CD3 monoclonal antibody, make that to become anti-CD3 monoclonal antibody concentration be the coating buffer of 5～10 μ g/ml, at 25cm
2Add the 5ml coating buffer in the training bottle, 4 ℃ spend the night or 37 ℃ of incubation 2h after, discard whole coating buffers and get final product;
(4) adhere to inducing and increasing of DC cell
Leaving on 6 orifice plates of adherent cell, every hole is added DC training liquid 3ml and is contained GM-CSF 800U/ml and IL-4500U/ml, in 37 ℃, 5%CO2 cultivated after 2 days, add α-Galcer 100ng/ml or CI (200ng/ml) in each hole, add totally 3 times every day 1 time, behind the continued stimulus 3 days, standby in 5-7 days collecting cells;
(5) DC and CIK mix to cultivate altogether and produce the DCCIK cell at 1: 5
Get the DC and the CIK cell that are cultured to 5-7 days, counting, centrifugal, collect DC and CIK cell respectively, transfer bicelluar density to be respectively 2 * 10 with the AIM-V serum-free medium
5With 1 * 10
6, press DC: CIK=1: 5 equal-volumes mix the back and move into 25cm
2Culture bottle 10ml/ bottle, in 37 ℃, 5%CO2 cultivates, and divides the bottle amplification culture 1 time every 48-72h by above cell density, can obtain 5 * 10 after 5 amplification culture
9～1 * 10
10The DCCIK cell;
(6) DCCIK cell preparation
Centrifugal collection 1～5 * 10
9Cell, centrifugal with 0.9% sodium chloride injection washing 2 times, abandon supernatant, cell is moved in the 250ml infusion bottle, add 2g human serum albumin and 250ml 0.9% sodium chloride injection, make the cell suspension preparation.
2, a kind of preparation method of human body immunocompetent cell DCCIK anti-tumor cell preparation is characterized in that this method comprises the following steps:
(1) preparation of PERIPHERAL BLOOD MONONUCLEAR CELL PBMC
Aseptic condition is got the tumor patient anticoagulation cirumferential blood with the blood system device down, after times dilutions such as normal saline, use lymphocyte separation medium Ficoll, 1.077, centrifugal 2000rpm * 25 ', separate PBMC, reuse Hanks liquid washing 2 times, mirror is the meter cell number down, regulates cell density with serum-free medium AIM-V at last and makes into 4 * 10
6/ ml cell suspension;
(2) non-adhesion separates with adherent cell
Cell suspension is moved into 6 orifice plate Nuclon, 37 ℃, 5%CO2, cultivate 2h, dash gently with pipet then and inhale cell, the non-adherent cell suspension is collected in induces the CIK cell standby in the centrifuge tube, adherent cell is just stayed on 6 orifice plates, and adding DC culture fluid 3ml/ hole is stand-by;
(3) non-adhesion CIK cell inducing and increasing
The non-adherent cell suspension inoculation in the centrifuge tube to the 25cm that contains IFN-γ 1000U/ml AIM-V culture fluid
2Culture bottle 10-15ml/ bottle is put 37 ℃, behind the 24-36h, the anti-CD3 monoclonal antibody 5 μ g/ml with the preparation of Hanks liquid is wrapped by 25cm in the 5%CO2 incubator
2Culture bottle adds final concentration IL-1 β 100U/ml AIM-V simultaneously, and IL-2300U/ml AIM-V continues to cultivate 48-72h, and mirror is counting down, and transferring cell density with CIK training liquid is 4 * 10
5/ ml by above-mentioned the same terms amplification 1 time, is cultured to 5-7 days and promptly collects the CIK cell standby every 48h;
Anti-CD3 monoclonal antibody method for coating: in AIM-V training liquid, add anti-CD3 monoclonal antibody, make that to become anti-CD3 monoclonal antibody concentration be the coating buffer of 5～10 μ g/ml, in the 25cm2 culture bottle, add the 5ml coating buffer, 4 ℃ spend the night or 37 ℃ of incubation 2h after, discard whole coating buffers and get final product;
(4) adhere to inducing and increasing of DC cell
Leaving on 6 orifice plates of adherent cell, every hole is added DC training liquid 3ml and is contained GM-CSF 800U/ml and IL-4500U/ml, in 37 ℃, 5%CO2 cultivated after 2 days, add α-Galcer 100ng/ml or CI (200ng/ml) in each hole, add totally 3 times every day 1 time, behind the continued stimulus 3 days, standby in 5-7 days collecting cells;
(5) DC and CIK mix to cultivate altogether and produce the DCCIK cell at 1: 5
Get the DC and the CIK cell that are cultured to 5-7 days, counting, centrifugal, collect DC and CIK cell respectively, transfer bicelluar density to be respectively 2 * 10 with the AIM-V serum-free medium
5With 1 * 10
6, press DC: CIK=1: 5 equal-volumes mix the back and move into 25cm2 culture bottle 10ml/ bottle, and in 37 ℃, 5%CO2 cultivates, and divides the bottle amplification culture 1 time every 48-72h by above cell density, can obtain 5 * 10 after 5 amplification culture
9～1 * 10
10The DCCIK cell;
(6) DCCIK cell preparation
Centrifugal collection 1～5 * 10
9Cell, centrifugal with 0.9% sodium chloride injection washing 2 times, abandon supernatant, cell is moved in the 250ml infusion bottle, add 2g human serum albumin and 250ml 0.9% sodium chloride injection, make the cell suspension preparation.
3, the preparation method of a kind of human immunity competent cell DCCIK cell preparation according to claim 1 and 2, it is characterized in that wherein said step (3), the inducing and increasing of non-adhesion CIK cell, the method that adopts the solid phase CD 3-resisting monoclonal antibody to stimulate, be included in and add anti-CD3 monoclonal antibody among the serum-free culture AIM-V, make that to become anti-CD3 monoclonal antibody concentration be the coating buffer of 5～10 μ g/ml, add culture bottle, 4 ℃ spend the night or 37 ℃ of incubation 2h after, incline behind whole coating buffers, move into and handled the stimulation that NA cell suspension is accepted anti-CD3 monoclonal antibody through IFN-γ.
4, a kind of human immunity competent cell DCCIK cell preparation preparation method according to claim 1 and 2, it is characterized in that wherein said step (4), DC inducing and increasing, remove in the DC culture fluid except that adding GM-CSF and IL-4, also in each culture hole, add α-Galcer 100ng/ml or CI 200ng/ml, every 24h 1 time, totally 3 times.
5, the application of a kind of human immunity competent cell DCCIK in the preparation antitumor drug.
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| CN103483454B (en) * | 2012-06-13 | 2015-05-27 | 深圳市阳溪生物科技有限公司 | Fusion protein used for preparing human cytokine induced killer cells |
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| CN103966163B (en) * | 2014-04-08 | 2016-07-06 | 安德生细胞生物（湖北）有限公司 | Enhancement mode DCIK cell preparation method and cell preparation thereof |
| CN106177931A (en) * | 2016-08-25 | 2016-12-07 | 河北利同康生物科技有限公司 | The double CTL high efficiency that blocks of immune detection point kills the preparation method of cell preparation |
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| CN108354946A (en) * | 2017-08-25 | 2018-08-03 | 肖定璋 | A kind of preparation method and application of human immunity composite factor |
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