CN1223675C - Antineoplastic immunocompetent cell-cell of DCIC, its preparing method and applications - Google Patents
Antineoplastic immunocompetent cell-cell of DCIC, its preparing method and applications Download PDFInfo
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Abstract
The present invention discloses DCIK cells as anti-tumor immunocompetent cells, a preparing method thereof and the application thereof. In the present invention, cell factors are mainly used for inducing DC cells and CIK cells, and the DC cells are impacted by tumor antigens; the DC cells and the CIK cells, which are impacted by the antigens, are mixed with each other according to a certain proportion for co-culture; consequently, a new immune effect cell group is obtained, which means that the DCIK cells are obtained. Compared with the CIK cells, the DCIK cells of the present invention have stronger proliferation activity and cytotoxic activity, and the specificity killing of malignant cells is displayed. The DCIK cells are mainly used in self tumor treatment, and metastasis and recurrence can be effectively prevented through immediate treatment after operations; the DCIK cells can also be used in auxiliary treatment of chemotherapy; in addition, the DCIK cells can also be used in the immune treatment of certain infectious diseases.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to a kind of antineoplastic immune active cells-DCIK cell, its preparation method and application.
Background technology
Malignant tumour is one of principal disease of current harm humans health, obtains the country of basic controlling in transmissible disease, and cardiovascular and cerebrovascular diseases and malignant tumour become the 1st and the 2nd of the cause of death respectively.Whole world New Development tumor cases in 2000 is about 1,010 ten thousand according to statistics, China about 1,800,000.What malignant tumour was died from the whole world in 2000 has 6,200,000 approximately, China about 1,200,000.In this 1,200,000 people, die from cancer of the stomach 240,000, the esophageal carcinoma 220,000, liver cancer 17.5 ten thousand, cervical cancer 100,000, lung cancer 130,000, colon/rectum cancer 90,000, leukemia 60,000 and other tumour 150,000.The main cancer of developed country is lung cancer, knot/rectum cancer, mammary cancer, cancer of the stomach and prostate cancer; Developing country is mainly cervical cancer, cancer of the stomach, liver cancer, the esophageal carcinoma, mammary cancer and lung cancer.
In tumor therapeuticing method, immunotherapy has become operative treatment, most important outside the chemotherapy and radiation, most promising treatment means.In in the past 20 years, two aspect researchs of tumour-specific active immunity treatment and adoptive immunotherapy all have very big development.
In adoptive immunotherapy, five kinds of noticeable immune effector cells have successively appearred.Lymphokine activated killer cell that Here it is (LAK cell) (Rosenberg SA, et al.N.Engl.J.Med.313:1485-1492,1985), tumor infiltrating lymphocyte (til cell) (Itoh K, et al.J Exp.Med.168:1419-1441,1988), anti-leukocyte differentiation antigen-3 monoclonal antibody (CD
3Monoclonal antibody) and interleukin II (IL-2) inductive killer cell AK-T cell (Uberti JP, et al.Clin.Immunol.Immunother, 70:234-240,1994), cytotoxic T cell (CTL) (ArugaA, et al.Int.J.Cancer, 49:19-24,1991) and cytokine induced kill cell (CIK cell) (Schmidt-wolf IGH, et al.J.Exp.Med.174:139-149,1991).Before for many years clinical and clinical study to find once to be considered to have LAK cell that huge immunotherapy is worth undesirable and cytotoxic activity is lower and it is short gradually by unfrequented to hold time because of its propagation performance.CTL and TIL, particularly CTL are the immune effector cell that is expected most theoretically, because of still not having effective amplification means and some other limiting factors at present not by wide clinical application.So people have shifted to back two kinds of effector cells---AK-T cell and CIK cell to hope gradually.AK-T cell and CIK cell all are to use anti-CD
3Monoclonal antibody is that stimulating factor excites the T lymphopoiesis, has identical proliferation activity.The CIK cell also depends on the participation of other cytokine, and therefore, the CIK cell has the cytotoxic activity stronger than AK-T cell.But the same with the LAK cell with the AK-T cell, the CIK cell does not have the characteristic of specific recognition and killing tumor cell yet.
In numerous tumour active specific immunotherapy methods of treatment, dendritic cell (DC) becomes focus in the active immunity treatment research in recent years as the research of tumor vaccine of new generation and application.In organism immune response, DC is topmost antigen presenting cell, have catch, processing treatment antigen and offer antigen molecule, express costimulatory molecules, discharge characteristics such as cytokine and interleukin-to the T lymphocyte.Therefore, DC plays main immunoregulation effect in the tumour cell immunne response, is the most important immune accessory cell of tumour active specific immunotherapy.
When DC and CIK co-culture of cells, can interact each other and induce than CIK cell has stronger proliferation activity, the active cell mass of higher tumor cytotoxicity (Marten A et al.J.Immunother 24 (6): 502-510,2001).But this cell is to the same antigen-specific that lacks with the CIK cell of the attack of malignant cell.People such as Peter M once found, when the DC cell through impacting back and neoplasm invasiveness lymphocyte (TIL) with tumor associated antigen when cultivating altogether, the effector cell who induces has ability (the Peter M. that significantly specificity of malignant cell is attacked, et al.Clin.Cancer Res.5:445-454,1999).But this immune effector cell amplification is limited in one's ability, and TIL source difficulty.
Summary of the invention
The objective of the invention is to improve antineoplastic immune effector cell in the prior art at proliferation activity, cytotoxic activity and to the deficiency of the vigor of tumour cell specific recognition and attack, thereby develop a kind of new antineoplastic immune effector cell and preparation method thereof.
For reaching this purpose, the present invention is on the basis of people such as Marten and Peter research, use cytokine induction DC and CIK cell respectively, impact DC with tumour antigen then, to mix by a certain percentage with the CIK cell through the DC that antigen impacted and carry out common cultivation, obtain a kind of new immune effector cell group, we name the cell into DCIK with this immune effector cell group who uses the DC that impacted through tumour antigen and homology CIK co-culture of cells to be produced.
The concrete preparation method of DCIK cell is as follows:
One, the preparation of peripheral blood mononuclear cell (PBMC):
With the anticoagulation cirumferential blood of physiological saline two-fold dilution 20～50ml tumour patient, be that 1.077 lymphocyte separation medium separates PBMC with proportion.The rotary head that separates used whizzer is a swing bucket rotor, and centrifugal speed is 2000rpm, centrifugal 20～25 minutes.Wash PBMC 2 times with the Hanks balance liquid then, carry out the microscopically cell counting again, regulate cell density with lymphocyte culture fluid AIM-V at last and become 3～5 * 10
6The cell suspension of/ml.
Two, separating of peripheral blood lymphocyte (PBL) and adherent cell:
Aforementioned cell suspension is moved into 6 orifice plates, and every hole 3-5ml puts 37 ℃, 5%CO
2Saturated humidity incubator incubation 2 hours dashes gently with transfer pipet then and inhales cell, and non-adhesion PBL suspension is collected in the 50ml centrifuge tube, and the adherent cell that stays in 6 orifice plates induces DC to use.
Three, the inducing and increasing of CIK cell:
Adding recombinant human gamma-interferon (IFN-γ) final concentration in above-mentioned PBL suspension is 1000u/ml, and moving into surface-area then is 25cm
2Culturing bottle in, every bottled amount 10ml places 37 ℃, 5%CO
2Incubation is 24 hours in the saturated humidity incubator.Adopt solid phase CD then
3The method that monoclonal antibody stimulates moves into CD with whole cell suspensions
3The monoclonal antibody bag is by the 25cm that crosses
2In the culturing bottle, add recombinant human interleukin--1 (IL-1) and recombinant human interleukin--2 (IL-2) again, final concentration is respectively 100u/ml and 500u/ml; Or adopt the liquid phase stimulus method, promptly directly add CD
3Monoclonal antibody, IL-1 and IL-2, concentration is respectively 50ng/ml, 100u/ml and 500u/ml.Continue to cultivate microscopically cell counting after 48～72 hours, using CIK cell culture fluid (AIM-V contains IL-1 and IL-2, and concentration is respectively 100u/ml and 500u/ml) to regulate cell density then is 3 * 10
5/ ml is assigned to 25cm
2In the culturing bottle, every bottle of 10ml carries out enlarged culturing.After this, every 48 or 72 hours once by above-mentioned the same terms enlarged culturing.It is stand-by to collect the CIK cell when being cultured to the 8th day.
CD in the above-mentioned steps
3Monoclonal antibody method for coating: in AIM-V, add CD
3Monoclonal antibody, making becomes CD
3Monoclonal antibody concentration is the coating buffer of 5-10 μ g/ml.At 25cm
2Add the 5ml coating buffer in the culturing bottle, 4 ℃ are spent the night or 37 ℃ of incubations after 2 hours, discard whole coating buffer bodies and get final product.
Four, DC induces and increases:
Every hole adds DC nutrient solution 3ml in aforementioned 6 orifice plates that leave adherent cell.The DC nutrient solution is recombinant human granulocyte-macrophage clone's stimulating factor (GM-CSF) and interleukin 4 (IL-4), and concentration is respectively 800u/ml and 500u/ml.Put 6 orifice plates in 37 ℃, 5%CO
2The saturated humidity incubator was cultivated 5 days, added from body oncocyte lysate in each hole then, and making the lysate final concentration of protein is 10-50 μ g/ml, continued to cultivate after 2～3 days, dashed with transfer pipet and inhaled and collect non-adhesion and half adhesivity DC is stand-by.
Above-mentioned preparation method: the blood of using the fresh tumor tissues of Hank ' s balance liquid flush away from body oncocyte lysate, wipe out necrotic tissue, reticular tissue and fatty tissue, stay fresh tumor tissue, shred the back and grind with homogenizer, multigelation is 5 times again, add an amount of Hank ' s balance liquid, centrifugal 10 minutes of 3000rpm discards precipitation, and supernatant sampling is surveyed behind the protein concentration in-20 ℃ of storages.Store the lysate protein concentration and should not be lower than 1mg/ml, be lower than this concentration and need make concentration.
Five, DC and CIK co-culture of cells prepare the DCIK cell:
Cultivate 7～8 days DC and CIK cell, counting respectively, centrifugal abandon supernatant after, be 6 * 10 with CIK nutrient solution adjusting cell density respectively
5/ ml and 2 * 10
5/ ml, equal-volume mix the back and move into 25cm
2In the culturing bottle, every bottle of 10ml.This moment, total cell density was 4 * 10
5/ ml, CIK: DC=3: 1.Put 37 ℃, 5%CO
2The saturated humidity incubator is cultivated, and divides the bottle enlarged culturing once every 48～72 hours by above cell density.
Used suction pipe and centrifuge tube are polypropylene (polypropylene) material in the above preparation process, and 6 orifice plates and culturing bottle are polystyrene (polystyrene) material.In addition, cultivate vessel, should adjust above-mentioned cell suspension inoculation amount according to the culture area size if use other specifications instead.
Above-mentionedly cultivate altogether the DCIK cell that obtains and LAK, the CIK cell is the same is a heterogeneous body cell mass, its main biological characteristics has:
(1) cell is formed: the T lymphocyte accounts for more than 98% and a small amount of DC in the DCIK cell.Each subgroup proportion has individual difference and certain variation range is arranged because of vitro culture time length in the T lymphocyte.Be generally CD
3 +CD
4 +T cell 20-40%, CD
3 +CD
8 +T cell 50-80%, CD
3 +CD
56T cell (NKT cell) 10-50%, and the CTL (CD that exists probably
8 +);
(2) proliferation activity height: the DCIK cell-proliferation activity is more powerful than CIK cell.After amplification in vitro 3-4 week, the DCIK cell is distinguished big 2-4 times and 20-40 times than homology CIK cell and LAK cell;
(3) release cytokine amount is big: the amount of DCIK cell release IFN-γ is 3.9 times of homology CIK cell;
(4) cytotoxicity height: compare with homology CIK cell, the DCIK cells in vitro exceeds 10-20% to the lethal effect of specificity target cell, and performance is to the specific killing of malignant cell.People's carcinoma animal model treatment experiment shows that the average survival time of DCIK treated animal is 1.3 times of CIK treated animal.
The DCIK cell is mainly used in from the body oncocyte, and postoperative is executed immediately and controlled effectively the anti-rotation recurrence of being shifted elsewhere for garrison duty, and also can make chemotherapeutic assisting therapy, and in addition, the DCIK cell also can be used for the immunotherapy of some communicable disease.
Embodiment
Below by specific embodiment, the invention will be further described.
1. the preparation of peripheral blood mononuclear cell (PBMC)
With the anticoagulation cirumferential blood of physiological saline two-fold dilution 20～50ml tumour patient, be that 1.077 lymphocyte separation medium separates PBMC with proportion.The rotary head that separates used whizzer is a swing bucket rotor, and centrifugal speed is 2000rpm, centrifugal 20～25 minutes.Wash PBMC 2 times with the Hanks balance liquid then, carry out the microscopically cell counting again, regulate cell density with lymphocyte culture fluid AIM-V at last and become 3～5 * 10
6The cell suspension of/ml.
2. separating of peripheral blood lymphocyte (PBL) and adherent cell:
Cell suspension is moved into 6 orifice plates, and every hole adds 3-5ml, puts 37 ℃, 5%CO
2Saturated humidity incubator incubation 2 hours dashes gently with transfer pipet then and inhales cell, and non-adhesion PBL suspension is collected in the 50ml centrifuge tube, and the adherent cell that stays in 6 orifice plates induces DC to use.
3.CIK the inducing and increasing of cell:
Adding recombinanthumanifn-'s final concentration in above-mentioned PBL suspension is 1000u/ml, and moving into surface-area then is 25cm
2Culturing bottle in, every bottled amount 10ml places 37 ℃, 5%CO
2Incubation is 24 hours in the saturated humidity incubator.Afterwards, every bottle is added mouse-anti people CD
3Monoclonal antibody, IL-1 and IL-2, final concentration is respectively 50ng/ml, 100u/ml and 500u/ml; Perhaps adopt solid phase CD
3The method that monoclonal antibody stimulates is about to whole cell suspensions and moves into CD
3The monoclonal antibody bag is by the 25cm that crosses
2In the culturing bottle, add IL-1 and IL-2 again, final concentration is respectively 100u/ml and 500u/ml.Continue to cultivate microscopically cell counting after 48～72 hours, using CIK cell culture fluid (AIM-V contains IL-1 and IL-2, and concentration is respectively 100u/ml and 500u/ml) to regulate cell density then is 3 * 10
5/ ml is assigned to 25cm
2In the culturing bottle, every bottle of 10ml carries out enlarged culturing.After this, every 48 or 72 hours once by above-mentioned the same terms enlarged culturing.It is stand-by to collect the CIK cell when being cultured to the 8th day.
CD in the above-mentioned steps
3Monoclonal antibody method for coating: in AIM-V, add CD
3Monoclonal antibody, making becomes CD
3Monoclonal antibody concentration is the coating buffer of 5-10 μ g/ml.At 25cm
2Add the 5ml coating buffer in the culturing bottle, 4 ℃ are spent the night or 37 ℃ of incubations after 2 hours, discard whole coating buffer bodies and get final product.
4.DC induce and increase:
In aforementioned 6 orifice plates that leave adherent cell, add DC nutrient solution (contain GM-CSF and IL-4 among the AIM-V, concentration is respectively 800u/ml and 500u/ml), every hole 3ml, 37 ℃, 5%CO
2The saturated humidity incubator was cultivated 5 days, added from body oncocyte lysate in each hole then, and making the lysate final concentration of protein is 10-50 μ g/ml, continued to cultivate after 2～3 days, dashed with transfer pipet and inhaled and collect non-adhesion and half adhesivity DC is stand-by.
Above-mentioned preparation method: the blood of using the fresh tumor tissues of Hank ' s balance liquid flush away from body oncocyte lysate, wipe out necrotic tissue, reticular tissue and fatty tissue, stay tumor tissue, shred the back and grind with homogenizer, multigelation is 5 times again, add an amount of Hank ' s balance liquid, centrifugal 10 minutes of 3000rpm discards precipitation, and supernatant sampling is surveyed behind the protein concentration in-20 ℃ of storages.Store the lysate protein concentration and be not less than 1mg/ml, otherwise need make concentration.
5.DC prepare the DCIK cell with the CIK co-culture of cells:
Cultivate 7～8 days DC and CIK cell, counting respectively, centrifugal abandon supernatant after, be 6 * 10 with CIK nutrient solution adjusting cell density respectively
5/ ml and 2 * 10
5/ ml, equal-volume mix the back and move into 25cm
2In the culturing bottle, every bottle of 10ml.This moment, total cell density was 4 * 10
5/ ml, CIK: DC=3: 1.Put 37 ℃, 5%CO
2The saturated humidity incubator is cultivated, and divides the bottle enlarged culturing once every 48～72 hours by above cell density.
Claims (4)
1, a kind of preparation method of DCIK cell is characterized in that may further comprise the steps:
The preparation of a, PBMC
Anticoagulation cirumferential blood with physiological saline two-fold dilution 20～50ml tumour patient, with proportion is that 1.077 lymphocyte separation medium separates PBMC, the rotary head that separates used whizzer is a swing bucket rotor, centrifugal speed is 2000rpm, centrifugal 20～25 minutes, wash PBMC 2 times with the Hanks balance liquid then, carry out the microscopically cell counting again, regulate cell density with lymphocyte culture fluid AIM-V at last and become 3～5 * 10
6The cell suspension of/ml;
Separating of b, PBL and adherent cell
Cell suspension is moved into 6 orifice plates, put 37 ℃, 5%CO
2Saturated humidity incubator incubation 2 hours dashes gently with transfer pipet then and inhales cell, and non-adhesion PBL suspension is collected in the centrifuge tube, and the adherent cell that stays in 6 orifice plates induces DC to use;
The inducing and increasing of c, CIK cell
Adding recombinanthumanifn-'s final concentration in above-mentioned PBL suspension is 1000u/ml, moves into then in the culturing bottle, places 37 ℃, 5%CO
2Incubation is 24 hours in the saturated humidity incubator, and afterwards, every bottle is added mouse-anti people CD
3Monoclonal antibody, IL-1 and IL-2, final concentration is respectively 50ng/ml, 100u/ml and 500u/ml; Perhaps adopt solid phase CD
3The method that monoclonal antibody stimulates is about to whole cell suspensions and moves into CD
3The monoclonal antibody bag is added IL-1 and IL-2 again by in the culturing bottle of crossing, and final concentration is respectively 100u/ml and 500u/ml, continues to cultivate microscopically cell counting after 48～72 hours, and regulating cell density with the CIK cell culture fluid then is 3 * 10
5/ ml is assigned to 25cm
2In the culturing bottle, carry out enlarged culturing, after this, every 48 or 72 hours by above-mentioned the same terms enlarged culturing once, it is stand-by to collect the CIK cell when being cultured to the 8th day;
D, DC induce and increase
In aforementioned 6 orifice plates that leave adherent cell, add the DC nutrient solution, 37 ℃, 5%CO
2The saturated humidity incubator was cultivated 5 days, added from body oncocyte lysate in each hole then, and making the lysate final concentration of protein is 10-50ug/ml, continued to cultivate after 2～3 days, dashed with transfer pipet and inhaled and collect non-adhesion and half adhesivity DC cell is stand-by;
E, DC and CIK co-culture of cells prepare the DCIK cell
Cultivate 7～8 days DC and CIK cell, counting respectively, centrifugal abandon supernatant after, be 6 * 10 with CIK nutrient solution adjusting cell density respectively
5/ ml and 2 * 10
5/ ml, equal-volume mix the back and move in the culturing bottle, put 37 ℃, 5%CO
2The saturated humidity incubator is cultivated, and presses 4 * 10 every 48～72 hours
5The initiator cell density of/ml is divided the bottle enlarged culturing once.
2. the preparation method of a kind of DCIK cell as claimed in claim 1 is characterized in that: inducing and increasing of described step c:CIK cell, adopt solid phase CD
3The step that monoclonal antibody stimulates is included in and adds CD among the AIM-V
3Monoclonal antibody, making becomes CD
3Monoclonal antibody concentration is the coating buffer of 5-10 μ g/ml, adds coating buffer in culturing bottle, and 4 ℃ are spent the night or 37 ℃ of incubations after 2 hours, discard whole coating buffer bodies, move into the PBL suspension of handling through IFN-γ then and accept CD
3Monoclonal antibody stimulates.
3. the preparation method of a kind of DCIK cell as claimed in claim 1, it is characterized in that: described steps d: DC inducing and increasing, also comprise the step of preparation from body oncocyte lysate, comprise the blood of using the fresh tumor tissues of Hank ' s balance liquid flush away, wipe out necrotic tissue, reticular tissue and fatty tissue, stay tumor tissue, shredding the back grinds with homogenizer, multigelation is 5 times again, adds an amount of Hank ' s balance liquid, centrifugal 10 minutes of 3000rpm, in-20 ℃ of storages, the protein concentration of storing the tumour cell lysate is not less than 1mg/ml behind collection supernatant and the sampling survey protein concentration.
4. the DCIK cell for preparing of the method for a claim 1.
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Families Citing this family (8)
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| CN101914497B (en) * | 2010-07-19 | 2013-10-30 | 山东迪博生物技术有限公司 | Clinical N-CIK cell culture and quality control and identification kit and application |
| CN102321577B (en) * | 2011-01-26 | 2013-09-11 | 深圳市中美康士生物科技有限公司 | Preparation method of antitumor adoptive immune cells and prepared immune cells |
| CN102321581B (en) * | 2011-09-13 | 2013-04-17 | 深圳市博泰生物医学科技发展有限公司 | Preparation method of ascites tumor cell sensitized DC-CIK |
| CN102357101A (en) * | 2011-10-31 | 2012-02-22 | 上海市胸科医院 | Application of dendritic cell combined with cytokine-induced killer cell in lung cancer treatment |
| CN103372204A (en) * | 2012-04-19 | 2013-10-30 | 孙勇 | External xenoliths vaccine combined dendritic cell cytokine induced killer (DC-CIK) new technology for cancer therapy |
| CN104651312A (en) * | 2015-02-06 | 2015-05-27 | 上海力沃生物科技有限公司 | Preparation method and application of efficient immunocompetent cell CpG-DCIK |
| CN105018424A (en) * | 2015-05-27 | 2015-11-04 | 贵州北科泛特尔生物科技有限公司 | DC-CIK cell and preparation method thereof |
| CN105039254B (en) * | 2015-08-31 | 2019-02-19 | 广州赛莱拉干细胞科技股份有限公司 | A kind of immunocyte and preparation method thereof |
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Assignee: ZMKS International Cancer Therapy Biotechnologies Co., Ltd. Assignor: Shanghai Institute of life Sciences, Chinese Academy of Sciences Contract record no.: 2010440020113 Denomination of invention: Antineoplastic immunocompetent cell-cell of DCIC, its preparing method and applications Granted publication date: 20051019 License type: Exclusive License Open date: 20030723 Record date: 20100719 |
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