CN101037670A - Effecter cell for preventing and curing CEA positive tumor and preparation method and application thereof - Google Patents
Effecter cell for preventing and curing CEA positive tumor and preparation method and application thereof Download PDFInfo
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- CN101037670A CN101037670A CNA2007100268655A CN200710026865A CN101037670A CN 101037670 A CN101037670 A CN 101037670A CN A2007100268655 A CNA2007100268655 A CN A2007100268655A CN 200710026865 A CN200710026865 A CN 200710026865A CN 101037670 A CN101037670 A CN 101037670A
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Abstract
The invention relates to a effect cell for precaution and treating CEA masculine tumor and preparing method and application thereof. The effect cell is prepared by using DC with CEA-rV to induce general culture CIK cell. The invention uses CEA-rV as differentia antigen to load DC to solve the problem of most patient with CEA masculine tumor can not obtain self tumor antigent, the CEA-rV can be prepared in laboratoy and safe, so easy to be popularized and applied; using self outside hemocyte of the patient to prepare DC and CIK to overcome infection of bleeding of the umbilicus and ''transplant resisted host'' reaction of patient, guarranting safety of clinical using, and improving validity of treating CEA masculine tumor; birth speed of CIK cell of the invention is more quickly, stronger killing function with less affection of immunity repressing, so the effect cell of the invention has better effect.
Description
Technical field
The present invention relates to the medical biotechnology field, effector cell of particularly a kind of prevention and treatment CEA positive tumor and its production and application.
Background technology
Tumour is the maximum killer of human health.The operative treatment of tumour though can effectively remove macroscopic tumour, does not still have effective methods of treatment to post operative recurrence and transfer.Radiation and chemotherapy is except that having serious toxic side effect, because many tumour cells are insensitive to it, its curative effect is also undesirable.The immune biotherapy of tumour is the new model of treatment and prevention of tumour, mainly is the immunity that strengthens tumour antigen by specific technological method, induces the killer cell and the humoral immune reaction of body generation antineoplastic specificity, with killing tumor cell.
(Carcinoembryonic Antigen CEA) is a kind of important tumor associated antigen to carcinomebryonic antigen.In human body epithelial cell malignant tumour, 90% gastroenteric tumor, carcinoma of the pancreas, nonsmall-cell lung cancer and about 50% mammary cancer more than 80%, the high expression level of CEA is all arranged, promptly above-mentioned tumour is the CEA positive.But because the antigenicity of CEA is very weak, can not activate the specific immune response of body effectively, make tumour cell can escape immunologic cytotoxicity, cause the mortality ratio of this class tumour patient higher.
For solving the very weak problem that causes tumour cell can escape immunologic cytotoxicity of CEA antigenicity, in the prior art CEA gene and vaccinia virus gene are recombinated, obtain CEA recombination bovine vaccine (CEA-recombinant Vaccinia Virus, CEA-rV), the antigenic high expression level of its existing CEA can stimulate body to produce CEA antibody again, experimental CEA positive tumor there are certain prevention and therapeutic action, but clinical application shows, merely injects CEA-rV, and its curative effect is desirable not enough.
(Dendritic Cell DC) is the strongest antigen presenting cell of present known function to dendritic cell, and the breeding of its energy significant stimulation T cells plays an important role in the biotherapy of tumour.Have in the prior art the DC cell of CEA-rV load, remove to induce the CD for preparing from Cord blood with CEA-rV+DC again with the Cord blood preparation
3The AK cell is prepared CEA-rV+DC+CD
3AK effector cell, promptly lotus has the DC inductive CD of CEA-rV
3The AK cell.Breadboard fundamental research shows that it has tangible fragmentation effect to the CEA positive tumor, but is not used for clinical application as yet.And, because DC cell and CD
3The AK cell all is by the Cord blood preparation, and Cord blood is the allosome source, and clinical application must detect various transmissible diseases, as acquired immune deficiency syndrome (AIDS), syphilis, hepatitis etc.In addition, variant cell is clinical to be used for patient, also wants HLA to join type, otherwise may produce the graft versus host rejection, so its clinical application is still immature, also has significant limitation.
Summary of the invention
One of purpose of the present invention is to provide the effector cell of a kind of prevention and treatment CEA positive tumor.
Another object of the present invention is to provide described effector cell's preparation method.
Another purpose of the present invention is to provide the application of effector cell in preparation prevention and treatment CEA positive tumor medicine of described prevention and treatment CEA positive tumor.
The effector cell of a kind of prevention provided by the invention and treatment CEA positive tumor is the inductive CIK of the DC institute cell (CEA-rV+DC+CIK cell) that lotus has CEA-rV.Preferably, described CIK cell is on the basis of LAK cell, prepares with IL-2, CD3 monoclonal antibody, IL-1 and four kinds of cytokine inductions cultivations of IFN-γ.In one embodiment of the invention, described DC and CIK cell prepare from peripheral blood.
The present invention also provides a kind of method for preparing effector cell of the present invention, comprises the steps:
(1) provides CEA-rV;
(2) preparation of DC: aseptic condition is gathered peripheric venous blood down, and routine is isolated mononuclearcell, and after pre-the cultivation, it is standby to collect suspension cell; Resuspended attached cell, the conventional cultivation makes DC;
(3) preparation of CIK cell:, make the CIK cell with the conventional cultivation of suspension cell collected in the step (2);
(4) lotus has the DC inductive CIK cell preparation of CEA-rV: add CEA-rV in DC, the conventional cultivation prepares the DC (CEA-rV+DC) that lotus has CEA-rV; Lotus has the DC of CEA-rV and the cultivation of CIK cytomixis to make the CEA-rV+DC+CIK cell.
The effector cell's of above-mentioned prevention and treatment CEA positive tumor preparation method, it is to cultivate by 1: 10 mixed to make the CEA-rV+DC+CIK cell that lotus has the DC of CEA-rV and CIK cell.
The application of DC inductive CIK cell in preparation treatment and prevention CEA positive tumor medicine that the present invention also provides lotus that CEA-rV is arranged.
The CIK cell is the abbreviation of cytokine induced kill cell (Cytokine Induced Killer Cells).The LAK cell is the abbreviation of lymphokine activated killer cell (Limphokine Activated Killer Cells).
Compared with prior art, the present invention has prepared the active effector cell who obviously improves of the specific killing of CEA positive tumor cell, for the specific immunity treatment of clinical CEA positive tumor provides the technological method of a renewal, overcome single with the unfavorable drawback of CEA-rV clinical efficacy.
Solved the rare problem of antigen with CEA-rV as specific antigen.The present invention has solved most of CEA positive tumor patient with CEA-rV as specific antigen load DC can't obtain the antigenic difficulty of autologous tumor, makes such patient increase a prevention and methods of treatment, has increased a hope of defeating tumour.Because of CEA-rV can prepare in a large number in the laboratory, safe in utilization again, so the supremacy clause of applying is arranged.
Adopt patient self peripheral blood cells to prepare DC and CIK, the transmissible disease of bleeding of the umbilicus and the hidden danger of " graft versus host " reaction have been overcome, guarantee the security of clinical use, improved the validity of treatment CEA positive tumor again, more helped the clinical application of this project.
The present invention with CIK cell preparation CEA-rV+DC+CIK effector cell with compared tangible advantage with CD3AK cell preparation CEA-rV+DC+CD3AK:
The CIK cell is that the T cell is induced the effector cell who produces down in IL-2, IL-1, IFN-Y and 4 kinds of factors of CD3 monoclonal antibody, has tangible CD3
+CD56
+Two positive marks.To be the T cell induce and produce in IL-2 and two kinds of factors of CD3 monoclonal antibody the CD3AK cell, do not have tangible CD3
+CD56
+Two positive signs.
The CIK cell is faster than CD3AK and LAK cell proliferation rate, approximately fast 100 times.
The CIK cell is stronger than the lethal effect of CD3AK and LAK cell, and is approximately strong 10 times.
The spectrum of knurl extremely of CIK cell is wider than CD3AK and LAK cell, and stronger to the tumor-killing effect of multidrug resistance.
The CIK cell is compared with the LAK cell with CD3AK, and it is less influenced by immunosuppression.
That is to say that we utilize the CEA-rV+DC+CIK effector cell of CIK cell preparation to compare with CEA-rV+DC+CD3AK effector cell with the CD3AK cell preparation, and significant difference is arranged in itself; More obvious advantage is arranged on function.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.
Embodiment one, CEA-rV+DC+CIK cell preparation.
1, the preparation of the structure of retroviral plasmid PLXSN-CEA and CEA-rV and W-VV (wild-type vaccinia virus): the structure of retroviral plasmid PLXSN-CEA, reference literature [journal of biological chemistry, 1997, the 8th national Biochemistry and Molecular Biology academic conference monograph: 257-259] carry out, a large amount of preparations of CEA-rV and W-VV and titer determination reference literature [Chinese biological chemistry and molecular biology, 1999,15 (1): 54-58] carry out.
2, the preparation of autologous peripheral blood DC and evaluation: aseptic condition is gathered peripheric venous blood 50-100ml down.Routine is isolated mononuclearcell (PBMC), and regulating cell concn with the RPMI-1640 substratum is 1 * 10
6/ ml adds 24 orifice plates, every hole 2ml, 37 ℃, 5%CO
2Cultivate 3h under the condition, collect suspension cell and prepare the CIK cell.Resuspended attached cell prepares DC: with containing GM-CSF (50ng/ml), and IL-4 (10ng/ml), the RPMI-1640 perfect medium of TNF-α (50ng/ml), regulating cell concn is 2 * 10
5/ ml, every hole 1ml is inoculated in 24 orifice plates, the conventional 3d that cultivates, half amount is changed liquid every other day later on.Under light microscopic, observe the metamorphosis in the DC growth course continuously, transmission electron microscope observing DC cellular form, flow cytometer detects the DC cell phenotype.
3. the preparation of autologous peripheral blood CIK cell: with containing IL-2 (1000u/ml), CD3Ab (5ug/ml), IL-1 (100u/ml), the RPMI-1640 perfect medium of IFN-γ (1000u/ml) and people AB serum (5%) or contain IL-2 (1000u/ml), CD3Ab (5ug/ml), IL-1 (100u/ml), suspension PBMC concentration to 5 * 10 that method is collected on the RPMI-1640 serum-free culture keynote of IFN-γ (1000u/ml)
6/ ml.The conventional preparation CIK cell of cultivating; Measure the CD3 of PBMC and CIK cell, CD4 equimolecular phenotype respectively with flow cytometer.
4.CEA-rV+DC+CIK cell preparation: when the DC of last method preparation cultivates 3d, add CEA-rV 10
4PFU/ml, the conventional preparation CEA-rV+DC that cultivates.When the CIK cell cultures 8d of last method preparation, CEA-rV+DC and CIK cell by 1: 10 mixed cultivate the CEA-rV+DC+CIK cell.
The application method of embodiment two, CEA-rV+DC+CIK cell
When the CEA-rV+DC+CIK cell number increases 1 * 10
9-10
10The time, the beginning collecting cell, vein feeds back patient.The using method of CEA-rV+DC+CIK cell suspension is identical with normal blood transfusion, feedback speed be 50-60 drip/minute.The jolting cell suspension was once gently in every 5-10 minute in the feedback process.Feed back the back and continue the normal saline flushing feedback pipe of input 50mL.Whenever 3-5 days once, 6 times one courses of treatment.
Embodiment three, CEA-rV+DC+CD3AK and CEA-rV+DC+CIK compare the specific killing effect of tumour cell
1. experiment material:
Target cell is divided 4 groups:
Erythroleukemia cell strain K562 is provided by Guangzhou Medical College clinical tumor research centre, is the strain of CEA negative cells;
Hepatoma cell strain cell strain BeL-7402 is provided by Guangzhou Medical College clinical tumor research centre, is the strain of CEA negative cells;
Colon cancer cell line LOVO is provided by Zhongshan University, is the strain of CEA positive cell;
Lung cancer cell line A549 is provided by Zhongshan University, is the strain of CEA positive cell.
The effector cell divides 3 groups:
CIK groups of cells, CEA-rV+DC+CD3AK groups of cells and CEA-rV+DC+CIK groups of cells, self-control, concentration all is 10 * 10
9PFU/ml.
2. experimental technique:
Respectively organize the killing activity of effector cell with mtt assay mensuration to 4 kinds of target cells.
With SPP11.0 statistics software data processing.Each effector cell's kill rate one-way analysis of variance method.
3. Data Processing in Experiment:
Experimental data is as shown in table 1, and each is organized data and is the empirical average value 10 times.
The various effector cells of table 1 to the kill rate of target cell (X ± S):
4. result and discussion:
3 kinds of effector cell: CIK, CEA-rV+DC+CD3AK and CEA-rV+DC+CIK all have obvious lethality to 4 kinds of target cells as can be seen from the table.CEA-rV+DC+CD3AK and CIK group relatively increase though CEA-rV+DC+CD3AK organizes than CIK the kill rate of each target cell, and not statistically significant (P＞0.05) illustrates that CEA-rV and DC do not have the effect of obvious raising CD3AK killing activity.
CEA-rV+DC+CIK compares with CEA-rV+DC+CD3AK, lethal effect to negative tumour cell K562 of CEA and BEL-7402 does not have considerable change (P＞0.05), but the specific killing effect of CEA positive tumor cell LOVO and A549 is truly had obvious raising (P＜0.01).Illustrate that CEA-rV+DC+CIK is more more obvious than CEA-rV+DC+-CD3AK to the specific killing effect of CEA positive tumor cell, effect is more superior.
Claims (6)
1. an effector cell who prevents and treat the CEA positive tumor is characterized in that described effector cell is the DC inductive CIK cell that lotus has CEA-rV.
2. effector cell as claimed in claim 1 is characterized in that described CIK cell is on the basis of LAK cell, is prepared from IL-2, CD3 monoclonal antibody, IL-1 and four kinds of cytokine inductions cultivations of IFN-Y.
3. effector cell as claimed in claim 1 is characterized in that described DC and CIK cell extract preparation from peripheral blood.
4. the effector cell's method for preparing prevention as claimed in claim 1 and treatment CEA positive tumor comprises the steps:
(1) provides CEA-rV;
(2) preparation of DC: aseptic condition is gathered peripheric venous blood down, and routine is isolated mononuclearcell, and after pre-the cultivation, it is standby to collect suspension cell; Resuspended attached cell, the conventional cultivation makes DC;
(3) preparation of CIK cell:, make the CIK cell with the conventional cultivation of suspension cell collected in the step (2);
(4) lotus has the DC inductive CIK cell preparation of CEA-rV: add CEA-rV in DC, the conventional DC that cultivates preparation lotus CEA-rV; Lotus has the DC of CEA-rV and the cultivation of CIK cytomixis to make the DC inductive CIK cell that lotus has CEA-rV.
5. the effector cell's of prevention as claimed in claim 4 and treatment CEA positive tumor preparation method, it is to cultivate by 1: 10 mixed to make the DC inductive CIK cell that lotus has CEA-rV that lotus has the DC of CEA-rV and CIK cell.
6. as the application of effector cell in preparation prevention and treatment CEA positive tumor medicine of claim 1 or the described prevention of claim 4 and treatment CEA positive tumor.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100268655A CN101037670A (en) | 2007-02-09 | 2007-02-09 | Effecter cell for preventing and curing CEA positive tumor and preparation method and application thereof |
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|---|---|---|---|
| CNA2007100268655A CN101037670A (en) | 2007-02-09 | 2007-02-09 | Effecter cell for preventing and curing CEA positive tumor and preparation method and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN102512448A (en) * | 2011-12-30 | 2012-06-27 | 江苏省疾病预防控制中心 | Application of DC-CIK (Dendritic Cell-Cytokine-Induced Killer) cells in preparation of medicine used for resisting HIV (Human Immunodeficiency Virus) infection |
| CN105002140A (en) * | 2015-09-01 | 2015-10-28 | 广州赛莱拉干细胞科技股份有限公司 | Culture method for enhancing killing activity and proliferation activity of LAK (fibroblast activation kinase) cells and application |
| CN109535241A (en) * | 2018-12-18 | 2019-03-29 | 北昊干细胞与再生医学研究院有限公司 | DC-CIK co-cultured cell and preparation method thereof, sensitising antigens and application |
-
2007
- 2007-02-09 CN CNA2007100268655A patent/CN101037670A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN102512448A (en) * | 2011-12-30 | 2012-06-27 | 江苏省疾病预防控制中心 | Application of DC-CIK (Dendritic Cell-Cytokine-Induced Killer) cells in preparation of medicine used for resisting HIV (Human Immunodeficiency Virus) infection |
| CN102512448B (en) * | 2011-12-30 | 2013-03-27 | 江苏省疾病预防控制中心 | Application of DC-CIK (Dendritic Cell-Cytokine-Induced Killer) cells in preparation of medicine used for resisting HIV (Human Immunodeficiency Virus) infection |
| CN105002140A (en) * | 2015-09-01 | 2015-10-28 | 广州赛莱拉干细胞科技股份有限公司 | Culture method for enhancing killing activity and proliferation activity of LAK (fibroblast activation kinase) cells and application |
| CN105002140B (en) * | 2015-09-01 | 2018-09-04 | 广州赛莱拉干细胞科技股份有限公司 | culture method for enhancing killing activity and proliferation activity of L AK (L-alanine kinase) cells and application |
| CN109535241A (en) * | 2018-12-18 | 2019-03-29 | 北昊干细胞与再生医学研究院有限公司 | DC-CIK co-cultured cell and preparation method thereof, sensitising antigens and application |
| CN109535241B (en) * | 2018-12-18 | 2021-01-08 | 北昊干细胞与再生医学研究院有限公司 | DC-CIK (dendritic cell-cytokine induced killer) co-culture cell, preparation method thereof, sensitizing antigen and application |
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Owner name: HEILONGJIANG TIANYI PHARMACEUTICAL CO., LTD. |
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Effective date of registration: 20101026 Address after: 510095, No. 78, cross Gang Road, Guangzhou, Guangdong Applicant after: Qi Yanchao Co-applicant after: Heilongjiang Tianyi Pharmaceutical Co., Ltd. Address before: 510095, No. 78, cross Gang Road, Guangzhou, Guangdong Applicant before: Qi Yanchao |
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Open date: 20070919 |