CN102978159A - Method for enhancing reproductive capacity of CIK (Cytokine Induced Killer) cell and improving tumor cell killing capacity - Google Patents
Method for enhancing reproductive capacity of CIK (Cytokine Induced Killer) cell and improving tumor cell killing capacity Download PDFInfo
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Abstract
The invention provides a method for enhancing the reproductive capacity of CIK (Cytokine Induced Killer) cells and improving tumor cell killing capacity. The method comprises the following steps of: sterilely collecting and separating the peripheral blood mononuclear cells of a healthy person; carrying out resuspension on the obtained peripheral blood mononuclear cells by using a Q007 culture medium; adding 24-26 ug/ml of PHA-L type phytohemagglutinin; and culturing for at least 15 days in the Q007 culture medium. The method provided by the invention has the beneficial effects that the reproductive capacity of the CIK cells and the cytotoxic effect are enhanced by adding 24-26 ug/ml of the PHA-L type phytohemagglutinin in a CIK cell culturing process, thereby enhancing the kill rate and the cure effect of the tumor cells.
Description
Technical field
The present invention relates to the cultural method of cytokine induced kill cell (CIK cell).
Background technology
Tumour is a large disease of harm humans life and health, has been listed as the second of crowd's disease death cause of disease at present.According to World Health Organization's statistics, there are 9,000,000 new cancers in the whole world every year, and 5,000,000 people are dead.The methods for the treatment of of tumour mainly is operation, radiation and chemotherapy at present.Wherein, surgical operation therapy is only applicable to infantile tumour, and toxic and side effects of chemoradiotherapy is larger.Therefore, developing safely and effectively, anti-tumor biological therapy of new generation will have extremely important Social benefit and economic benefit.
U.S. Stanford university has at first reported the killer cell that a class is induced by cytokine profiles, be cytokine induced kill cell (cy to kine-induced killer cells, CIK), be at present known to the strongest a kind of immune effector cell of killing activity.The effect of CIK immunocyte in bone marrow purging and adoptive immunotherapy is confirmed in experimentation on animals, and the clinical experiment of CIK immunocyte has also begun to carry out.（Ortaldo?J?R,Winkler-Pickett?R?T,Yagita?H,et?al.Comparat?ive?studies?of?CD3+and?CD3+CD56+cells:Exam-ination?of?morphology，functions,T?cellreceptor?rearrangement,and?por?e-forming?protein?expression.Cell?Immunol,1991,136:486-495.）；（S?chmidt-WolfI?G，?Negrin?R?S,Kiem?H?P，et?al.Us?e?ofa?SCIDm?ou?se/human?l?ymphoma?model?t?o?evalu?at?e?cyt?okine-in?duced?killer?cells?with?pot?ent?ant?itumor?cell?act?ivit?y[J].J?E?xp?Med,1991,174(1):139-149.）
Yet just quantity and the activity of the resulting CIK cell of method of cultivation CIK immunocyte are lower at present, and therefore low to the kill rate of tumour cell, result for the treatment of is relatively poor.
Summary of the invention
For this reason, technical problem to be solved by this invention is: the method that a kind of CIK of enhancing ability of cell proliferation and cytotoxicity are provided.
So, the invention provides the method that strengthens CIK ability of cell proliferation and cytotoxicity, comprise aseptic collection and separating health human peripheral blood single nucleus cell, with the mononuclearcell that obtains Q007 substratum re-suspended cell, add the PHA-L type phytohaemagglutinin of 24-26ug/ml, and in substratum, cultivated at least 15 days.
Preferably, the consumption of PHA-L type phytohaemagglutinin is 25ug/ml.
Preferably, with adjusting cell density at (2-3) * 10 behind the Q007 substratum re-suspended cell
6Individual/ml.
Particularly, described substratum is the substratum that contains recombinanthumanifn-γ, recombinant human IL-1 and recombinant human il-2, its cultural method is, the recombinanthumanifn-γ who added 250IU/ml at the 0th day, added the PHA-L type phytohaemagglutinin of 25ug/ml and the recombinant human IL-1 of 2.5ng/ml at the 1st day, the recombinant human il-2 who added 1000IU/ml at the 2nd day cultivates.
Preferably, the fresh recombinant human il-2 who in culturing process, added a 1000IU/ml every 2-3 days.
Preferably, the mononuclearcell that obtains is used Q007 substratum re-suspended cell after washing.
The invention has the beneficial effects as follows:
1, by in the culturing process of CIK cell, adding the PHA-L type phytohaemagglutinin of 24-26ug/ml, the PHA-L phytohaemagglutinin has higher mitogenesis effect with respect to the phytohaemagglutinin of other types, stimulates the T cell proliferation and differentiation to produce a large amount of effector T cells and cytotoxic T cell; Effector T cell secretion produces a large amount of cytokines (such as Interferon, rabbit etc.) killing tumor cell; But cytotoxic T cell direct killing tumour cell; The PHA-L phytohaemagglutinin can stimulate simultaneously the B cell transformation be plasmablast then proliferation and differentiation be plasmocyte, plasmocyte produces in a large amount of non-specific antibodies and tumour antigen, thereby multiplication capacity and the cytotoxicity of CIK cell have greatly been improved, through experimental verification, the CIK cell proliferation rate that the present invention cultivates reaches 140-150%.
2, adopting Q007 substratum re-suspended cell, its beneficial effect early stage is the differentiation that utilizes the CIK immunocyte more to be arranged and increase cytotoxicity
Description of drawings
Fig. 1 is the comparison diagram of CIK hyperplasia rate among the embodiment 1;
Fig. 2 be among the embodiment 1 the CIK cell to the kill rate comparison diagram of vitro culture cancer cell (hela, hepg2, ovar3, K562);
Embodiment
Below, describe the present invention in conjunction with specific embodiments.
At first, reagent and the consumptive material that need to use in specific implementation process of the present invention is as follows:
Table 1 main agents
| Use reagent and factor names | Specification | Producer | Working concentration |
| The Q007 substratum | The 4L/ bucket | PAA | |
| The human peripheral blood lymphocytes parting liquid | The 200ml/ bottle | Tianjin Hao ocean | |
| CD3McAb | 0.5mg/ | anogen | 50ng/ml |
| IL2 | 1,000,000 IU/ prop up | The two aigret medicine companies in Beijing | 1000IU/ml |
| IL1α | 10ug/ props up | PEPROTECH?ING | 2.5ng/ml |
| IFNγ | 1,000,000 IU/ prop up | The Shanghai clone is biological | 250IU/ml |
| CD28McAb | The 500ug/ bottle | Tong Lihai source, Beijing bio tech ltd | 2ug/ml |
| PHA-L | The 5mg/ bottle | SIGMA | 25ug/ml |
| CCK8 | The 5ml/ bottle | The green skies | |
| The trypan blue dye liquor | The 50ml/ bottle | 0.4% |
Main consumptive material comprises: blood taking bag, 50ml centrifuge tube, 10ml transfer pipet, 5ml transfer pipet, 3ml suction pipe, 1.5mlEP pipe, 96 orifice plates, 12 orifice plates, pipettor, rifle head, counter etc.
Embodiment 1
The aseptic collection healthy human peripheral blood is sent cell preparation experiment chamber back to and is tested in clean bench; Separate mononuclearcell with the lymph parting liquid, wash 2 times, with Q007 substratum re-suspended cell, adjust cell density at 3*10
6Then/ml carries out cultivation and the experiment contrast of CIK cell according to the following steps with method.
Experiment is divided into 1 blank group, and phytohaemagglutinin group and positive control CD3McAb group design are as follows:
Experiment is carried out in 12 orifice plates, and every hole inoculum size is 1ml
The blank group:
The 0th day: IFN-γ (250IU/ml)
The 1st day: IL-1(2.5ng/ml)
The 2nd day: IL-2(1000IU/ml)
Residue sky: IL-2(1000IU/ml)
Positive controls:
The 0th day: IFN-γ (250IU/ml)
The 1st day: CD3McAb(50ng/ml) 20ul, IL-1(2.5ng/ml)
The 2nd day: IL-2(1000IU/ml)
Residue sky: IL-2(1000IU/ml)
Phytohaemagglutinin group (PHA-L)
The 0th day: IFN-γ (250IU/ml)
The 1st day: PHA-L(25ug/ml), IL-1(2.5ng/ml)
The 2nd day: IL-2(1000IU/ml)
Residue sky: IL-2(1000IU/ml)
Remarks: the working concentration of every kind of interior each factor of numeric representation of factor back bracket.
Once add fresh culture (containing IL-2(1000IU/ml) every processing in 2-3 days in the culturing process), cultivate by density, add up simultaneously cell density, quantity and motility rate, at the 0th day, carried out killing experiments and verify its cytotoxicity in 15 days.Use cell counter to measure the density of cell suspension before each liquid feeding, cultivate according to density, and the actual volume behind the each liquid feeding of calculating record; With the motility rate of blood counting chamber trypan blue living cells count survey cell, through Trypan Blue, under inverted microscope, count not staining cell of Lan Ranji, namely dead cell is dyed to blueness, and viable cell is refused to dye.
The method of calculation of Cell viability and cell proliferation situation are:
Cell viability (%)=viable cell sum/(viable cell sum+dead cell sum) * 100%
Propagation multiple=[(cell suspension actual volume before the cell density x liquid feeding)-(suspension volume during inoculum density x inoculation)]/(suspension volume during inoculum density x inoculation)
Detect the effector cell to the situation of killing and wounding of target cell with CCK-8:
As target cell, to cultivate 7,11,15 days CIK cell action effect cell, target cell concentration is adjusted to 1 * 10 with culture of tumor cell respectively
4/ ml, effector cell's concentration is adjusted to 50 * 10
4/ ml concentration is inoculated in 96 well culture plates (with front uviolizing 2h with interior sterilization) by the cell of effect target after will cultivating than 50:1 and target cell, and target cell shifts to an earlier date 12 hours and inoculates, and makes it finish adherent process.Experimental port adds the effector cell, each 100ul of target cell, the target cell control wells adds target cell, each 100ul of nutrient solution, effector cell's control wells adds the effector cell, each 100ul of nutrient solution, each sample is all established 3 parallel holes, and establish blank hole (nutrient solution), put 37 ℃, 5%CO2, hatch in the cell culture incubator under the saturated humidity, behind the 48h, every hole adds 20ul CCK-8 solution (volume be nutrient solution 10%), enter in the incubator of same culture conditions, continued to hatch 4 hours, stop cultivating, take out culture plate, visual inspection has or not the albumen flocks, observes dyestuff dissolving crystallized situation under inverted microscope, if exist without viable cell and flocks, and dyestuff fully dissolves, and picking up survey instrument selection wavelength at enzyme linked immunological is that 450nm measures each hole light light absorption value.
The active method of calculation of effector cell:
Obtain the mean value of 3 parallel holes, calculate as follows the motility rate of CIK cell, represent with kill rate, cell killing activity %=[1-(experimental group OD value-effector cell organizes the OD value)/target cell group OD value] * l00%.
The comparison diagram of the CIK hyperplasia rate that finally obtains as shown in Figure 1, the CIK cell is to the kill rate comparison diagram of vitro culture cancer cell (hela, hepg2, ovar3, K562) as shown in Figure 2.
As shown in Figure 1, the CIK hyperplasia rate of phytohaemagglutinin group is 154%; The cancer cell kill rate of phytohaemagglutinin group is 79%.
Embodiment 2
The aseptic collection healthy human peripheral blood is sent cell preparation experiment chamber back to and is tested in clean bench; Separate mononuclearcell with the lymph parting liquid, with Q007 substratum re-suspended cell, adjust cell density at 2*106/ml, then carry out according to the following steps cultivation and the experiment contrast of CIK cell with method.
Experiment is divided into 1 blank group, and phytohaemagglutinin group and positive control CD3McAb group design are as follows:
Experiment is carried out in 12 orifice plates, and every hole inoculum size is 1ml
The blank group:
The 0th day: IFN-γ (250IU/ml)
The 1st day: IL-1(2.5ng/ml)
The 2nd day: IL-2(1000IU/ml)
Residue sky: IL-2(1000IU/ml)
Positive controls:
The 0th day: IFN-γ (250IU/ml)
The 1st day: CD3McAb(50ng/ml) 20ul, IL-1(2.5ng/ml)
The 2nd day: IL-2(1000IU/ml)
Residue sky: IL-2(1000IU/ml)
Phytohaemagglutinin group (PHA-L)
The 0th day: IFN-γ (250IU/ml)
The 1st day: PHA-L(24ug/ml), IL-1(2.5ng/ml)
The 2nd day: IL-2(1000IU/ml)
Residue sky: IL-2(1000IU/ml)
Remarks: the working concentration of every kind of interior each factor of numeric representation of factor back bracket.
The method according to embodiment 1 of cultivating after 15 days detects and calculates Cell viability and cell proliferation situation, and the CIK hyperplasia rate of PHA-L type phytohaemagglutinin group is 145%; The cancer cell kill rate of phytohaemagglutinin group can be near 75%.
Embodiment 3
The aseptic collection healthy human peripheral blood is sent cell preparation experiment chamber back to and is tested in clean bench; Separate mononuclearcell with the lymph parting liquid, wash 2 times, with Q007 substratum re-suspended cell, adjust cell density at 3*10
6Then/ml carries out cultivation and the experiment contrast of CIK cell according to the following steps with method.
Experiment is divided into 1 blank group, and phytohaemagglutinin group and positive control CD3McAb group design are as follows:
Experiment is carried out in 12 orifice plates, and every hole inoculum size is 1ml
The blank group:
The 0th day: IFN-γ (250IU/ml)
The 1st day: IL-1(2.5ng/ml)
The 2nd day: IL-2(1000IU/ml)
Residue sky: IL-2(1000IU/ml)
Positive controls:
The 0th day: IFN-γ (250IU/ml)
The 1st day: CD3McAb(50ng/ml) 20ul, IL-1(2.5ng/ml)
The 2nd day: IL-2(1000IU/ml)
Residue sky: IL-2(1000IU/ml)
Phytohaemagglutinin group (PHA-L)
The 0th day: IFN-γ (250IU/ml)
The 1st day: PHA-L(26ug/ml), IL-1(2.5ng/ml)
The 2nd day: IL-2(1000IU/ml)
Residue sky: IL-2(1000IU/ml)
Remarks: the working concentration of every kind of interior each factor of numeric representation of factor back bracket.
The method according to embodiment 1 of cultivating after 15 days detects and calculates Cell viability and cell proliferation situation, and the CIK hyperplasia rate of PHA-L type phytohaemagglutinin group is 148%; The cancer cell kill rate of phytohaemagglutinin group can be near 76%.
The above only is preferred embodiment of the present invention, and is in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1. strengthen the method for CIK ability of cell proliferation and cytotoxicity, comprise aseptic collection and separating health human peripheral blood single nucleus cell, it is characterized in that: with the mononuclearcell that obtains Q007 substratum re-suspended cell, add the PHA-L type phytohaemagglutinin of 24-26ug/ml, and in substratum, cultivated at least 15 days.
2. the method for enhancing CIK ability of cell proliferation according to claim 1 and cytotoxicity is characterized in that the consumption of PHA-L type phytohaemagglutinin is 25ug/ml.
3. the method for enhancing CIK ability of cell proliferation according to claim 1 and cytotoxicity is characterized in that, with adjusting cell density at (2-3) * 10 behind the Q007 substratum re-suspended cell
6Individual/ml.
4. the method for enhancing according to claim 2 CIK ability of cell proliferation and cytotoxicity, it is characterized in that, described substratum is the substratum that contains recombinanthumanifn-γ, recombinant human IL-1 and recombinant human il-2, its cultural method is, the recombinanthumanifn-γ who added 250IU/ml at the 0th day, added the PHA-L type phytohaemagglutinin of 25ug/ml and the recombinant human IL-1 of 2.5ng/ml at the 1st day, the recombinant human il-2 who added 1000IU/ml at the 2nd day cultivates.
5. the method for enhancing CIK ability of cell proliferation according to claim 4 and cytotoxicity is characterized in that, added the fresh recombinant human il-2 of a 1000IU/ml in culturing process every 2-3 days.
6. the method for enhancing CIK ability of cell proliferation according to claim 1 and cytotoxicity is characterized in that, the mononuclearcell that obtains is used Q007 substratum re-suspended cell after washing.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103385890A (en) * | 2013-07-17 | 2013-11-13 | 崔澂 | A preparation method for an anti-aging biological cell preparation |
| CN103740643A (en) * | 2014-01-21 | 2014-04-23 | 山东省齐鲁干细胞工程有限公司 | In-vitro induction culture method for MHC (Myosin Heavy Chain) restrictive cytotoxic T cells |
| CN104673751A (en) * | 2015-03-24 | 2015-06-03 | 刘慧玉 | Efficient CIK cell culturing method |
| CN105713874A (en) * | 2016-01-29 | 2016-06-29 | 深圳市中美康士生物科技有限公司 | Anti-tumor associated antigen WT1 specificity CTL and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101519646A (en) * | 2009-02-06 | 2009-09-02 | 上海德嘉生物科技有限公司 | CIK cell, as well as preparation method and cell preparation thereof |
| CN102321577A (en) * | 2011-01-26 | 2012-01-18 | 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 |
-
2012
- 2012-11-22 CN CN2012104770478A patent/CN102978159A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101519646A (en) * | 2009-02-06 | 2009-09-02 | 上海德嘉生物科技有限公司 | CIK cell, as well as preparation method and cell preparation thereof |
| CN102321577A (en) * | 2011-01-26 | 2012-01-18 | 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 |
Non-Patent Citations (1)
| Title |
|---|
| 秦福丽等: "PHA对CIK细胞增殖和细胞毒作用影响的研究", 《中国实验血液学杂志》, vol. 13, no. 2, 31 December 2005 (2005-12-31), pages 118 - 120 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103385890A (en) * | 2013-07-17 | 2013-11-13 | 崔澂 | A preparation method for an anti-aging biological cell preparation |
| CN103385890B (en) * | 2013-07-17 | 2015-09-23 | 崔澂 | A kind of preparation method of anti-ageing biological cell preparation |
| CN103740643A (en) * | 2014-01-21 | 2014-04-23 | 山东省齐鲁干细胞工程有限公司 | In-vitro induction culture method for MHC (Myosin Heavy Chain) restrictive cytotoxic T cells |
| CN104673751A (en) * | 2015-03-24 | 2015-06-03 | 刘慧玉 | Efficient CIK cell culturing method |
| CN104673751B (en) * | 2015-03-24 | 2018-04-10 | 刘慧玉 | A kind of efficiently CIK cell cultural method |
| CN105713874A (en) * | 2016-01-29 | 2016-06-29 | 深圳市中美康士生物科技有限公司 | Anti-tumor associated antigen WT1 specificity CTL and preparation method thereof |
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