CN104830769B - Butylated hydroxy anisole is to the application in central memory t cell amplification in vitro - Google Patents
Butylated hydroxy anisole is to the application in central memory t cell amplification in vitro Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides the application that butylated hydroxy anisole is expanded in vitro to central memory t cell, the concentration of the butylated hydroxy anisole is 5 ~ 40uM, and optimum concentration is 20uM, and the central memory t cell is CD4+T cell.Application of the butylated hydroxy anisole in vitro in T cell culture is made public for the first time in the present invention, method safety is effectively, with low cost, and good technical support is provided for the extensive development of adoptive immunotherapy.
Description
Technical field
The invention belongs to central memory t cell Amplification Technologies field, more particularly, to butylated hydroxy anisole
To the application in central memory t cell amplification in vitro.
Background technology
Butylated hydroxy anisole(English:Butylated hydroxyanisole, abbreviation BHA), it is a kind of antioxidant,
For two kinds of isomer 2- tertiary butyl-4-hydroxies anisoles and the mixture of 3- tertiary butyl-4-hydroxy anisoles, BHA master
It is in food to want purposes(Including animal foodstuff), packaging for foodstuff, play antioxidant and preservative in cosmetics and oil product
Effect.In addition, it is also used for the anti-oxidant of medicine, such as Accutane, Simvastatin.
Because T cell plays an important role in immune system, increasing research group is attempted with T cell
The mode for the treatment of of adopting carries out oncotherapy.Central memory t cell(TCM)Ability with self-renewing, and response
Time is short, long action time, to the Small side effects of human body.This year, multiple seminar's reports find the T with memory function
After cell is fed back in tumor patient body, therapeutic effect is substantially and the continued treatment time is long, it is possible to reduce the pain of patient with
And the excessive bio-safety risk of reduction cell injuring model number of times.But relative populations are less in human body, and due to external
The T cell of culture can make T cell lose vigor for a long time, and most cell differentiations are the effector cell of terminal differentiation so that
Feed back the internal T cell time-to-live too short, it is impossible to produce the function of lasting killing tumor cell, therefore how to obtain in vitro
Obtain the new study hotspot that substantial amounts of memory t cell is cellular immunotherapy.
At present, external many research groups are applied to the external of T cell by building artificial antigen presenting cells
Culture, improves the time-to-live of T cell, for adoptive immunotherapy, but to obtain the T cell of sufficient amount, and technical requirements are complicated,
The success rate of culture is relatively low.When carrying out T cell treatment, main policies are to first pass through clonal expansion, and obtain sufficient amount has
The T cell of killing ability, can be co-cultured with the presenting cells such as DC, can also be directly with associated tumor antigen stimulation cell clone
Amplification, is obtained with the specific T cell of particular tumor antigens.The T cell that such a method culture is obtained turns base without any
Because of modification, security is higher.But the cell of this tumour high response needs to be expanded to the 10 of clinical needs9-1011Therapeutic dose
Quantity number, process duration is long, and the time, which is also one, for tumor patient needs the factor of more considerations.And
And the time of in vitro culture is long, cell is easily ageing, and vigor declines, mostly the cell of terminal differentiation, there is preferable effect in vitro
Really, but in vivo the time-to-live is shorter, and therapeutic effect is impacted.
The content of the invention
The technical problem to be solved in the present invention is to overcome the shortcomings of existing central memory t cell Amplification Technologies, is carried
For butylated hydroxy anisole to the application in central memory t cell amplification in vitro.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The invention provides the application that butylated hydroxy anisole is expanded in vitro to central memory t cell.
Preferably, the concentration of the butylated hydroxy anisole is 5 ~ 40uM.
It is highly preferred that the concentration of the butylated hydroxy anisole is 20uM.
Preferably, the central memory t cell is CD4+T cell.
Preferably, the extracting method of the central memory t cell is first by periphery component blood with containing 2% BSA and 0.5%
EDTA PBS presses 1:4 are well mixed;Then blood after dilution be slowly added to the top of lymphocyte separation medium, the two
Ratio is 1:1;Centrifuge the min of 300 × g 30;It is careful to draw monocyte, insert in another centrifuge tube, add containing for 5 times of volumes
0.5% BSA and 2% EDTA PBS dilutions, after being well mixed, the min of 300 × g 15;Repeat previous step once;It is incubated CD4
+ T cell the moon selects primary antibody, 15 min;Diluted with the PBS containing 0.5% BSA and 2% EDTA of 10 times of volumes, after being well mixed,
300×g 12 min;It is incubated the secondary antibody with magnetic bead, 30 min;Cross post and carry out cell sorting, obtain CD4+ T cells.
Preferably, the cultural method of the central memory t cell is in 5% CO2, will under the conditions of saturated humidity and 37 DEG C
5×105The CD4 in individual/hole+T cell is placed in RPMI1640 of 1 mL containing 10% hyclone and cultivated.
Compared with prior art, the present invention has advantages below and beneficial effect:
Present invention firstly discloses the application in butylated hydroxy anisole in vitro T cell culture, method safety is effective, into
This is cheap, and good technical support is provided for the extensive development of adoptive immunotherapy.
Brief description of the drawings
Fig. 1 is to CD4 when BHA concentration is 20uM+CD4 in T cell+The detection of TCM proportions.
Fig. 2 is the BHA of various concentrations to central Memorability CD4+The ratio expanding effect of T cell.
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment, but embodiment does not do any type of to the present invention
Limit.Unless stated otherwise, the reagent of the invention used, method and apparatus is the art conventional reagent, methods and apparatus.
Unless stated otherwise, agents useful for same and material of the present invention are purchased in market.
Embodiment 1
1st, test material prepares
The periphery component blood of people is provided by Guangzhou Blood Center, and the blood sample of 24 parts of normal persons has been randomly selected in experiment
This.
Main agents:Hyclone is purchased from GIBCO companies;RPMI1640 culture mediums are purchased from INVERTROGEN companies;
BHA is purchased from INVERTROGEN companies;Flow cytomery antibody anti-CD45RA-Texas Red, anti-CCR7-
AF700 anti-CD62L-PE-cy7 be purchased from BD companies;Lymphocyte separation medium is limited purchased from the positive biological products of Tianjin Hao
Responsible company;CD4+T cell Solid phase magnetic bead is purchased from BD companies.
Main laboratory apparatus:Table model high speed centrifuge(Eppendorf Centrifuge 5810R), flow cytometer
(Beckman Coulter companies)、CO2Cell culture incubator(Thermo SCIENTIFIC), Biohazard Safety Equipment(Thermo
SCIENTIFIC), inverted biologic microscope(Leica), magnetic bead sorting magnetic frame(BD Pharmigen).
Cell culture
2.1 cell extractions are separated
By periphery component blood and PBS(Containing 2% BSA, 0.5% EDTA)By 1:4 are well mixed;Then after diluting
Blood be slowly added to the top of lymphocyte separation medium, the ratio of the two is 1:1;Centrifuge the min of 300 × g 30;It is careful to draw
Monocyte, is inserted in another centrifuge tube, adds the PBS of 5 times of volumes(Containing 0.5% BSA, 2% EDTA)Dilution, is well mixed
Afterwards, the min of 300 × g 15;Repeat previous step once;It is incubated CD4+ T cell the moon and selects primary antibody, 15 min;The PBS of 10 times of volumes
(Containing 0.5% BSA, 2% EDTA)Dilution, after being well mixed, the min of 300 × g 12;It is incubated the secondary antibody with magnetic bead, 30 min;
Cross post and carry out cell sorting, obtain CD4+ T cells, add the RPMI1640 containing 10% hyclone and cell is resuspended, count cell
Cell is adjusted afterwards to required concentration.
2.2 condition of culture
In 5% CO2, saturated humidity and 37oUnder C, by 5 × 105The CD4 in individual/hole+T cell is placed in 1 mL containing 10% tire ox blood
Cultivated in clear RPMI1640.
Plating cells and agent-feeding treatment
3.1 bed board
By the CD4 sorted out+T cell is layered in 12 orifice plates, according to experimental design, be 5 per cell number expected from hole ×
105。
3.2 agent-feeding treatment
To the CD4 after bed board+T cell carries out 5 kinds of different processing, and every kind of processing sets three multiple holes as parallel right
According to, and at the 4th day of cell culture, the 7th day, the 10th day, apply within the 13rd day corresponding stimulate.
Cell subsets analysis and the detection of cell quantity
Cell culture, after 15 days, different donors are drawn respectively(n=24)Culture hole in cell, use cell counter
Counted;5 × 10 are drawn in the corresponding culture hole of slave phase5Individual cell, the min of 300 × g 15, cell is washed with PBS twice,
It is incubated detection CD4+TCM streaming antibody(CD45RA、CCR7、CD62L)Half an hour;Wash streaming antibody off, add PBS dilute
Respective concentration is released, with flow cytomery.Control group and the CD4 that experimental group is same donor+ T cells, Mei Geshi
Between put and take the cell of different donors to be detected respectively.
Statistics software and statistical method
Statistical analysis is carried out using SPSS13.0 analysis softwares.All results of measurement data use mean ± standard deviation table
Show.Experimental group is compared with control group to be examined using t, P<0.01 is to have significant difference.
Experimental result
Flow cytometer detections of 6.1 BHA to CD4+TCM proportions in CD4+T cells
In order to determine the original ratio for adding TCM in the CD4+ T cells that BHA is stimulated, by CD4+T cells are with 5 × 106
Individual/hole is laid in 24 orifice plates, adds anti-CD3 antibody, anti-CD28 antibody, IL-7 and IL-15.Control group and experimental group
Streaming antibody labeled cells are used in 15 d, are then detected sample by flow cytometer.Negative from CD45RA
In cell, it is the CD4 to be observed to choose the double positive cells of CD62L and CCR7+TCM.According to above by flow cytometer
Sort CD4+TCM method, to CD4+TCM is in CD4+Ratio in T cell detected, as shown in Figure 1.
The BHA of 6.2 various concentrations is for CD4+ The detection of T cell expanding effect
The time point that selection BHA handles the 15th day is detected, is added without the CD4 of BHA control group+TCM cells are accounted for
Total CD4+The ratio of T cell is 16.06%;BHA concentration is that 5uM ratios are 22.59%;The ratio that BHA concentration is 10uM is
25.91%;The ratio that BHA concentration is 20uM is 29.09%;The ratio that BHA concentration is 40uM is 28.33%.Test result indicates that,
The BHA of various concentrations is for CD4+There is dose-dependent trend in the amplification effect of T cell, such as in the case of less than 20uM
Shown in Fig. 2.
Interpretation of result
We promote CD4 using small-molecule drug BHA first+The amplification of TCM cells in vitro.Experimental group is compared with control group
Compared to CD4+The ratio of TCM cells is in CD4+Have in T cell and significantly raise.
Among the immune treatment of adopting of T cell, if substantial amounts of adoptive transfer T cell can produce radical response, in experiment
During, we be have found in the case where T cell sum is constant, and substantial amounts of T cells are converted into CD4+TCM cells
Method.We, which also further have found BHA, in experiment can promote CD4+The optimum concentration that TCM is expanded in vitro:20uM.I
Find, using relatively low-dose BHA handle cell after, CD4+ TCM cells are in CD4+Lift exists aobvious in ratio in T cell
The dose-dependant trend of work;And lift effect when BHA concentration reaches 20uM, in ratio and substantially tend towards stability(Fig. 2), this may
It is due to that drug concentration is excessive so that the growing environment of cell there occurs larger change and have impact on the normal growth of cell.
In this experiment, counted after choose BHA processing cells 15 days and detection is due to the CD4 in peripheral blood+TCM's rises
Beginning quantity is considerably less, CD4+T cell needs longer time could be under anti-CD3 and anti-CD28 Co stituations gradually
Form more CD4+TCM.Experimental result also indicates that selection culture long period rear carries out detection and can obtain more preferable effect
(Fig. 2).
BHA is a kind of conventional food and medicine antioxidant.BHA T cell cultures in vitro are elaborated in the present invention first
In brand-new application, provide good technical support for the extensive development of adoptive immunotherapy, its molecular mechanism needs subsequently
Further research.
Claims (6)
1. the application that butylated hydroxy anisole is expanded in vitro to central memory t cell.
2. application according to claim 1, it is characterised in that the concentration of the butylated hydroxy anisole is 5 ~ 40 μM.
3. application according to claim 2, it is characterised in that the concentration of the butylated hydroxy anisole is 20 μM.
4. the application according to claims 1 to 3 any one, it is characterised in that the central memory t cell is CD4+
T cell.
5. application according to claim 1, it is characterised in that the extracting method of the central memory t cell is first will
Periphery component blood presses 1 with the PBS containing 2% BSA and 0.5% EDTA:4 are well mixed;Then the blood after dilution is slowly added to
The top of lymphocyte separation medium, the ratio of the two is 1:1;Centrifuge the min of 300 × g 30;It is careful to draw monocyte, insert
In another centrifuge tube, the dilutions of the PBS containing 0.5% BSA and 2% EDTA of 5 times of volumes are added, after being well mixed, 300 × g are centrifuged
15 min;Repeat previous step once;It is incubated CD4+T cell the moon selects primary antibody, 15 min;Contain 0.5% BSA with 10 times of volumes
Diluted with 2% EDTA PBS, after being well mixed, centrifuge the min of 300 × g 12;It is incubated the secondary antibody with magnetic bead, 30 min;Cross
Post carries out cell sorting, obtains CD4+T cell.
6. the application that butylated hydroxy anisole according to claim 1 is expanded in vitro to central memory t cell, its
It is characterised by, the cultural method of the central memory t cell is in 5% CO2, under the conditions of saturated humidity and 37 DEG C by 5 × 105
The CD4 in individual/hole+T cell is placed in RPMI1640 of 1 mL containing 10% hyclone and cultivated.
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Citations (1)
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CN103379921A (en) * | 2010-12-17 | 2013-10-30 | 人类起源公司 | Treatment of spinal cord injury and traumatic brain injury using amnion derived adherent cells |
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US20040203142A1 (en) * | 2003-04-14 | 2004-10-14 | Reliance Life Sciences Pvt. Ltd. | Growth of neural precursor cells using umbilical cord blood serum and a process for the preparation thereof for therapeutic purposes |
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CN103379921A (en) * | 2010-12-17 | 2013-10-30 | 人类起源公司 | Treatment of spinal cord injury and traumatic brain injury using amnion derived adherent cells |
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Cheryl E.Rockwell等.Th2 Skewing by Activation of Nrf2 in CD4+ T Cells.《The Journal of Immunology》.2012,第188卷第1630-1637页. * |
Fang-Ming Hung等.Butylated hydroxyanisole affects immunomodulation and promotes macrophage phagocytosis in normal BALB/c mice.《Molecular Medicine Reports》.2012,第5卷第683-687页. * |
John Whysner等.Butylated Hydroxyanisole Mechanistic Data and Risk Assessment:Conditional Species-Specific Cytotoxicity,Enhanced Cell Proliferation,and Tumor Promotion.《Pharmacology & Therapeutics》.1996,第71卷(第1-2期),第137-151页. * |
P.A.E.L.Schiderman等.Induction of oxidative DNA damage and enhancement of cell proliferation in human lymphocytes in vitro by butylated hydroxyanisole.《 Carcinogenesis 》.1995,第16卷(第3期),第507-512页. * |
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