CN104830768B - Alprenolol is to the application in central memory t cell amplification in vitro - Google Patents
Alprenolol is to the application in central memory t cell amplification in vitro Download PDFInfo
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- 238000000338 in vitro Methods 0.000 title claims abstract description 15
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Abstract
The invention provides the application that alprenolol expands in vitro to central memory t cell, the concentration of the alprenolol is 5 ~ 20uM, and optimum concentration 10uM, the central memory t cell is CD4+T cell.Application of the alprenolol in vitro in T cell culture is made public for the first time in the present invention, method safety is effective, and cost is cheap, 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 alprenolol to center
Application in memory t cell amplification in vitro.
Background technology
Alprenolol（English：Alprenolol）, there is the work that adrenaline beta receptor position competitively suppresses catecholamine
With.The convergent force of heart is declined with contraction speed by weakening or preventing beta receptor excited, pass through the conduction speed of conducting system
Degree slows down, make heart to motion or stress habituation.Therefore, for anginal treatment, myocardium keto consumption, increase fortune are lowered
Dynamic tolerance.Adrenergic due to blocking heartpacer current potential is excited therefore for treating arrhythmia cordis.Alprenolol passes through
Maincenter, adrenergic neuron blockade, anti-renin activity and cardiac output attenuating etc. reduce blood pressure, suitable for treating high blood
Pressure.For treating pheochromocytoma and hyperthyroidism, the activity of β 1 and beta 2 receptor is set to be in holddown.
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.In this year, multiple seminar's reports, find the T with memory function
After cell is fed back in tumor patient body, therapeutic effect is obvious and the continued treatment time is grown, it is possible to reduce the pain of patient with
And reduce the excessive bio-safety risk of cell injuring model number.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
It is too short to feed back the internal T cell time-to-live, can not 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, it can be co-cultured with presenting cells such as DC, directly can also stimulate cell clone with associated tumor antigen
Amplification, is obtained with the specific T cell of particular tumor antigens.The T cell that such a method culture obtains turns base without any
Because modification, security are 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 is also a factor for needing more to consider for tumor patient.And
And the time length of in vitro culture, cell is easily ageing, and vigor declines, mostly the cell of terminal differentiation, there is preferable effect in vitro
Fruit, but the time-to-live is shorter in vivo, 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 alprenolol 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 alprenolol expands in vitro to central memory t cell.
Preferably, the concentration of the alprenolol is 5 ~ 20uM.
It is highly preferred that the concentration of the alprenolol is 10uM.
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 is well mixed；Then blood after dilution is 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, 5 times of volumes of addition contain
0.5% BSA and 2% EDTA PBS dilute, 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 cell.
Preferably, the cultural method of the central memory t cell is in 5% CO2, saturated humidity and 37oBy 5 under the conditions of C
×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 alprenolol in vitro T cell culture, method safety is effective, and cost is low
It is honest and clean, provide good technical support for the extensive development of adoptive immunotherapy.
Brief description of the drawings
Fig. 1 is to CD4 when alprenolol concentration is 10uM+CD4 in T cell+The detection of TCM proportions.
Fig. 2 is the alprenolol of various concentrations to central Memorability CD4+The ratio expanding effect of T cell.
Embodiment
Further illustrate the present invention below in conjunction with specific embodiment, but embodiment the present invention is not done it is any type of
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；
Alprenolol 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 purchased from Tianjin Hao sun biology systems
Product Co., Ltd；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 separate
By periphery component blood and PBS（Containing 2% BSA, 0.5% EDTA）By 1:4 is 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, insert in another centrifuge tube, add the PBS of 5 times of volumes（Containing 0.5% BSA, 2% EDTA）Dilution, it 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 and contains 10% tire ox blood
Cultivated in clear RPMI1640.
Plating cells and agent-feeding treatment
3.1 bed board
The CD4 that will be sorted out+T cell is layered in 12 orifice plates, and according to experimental design, cell number expected from per hole is 5 ×
105。
3.2 agent-feeding treatment
To the CD4 after bed board+T cell carries out 4 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 is analyzed and the detection of cell quantity
After cell culture 15 days, different donors are drawn respectively（n=24）Culture hole in cell, entered with cell counter
Row counts；5 × 10 are drawn in culture hole corresponding to slave phase5Individual cell, the min of 300 × g 15, wash cell twice with PBS, incubate
Educate detection CD4+TCM streaming antibody（CD45RA、CCR7、CD62L）Half an hour；Wash streaming antibody off, add PBS dilutions
To respective concentration, with flow cytomery.Control group and the CD4 that experimental group is same donor+ T cells, each time
Point takes 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 examined compared with control group using t, P<0.01 is to have significant difference.
Experimental result
Flow cytometer detection of 6.1 alprenolols to CD4+TCM proportions in CD4+T cells
In order to determine to add the original ratio of TCM in the CD4+ T cells of alprenolol stimulation, by CD4+T cells are with 5
×106Individual/hole is laid in 24 orifice plates, adds anti-CD3 antibody, anti-CD28 antibody, IL-7 and IL-15.Control group
(NC) and experimental group uses streaming antibody labeled cells in 15 d, is then detected sample by flow cytometer.From
In cell negative CD45RA, it is the CD4 to be observed to choose the double positive cells of CD62L and CCR7+TCM.According to above-mentioned logical
Overflow-type cell instrument sorts CD4+TCM method, to CD4+TCM is in CD4+Ratio in T cell is detected, such as Fig. 1 institutes
Show.
The alprenolol of 6.2 various concentrations is for CD4+ The detection of T cell expanding effect
The time point that selection alprenolol handles the 15th day is detected, and is added without the control group (NC) of alprenolol
CD4+TCM cells account for total CD4+The ratio of T cell is 16.06%；Alprenolol concentration is that 5uM ratios are 27.26%；A Puluo
The ratio that your concentration is 10uM is 32.81%；The ratio that alprenolol concentration is 20uM is 34.44%.It is test result indicates that different
The alprenolol of concentration is for CD4+There is dose-dependent trend in the amplification effect of T cell, such as in the case of less than 10uM
Shown in Fig. 2.
Interpretation of result
We promote CD4 using small-molecule drug alprenolol first+The amplification of TCM cells in vitro.Experimental group is more right
CD4 is compared according to group+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, testing
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 alprenolol, in experiment can promote CD4+The optimum concentration that TCM is expanded in vitro：
20uM.It was found that after using the alprenolol processing cell of relatively low-dose, CD4+ TCM cells are in CD4+Ratio in T cell
In example there is significant dose-dependant trend in lift；And lift positive effect when the concentration of alprenolol reaches 20uM, in ratio and become
In gentle（Fig. 2）, this is probably so that the growing environment of cell influences there occurs larger change because drug concentration is excessive
The normal growth of cell.In this experiment, counted after choosing alprenolol processing 15 days of cell and detection be by
In the CD4 in peripheral blood+TCM starting quantity is considerably less, CD4+T cell need longer time could anti-CD3 with
More CD4 is gradually formed under anti-CD28 Co stituations+TCM.Experimental result also indicates that selection culture long period rear
More preferable effect can be obtained by carrying out detection（Fig. 2）.
Alprenolol is clinically usually used in hypertension, angina pectoris, arrhythmia cordis, and A Puluo is elaborated first in the present invention
Your application brand-new in T cell culture in vitro, good technical support is provided for the extensive development of adoptive immunotherapy, its
Molecular mechanism needs follow-up further research.
Claims (5)
1. the application that alprenolol expands in vitro to central memory t cell, the central memory t cell is CD4+T is thin
Born of the same parents.
2. application according to claim 1, it is characterised in that the concentration of the alprenolol is 5~20 μM.
3. application according to claim 2, it is characterised in that the concentration of the alprenolol is 10 μM.
4. 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 is well mixed；Then the blood after dilution is slowly added to
The top of lymphocyte separation medium, the ratio of the two are 1:1；Centrifuge 300 × g 30min；It is careful to draw monocyte, insert another
In one centrifuge tube, the dilutions of the PBS containing 0.5%BSA and 2%EDTA of 5 times of volumes, after being well mixed, 300 × g 15min are added；
Repeat previous step once；It is incubated CD4+T cell the moon selects primary antibody, 15min；Contain 0.5%BSA and 2%EDTA with 10 times of volumes
PBS dilution, be well mixed after, 300 × g 12min；It is incubated the secondary antibody with magnetic bead, 30min；Cross post and carry out cell sorting,
Obtain CD4+T cell.
5. application according to claim 1, it is characterised in that the cultural method of the central memory t cell is 5%
CO2, under the conditions of saturated humidity and 37 DEG C by 5 × 105The CD4 in individual/hole+T cell is placed in 1mL and contains 10% hyclone
Cultivated in RPMI1640.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0772677B1 (en) * | 1994-08-17 | 2009-06-03 | Novartis Vaccines and Diagnostics S.r.l. | T cell activation |
| CN102732480A (en) * | 2012-06-11 | 2012-10-17 | 中山大学 | Application of N-acetylcysteine in in-vitro amplification of absolute quantity of central memory T cells |
| CN102925411A (en) * | 2012-11-30 | 2013-02-13 | 中山大学 | Application of vitamin C in in-vitro expansion of number of effector memory T cells |
-
2015
- 2015-05-07 CN CN201510228511.3A patent/CN104830768B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0772677B1 (en) * | 1994-08-17 | 2009-06-03 | Novartis Vaccines and Diagnostics S.r.l. | T cell activation |
| CN102732480A (en) * | 2012-06-11 | 2012-10-17 | 中山大学 | Application of N-acetylcysteine in in-vitro amplification of absolute quantity of central memory T cells |
| CN102925411A (en) * | 2012-11-30 | 2013-02-13 | 中山大学 | Application of vitamin C in in-vitro expansion of number of effector memory T cells |
Non-Patent Citations (3)
| Title |
|---|
| b-Adrenoceptor inverse agonists in asthma;Burton F Dickey et al;《Current Opinion in Pharmacology 》;20101231;第10卷;第254-259页 * |
| 可能诱发和加重银屑病的药物;靳培英;《中华皮肤科杂志》;20050731;第38卷(第7期);第462-464页，尤其是第462页第2段 * |
| 治未病理论在复发型寻常型银屑病防治中的应用研究;王建青;《山东中医药大学博士学位论文》;20111215;第1-4页，尤其是第3页 * |
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