CN110331131A - A kind of Treg cell abductive approach of optimization - Google Patents
A kind of Treg cell abductive approach of optimization Download PDFInfo
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- CN110331131A CN110331131A CN201910674208.4A CN201910674208A CN110331131A CN 110331131 A CN110331131 A CN 110331131A CN 201910674208 A CN201910674208 A CN 201910674208A CN 110331131 A CN110331131 A CN 110331131A
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- 210000003289 regulatory T cell Anatomy 0.000 title claims abstract description 23
- 238000005457 optimization Methods 0.000 title claims abstract description 11
- 210000004027 cell Anatomy 0.000 claims abstract description 48
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 8
- 230000006698 induction Effects 0.000 claims abstract description 7
- 210000004369 blood Anatomy 0.000 claims abstract description 5
- 239000008280 blood Substances 0.000 claims abstract description 5
- 238000000432 density-gradient centrifugation Methods 0.000 claims abstract description 5
- 210000005087 mononuclear cell Anatomy 0.000 claims abstract description 5
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 claims description 20
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 claims description 20
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 claims description 20
- 239000011324 bead Substances 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 8
- 238000010186 staining Methods 0.000 claims description 8
- 108010002350 Interleukin-2 Proteins 0.000 claims description 7
- 102000004887 Transforming Growth Factor beta Human genes 0.000 claims description 7
- 108090001012 Transforming Growth Factor beta Proteins 0.000 claims description 7
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 claims description 7
- 239000006285 cell suspension Substances 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 4
- 239000000725 suspension Substances 0.000 claims description 4
- 239000000654 additive Substances 0.000 claims description 3
- 230000000996 additive effect Effects 0.000 claims description 3
- 238000007689 inspection Methods 0.000 claims 1
- 230000001939 inductive effect Effects 0.000 abstract description 8
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241001085205 Prenanthella exigua Species 0.000 description 1
- 230000005784 autoimmunity Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
- C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/15—Transforming growth factor beta (TGF-β)
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- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2302—Interleukin-2 (IL-2)
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/515—CD3, T-cell receptor complex
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Abstract
The present invention provides a kind of Treg cell abductive approach of optimization, comprising the following steps: S1, acquires whole blood out of volunteer body, then obtains mononuclearcell (PBMC) using density-gradient centrifugation method；S2, preparation CD4+CD25- cell: S3, induction generate Treg cell.The present invention is optimized by the inducing expression system to existing Treg cell, and one of foundation is more efficient, more convenient and fast inducing expression technology.A large amount of experimental period has been saved, highly shortened the period of inducing expression.
Description
Technical field
The present invention relates to cell technology field, the Treg cell abductive approach of specially a kind of optimization.
Background technique
Treg (regulatory T cells) cell is a kind of extremely important immunity regulatory cell, is attempted to utilize in recent years
Treg cell regulates and controls the adjustment effect of immune system to autoimmunity disease, tumour and other immune-related diseases,
Maintain the immunologic balance of body.But the content of Treg cell in vivo is few, can not be separated to from peripheral blood enough thin
Born of the same parents, therefore, obtaining Treg cell from other approach just seems meaningful.
Summary of the invention
Technical problem solved by the invention is to provide a kind of Treg cell abductive approach of optimization, to solve above-mentioned back
The problems in scape technology.
Technical problem solved by the invention is realized using following technical scheme: a kind of Treg cell induction side of optimization
Method, comprising the following steps:
S1, whole blood is acquired out of volunteer body, then obtain mononuclearcell (PBMC) using density-gradient centrifugation method；
S2, preparation CD4+CD25- cell:
1) CD4+ cell, is isolated from PBMC using immunomagnetic beads；
2) CD4+CD25+ cell, is removed from the CD4+ cell 1) obtained using CD25 key player on a team's magnetic bead；
3) it, is sent out using double fluorescent stainings and the solution after 2) removal removal CD4+CD25+ cell is dyed, cocurrent
The ratio of CD4, CD25 cell in formula test sample；
S3, induction generate Treg cell:
1), make 1 × 105cells/ml of cell concentration using 1640+10%FBS culture medium suspension CD4+CD25- cell；
2), taking the concentration obtained in 10ml step 5 is that the cell suspension of 1 × 105cells/ml is inoculated into culture bottle
In；
3), anti-CD3, IL-2 and TGF-β are added into the culture medium in culture bottle in 2) within second day；
4) it, collects within the 6th day and passes through aquicultural cell in 3), and take the double fluorescent stainings of part cell, flow cytometer detection sample
The ratio of CD4, CD25 cell in product.
Preferably, the culture bottle in the S3 is 25 culture bottle of T.
Preferably, described anti-CD3, IL-2 and the additive amount of TGF-β are respectively 10ug/ml, 100U/ml and 10ng/
ml。
Be compared to open technology, there are following advantages by the present invention: the present invention passes through the induction to existing Treg cell
Expression system optimizes, and one of foundation is more efficient, more convenient and fast inducing expression technology.A large amount of experimental period has been saved,
It highly shortened the period of inducing expression.
Specific embodiment
In order to make, technological means of the invention, creation characteristic, workflow, application method reach purpose and effect is easy to bright
White understanding, below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
Embodiment 1
A kind of Treg cell abductive approach of optimization, comprising the following steps:
S1, whole blood is acquired out of volunteer body, then obtain mononuclearcell (PBMC) using density-gradient centrifugation method；
S2, preparation CD4+CD25- cell:
1) CD4+ cell, is isolated from PBMC using immunomagnetic beads；
2) CD4+CD25+ cell, is removed from the CD4+ cell 1) obtained using CD25 key player on a team's magnetic bead；
3) it, is sent out using double fluorescent stainings and the solution after 2) removal removal CD4+CD25+ cell is dyed, cocurrent
The ratio of CD4, CD25 cell in formula test sample；
S3, induction generate Treg cell:
1), make 1 × 105cells/ml of cell concentration using 1640+10%FBS culture medium suspension CD4+CD25- cell；
2), taking the concentration obtained in 10ml step 5 is that the cell suspension of 1 × 105cells/ml is inoculated into culture bottle
In；
3), anti-CD3, IL-2 and TGF-β are added into the culture medium in culture bottle in 2) within second day；
4) it, collects within the 6th day and passes through aquicultural cell in 3), and take the double fluorescent stainings of part cell, flow cytometer detection sample
The ratio of CD4, CD25 cell in product.
Embodiment 2
A kind of Treg cell abductive approach of optimization, comprising the following steps:
S1, whole blood is acquired out of volunteer body, then obtain mononuclearcell (PBMC) using density-gradient centrifugation method；
S2, preparation CD4+CD25- cell:
1) CD4+ cell, is isolated from PBMC using immunomagnetic beads；
2) CD4+CD25+ cell, is removed from the CD4+ cell 1) obtained using CD25 key player on a team's magnetic bead；
3) it, is sent out using double fluorescent stainings and the solution after 2) removal removal CD4+CD25+ cell is dyed, cocurrent
The ratio of CD4, CD25 cell in formula test sample；
S3, induction generate Treg cell:
1), make 1 × 105cells/ml of cell concentration using 1640+10%FBS culture medium suspension CD4+CD25- cell；
2), taking the concentration obtained in 10ml step 5 is that the cell suspension of 1 × 105cells/ml is inoculated into culture bottle
In；
3), anti-CD3, IL-2 and TGF-β are added into the culture medium in culture bottle in 2) within second day；
4) it, collects within the 6th day and passes through aquicultural cell in 3), and take the double fluorescent stainings of part cell, flow cytometer detection sample
The ratio of CD4, CD25 cell in product.
Preferably, the culture bottle in the S3 is 25 culture bottle of T.
Preferably, described anti-CD3, IL-2 and the additive amount of TGF-β are respectively 10ug/ml, 100U/ml and 10ng/
ml。
The present invention is optimized by the inducing expression system to existing Treg cell, and one of foundation is more efficient, more
Convenient and fast inducing expression technology.A large amount of experimental period has been saved, highly shortened the period of inducing expression.
The above shows and describes the basic principle, main features and advantages of the invention.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.Claimed range of the invention by appended claims and
Its equivalent thereof.
Claims (3)
1. a kind of Treg cell abductive approach of optimization, which comprises the following steps:
S1, whole blood is acquired out of volunteer body, then obtain mononuclearcell (PBMC) using density-gradient centrifugation method；
S2, preparation CD4+CD25- cell:
1) CD4+ cell, is isolated from PBMC using immunomagnetic beads；
2) CD4+CD25+ cell, is removed from the CD4+ cell 1) obtained using CD25 key player on a team's magnetic bead；
3) it, is sent out using double fluorescent stainings and the solution after 2) removal removal CD4+CD25+ cell is dyed, parallel type inspection
The ratio of CD4, CD25 cell in sample；
S3, induction generate Treg cell:
1), make 1 × 105cells/ml of cell concentration using 1640+10%FBS culture medium suspension CD4+CD25- cell；
2), taking the concentration obtained in 10ml step 5 is that the cell suspension of 1 × 105cells/ml is inoculated into culture bottle；
3), anti-CD3, IL-2 and TGF-β are added into the culture medium in culture bottle in 2) within second day；
4) it, collects within the 6th day and passes through aquicultural cell in 3), and take the double fluorescent stainings of part cell, in flow cytometer detection sample
The ratio of CD4, CD25 cell.
2. a kind of Treg cell abductive approach of optimization according to claim 1, it is characterised in that: the culture in the S3
Bottle is 25 culture bottle of T.
3. a kind of Treg cell abductive approach of optimization according to claim 1, it is characterised in that: the anti-CD3,
IL-2 and the additive amount of TGF-β are respectively 10ug/ml, 100U/ml and 10ng/ml.
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| CN201910674208.4A CN110331131A (en) | 2019-07-25 | 2019-07-25 | A kind of Treg cell abductive approach of optimization |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101126076A (en) * | 2007-07-12 | 2008-02-20 | 郑颂国 | TGF-beta induced adjustment T cell and its forming method and application |
| CN104278012A (en) * | 2014-10-11 | 2015-01-14 | 湖南赛诺生物科技有限责任公司 | Adult regulatory T cell in-vitro amplification culture medium and application method thereof |
| CN107083360A (en) * | 2017-05-23 | 2017-08-22 | 华中科技大学同济医学院附属同济医院 | A kind of method of the external evoked non-specific regulatory T cells of amplification human antigen |
| CN108004209A (en) * | 2017-12-11 | 2018-05-08 | 山东省齐鲁干细胞工程有限公司 | A kind of bleeding of the umbilicus regulatory T cells amplification in vitro method |
-
2019
- 2019-07-25 CN CN201910674208.4A patent/CN110331131A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101126076A (en) * | 2007-07-12 | 2008-02-20 | 郑颂国 | TGF-beta induced adjustment T cell and its forming method and application |
| CN104278012A (en) * | 2014-10-11 | 2015-01-14 | 湖南赛诺生物科技有限责任公司 | Adult regulatory T cell in-vitro amplification culture medium and application method thereof |
| CN107083360A (en) * | 2017-05-23 | 2017-08-22 | 华中科技大学同济医学院附属同济医院 | A kind of method of the external evoked non-specific regulatory T cells of amplification human antigen |
| CN108004209A (en) * | 2017-12-11 | 2018-05-08 | 山东省齐鲁干细胞工程有限公司 | A kind of bleeding of the umbilicus regulatory T cells amplification in vitro method |
Non-Patent Citations (1)
| Title |
|---|
| 张少鹏 等: "人CD4+CD25-CD45RA+T细胞和CD4+CD25-T细胞诱导获得iTreg细胞的方法学研究", 《中国免疫学杂志》 * |
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