CN110184237A - Abdominal cavity cell inducing T cell is divided into the purposes of regulatory T cells - Google Patents
Abdominal cavity cell inducing T cell is divided into the purposes of regulatory T cells Download PDFInfo
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- CN110184237A CN110184237A CN201910434339.5A CN201910434339A CN110184237A CN 110184237 A CN110184237 A CN 110184237A CN 201910434339 A CN201910434339 A CN 201910434339A CN 110184237 A CN110184237 A CN 110184237A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
- C12N2506/115—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells from monocytes, from macrophages
Abstract
The present invention relates to the purposes that abdominal cavity cell inducing T cell is divided into regulatory T cells, belong to field of medicaments.For solve the problems, such as to obtain from tissue enough regulatory T cells for treat it is very difficult, acquisition methods are few, the present invention provides the purposes that abdominal cavity cell inducing T cell is divided into regulatory T cells.The present invention passes through the expression of the detection various markers in abdominal cavity cell surface first, it was demonstrated that abdominal cavity cell is the mixture of a variety of inhibitory cells such as B cell, macrophage and T cell, may have immunosuppressive action.Then abdominal cavity cell and T cell are co-cultured, as a result CD3+CD4+CD25+Foxp3+ regulatory T cells obviously increase, show abdominal cavity cell can inducing T cell to regulatory T cells break up, promote the generation of regulatory T cells, provide new method for the preparation of regulatory T cells.
Description
Technical field
The present invention relates to the purposes that abdominal cavity cell inducing T cell is divided into regulatory T cells, belong to field of medicaments.
Background technique
Regulatory T cells (RegulatoryT cell, Treg) are during treating inflammation and autoimmune disease
With good immunosuppressive action, but obtained from tissue enough regulatory T cells for treat be very difficult.
Therefore, a kind of method is studied to induce the generation of regulatory T cells very necessary.
Abdominal cavity cell (peritoneal cell, PC) is a kind of important cells group for being present in abdominal cavity.In recent years research
It was found that B1a cell is distributed mainly on abdominal cavity and thoracic cavity, it is positive to show as CD5 and CD11b.B1a cell can secrete IL-10, and
It plays an important role in mucosal immunity.In addition, abdominal cavity cell expresses CD206mRNA strong positive, and CD206 is M2 type macrophage
Characteristic mark, M2 type macrophage can be reduced inflammatory cytokine and promote the generation of IL-10 and TGF-β, to control
Treat chronic diseases associated with inflammation.Clinically abdominocentesis is a minor operation that is simple, safe, quick and not needing anesthesia,
So abdominal cavity, which provides one, can largely obtain abdominal cavity cell, the ring including inhibition abdominal cavity B cell and peritoneal macrophage
Border.
Until up to now, there are no any reports generated about abdominal cavity cell induction regulatory T cells.
Summary of the invention
The present invention is directed at least solve one of the technical problems existing in the prior art.For this purpose, it is an object of the invention to
The purposes that abdominal cavity cell inducing T cell is divided into regulatory T cells is provided.Another object of the present invention is to provide regulatory Ts
The preparation method of cell.
The present invention provides the purposes that abdominal cavity cell inducing T cell is divided into regulatory T cells.
Further, the method for the induction is to co-culture abdominal cavity cell and T cell.
Further, the T cell is spleen t-cell.
Further, the T cell is CD4+T cell.
Further, the abdominal cavity cell is the mixture comprising B cell, macrophage and T cell.
Preferably, for the abdominal cavity cell mainly by the B cell of 50+3%, the macrophage of 41+5% and the T of 4+1% are thin
Born of the same parents' composition.
Further, the abdominal cavity cell is Peritoneal Cells of Mice.
The present invention provides the preparation methods of regulatory T cells: being generated with the differentiation of abdominal cavity cell inducing T cell.
Further, the method for the induction is to co-culture abdominal cavity cell and T cell.
Further, the co-cultivation meets at least one of following:
It is co-cultured in 37 degrees Celsius, the incubator of 5% carbon dioxide；
Culture solution is RPMI-1640 culture solution；
Cultivating cell concentration is 2 × 106/ml；
Abdominal cavity cell: the quantitative proportion of T cell is 1:(1~3)；
Preferably, abdominal cavity cell: T cell quantitative proportion is 1:1.
Further, the preparation method meets at least one of following:
The T cell is spleen t-cell；
The T cell is CD4+T cell；
The abdominal cavity cell is the mixture comprising B cell, macrophage and T cell；
The abdominal cavity cell is mainly by the B cell of 50+3%, the macrophage of 41+5% and the T cell composition of 4+1%；
The abdominal cavity cell is Peritoneal Cells of Mice.
The present invention passes through the expression of the detection various markers in abdominal cavity cell surface first, it was demonstrated that abdominal cavity cell is B thin
The mixture of a variety of inhibitory cells such as born of the same parents, macrophage and T cell, may have immunosuppressive action.Then abdominal cavity is thin
Born of the same parents co-culture with T cell, and as a result CD3+CD4+CD25+Foxp3+ regulatory T cells obviously increase, and show abdominal cavity cell energy
Enough inducing T cells break up to regulatory T cells, promote the generation of regulatory T cells, provide for the preparation of regulatory T cells
New method.
Detailed description of the invention
Fig. 1 is the cell constituent and surface marker testing result figure of abdominal cavity cell in embodiment 1；
Fig. 2 is Flow cytometry CD3+CD4+CD25+Foxp3+ regulatory T cells and CD3+CD4+ in embodiment 2
IL-17+Th17 cell percentage result figure.
Specific embodiment
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment
Part, it described technology or conditions or is carried out according to the literature in the art according to product description.Agents useful for same or instrument
Production firm person is not specified in device, and being can be with conventional products that are commercially available.Used experimental method is such as without special theory
Bright is conventional method.Reagent used in embodiment and instrument are as follows:
One, main agents
1, RPMI-1640 (GibcoBRL company)；Fetal calf serum (GibcoBRL company)；Mycillin.
2, streaming antibody: anti-CD19, anti-F4/80, anti-CD11b, anti-B220, anti-CD3, anti-
CD4, anti-CD5, anti-IL-17, anti-CD25, anti-Foxp3 (are all from BD Pharmingen and eBioscience
Company).
Two, instrument
1, superclean bench；
2,37 DEG C of constant temperature, 5%CO2Cell incubator (Thermo, USA)；
3,15ml centrifuge tube (BD Filcon company), 50ml centrifuge tube (BD Filcon company), 100mm Tissue Culture Dish
(WHB company)；
4, inverted microscope (Olympus)；Just setting microscope (Leica)；
5, low-temperature and high-speed centrifuge (Thermo, USA)；
6, micro adjustable pipette (Eppendorf company)；
7, refrigerator, cell counting board, eye scissors, tweezers.
The composition and properties study of 1 abdominal cavity cell of embodiment
One, the acquisition of abdominal cavity cell
1, dislocation of cervical vertebra method puts to death 6-7 week old mouse, impregnates 1-2 minutes in 75% alcohol, moves into super-clean bench, dorsal position is solid
Due in dissection plate, alcohol disinfecting skin of abdomen.
2, the RPMI-1640 culture medium injection abdominal cavity that 25g syringe needle extracts 8mL pre-cooling is loaded into 10ml syringe, gently rubs abdomen
Then portion cuts off an osculum in neck with eye scissors, from osculum along ventrimeson abdominal cut skin, exposure abdomen muscle layer.
3, under germ-free condition, cell suspension is drawn with 10ml syringe, is moved into centrifuge tube.
4, after counting, 1200rpm is centrifuged 5min, and cell is resuspended and is diluted to purpose concentration in RPMI-1640 culture solution i.e.
It can.
Two, the composition and feature of Flow cytometry abdominal cavity cell
It is incubated for using streaming antibody anti-F4/80, anti-B220, anti-CD11b, anti-CD3 and anti-CD5 new
Fresh abdominal cavity cell, the expression of the various markers in flow cytomery abdominal cavity cell surface.
Experiment sets 3 repetitions altogether.Experimental result is shown in Fig. 1, wherein Figure 1A shows the cell constituent of abdominal cavity cell, figure
1B shows the surface marker of abdominal cavity cell.
The result shows that: abdominal cavity cell is mainly by the B cell of 50+3%, the macrophage of 41+5% and the T cell group of 4+1%
At.It is worth mentioning that abdominal cavity B cell surface height expresses CD5 and CD11b, illustrate abdominal cavity B cell based on B1a cell.Thus
As it can be seen that abdominal cavity cell is the mixture of a variety of inhibitory cells, there may be immunosuppressive action.
2 abdominal cavity cell of embodiment induces CD4+T cell differentiation as the effect of regulatory T cells
Utilize 1ml 2 × 106The abdominal cavity cell and 1ml 2 × 10 that/ml is obtained according to embodiment 16The spleen t-cell of/ml exists
37 degrees Celsius, the incubator of 5% carbon dioxide co-cultures 6 or 12 hours, CD3+CD4+ in Flow cytometry CD4+T cell
CD25+Foxp3+ regulatory T cells and CD3+CD4+IL-17+Th17 cell percentage.Experiment sets 3 repetitions altogether.Experiment
As a result see Fig. 2.
Fig. 2 shows that abdominal cavity cell and spleen t-cell co-culture 6 or 12 hours, CD3+CD4+CD25+Foxp3+ regulatory T
Cell obviously increases, and CD3+CD4+IL-17+Th17 cell significantly reduces.Above the results showed that abdominal cavity cell and T cell
It co-cultures, CD4+T cell can be effectively facilitated and broken up to regulatory T cells, to promote the generation of regulatory T cells, simultaneously
Inhibit the generation of helper T lymphocyte 17 (T helper cell 17, Th17).
Claims (10)
1. the purposes that abdominal cavity cell inducing T cell is divided into regulatory T cells.
2. purposes as described in claim 1, it is characterized in that: the method for the induction is to co-culture abdominal cavity cell and T cell.
3. purposes as claimed in claim 1 or 2, it is characterized in that: the T cell is spleen t-cell.
4. the purposes as described in claims 1 to 3 any one, it is characterized in that: the T cell is CD4+T cell.
5. purposes as claimed in claim 1 or 2, it is characterized in that: the abdominal cavity cell is comprising B cell, macrophage and T
The mixture of cell；Preferably, the abdominal cavity cell is mainly by the B cell of 50+3%, the macrophage and 4+1% of 41+5%
T cell composition.
6. the purposes as described in 1,2,5 any one of claim, it is characterized in that: the abdominal cavity cell is that mouse peritoneal is thin
Born of the same parents.
7. the preparation method of regulatory T cells generates it is characterized in that: being broken up with abdominal cavity cell inducing T cell.
8. preparation method as claimed in claim 7, it is characterized in that: the method for the induction is to be total to abdominal cavity cell and T cell
Culture.
9. preparation method as claimed in claim 8, it is characterized in that: at least one of below co-cultivation satisfaction:
It is co-cultured in 37 degrees Celsius, the incubator of 5% carbon dioxide；
Culture solution is RPMI-1640 culture solution；
Cultivating cell concentration is 2 × 106/ml；
Abdominal cavity cell: the quantitative proportion of T cell is 1:(1~3)；
Preferably, abdominal cavity cell: T cell quantitative proportion is 1:1.
10. the preparation method as described in claim 7~9 any one, it is characterized in that: meeting at least one of following:
The T cell is spleen t-cell；
The T cell is CD4+T cell；
The abdominal cavity cell is the mixture comprising B cell, macrophage and T cell；
The abdominal cavity cell is mainly by the B cell of 50+3%, the macrophage of 41+5% and the T cell composition of 4+1%；
The abdominal cavity cell is Peritoneal Cells of Mice.
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| CN201910434339.5A CN110184237B (en) | 2019-05-23 | 2019-05-23 | Application of abdominal cavity cell to induce differentiation of T cell into regulatory T cell |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20130106320A (en) * | 2012-03-19 | 2013-09-27 | 가톨릭대학교 산학협력단 | Composition for treatment or prevention of transplant rejection or autoimmune diseases comprising myeloid derived suppressor cell |
| CN104342404A (en) * | 2013-08-09 | 2015-02-11 | 中国人民解放军军事医学科学院基础医学研究所 | New method for obtaining regulatory macrophage |
| CN105087477A (en) * | 2015-05-21 | 2015-11-25 | 王松灵 | Application of mesenchymal stem cell modified by miR-21 antisense nucleotide |
| CN106581064A (en) * | 2015-10-20 | 2017-04-26 | 四川大学 | Applications of abdominal cavity cells in treatment of immune diseases |
| CN108004208A (en) * | 2017-12-01 | 2018-05-08 | 南京爱瑞生物科技有限公司 | A kind of method of external evoked T cells with antigenic specificity |
-
2019
- 2019-05-23 CN CN201910434339.5A patent/CN110184237B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20130106320A (en) * | 2012-03-19 | 2013-09-27 | 가톨릭대학교 산학협력단 | Composition for treatment or prevention of transplant rejection or autoimmune diseases comprising myeloid derived suppressor cell |
| CN104342404A (en) * | 2013-08-09 | 2015-02-11 | 中国人民解放军军事医学科学院基础医学研究所 | New method for obtaining regulatory macrophage |
| CN105087477A (en) * | 2015-05-21 | 2015-11-25 | 王松灵 | Application of mesenchymal stem cell modified by miR-21 antisense nucleotide |
| CN106581064A (en) * | 2015-10-20 | 2017-04-26 | 四川大学 | Applications of abdominal cavity cells in treatment of immune diseases |
| CN108004208A (en) * | 2017-12-01 | 2018-05-08 | 南京爱瑞生物科技有限公司 | A kind of method of external evoked T cells with antigenic specificity |
Non-Patent Citations (4)
| Title |
|---|
| LUZ-CRAWFORD等: "Mesenchymal Stem Cells Generate a CD4+CD25+Foxp3+ Regulatory T Cell Population During the Differentiation Process of Th1 and Th17 Cells", 《STEM CELL RESEARCH & THERAPY》 * |
| 居颂光等: "《医学免疫学实验技术》", 28 February 2011 * |
| 王坤鹏等: "小鼠腹腔巨噬细胞对调节性T细胞体外诱导的影响", 《南京医科大学学报》 * |
| 陈祚珈等: "FOXP3+ 调节性T 细胞", 《生命科学》 * |
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