CN106754707A - A kind of separation method of intestines gamma delta T cells - Google Patents
A kind of separation method of intestines gamma delta T cells Download PDFInfo
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- CN106754707A CN106754707A CN201710084479.5A CN201710084479A CN106754707A CN 106754707 A CN106754707 A CN 106754707A CN 201710084479 A CN201710084479 A CN 201710084479A CN 106754707 A CN106754707 A CN 106754707A
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- 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
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The invention discloses a kind of separation method of intestines gamma delta T cells, comprise the following steps：1) IL of animal, is collected, supernatant, physiological saline suspension IL are removed in centrifugation；2), the IL being collected into the anti-gamma delta T CR antibody labelings of fluorescence labeling, it is subsequently adding the anti-FITC monoclonal antibody of anti-fluorescent microbead mark, then the optimization sorting of magnetic cell sorter is gone up, the cell of positive selection is collected, that is, obtains gamma delta T cells.The present invention by mesenteric lymph duct intubation directly, efficiently harvest IL, then intestines gamma delta T cells are further separated by magnetic cell instrument easy to operate, it is time-consuming short, whole separation method is simple, efficient, harvesting quantity is more, active high, purity is high, is conducive to playing the biological function of intestines gamma delta T cells.
Description
Technical field
The present invention relates to a kind of cell isolation method, specially a kind of separation method of intestines gamma delta T cells
Background technology
Gamma delta T cells, be otherwise known as I type T cells, is the unique T cell hypotype of a class phenotype and function, it and α β T cells
Collectively constitute CD3+T cell.Gamma delta T cells have anti-infective, antineoplastic important function, and it also take part in microorganism infection
Immune defense, additionally, gamma delta T cells are to maintaining mucosa-immune stable state to play an important roll.But, for a long time, because gamma delta T is thin
Born of the same parents are distributed seldom in peripheral blood, only account for the 0.5%-5% of T cell sum, separate relatively difficult；And gamma delta T cells are in intestinal mucosa
It is widely distributed.
After existing document report is using animal is put to death at present, small intestine (including jejunum and ileum) tissue, machine is quickly removed
Tool vibrates and the discrete intestinal tissue of chemical action, copper mesh filtering, with the discontinuous Percoll gradients single spins of 40%-70% and elder generation
It is centrifuged with 30%Percoll and is centrifuged with 40%-70%Percoll two step methods again, draw and remove white cellular layer, separates total pouring
Bar cell.It is subsequently adding immunofluorescence label TCR γ anti-δs mark, magnetic cell instrument sorting gamma delta T cells, FCM analysis
Gamma delta T cells purity.But the method step is more, mechanical oscillation and chemical action are big to cellular damage, separate total cell quantity
Few, gamma delta T cells activity is low and purity is low, using being restricted.
The content of the invention
The technical problem to be solved in the present invention is that the separation method needs for overcoming existing intestines gamma delta T cells shake by machinery
Swing and chemical action is big to cellular damage, separate the few defect of total cell quantity, there is provided a kind of separation method of intestines gamma delta T cells.
In order to solve the above-mentioned technical problem, the invention provides following technical scheme：
A kind of separation method of intestines gamma delta T cells, comprises the following steps：
1) IL of animal, is collected, supernatant, physiological saline suspension IL are removed in centrifugation；
2), the IL being collected into the anti-gamma delta T CR antibody labelings of fluorescence labeling, is subsequently adding anti-fluorescent microbead
The anti-FITC monoclonal antibody of mark, then goes up the optimization sorting of magnetic cell sorter, collects the cell of positive selection, that is, obtain
Gamma delta T cells.
Further, the step 1) the middle IL that animal is collected using mesenteric lymph duct intubation.
Further, the step 2) the middle anti-gamma delta T CR labeling of monoclonal antibodies receipts using fluorescein isothiocynate crosslinking
The IL for collecting, every 1 × 108Individual IL adds the anti-gamma delta T CR Dan Ke that 100 μ l fluorescein isothiocynates are crosslinked
Grand antibody, 4 DEG C are incubated 30 minutes, are washed 2 times with buffer solution.
Further, the step 2) in every 1 × 108Individual IL adds the anti-of the anti-fluorescent microbead marks of 100 μ l
FITC monoclonal antibodies, 4 DEG C are incubated 15 minutes, are washed 1 time with buffer solution.
The key that intestines gamma delta T cells are separate is the active IL that obtain a large amount of high-purities.Mesenteric lymph manifold
Collect whole gut-active lymphocyte, purity is 100%, not including other any kind of cells.Therefore, drenched by mesenterium
Hand shaft intubation is collected IL and ensure that a large amount of high-purity activity lymphocytes of acquisition in the short time, it is only necessary to a step
It is rapid i.e. separable, make the multiple step such as tissue homogenate, filtering, centrifugation so as to avoid mechanical oscillation repeatedly and chemical action
Caused cell death, competent cell quantity declines.Additionally, compared to the lymph node in separating whole intestinal tissue or separation intestinal tissue
All inevitably mix other types of cell so that lymphocyte purity declines, the final TCS amount for harvesting compared with
It is low.This method can obtain the total more activated lymphocytes of high-purity, and time-consuming short.
The present invention by mesenteric lymph duct intubation directly, efficiently harvest IL, it is then thin by magnetic-type
It is easy to operate that born of the same parents' instrument further separates intestines gamma delta T cells, time-consuming short, and whole separation method is simple, efficiently, and harvesting quantity is more,
Active high, purity is high, is conducive to playing its biological function.
Specific embodiment
The preferred embodiments of the present invention are illustrated below, it will be appreciated that preferred embodiment described herein is only used
In the description and interpretation present invention, it is not intended to limit the present invention.
Embodiment 1
A kind of separation method of intestines gamma delta T cells, comprises the following steps：
1) IL of animal, is collected, supernatant, physiological saline suspension IL are removed in centrifugation；
Selection weighs about 300 grams of male Wistar rat as materials object.With amytal (2.5%, 100mg/
Kg) intraperitoneal injection, anesthetized rat.It is preoperative to give the life of rat injection 0.9% by micro computer infusion pump (German B.Braun companies)
Reason salt solution (0.3ml/h).Anesthetized rat is placed on operating table, rat abdominal cavity is cut open along ventrimeson, push stomach, small intestine to abdominal cavity
Left side, liver pushes upper right side to, exposes ligamentum hepatoduodenale.0.5% platform is expected into blue injection small bowel, in 1 minute, it is seen that
Blue mesenteric lymph duct is dyed in ligamentum hepatoduodenale, external diameter about 0.1cm is about 0.3~0.5cm, under normal circumstances, intestines
Mesentery lymphatic vessel is water white transparency, takes this to may determine that the position of mesenteric lymph duct, i.e., positioned at inferior caval vein left side, right kidney is quiet
Arteries and veins offside, with superior mesenteric artery with row, moves towards in left and right row.In the free inferior caval vein in vena renalis dextra top, and on right side
Stomach wall is perforated.By an external diameter 0.1cm, the plastic tube of 6~8cm is about full of heparin, one end is cut into inclined-plane, and through right side stomach wall
Hole, liver lower section and inferior caval vein lower section, mesenteric lymph duct is inserted with its beveled end, and depths is reached as far as possible, and the other end is used
The centrifuge tube access intestines lymph of 1.5ml.The intestines lymph of collection expects that blue dyeing carries out viable count with 0.2%, aobvious being inverted
The lymphocyte number in each pipe is counted under micro mirror.The intestines lymph that will be collected is centrifuged through 500g × 5min, removes supernatant, 0.9%
Physiological saline suspension lymphocyte.
2), the IL that the anti-gamma delta T CR labeling of monoclonal antibodies being crosslinked using fluorescein isothiocynate are collected into,
Every 1 × 108Individual IL adds the anti-gamma delta T CR monoclonal antibodies that 100 μ l fluorescein isothiocynates are crosslinked, and 4 DEG C are incubated 30 points
Clock, is washed 2 times with buffer solution；Then the anti-FITC monoclonal antibody of anti-fluorescent microbead mark, every 1 × 10 are subsequently adding8Individual intestines drench
Bar cell adds the anti-FITC monoclonal antibody of the anti-fluorescent microbead marks of 100 μ l, and 4 DEG C are incubated 15 minutes, are washed 1 time with buffer solution.So
The optimization sorting of magnetic cell sorter is gone up afterwards, the cell of positive selection is collected, that is, obtain gamma delta T cells.
Every 2 × 105The anti-gamma delta T CR antibody that individual cell is marked with PE is dyeed, and flow cytometer uses CellQuest
Software analysis data, calculates intestines gamma delta T cells purity.
Finally it should be noted that：The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention,
Although being described in detail to the present invention with reference to the foregoing embodiments, for a person skilled in the art, it still may be used
Modified with to the technical scheme described in foregoing embodiments, or equivalent is carried out to which part technical characteristic.
All any modification, equivalent substitution and improvements within the spirit and principles in the present invention, made etc., should be included in of the invention
Within protection domain.
Claims (4)
1. a kind of separation method of intestines gamma delta T cells, it is characterised in that comprise the following steps：
1) IL of animal, is collected, supernatant, physiological saline suspension IL are removed in centrifugation；
2), the IL being collected into the anti-gamma delta T CR antibody labelings of fluorescence labeling, is subsequently adding anti-fluorescent microbead mark
Anti-FITC monoclonal antibody, then go up the optimization sorting of magnetic cell sorter, collect the cell of positive selection, that is, obtain gamma delta T
Cell.
2. the separation method of intestines gamma delta T cells as claimed in claim 1, it is characterised in that the step 1) in use mesenterium
Lymphatic vessel intubation collects the IL of animal.
3. the separation method of intestines gamma delta T cells as claimed in claim 2, it is characterised in that the step 2) in use different sulphur cyanogen
The IL that the anti-gamma delta T CR labeling of monoclonal antibodies of sour fluorescein crosslinking are collected into, per l × 108Individual IL adds
The anti-gamma delta T CR monoclonal antibodies of 100 μ l fluorescein isothiocynates crosslinking, 4 DEG C are incubated 30 minutes, are washed 2 times with buffer solution.
4. the separation method of intestines gamma delta T cells as claimed in claim 2, it is characterised in that the step 2) in per l × 108It is individual
IL adds the anti-FITC monoclonal antibody of the anti-fluorescent microbead marks of 100 μ l, and 4 DEG C are incubated 15 minutes, and 1 is washed with buffer solution
It is secondary.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1788078A1 (en) * | 2004-07-08 | 2007-05-23 | Medinet., Co. Ltd. | Dentritic cell drug containing the dentritic cell, therapeutic method using the dentritic cell and method of culturing gamma delta t cell |
| CN104357393A (en) * | 2014-11-14 | 2015-02-18 | 中国农业科学院饲料研究所 | Isolated culture method of chicken intestinal tract epithelial GammaDeltaT cells |
-
2017
- 2017-02-16 CN CN201710084479.5A patent/CN106754707A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1788078A1 (en) * | 2004-07-08 | 2007-05-23 | Medinet., Co. Ltd. | Dentritic cell drug containing the dentritic cell, therapeutic method using the dentritic cell and method of culturing gamma delta t cell |
| CN104357393A (en) * | 2014-11-14 | 2015-02-18 | 中国农业科学院饲料研究所 | Isolated culture method of chicken intestinal tract epithelial GammaDeltaT cells |
Non-Patent Citations (3)
| Title |
|---|
| LAN CHENG ET AL.,: ""Mouse γδ T cells are capable of expressing MHC class II molecules,and of functioning as antigen-presenting cells"", 《JOURNAL OF NEUROIMMUNOLOGY》 * |
| 叶月芳等: ""小肠上皮内γδT细胞的分离和纯化"", 《第二届浙江省消化病学术大会论文汇编》 * |
| 杨辉: ""胃肠多肽调节多器官功能障碍综合征时肠淋巴细胞归巢"", 《中国优秀博硕士学位论文全文数据库(博士)医药卫生科技辑》 * |
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Application publication date: 20170531 |