CN107153115A - A kind of method and kit that testing inspection is co-cultured for mixed lymphocytes - Google Patents
A kind of method and kit that testing inspection is co-cultured for mixed lymphocytes Download PDFInfo
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- CN107153115A CN107153115A CN201710395871.1A CN201710395871A CN107153115A CN 107153115 A CN107153115 A CN 107153115A CN 201710395871 A CN201710395871 A CN 201710395871A CN 107153115 A CN107153115 A CN 107153115A
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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Abstract
The invention discloses a kind of method and kit that testing inspection is co-cultured for mixed lymphocytes.The method and kit of the present invention can be used for mixed lymphocytes to co-culture the detection tested, suitable for the tissue matching test before organ transplant, the immunologic function detection of immunocompromised patients, the immunological rejection effect detection of medicine and graft, other cells or environmental stimuli thing are detected for the influence of T lymphocyte activations, drug screening to influenceing T cell activation ability, and other immunological regulation experimental studies etc..The method and kit of the present invention is based primarily upon flow cytometry assay, and required sample size is few；It is simple to operate, it is workable；Accuracy is high；It is repeatable strong；Testing result is clear, high specificity, it is easy to analyze；Cost is low, and generalization is strong.
Description
Technical field
The invention belongs to inspection technology field, and in particular to a kind of side that testing inspection is co-cultured for mixed lymphocytes
Method and kit.
Background technology
When the lymphocyte of the different individual of two heredity is mixed into co-incubation in vitro, because of the tissue compatible on its surface
Property antigen it is different and mutually stimulate, other side's lymphocytic cell division can be caused to breed and convert, this reaction is referred to as two-way mixing and drenched
Bar cell culture (two way MLC)；If the lymphocyte of a wherein side is first handled with mitomycin C (mytomycine C)
Or radiation exposure is allowed to DNA in cell and loses replication capacity, but remain to stimulate the opposing party's lymphocyte to convert, be referred to as unidirectional
Heart xenotransplantaion (one way MLC).It is thin that major histocompatibility complex (MHC) and/or M locus genes are encoded
Cellular surface antigen is the main stimulator of this reaction, and reacting cells are T cell.According to the intensity of lymphocyte reaction, it can comment
Respond of the difference degree and lymphocyte of both valencys histocompatibility antigen to homogeneous variant cell.Therefore, mix
Lymphocyte reaction is usually used in the tissue matching before organ transplant, to determine the degree that acceptor is compatible with donor MHC；And medicine
And the immunological rejection effect detection of graft, to assess its application risk.Because heart xenotransplantaion medium size lymphocyte connects
Activated, bred by the stimulation of allogenic antigen, the cell factor of generation huge number, promotion NK,
Tumor-infiltrating lymphocytes and cytotoxic T lymphocyte etc. kill the differentiation of cell, therefore are that immunological regulation is ground again
The experimental model commonly used in studying carefully.In addition, mixed lymphocyte reaction (MLP) can also be used to detect other cells or the effect of environmental stimuli thing
Under, the proliferative conditions of T cell.In short, compared with control group, T cell, which increases, means that determinand can promote T cell
Reaction；T cell propagation, which is reduced, then means that determinand can suppress t cell responses.
But at present, mixed lymphocytes do not co-culture the finished product kit of testing inspection both at home and abroad, cause each doctor
Institute, colleges and universities, scientific research institutions, third party inspection mechanism carry out difficult during this testing inspection, it is necessary to carry out a large amount of previous experiments
Condition is groped；And due to the not equal reason of bibliography, institute is experimentally different when may cause the different institutions to be detected,
Therefore testing result otherness is very big.Therefore, it is necessary to provide a kind of side that testing inspection is co-cultured for mixed lymphocytes
Method and kit are to overcome drawbacks described above.
The content of the invention
It is an object of the invention to for not having mixed lymphocytes to co-culture related reagent on current domestic and international market
The present situation of box, researches and develops a kind of new kit that testing inspection is co-cultured for mixed lymphocytes.The present invention method and
Kit be based on hydroxyl fluorescein diacetate succinimide fat (Carboxyfluorescein diacetate,
Succinimidyl ester, CFSE) dyeing combination flow cytometry assay detection mixed lymphocytes co-cultivation result, it is led
It is (referring to Fig. 1) to want principle：Anticipating stimulation cell using mitomycin C (Mitomycin C) (stimulates cell and second
Lymphocytic cell surface major histocompatibility complex is different, can be lymphocyte or other cells, and such as Epstein-Barr virus is converted
B lymphoblasts (such as N23 cell lines, by cloning), HTC cells or PBMC cells etc.), lose DNA in stimulation cell
De-duplicate ability, is no longer bred, but remains to stimulate second of lymphocyte (can be lymphocyte or the T isolated and purified
Lymphocyte etc.) activation, further, locate in advance with Mitomycin C using second of lymphocyte of CFSE marks, and by it
The stimulation cell co-culture of reason is after 3-5 days, you can second of the lymphocyte marked by Flow cytometry through CFSE
Proliferative conditions.Technical scheme is specific as follows：
According to the first aspect of the invention, a kind of method that testing inspection is co-cultured for mixed lymphocytes, including
Following steps：
The first step, will stimulate cell to be handled using mitomycin C；
Second step, CFSE fluorescent dyeings are utilized by second of lymphocyte；
3rd step, stimulates cell and second of lymph through CFSE fluorescent dyeings thin by what mitomycin C was handled
Born of the same parents' co-incubation；
4th step, uses the proliferative conditions of second of lymphocyte of Flow cytometry；
Wherein, the stimulation cell is different from second of lymphocytic cell surface major histocompatibility complex.
According to the present invention, the stimulation cell includes lymphocyte or other cells, and other described cells include Epstein-Barr virus
B lymphoblasts, HTC cells or PBMC cells of conversion etc., second of lymphocyte include lymphocyte or separated pure
T lymphocytes of change etc..
According to the present invention, the final concentration of 5-100 μ g/ml of the mitomycin C, it is preferred that the end of the mitomycin C
Concentration is 10 μ g/ml.
According to the present invention, final concentration of 1-10 μM of the CFSE fluorescent dyes, it is preferred that the CFSE fluorescent dyes
Final concentration of 5 μM.
According to the present invention, the stimulation cell and second through CFSE fluorescent dyeings of the mitomycin C processing
Lymphocyte is according to 1:1-1:20 ratio co-incubation.
According to the present invention, the time of the co-incubation is 3-5 days.
According to the second aspect of the invention, a kind of kit that testing inspection is co-cultured for mixed lymphocytes, institute
State kit and include mitomycin C and CFSE fluorescent dyes.
According to the present invention, the final concentration of 5-100 μ g/ml of the mitomycin C, it is preferred that the end of the mitomycin C
Concentration is 10 μ g/ml.
According to the present invention, final concentration of 1-10 μM of the CFSE fluorescent dyes, it is preferred that the CFSE fluorescent dyes
Final concentration of 5 μM.
The method and kit of the present invention can be used in mixed lymphocytes and co-culture testing inspection, have the following advantages that：
1) it is simple to operate, it is only necessary to carry out simple operations according to product description, without special experiment skill, can grasp
The property made is strong；
2) proliferative conditions of lymphocyte are detected using CFSE decoration method combinations flow cytometry, accuracy is high；
3) flow cytometer detection method can truly proliferative conditions of responder cell directly perceived, not by operator's subjective judgement shadow
Ring, reproducibility of results is strong；
4) sample size is few needed for, and testing result is clear, high specificity, it is easy to analyze；
5) cost is low, and generalization is strong.
The invention provides the method for the detection that experiment is co-cultured available for mixed lymphocytes and kit, it is adaptable to device
The immunological rejection of tissue matching test before official's transplanting, the immunologic function detection of immunocompromised patients, medicine and graft is made
With detection, other cells or environmental stimuli thing are detected for the influence of T lymphocyte activations, to influence T cell activation ability
Drug screening, and other immunological regulation experimental studies etc..The method and kit of the present invention is not limited only to the inspection of human sample
Survey, it may also be used for the detection that other species mixed lymphocytes are co-cultured, including mouse, rat, rabbit, dog, ox, pig etc., have
Extensive industrial application value.
Brief description of the drawings
Fig. 1 is the principle schematic of the method for the present invention.
Fig. 2 is the operating instruction schematic diagram of embodiment 1.
Fig. 3 is the experimental result schematic diagram of embodiment 1.
Fig. 4 is the operating instruction schematic diagram of embodiment 2.
Fig. 5 is the experimental result schematic diagram of embodiment 2.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described.It should be understood that following examples are merely to illustrate this
Invention is not for limitation the scope of the present invention.
Embodiment 1
Tissue matching test before subject organ transplant can detect by the method and kit of the present invention, with judge by
Whether examination person is suitable to receive this organ transplant, the risk repelled after prediction transplanting.Carried it is generally necessary to obtain organ transplant
Donor (hereinafter referred to as donor) and the fresh peripheral venous blood of transplant recipients' (hereinafter referred to as acceptor) are tested.
As shown in Fig. 2 the concrete operation step of the present embodiment is as follows：
The first step, the preparation of donor lymphocyte：
1) donor fresh peripheral venous blood is taken, with PBS according to 1：1 dilution proportion.Along tube wall blow slowly drip superposition fill
(layering of destruction liquid level is careful not in the test tube of the lymphocyte separation medium of 1/2 volume), then 2000rpm horizontal centrifugals
20min.It is divided into 4 layers in pipe, blood plasma, mononuclearcell, granular leucocyte, red blood cell is followed successively by from top to bottom.Stretched with capillary
Into mononuclearcell layer (on the interface for being located at cell separation liquid and blood plasma), whole cells are gently suctioned out along tube wall.Then use
PBS is washed twice, and each 1500rpm centrifuges 10min.Trypan Blue, tally is counted.It is 1~2 × 10 to adjust cell density6/
Ml, centrifugation, leaves and takes precipitation.
2) 1 μ l mitomycins (the μ g/ml of final concentration 10) and 1ml PBS, 37 DEG C of water-bath 30min are added into above-mentioned cell,
PBS centrifuge washings 2 times.
3) it is 1 × 10 with the liquid of the RPMI 1640 adjustment cell density containing 10%FBS7/ ml, labeled as D.
Second step, the preparation of recipient lymphocytes：
1) acceptor fresh peripheral venous blood is taken, with PBS according to 1：1 dilution proportion.Along tube wall blow slowly drip superposition fill
(layering of destruction liquid level is careful not in the test tube of the lymphocyte separation medium of 1/2 volume), then 2000rpm horizontal centrifugals
20min.It is divided into 4 layers in pipe, blood plasma, mononuclearcell, granular leucocyte, red blood cell is followed successively by from top to bottom.Stretched with capillary
Into mononuclearcell layer (on the interface for being located at cell separation liquid and blood plasma), whole cells are gently suctioned out along tube wall.Then use
PBS is washed twice, and each 1500rpm centrifuges 10min.Trypan Blue, tally is counted.
2) above-mentioned cell is resuspended with 1640 culture mediums containing 5%FBS, adjustment cell concentration is 1 × 107/ml.Add
Final concentration of 5 μM of CFSE solution, 37 DEG C of lucifuge water-bath 5min.Then washed 2 times using the PBS containing 5%FBS of precooling.
3) above-mentioned cell is resuspended with the PRMI 1640 containing 10%FBS, adjustment cell density is 1 × 107/ ml, is labeled as
R。
3rd step, heart xenotransplantaion
The above-mentioned R prepared and D is added into 96 orifice plates by following packet, 37 DEG C, 5%CO are put2Incubator in cultivate
3-5 days.
Experimental group：R(50μl)+D(50μl)；
Control group：R(50μl)；
Every group of 3 multiple holes, blank control group is the μ L of RPMI 1640 culture mediums 100.
4th step, the proliferative conditions of Flow cytometry lymphocyte
By flow cytometry, lymphocyte populations are irised out by gating, and analyze the proliferative conditions of lymphocyte.As a result with
Stimulus index (Stimulation indices, SI) expression, SI=experimental groups lymphopoiesis percentage-control group lymph
Cell breeds percentage.
Testing result is as shown in figure 3, cellular control unit has no or only a small amount of lymphopoiesis, and experimental group has a large amount of pouring
Bar cell is bred.If SI values are more than 10, point out donor not to be inconsistent with both acceptors distribution type, be not suitable for carrying out follow-up organ
Transplanting.
Embodiment 2
Subject T lymphocyte functions can detect by the method and kit of the present invention whether low, it usually needs obtain
The fresh peripheral venous blood of subject is taken to be tested.
As shown in figure 4, the concrete operation step of the present embodiment is as follows：
The first step, stimulates the preparation of cell：
1) conventional stimulation cell has B lymphoblasts (such as N23 cell lines, by cloning), the HTC that Epstein-Barr virus is converted
Cell or PBMC cells etc..By taking N23 cells as an example, the N23 cells in exponential phase are taken, are resuspended in after centrifugation fresh complete
In culture medium, Trypan Blue, tally is counted.
2) 1 μ l mitomycins (the μ g/ml of final concentration 10) and 1ml PBS, 37 DEG C of water-bath 30min are added into above-mentioned cell,
PBS centrifuge washings 2 times.
3) it is 1 × 10 with the liquid of the RPMI 1640 adjustment cell density containing 10%FBS7/ml.Labeled as S.
Second step, the preparation of subject's peripheral blood PBMC cells：
1) individual subject's fresh peripheral venous blood is taken, with PBS according to 1：1 dilution proportion.Along tube wall blow slowly drip fold
Plus fill 1/2 volume lymphocyte separation medium test tube in (be careful not to destruction liquid level layering), then 2000rpm levels from
Heart 20min.It is divided into 4 layers in pipe, blood plasma, mononuclearcell, granular leucocyte, red blood cell is followed successively by from top to bottom.Use capillary
(on the interface for being located at cell separation liquid and blood plasma) is extended in mononuclearcell layer, whole cells are gently suctioned out along tube wall.Then
Washed with PBS twice, each 1500rpm centrifuges 10min.Trypan Blue, tally is counted.
2) above-mentioned cell is resuspended with 1640 culture mediums containing 5%FBS, adjustment cell concentration is 1 × 107/ml.Add
Final concentration of 5 μM of CFSE solution, 37 DEG C of lucifuge water-bath 5min.Then washed 2 times using the PBS containing 5%FBS of precooling.
3) above-mentioned cell is resuspended with the PRMI 1640 containing 10%FBS, adjustment cell density is 1 × 107/ ml, is labeled as
U。
3rd step, heart xenotransplantaion
By the S prepared and U according to 20:1 ratio, following packet adds 96 orifice plates, puts 37 DEG C, 5%CO2Culture
Cultivated 3-5 days in case.
Experimental group：U(2×105Individual cell)+S (1 × 104Individual cell)；
Control group：U(2×105Individual cell)
Every group of 3 multiple holes, blank control group is the μ L of RPMI 1640 culture mediums 200.
4th step, the proliferative conditions of Flow cytometry lymphocyte
Using CD3, CD8 streaming antibody labeling culture cell, by flow cytometry, all kinds of T lymphs are irised out by gating
Cell mass, and analyze the proliferative conditions of T lymphocytes.As a result represented with stimulus index (Stimulation indices, SI),
SI=experimental groups lymphopoiesis percentage-control group lymphopoiesis percentage.
Testing result as shown in figure 5, for the normal Healthy People of T lymphocyte functions, cellular control unit without or
Only a small amount of lymphopoiesis, experimental group has a large amount of lymphopoiesis；For T lymphocyte function defect patients,
Control group and experimental group cell have no or only a small amount of lymphopoiesis.
Claims (10)
1. a kind of method that testing inspection is co-cultured for mixed lymphocytes, it is characterised in that comprise the following steps：
The first step, will stimulate cell to be handled using mitomycin C；
Second step, CFSE fluorescent dyeings are utilized by second of lymphocyte；
3rd step, the stimulation cell that mitomycin C is handled is total to second of lymphocyte through CFSE fluorescent dyeings
With culture；
4th step, the proliferative conditions of second of lymphocyte are detected using flow cytometry；
Wherein, the stimulation cell is different from second of lymphocytic cell surface major histocompatibility complex.
2. the method as described in claim 1, it is characterised in that the stimulation cell includes lymphocyte or other cells, institute
Stating second of lymphocyte includes lymphocyte or the T lymphocytes isolated and purified.
3. method as claimed in claim 2, it is characterised in that the B lymphs that other described cells include Epstein-Barr virus conversion are female thin
Born of the same parents, HTC cells or PBMC cells.
4. the method as described in claim 1, it is characterised in that the final concentration of 5-100 μ g/ml of the mitomycin C, preferably
, the final concentration of 10 μ g/ml of the mitomycin C.
5. the method as described in claim 1, it is characterised in that final concentration of 1-10 μM of the CFSE fluorescent dyes, preferably
, final concentration of 5 μM of the CFSE fluorescent dyes.
6. the method as described in claim 1, it is characterised in that the stimulation cell of the mitomycin C processing with through CFSE
Second of lymphocyte of fluorescent dyeing is according to 1:1-1:20 ratio co-incubation.
7. the method as described in claim 1, it is characterised in that the time of the co-incubation is 3-5 days.
8. a kind of kit that testing inspection is co-cultured for mixed lymphocytes, it is characterised in that the kit includes silk
Rimocidin C and CFSE fluorescent dye.
9. method as claimed in claim 8, it is characterised in that the final concentration of 5-100 μ g/ml of the mitomycin C, preferably
, the final concentration of 10 μ g/ml of the mitomycin C.
10. method as claimed in claim 8, it is characterised in that final concentration of 1-10 μM of the CFSE fluorescent dyes, preferably
, final concentration of 5 μM of the CFSE fluorescent dyes.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115433709A (en) * | 2022-06-13 | 2022-12-06 | 南京艾尔普再生医学科技有限公司 | In-vitro experimental model for predicting myocardial cell transplantation immune rejection |
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| CN103237887A (en) * | 2010-10-06 | 2013-08-07 | 车比奥及戴奥斯泰有限公司 | Embryonic stem cell-derived cardiomyocytes and cell therapy product using same as active ingredient |
| US20160018371A1 (en) * | 2013-03-13 | 2016-01-21 | Platypus Technologies, Llc | Detection of gas-phase analytes using liquid crystals |
| CN105950548A (en) * | 2016-05-26 | 2016-09-21 | 南京医科大学附属南京儿童医院 | Method for efficiently obtaining beating functional cardiomyocytes from mesenchymal stem cells |
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2017
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| CN103237887A (en) * | 2010-10-06 | 2013-08-07 | 车比奥及戴奥斯泰有限公司 | Embryonic stem cell-derived cardiomyocytes and cell therapy product using same as active ingredient |
| US20160018371A1 (en) * | 2013-03-13 | 2016-01-21 | Platypus Technologies, Llc | Detection of gas-phase analytes using liquid crystals |
| CN105950548A (en) * | 2016-05-26 | 2016-09-21 | 南京医科大学附属南京儿童医院 | Method for efficiently obtaining beating functional cardiomyocytes from mesenchymal stem cells |
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