CN103614337B - The foundation of IgE mediating mast cell retting conditions model - Google Patents
The foundation of IgE mediating mast cell retting conditions model Download PDFInfo
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Abstract
The invention provides a kind of method setting up people's mast cell degranulation model, specifically comprise step: 1) mast cell line cell is provided, detect the growth curve of cell; 2) typical curve that one or more index quality testings are surveyed is set up; 3) activation step 1) described in mast cell line cell, by activation mast cell line cell centrifugal at 900rpm × 5min, get supernatant liquor as detection sample; 4) use as step 2) as described in the detection sample of one or more index quality testings survey as described in step 3).The present invention still further provides the people's mast cell degranulation model set up according to aforesaid method.Method and people's mast cell degranulation model of the people's of foundation mast cell degranulation model provided by the invention effectively reduce cost, and obviously shorten the time of setting up people's mast cell degranulation model, improve working efficiency.
Description
Technical field
The present invention relates to the research field of I type allergy, be specifically related to the foundation of the mast cell degranulation model of IgE mediation.
Background technology
I type allergy outbreak rapidly, strongly, but is disappeared also soon, therefore also known as type Ⅰ reaction, causing primarily of IgE mediation in serum, is a kind of allergic disorder the most common clinically.There are obvious individual difference and hereditary predisposition.The generation of I type allergy is often divided into two stages: sensitisation phase and Fa Min stage.
At the sensitisation phase of the generation of I type allergy, allergen enters body, activates CD4 ' Th2 cell and B cell, inducing producing specificity IgE antibody (a few peoples produce IgC4).Its Fc section can with mastocyte and basophil film surface bonding, make body be in sensitization.Being specially allergen loses into body by approach such as respiratory tract, digestive tube, injections, activate CD4 ' Th2 cell, the cytokines such as the IL-4 of CD4 ' Th2 cell and secretion thereof can induce allergen specific B cell proliferation to be divided into the plasmocyte producing specific IgE antibody, thus produce specific IgE.IgE is adsorbed in mastocyte and basophil cellular surface.
Mastocyte is mainly distributed in reticular tissue and Submucosa, and basophil is mainly distributed in peripheral blood.This two classes cell surface all expresses high-affinity IgEFc acceptor, can be combined, make body be in sensitization with the Fc section of IgE.
The allergy of I type generation send out quick the stage, when identical allergen enters body again, IgEFab section specific binding surperficial with sensitization target cell (i.e. mastocyte and basophilic granulocyte), trigger the cytolemma change of target cell (i.e. mastocyte and basophilic granulocyte), make its retting conditions and synthesize new active media.These media act on corresponding effector, cause effector pathological change.The active media of mastocyte and the rear release of basophil activation mainly contains histamine, kininogenase, leukotriene, PGD2, platelet activation factor.
In order to study the allergy of I type and suppress the anaphylactic disease mediated by IgE usually to need to utilize the mastocyte that separant induction is cultivated from people's marrow and bleeding of the umbilicus to set up people's mast cell degranulation model, but, the mastocyte that separant induction is cultivated from people's marrow and bleeding of the umbilicus, cost intensive, culture cycle are longer, therefore how the immortal human mast cell line of acquisition is used for anaphylaxis research, setting up the people's mast cell degranulation model mediated by IgE is the anaphylactic disease urgent problem that research I type allergy and suppression are mediated by IgE.
Summary of the invention
For above-mentioned needs, the invention provides a kind of method setting up people's mast cell degranulation model based on immortalization mast cell line, its technical scheme is specific as follows:
Set up the method for people's mast cell degranulation model, the steps include:
1) mast cell line cell is provided, detects the growth curve of cell;
2) typical curve that one or more index quality testings are surveyed is set up;
3) activation step 1) described in mast cell line cell, by activation mast cell line cell centrifugal at 900rpm × 5min, get supernatant liquor as detection sample;
4) use as step 2) as described in the detection sample of one or more index quality testings survey as described in step 3);
Wherein, the activation step 1 described in step 3)) described in the step of mast cell line cell be:
A) will be resuspended with tyrode after described mast cell line cell centrifugation, and to adjust cell concn be 1 × 10
6cell/ml;
B) get 5 parts of cell suspensions, add respectively Biotin-IgE make the final concentration of described Biotin-IgE be respectively 0,50,100,500,1000ng/ml;
C) by as step b) as described in added Biotin-IgE respectively cell suspension be placed in 37 DEG C and hatch 2 hours;
D) will as step c) as described in hatch after 5 parts of cell suspension centrifugal segregation supernatants, obtain 5 parts of cell precipitation things;
E) to steps d) described in 5 parts of cell precipitation things in add tyrode respectively, and add Streptavidin respectively, the final concentration of Streptavidin described in making every part is 1000ng/ml, mixing re-suspended cell, obtains 5 parts of cell re-suspension liquid;
F) by step e) described in 5 parts of cell re-suspension liquid 37 DEG C hatch half an hour, piping and druming cell to suspend, 900g × 5min is centrifugal.
Preferably, the mast cell line cell described in step 1) is LAD2 cell.
Further preferably, step 2) described in index thing be histamine and/or Hex.
The present invention still further provides the people's mast cell degranulation model according to aforesaid method establishment.
Successfully the immortal human mast cell line of acquisition is used for anaphylaxis research by the method for the people's of foundation mast cell degranulation model provided by the invention and people's mast cell degranulation model, significantly reduce the anaphylactic disease for research I type allergy and suppressing is mediated by IgE and the research cost of the people's mast cell degranulation model mediated by IgE set up, and shorten the time of setting up the people's mast cell degranulation model needs mediated by IgE, improve working efficiency.
The mastocyte of separation and purification inductive formation from people's venous blood or bleeding of the umbilicus, induction duration is generally 7-8 week, and the factor required in inducing culture process, except STEM CELL FACTOR, also needs interleukin-13 and interleukin 6, toxigenic capacity is high and the cycle is long, limits its application.
In LAD2 cell cultivation process, contriver finds that cell doubling time is 10 days, with
nagaiKgrowth conditions Deng people's report in 2012 is consistent.Between passage incubation period, the good cell of state generally can be laid in bottom Tissue Culture Flask uniformly, and the slightly poor cell of state may be assembled agglomerating, blows and beats gently and cell state can recover gradually after continuing to cultivate.
Retting conditions is the mark of mast cells activation, and histamine is the main inflammatory mediator of mast cell degranulation.Because histamine molecular weight is little, non-immunogenicity, in blood plasma, the transformation period is shorter, there is certain difficulty in clinical detection.The method that current test in laboratory histamine is conventional has enzyme-linked immunosorbent assay, spectrophotofluorimetry, radio immunoassay.The superfine employing spectrophotofluorimetry of Guo Yong, detect after utilizing histamine sample and o-Xylol (OPT) to hatch altogether, stable not (the Guo Yongchao of the method result, Li Zhenxing, Lin Hong. histamine, tryptase, the Hex mutual relationship [J] in mastocyte release in vitro process. cell and molecular immunology magazine, 2009,259 (12): 1073-1075).Although radioimmunology susceptibility is high, but complicated and time consumption, particularly each laboratory all needs to be separated methyltransgerase (Sun Renshan voluntarily, Liu Rongqing. the mark [J] of mast cell degranulation. foreign medical science clinical biochemistry and ecsomatics fascicle, 2001,22 (2): 100-101).The euzymelinked immunosorbent assay (ELISA) of the present invention's application, after histamine sample acylations, interpolation histamine alkalescence enzyme connection thing is hatched altogether and is detected, and detected result is reliable and stable.Histamine release level raises gradually along with the rising of Biotin-IgE concentration, can be applied to the mast cell degranulation research of IgE mediation.Histamine and Hex are the conventional Testing index of mast cell degranulation.Detect mast cell degranulation and can also use tryptase, prostaglandin(PG), interleukin, TNF etc.; the LAD2 cell degranulation model of the IgE mediation that the present invention preferably utilizes histamine and Hex two indices successfully to set up, lays a good foundation for studying the allergy of I type further.
The RBL-2H3 cell that the more mast cell line of current application has rat basophilic granulocyte to originate, mouse hypertrophy cell knurl P815 cell, people's mast cell line has HMC-1 cell and LAD2 cell.Expression level is low, granule content is few for the IgE high-affinity receptor (Fc ε RI) of people mast cell line HMC-1.Recently the mast cell line LAD2 cell derived set up is in the marrow of a mastocytosis patient, built by NIH's anaphylactic disease laboratory (laboratoryofallergicdiseases) and be tied to form merit, confirm that this cell almost comprises the biological property of complete people's original cuiture mastocyte from aspects such as phenotype, caryogram and Anaphylactic mediators, be more suitable for the research carrying out the allergy of I type.
This mast cell line of culture & identification of the present invention, establishes the people's mast cell degranulation model mediated by IgE.
Accompanying drawing explanation
The form of the LAD2 cell that Fig. 1 a observes under being depicted as common inverted microscope (× 200);
Fig. 1 b is depicted as after 0.5% Toluidine blue staining, the form of the LAD2 cell observed under inverted microscope (× 200);
Fig. 1 c is depicted as after improvement Giemsa staining, the form of the LAD2 cell observed under inverted microscope (× 200);
Fig. 1 d is depicted as the form of the LAD2 cell that LAD2 cell transmission electron microscope (× 8000) is observed;
Figure 2 shows that LAD2 cell growth curve;
Figure 3 shows that the typical curve measuring histamine, wherein correlation coefficient r=0.99945;
Figure 4 shows that the rising along with sensitization concentration, the bar graph that the emission levels of histamine raises gradually;
Along with the rising of sensitization concentration shown in Fig. 5, the bar graph that the emission levels of Hex raises gradually.
Embodiment
In order to make the object of the invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Reagent and cell
Biotin-IgE(USBiological), cell culture medium (STEMPRO-34SFMCompleteMedium), LAD2 cell (No.3 Hospital Attached to No.3 Military Medical Univ., P.L.A.).RhMGF (Peprotech), glutamine, mycillin solution (Gibco), histamine detection kit (BECKMAN), MTT (the green skies), Jim Sa modified form dye liquor, beta-amino hexose, citric acid, Trisodium Citrate (sigma), the glutaraldehyde (commercially available prod, analytical pure) of 2.5%, Streptavidin toluidine blue, dimethyl sulfoxide (DMSO), trypan blue, toluidine blue, the raw work in TritionX-100(Shanghai).Preparation calcic tyrode (Tyrode) and Na
2cO
3/ NaHCO
3reagent needed for damping fluid is domestic analytical pure.
Instrument
Carbon dioxide cell incubator (Thermo), low temperature desk centrifuge (BECKMAN), inverted microscope (OLYMPUSIX70), transmission electron microscope (HIT H-7500).
The cultivation of embodiment 1LAD2 cell
LAD2 cell cultures in the culturing bottle of 50ml, every bottle of 5ml perfect medium.Perfect medium comprises StemPro-34NutrientSupplement, 100IU/ml penicillin, 100ug/ml Streptomycin sulphate, 2mM glutamine, 100ng/ml rhMGF.Cell density should maintain 0.5x10
6-1x10
6/ ml, half amount changes fresh culture weekly.Utilize six 96 orifice plates, 10000 cells/well, 200ul substratum/hole, every 3 days half amount replaced medium, and utilize mtt assay to detect the growth curve of cell.
The histological stain of embodiment 2LAD2 cell and electron microscopic observation
Observation of cell grown form under inverted microscope, trypan blue detects vigor, Giemsa staining, Toluidine blue staining, Electronic Speculum Ultrastructural observation.
With reference to Fig. 1 a-Fig. 1 d, observe under common inverted microscope, LAD2 cell is irregular cycle, and diameter is about 18-22um(as shown in Figure 1a).After 0.5% Toluidine blue staining, the nucleus of LAD2 cell is Dark grey, and cytoplasmic granule is in light grey (as shown in Figure 1 b); After improvement Giemsa solution, the nucleus of LAD2 cell is Dark grey, and cytoplasmic granule is in light grey (as illustrated in figure 1 c); Under transmission electron microscope, the gathering around of the nucleus (in Fig. 1 d with N indicate) of LAD2 cell is differed in size particle (as shown in Figure 1 d) in a large number.
Embodiment 3 sets up the typical curve that histamine detects
Carry out according to histamine detection kit specification sheets, be summarized as follows: get 6 1.5mlEP pipes, add the acyl group damping fluid of 25ul respectively, add the histamine standard substance of 100ul respectively, concentration is respectively 0nM, 1nM, 3nM, 10nM, 30nM, 100nM.Add 25ul acyl reagent respectively and vortex immediately.Inhale 50ul acylations standard substance to antibody bag by hole.Add the enzyme connection thing in 200ul/ hole.Put 4 DEG C of refrigerators and hatch 18h.Manually wash the substrate that plate respectively adds 200ul for three times afterwards.18-25 DEG C of lucifuge, rocks while hatch 30min.Add the stop buffer of 50ul.Read absorbancy OD value at 405nm place, each concentration standards does a multiple hole.CurceExpert1.3 Software on Drawing typical curve.
In mtt assay determination experiment, contriver utilizes the relation that in testing process, formazan growing amount is directly proportional to viable count, infers the number viable cell according to optical density(OD) OD value.Result display LAD2 cell doubling time is 10 days (as shown in Figure 2).
The principle of work of histamine test kit is competitive ELISA, and Histamine concentrations is higher, and OD value is lower.The concentration of histamine standard substance is respectively 0nM, 1nM, 3nM, 10nM, 30nM, 100nM.With OD value for ordinate zou, Histamine concentrations is X-coordinate, and drawing standard curve is as Fig. 3.
The activation of the LAD2 cell of embodiment 4IgE mediation
Resuspended with tyrode after LAD2 cell centrifugation, adjustment cell concn is 1 × 10
6cell/ml, get 5 1.5mlEP pipe, often pipe adds 0.5ml cell suspension, add successively Biotin-IgE make its final concentration be respectively 0,50,100,500,1000ng/ml, hatch 2 hours for 37 DEG C.Centrifugal remove supernatant after, add tyrode 0.5ml, then add Streptavidin 5ul respectively, make its final concentration be 1000ng/ml, mixing re-suspended cell, hatch half an hour for 37 DEG C.Piping and druming cell is to suspending, and 900g × 5min is centrifugal.Experiment negative control: use ddH
2o replaces Biotin-IgE; Total Hex release: after 0.5ml cell suspension 1000rpm × 5min is centrifugal, after adding 0.5mlTritionX-100 reaction 30min, 900g × 5min centrifuging and taking supernatant is as detection sample.All adopt 3 parallel laboratory test pipes in experiment, repeat experiment three times.
Embodiment 5 histamine detects
Acylations and the enzyme of histamine detection sample join the step of step with embodiment 3.
Embodiment 6 Hex detects
Aspirate supernatant 30ul is on 96 orifice plates, and the 1mM hexosamine and the 30ul cell conditioned medium liquid that add 50ul react 2 hours in 37 DEG C of incubators.With the Na2CO of 0.1mol/L after 2h
3/ NaHCO
3termination reaction, and detect the sightseeing degree under 410nm by microplate reader.
Adopt in experiment raise gradually Biotin-IgE sensitization concentration (0-1000ng/ml), mastocyte is activated with Streptavidin (1000ng/ml) after 2 hours, result display is along with the rising of sensitization concentration, the emission levels of histamine and Hex raises gradually, and reaches plateau (Fig. 4, Fig. 5) in 500-1000ng/ml.The calculation formula of Hex release rate is: Hex release rate %=stimulates Hex burst size/total Hex burst size %.
Embodiment 7 statistical procedures
Adopt SPSS13.0 statistical software to carry out data analysis, data are used
represent, compare employing two sample average t between 2 groups and check.P < 0.01 points out difference to have significance.P>0.05 prompting is without obvious statistical significance.
The foregoing is only preferred embodiment of the present invention, be not used for limiting practical range of the present invention; If do not depart from the spirit and scope of the present invention, the present invention is modified or equivalent to replace, in the middle of the protection domain that all should be encompassed in the claims in the present invention.
Claims (2)
1. set up a method for the people's mast cell degranulation model mediated by IgE, it is characterized in that, comprise step:
1) cultivate LAD2 cell in perfect medium, obtain LAD2 mast cell line cell, detect the growth curve of this cell;
2) typical curve of the detection of histamine and/or Hex index thing is set up;
3) adjusting LAD2 cell concn is 1 × 10
6cell/ml, activation, by centrifugal at 900rpm × 5min for the mast cell line cell of activation, gets supernatant liquor as detection sample;
The step of described activation is:
A) by resuspended with tyrode after described LAD2 cell centrifugation, adjustment cell concn is 1 × 10
6cell/ml;
B) get 5 parts of cell suspensions, add respectively Biotin-IgE make the final concentration of described Biotin-IgE be respectively 0,50,100,500,1000ng/ml;
C) by as step b) as described in added Biotin-IgE respectively cell suspension be placed in 37 DEG C and hatch 2 hours;
D) will as step c) as described in hatch after 5 parts of cell suspension centrifugal segregation supernatants, obtain 5 parts of cell precipitation things;
E) to steps d) described in 5 parts of cell precipitation things in add tyrode respectively, and add Streptavidin respectively, the final concentration of Streptavidin described in making every part is 1000ng/ml, mixing re-suspended cell, obtains 5 parts of cell re-suspension liquid;
F) by step e) described in 5 parts of cell re-suspension liquid 37 DEG C hatch half an hour, piping and druming cell to suspend, 900g × 5min is centrifugal;
4) use as step 2) as described in index quality testing survey detection sample as described in step 3), during detection, concentration is the Streptavidin activation mastocyte of 1000ng/ml.
2. the LAD2 people's mast cell degranulation model mediated by IgE of a method establishment according to claim 1.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1575335A (en) * | 2001-10-22 | 2005-02-02 | 国家农艺研究院 | Pig mast cell cultures and uses thereof |
| WO2010105215A2 (en) * | 2009-03-12 | 2010-09-16 | University Of South Florida | Human mast cell line and uses thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1575335A (en) * | 2001-10-22 | 2005-02-02 | 国家农艺研究院 | Pig mast cell cultures and uses thereof |
| WO2010105215A2 (en) * | 2009-03-12 | 2010-09-16 | University Of South Florida | Human mast cell line and uses thereof |
Non-Patent Citations (4)
| Title |
|---|
| Proteomic Analysis of IgE-Mediated Secretion by LAD2 Mast Cells;Matthew C. Gage et al.;《Journal of Proteome Research》;20090529;第4117页左栏第3段 * |
| 三种细胞用于建立体外肥大细胞脱颗粒模型的比较研究;高婕;《中国优秀硕士学位论文全文数据库医药卫生科技辑》;20130415(第4期);中文摘要第II页到第III页方法1-2,正文第21页1.1节细胞系 * |
| 大鼠腹腔肥大细胞与RBL-2H3细胞脱颗粒方法的建立及其在中药注射剂安全性评价上的应用;李咏梅等;《中国实验方剂学杂志》;20111130;第17卷(第22期);152-157 * |
| 肥大细胞系LAD2的培养及肥大细胞脱颗粒模型的建立;杨永强等;《中国皮肤性病学杂志》;20140115;第28卷(第1期);26-29 * |
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