CN101149371B - Mouse immune state monitoring reagent and its preparing process - Google Patents
Mouse immune state monitoring reagent and its preparing process Download PDFInfo
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Abstract
This invention provides a kind of mouse immunity state test reagent, this reagent is PBS suspension of mouse spleen cell in two inbreeding system which is stained by CFSE, the total concentration of the two kind of mouse spleen cell is 0.5-1 multiply 10 to the power 8 per ml, the proportion is 1:1, fluorescence tinction differential concentration is 1:20. Mainline the reagent in the model mouse; it can judge the immunity state of the model mouse by the change of the proportion of the two cell in this reagent when letting blood to test.
Description
Technical field
The present invention relates to medical configuration product, be specifically related to derive from the medical configuration product of animal material.
Background technology
Graft-rejection (transplantation rejection) is the graft function that immune response caused forfeiture or receptor's body damage of being induced by transplantation antigen, is a crucial difficult problem of making every effort to overcome clothes due to the main cause of clinical transplantation failure and the transplantation immunology.In homograft, even operation is very successful, graft also can because of rejection can not long-term surviving.Therefore, graft rejection is the emphasis of medical circle research always, and in the research process of graft rejection, the acceptor immune state detects the link that is absolutely necessary.But still do not have a kind of reliable reagent/method can detect the acceptor immune state at present, what existing acceptor immune state detected mainly is to reflect immune state indirectly by detecting some amynologic index, all has different defectives.
The index that detects commonly used has following several:
1, periphery blood T cell counting.Studies show that CD4/CD8 ratio homologous transplantation rejection is relevant.Rejection often betides among the low patient of CD4/CD8 ratio, with monoclonal antibody immunity fluorescence method or cells were tested by flow cytometry T cell and subgroup thereof, before the clinical symptoms of acute cellular rejection occurred, T total cellular score and CD4/CD8 ratio raise, and ratio reduces during cytomegalovirus infection; Ratio difference of each family report, it is generally acknowledged when ratio greater than 1.2 the time, the indication acute cellular rejection is about to generation; Ratio is very big less than 1.08 possibility of infection.If can carry out dynamic monitoring, the antidiastole of acute cellular rejection and infection is had important value.Yet, also there are some scholars to think, CD4/CD8 ratio and rejection do not have direct relation, and each stand one's own ground comes from the kind type diversity (heterogeneity) of CD4 and CD8 subgroup for different results of study.For example: CD8 can be divided into inhibition type and cell toxicant type, also has part NK cell also to express CD8 antigen.Equally, CD4 also can be divided into adult form and primary tape according to the expression of CD29 and CD45, and both having made is to have distinguished various hypotypes, and the relation of CD4, CD8 homologous transplantation rejection is still not obvious, and further research awaits.
2, killer cell CTL and NK cytoactive are measured.Transplant the application of back because of immunodepressant, the activity of killer cell is suppressed, but can obviously increase before acute cellular rejection.Method commonly used at present is in vitro test, get after the deactivation of donor lymphocyte as irritation cell, separate receptor's lymphocyte and make reacting cells, two kinds of mixing with cells are directly done unidirectional mixed lymphocytes test, the result who records is Tc cell and the coefficient result of NK cell.
3, MHC Molecular Detection.The target antigen that organ transplant rejection is discerned mainly is the MHC molecule that is expressed in the graft cell surface.The MHC molecule can be by direct way with approach be by receptor T cell recognition indirectly, and it has important effect in organ transplant rejection.The MHC molecule extensively distributes in vivo, and MHC has 2 kinds of forms in the human body: the one, be present in nearly all histocyte surface with the membranous antigen form, and the 2nd, be present in soluble form in multiple body fluid such as serum, urine, seminal fluid, milk, saliva and the juice.Serum soluble MHC Molecular Detection is more convenient, detects serum soluble HLA-antigen levels with ELISA, can point out rejection.Serum soluble MHC Molecular Detection is used more at home at present.With respect to serum soluble MHC molecule, it is less that PERIPHERAL BLOOD MONONUCLEAR CELL surface MHC molecule is subjected to ectocine.When PERIPHERAL BLOOD MONONUCLEAR CELL took place to repel at post-transplantation, surface expression MHC molecular level increased, and research mainly is the MHC-II molecule at present.That is that all right is ripe in the detection of cell surface MHC molecule, and flow cytometry is the method for using early, and the molecular biology method particularly application of fluorescence real-time quantitative polymerase chain reaction (PCR) makes the detection of cell surface MHC molecule use day by day extensive.In human HLA-DR as the lymphocytic activate sign of T, HLA-DR positiveization T lymphocyte increased when rejection took place, and also had document to point out that the increase of HLA-DQ positiveization monocyte can be used as the validity index of transplanting the acute rejection early diagnosis in the recent period.But the monocyte in the human tip blood nearly all is HLA-DR, HLA-DQ, CD80, CD86, CD40 etc. in the process of function maturation are the guide as antigen presenting cell, HLA-DR shows cell surface, so a part of scholar thinks that with this index as the immune response initial stage be more rational.
4, cytokine assay.Serum il-2R measures, and can disengage IL-2R behind the t cell activation, and the serum content of IL-2R raises when acute cellular rejection and virus infections, increases the most obvious during with cytomegalovirus infection.And the content significant difference of serum il-2R between individuality does not have the diagnostic criteria of generally acknowledging, has limited its Clinical Application, and dynamic measurement can overcome this shortcoming.Cellular immunity is being brought into play main effect in rejection, studies show that at present Th1/Th2 lymphocytic be equilibrated in the immunclogical response of transplantation quite important.During transplanted kidney generation acute rejection, antigen presenting cell identification exotic antigen is through signal transmission and Th cell activation.The Th1 lymphocyte can impel propagation, differentiation, the maturation of TDTH cell and Tc cell by cell factors such as secretion of gamma-IFN, IL-2, IL-12.Thereby inspire cellullar immunologic response, finally cause acute transplant rejection.The Th2 lymphocyte can be by discharging TL-4, and cell factors such as TL-10 stimulate B cell proliferation and produce specific antibody, thus the certain effect of performance in the chronic transplant rejection reaction.The research of part zoografting tolerance in recent years prompting strengthens the same expression that can suppress the Th1 cell often of Th2 lymphocyte function, thereby helps the foundation of peripheral tolerance.By mensuration, can react immune state to Th1/Th2 lymphocyte relevant cell factor.Also be one of immune state monitoring research focus at present.
5, the mensuration of Perforin, granzyme B, CD40L and FasL.The expression of cellulotoxic effect molecule is becoming the sensitivity of diagnosing acute rejection and special index.During acute rejection in peripheral blood lymphocyte or the Perforin in the graft local organization, granzyme B and CD40L gene, express all and obviously raise.
6, chimera state or gene therapy might become in the future the index of immune detection in tolerance induction.Chimerism is meant and has the cell with graft antigen active, i.e. chimera (microchimerism) in the recipient's body.Starzl etc. think that chimera may be one of reason of keeping immune tolerance.There is data to show that chimeric clinically disappearance is relevant with the generation of chronic rejection.By a large amount of research, it is found that to be entrenched in to transplant in different period of back and the different transplant organ all can occur, in acute and chronic repulsion, also can occur.
7, circulating antibody detects.Panel reaction antibody (PRA) is meant the anti-hla antibody in the organ transplant recipients body, because the diversity of humam leucocyte antigen, the corresponding antibody kind also is diversified.In 2 weeks of post-transplantation, anti-hla antibody can appear in the recipient's serum.The PRA height of preoperative and postoperative is obviously relevant with the postoperative acute rejection, so dynamic measurement PRA level is particularly important, also is a monitoring index of extensively being admitted and using at present.The application of PRA is existing for many years, but the specificity that adopts enzyme-linked immunosorbent assay antibody recently kind surplus in the of 40 nearly.For diagnostic analysis brings difficulty.
Additive method such as SC mensuration etc. also have report, but its meaning is imprecise.
Detection method commonly used at present:
1,51Cr release test.The present the most frequently used standard test method of detection specificity T lymphocytotoxicity test (LCT) is still the 51Cr release test, and this law result is accurate, good reproducibility; But also have the following disadvantages: 1. use radioactive 51Cr that the danger of r gamma ray activity is arranged, be unfavorable for safe operation and Waste disposal, and need the particular assay instrument; 2. the spontaneous release rate height of 51Cr, some cell marking problem and susceptibility are low etc., often influence result's judgement greatly because of different target cell labeling effciencies change difference; 3. the 51Cr half life period (27.8 days), can't be used for the animal experiment that repeatedly to measure; 4. it is short and the test operation step is many that cell is educated the time altogether, can not measure in the individual cells level.Therefore people attempt to seek the CTL activity determination method that can substitute the 51Cr method for releasing always for many years, as adopting sensitive reliable, the simple heterotope determination method of technology such as fluorescence labeling, flow cytometry and reporter gene.And the 51Cr release test is in vitro test, and CTL and NK cell activity are just measured in test itself, and only reacting cells immune state to a certain extent can not react immune environment complicated in the body comprehensively.
2, adopt the ELISA method with the cytokine concentration in the acceptor serum as monitoring index, and the approval that has obtained some scholars with the validity of the method for cytokines mRNA in the RT-PCR method detection graft biopsy specimen, but cell factor is the network of a complexity, and the single cell factor can not reflect immune state comprehensively.
3, solid-phase enzyme-linked immune spot test (Enzyme-linked Immunospot Assay ELISPOT): ELISPOT can T cells with antigenic specificity reaction in external quantitative measurement patient P BMC (the T cell is produced by antigenic stimulus and discharges IFN-γ) by the secretion of the cell factor (as IFN-γ) of detection antigen induction.This law is between the T of peptide specific secretion of gamma-IFN cell quantity and cytotoxic activity, relevant with the 51Cr method for releasing of standard good, be a kind of in clinical testing the monitoring patient to the sensitivity of the CTL of tumour antigen or transplantation antigen or Th-cell effect, accurate, inexpensive, method.But only can pass through cell factor indirect reaction immune state.
4, Luminex: be a multi-functional liquid-phase chip analysis platform, be generally used for researchs such as immunoassay, nucleic acids research, enzymatic analysis, acceptor and part discriminance analysis.Liquid-phase chip technology can detect a plurality of cell factors simultaneously.
5, Protocols in Molecular Biology: in recent years, be accompanied by the progress of cellular elements biology techniques, the detection of mRNA becomes possibility in the graft, and molecular level has been brought up in the research of immune response process, and the value of immune indexes in diagnosis receives publicity.
6, low cytometric analysis: along with the progress of low cytometric analysis, the research of cellular antigens is further developed.Utilize the method for interior Th1 of CD3, CD4, CD8 and cell and Th2 multiple staining to find, use low cytometric analysis and in the case of acute rejection, can monitor activate initial stage T lymphocyte (CD69), IL-2 positive t lymphocytes (T) increase, IL-4/IL-10 positive t lymphocytes (Th2) reduces in the cell, and Th1/Th2 is unbalance during rejection.
Because immunoreactive complicacy, though various detection techniques have had significant progress, though above-mentioned immunological detection method detects levels of precision and improves constantly, but its limitation is arranged all, rely on single index to be not enough to judge the immune state of body complexity, set up a kind of joint assessment pattern (pattern/profile) on many indexs basis and monitor that to transplant the back acceptor state of exempting from service be the difficult problem that immunologist and transplanting worker need solve.Mainly a situation arises (pathological biopsy) and the graft function situation is assessed by rejection to weigh at present the usefulness of rejection and immunosuppressive therapy after the organ transplant clinically.But graft living tissue pathologic finding also has its limitation: one. it is a kind of damaging inspection method that biopsy detects, normal survival to graft has certain influence, can not take sample repeatedly, in addition, take that sample is very few can to influence the correctness that pathological section is read sheet again.They are two years old. and the biopsy pathological examination is the non-quantitation index, influences the accuracy of its clinical diagnosis, and pathological examination is read the sheet easily person's the subjective factor and the influence of technical merit produce bias, lack objectivity.They are three years old. and check pathological section can't be done functional judge to the wellability lymphocyte, be unfavorable for the early diagnosis of rejection. lose the receptor of function the early stage or graft of rejection generation, even show as the receptor of stable type clinically, the pathological biopsy section all can be seen a large amount of T lymphocytic infiltrations, or only shows as spot T cellular infiltration.
Summary of the invention
The invention provides a kind of mouse immune state monitoring reagent, can quick, intuitive and accurate monitoring transfer operation after the immune state of mouse.
The present invention realizes that the technical scheme of above-mentioned purpose is:
A kind of mouse immune state monitoring reagent, this reagent is through diacetyl Fluoresceincarboxylic acid-succinimide ester (carboxyfluorescein diacetate succinimidyl ester, CFSE) the PBS suspension of the mice spleen cell of Ran Se two kinds of inbred strais, wherein the total concentration of two kinds of mice spleen cells is 0.5～1 * 10
8Individual/ml, the ratio of the two is 1: 1, and the fluorescent dye concentration difference is 1: 20.
The preparation method of the suspension of immune state monitoring of the present invention, this method is made up of following steps:
1) gets the mice spleen cell of two kinds of inbred strais respectively, suspend and adjust cell concentration, obtain two kinds of concentration and be 10 with PBS
7The cell suspension of individual/mL;
2) with concentration be the dimethyl sulfoxide solution of CFSE of 10mM after with 50 times PBS dilution 200 μ M CFSE working fluids;
3) add the CFSE working fluid of step 2 gained in a kind of cell suspension of step 1 gained, the final concentration that makes CFSE is 0.3 μ M, the CFSE working fluid that adds step 2 gained in the another kind of cell suspension of step 1 gained, making the CFSE final concentration is 6 μ M, is to hatch dyeing 15～20 minutes under 37 ℃ in temperature then;
4) collect staining cell in the cell suspension of step 3 gained respectively, be resuspended in PBS with after 4 ℃ of PBS washings of 0.01M 2～3 times;
5) in two kinds of staining cell suspensions of 4 of 1: 1 ratio blend steps of cell number, and to adjust the cell total concentration with PBS be 0.5～1 * 10
8Individual/mL, get final product.
In actual production process, need be to the suspension sampling Detection and the monitoring of immune state monitoring of the present invention.Concrete detection method is as follows:
Get 0.5ml adding test tube after getting reagent mixing of the present invention, add 0.4% of 0.5ml again and expect blue dye liquor, mixing left standstill 1-2 minute.Simultaneously, be ready to blood cell counting plate and cover glass.With reagent sucking-off of the present invention a little, drip at the cover plate edge in right amount, reagent of the present invention is full of between cover plate and the tally.After leaving standstill 0.5 minute, observe down with being inverted the optical microscope mirror: on the left of count plate four big lattice total cellular score, line ball cell are only counted and top; Be calculated as follows then: the big lattice total cellular score of cell number/ml=4/4 * 10000, add up the cell total concentration respectively, and dead cell (mazarine) concentration, deduct the concentration that dead cell concentration is suspension of the present invention with the cell total concentration.With the rate of dyeing of Flow cytometry reagent of the present invention, the negative reference of cell that PBS handles with reagent sample up flow type cell instrument of the present invention, is set 488nm to excite/the 525nm filtration, and measuring the stained positive cell proportion needs greater than 95%.
The mice spleen cell of two kinds of inbred strais of the present invention, wherein a kind of is test cell, come from mouse individuality with the same inbred strais of transplantation donor, this cell and graft are homogenic, antigenicity and graft are identical, can be reflected the immune attack that graft is suffered exactly by the immune response specific killing that produces because of transplanting; Another kind is the confidential reference items photo cell, come from mouse individuality with the same inbred strais of transplant recipient, homogenic with acceptor, as the confidential reference items of immune state monitoring, be used to eliminate because clasmatosis, death, a series of cells error that non-specific loss such as natural death brings in external dyeing processing and body such as go back to the nest.In the present invention, the any cell of special provision is not a test cell, any cell is the confidential reference items photo cells, this is that selection by the acceptor of transfer operation and donor is determined: with the mouse cell of the same inbred strais of acceptor be the confidential reference items photo cell, with the mouse cell of the same inbred strais of donor be donorcells.
After reagent of the present invention is arrived the transplantation model mouse by intravenous injection, can change the immune state of judging the transplantation model mouse by the ratio of two kinds of cells in the blood drawing detection different time sections reagent of the present invention.Generally speaking, if the ratio of 2～4 hours two kinds of cells in injection back promptly shows notable difference (test cell sharply descends, and the confidential reference items photo cell then changes small), illustrate that acceptor mouse body immune system is in high repulsion state to graft.Because two kinds of cells in the reagent of the present invention all pass through the dyeing of fluorescent dye variable concentrations of the same race, can detect the ratio variation of two kinds of fluorecytes in the acceptor easily and intuitively by flow cytometer.
Mouse immune state detectable of the present invention, can be intuitively, accurately, comprehensively the back immune state is transplanted in monitoring: (1) by with interior direct contrast with reference to recipient cell, the size of area can quicklook under the peak of the flow cytometer detected peaks of compare test cell and confidential reference items photo cell knows does not need testing result to do complicated calculating again and can understand the immune situation of acceptor to graft; (2) the fluidic cell test can be accurate to individual cells level, reliable results; (3) antigenicity of test cell and graft are identical, and the result can accurately reflect the immune attack that graft is suffered; The setting up of interior reference eliminated cell because broken, dead, a series of cells error that non-specific loss such as natural death brings in external dyeing processing and body such as go back to the nest; (4) reflect immune state by the specificity loss of test cell, rather than the rising or the reduction of the unilateral some cell factors of dependence, perhaps what of certain a group cell, active height judges that its result is more comprehensively, accurately.
To prove that immune state monitoring reagent of the present invention can detect the immune response state of transplanting the back acceptor effectively by zoopery below.
One experiment material
(1) animal used as test
Health, cleaning level, inbred strais C57BL/6j (C57; H-2b) female mice, 6 ~ 8 ages in week, body weight 16 ~ 22g (the animal used as test certification of fitness number: SCXK 2006-0015,2006B008,2005A044), as donor.BALB/c (BALB; H-2d) ages in female mice 6～8 week, and body weight 16～22g (the animal used as test certification of fitness number: SCXK 2006-0015,2006B008,2005A046), as acceptor.Available from Nanfang Medical Univ's Experimental Animal Center.The SPF level is raised.Observed for 1 week before the experiment, standard feed is fed, and drinking-water arbitrarily.
(2) main agents
1, CFDA-SE Cell Tracer Kit:Molecularprobes company (U.S.)
2, dimethyl sulfoxide (DMSO): Sigma
3, paraformaldehyde: Guangzhou Chemical Reagent Factory
4,0.5% trypan blue solution: Guangzhou Chemical Reagent Factory
5, distilled water: the court centers for making of pharmaceutical preparations
6, ammonium chloride: Guangzhou Chemical Reagent Factory
7, erythrocyte cracked liquid: precision takes by weighing NH
4Cl 0.83g, KHCO
30.1g and EDTA
2Na 0.004g is dissolved in the 1000ml tri-distilled water.
8, PBS damping fluid (0.1M): precision takes by weighing NaCl 8.00g, KCl 0.20g, Na
2HPO
42.08g, KH
2PO
40.20g be dissolved in the 1000ml tri-distilled water.
(3) main experimental apparatus
1, the electronic analytical balance ER120A U.S.
2, table model high speed centrifuge TDL-50B Shanghai
3, the superclean bench Thermo Forma U.S.
4, CO
2The incubator SL-JC-2323 U.S.
5, OLYMPUS phase microscope CK-2 Japan
6, OLYMPUS photographic system PM-CBAD Japan
7, refrigerated centrifuge Eppendorf Germany
8, micro sample adding appliance Eppendorf Germany
9, the constant water bath box Grant U.S.
10, stream Schwann Cells instrument EPICS ELITE Beckman-Coulter company
11, fluorescence inverted microscope LEICA Germany
12, pH acidometer pHS-25 type Shanghai
Two, experimental technique
1, test grouping
After setting up C57-BALB/c mouse skin transplant rejection model, tail vein injection reagent of the present invention (preparation method sees embodiment 1), be divided into 7 groups by different time points behind the infusion, be respectively 1h, 2h, 4h, 10h, 1d, 2d, 6d group, every group of 5 model mouses, other establishes isograft BALB/c-BALB/c control group.
2, the foundation of dermatoplasty model
Press inbred mouse dermatoplasty national standard, and carried out suitable improvement according to the experimental observation needs, step is as follows:
1) test material is placed 121 ℃, 40min autoclaving in the pressure cooker.
2) get 10 of inbred strais C57 mouse at random, 35 of BALB/c mouse.
3) body weight is numbered and taken by weighing to every animal respectively, detail record family name, sex, birthdate, pedigree and other features.
4) prepare 0.7% amobarbital sodium solution with stroke-physiological saline solution.
5) adopt the intraperitoneal injection of anesthesia animal.The every 10g body weight injection of mouse 0.1mL.
6) at first anaesthetize C57 for the skin mouse, treat that Animal Anesthesia is lost consciousness after, its back is placed on the fixed head up, fixedly animal is sterilized behind the preserved skin and with 3% tincture of iodine cotton balls and 75% cotton ball soaked in alcohol.
7) cut about the skin of diameter 5mm～10mm each one at the back.
8) skin graft that shears is turned the double dish of putting into a small amount of physiological saline, use eye scissors, cut hypodermis lightly to corium, standby with stroke-physiological saline solution flushing back then.
9) anesthesia acceptor BALB/c mouse.
10) contrary cant and does not have fixedly skin edge of wound suture line with 5-0 to transplanting the C57 skin graft in the back.
11) cover the gauze piece that was coated with vaseline and novocillin, 3～4 layers, fix the degree of tightness appropriateness with the wide strapping of 1cm.
12) after operation finishes to treat that animal revives, animal is put into the mouse box, and hang up mark card.
13) carry out the control group dermatoplasty with method, donor skin is same inbred strais BALB/c mouse.
3, reagent infusion of the present invention
1) during fixedly mouse cage was put with mouse, only the mouse tail appeared;
2) emerge in worm water mouse tail makes the tail phlebectasia;
3) behind 75% alcohol disinfecting, only puncture the about 0.5mL/ of successfully back injection reagent total amount of the present invention with 4 number sword-shaped needles, every injected in mice total cellular score is about 5～10 * 10
7Individual.Injection quantity can suitably reduce to 1 * 10
7Individual.
4, positive cell ratio in the flow cytometry mouse peripheral blood lymphocyte
1) collection of specimens is got blood, each about 200 μ L with the pili tubule puncture mouse endocanthion that disappears.
2) fluidic cell specimen preparation whole blood erythrocytolysis method operation steps is as follows:
A. get whole blood 100 μ l, put into the 5ml test tube.
B. use 2ml ammonium chloride solution lyse red blood cells at room temperature, about 5min.If red blood cell is dissolved fully, solution is changed to transparent by muddiness.The grasp of noting dissolution degree is very important: if dissolving is undue, then cause on the leucocyte antigen destroyed, perhaps some responsive cell death and cause the change of ratio.If dissolving is not thorough, a large amount of red blood cells can influence lymphocytic analysis.This is because lymphocyte is approaching with red blood cell mass on analysis image, goes out the lymphocyte group time at frame, can confine the part red blood cell in lymphocyte.And erythrocytolysis is not enough or affect analysis result excessively to a great extent.
C. red blood cell is thoroughly dissolved, and adds 0.5% formaldehyde of 1ml PBS dilution, and is centrifugal immediately, cleans with the cold BPS of 3～4ml.Centrifugal 250 * g (about per minute 1500 changes), 5min washes 3 times.Note at every turn with suction pipe the supernatant sucking-off can not be toppled over liquid.
D. with the cell suspension of finishing dealing with in 0.1% formalin of 0.5～1ml.Put 4 ℃, cover light, wait to be analyzed.Cell after fixing can be preserved a week in refrigerator.Can not fix if detect immediately.
E. go up machine on the machine testing: open flow cytometer and argon laser source (488nm), preheating 20min, the instrument auto-initiation, carry out light path and stream standard with the standard fluorescence microballoon, the coefficient of variation of all fluorescence signals (CV) is all less than 2%, in forward scattering light (FS) and side scattered light (SS) scatter diagram, draw a circle to approve cell mass, the selection lymphocyte is an analytic target, remove cell fragment, determine negative scope of fluorescence and PMT voltage standard with negative control, color compensating, every part of sample is measured 10000 cells, and total data is obtained and is analyzed with flow cytometer and software SYSTEMII.
F. kill rate calculates: specific killing rate (R)=1-donor cells/recipient cells * 100%
5, statistical analysis
All data are shown mean ± standard deviation.Use SPSS13.0 and carry out statistical analysis, relatively with the t check, p＜0.05 is thought statistical significance to the kill rate mean between group.
Three, experimental result
1. the dermatoplasty model is observed
1.1 after transplanting one week of back, find since acute cellular rejection C57 skin graft periphery begin shrivelled, come off, two weeks after the dermatoplasty (13 days) cutify come off C57-BALB/c dermatoplasty repulsion modelling fully.
1.2 the dermatoplasty of control group BALB/c homology, the skin graft decrustation, operative site is smooth, have virgin wool to grow.
2. streaming result
2.1 two groups kill rate situation as shown in Figure 1
1) ratio at model group and control group changes different behind two kinds of cell infusions, behind model group and control group infusion reagent 1h of the present invention, 2h, 4h, 10h, 1d, the 2d to the specific killing rate of fluorescent dye C57 mouse boosting cell there were significant differences (t=39.5,41.1,26.2,27.4,15.8,5.5, all P＜0.01).Behind the infusion in one hour and the two hours model group kill rate of fluorescent dye C57 mouse boosting cell respectively up to 87.4% ± 4.5%, 92.2% ± 3.9%.
2) cell proportion of homology dermatoplasty control group mice when 1～4 hour peripheral blood streaming testing result shows that the C57/BALB ratio still remains on injection behind injection reagent of the present invention is about 1: 1, BALB/c mouse does not show has the specific killing effect to allogene C57 splenocyte, slightly kill and wound and just showed in 10 hours the C57 splenocyte had, As time goes on lethal effect obviously strengthens then, is all killed to back three days C57 cells of injection.The cells in vivo toxicity test show homology dermatoplasty BALB/c mouse to allogene C57 cell by not the killing and wounding of beginning, along with the prolongation of time was was in vivo just all killed and wounded after three days, be a process of killing gradually.
3) process of killing and wounding in the dermatoplasty model body is then different with control group, inject promptly to show significantly in back 1 hour and kill and wound, the C57/BALB ratio reduces, and the C57 cell obviously is killed, inject back 2 hours C57 cells and just removed fully substantially, BALB show to donorcells fierce kill and wound characteristic.
2.2 the BALB/c mouse immune state change procedure that two treated animals experiment streaming figure can intuitively react:
Shown in Fig. 2～17: 1) homology dermatoplasty control group BALB/c mouse is a little less than melting rejection with the internal specific cell in 10 hours behind the injection allogene C57 splenocyte, and peripheral blood C57 splenocyte still remained 83% in 10 hours.Along with the prolongation BALB/c mouse of time begins C57 splenocyte rejection is strengthened gradually, the C57 splenocyte is after 1 o'clock fast cleaved 64%.Behind the spleens cell injection the 3rd day, allogene C57 splenocyte was eliminated substantially, only remains 5%.2) and isogenic BALB/c splenocyte to perfusion back the 6th day still higher level exist, and brightness does not reduce.3) model group C57 splenocyte behind infusion 1 hour just only surplus 7%, 2～4 hours residues 5%, repelled fully substantially, and homogenic BALB/c splenocyte only slowly descends with control group is the same.4) two groups of results show that the cells in vivo toxicity test promptly showed notable difference in 2-4 hour behind injection reagent of the present invention, select this time point can accurately judge two groups of residing immune states of mouse, model group mouse kill rate reached 95% in two hours, illustrate that the mouse body immune system is in high repulsion state to the donor specific splenocyte, and homology dermatoplasty control group mice injection reagent of the present invention after two hours the cells in vivo kill rate only be 5%, showing does not in a short time have tangible specific killing effect to heterogenote.Therefore reagent of the present invention can be judged the specific immunity state of vivo immuning system accurately, be to repel or tolerance status promptly to a certain donor, selecting for use reagent of the present invention to inject back 2 hours is optimum time point as the monitoring point, can satisfy the requirement of clinical fast detecting.
2.3 we have carried out the own control experiment for the stability of further research reagent of the present invention, because same mouse got the restriction of blood number of times, every mouse is got the blood time point and can only set at most 5 times, experimental animal adopts not capable dermatoplasty blank BALB/c mouse and C57-BALB/C dermatoplasty to repel model, and streaming result is as follows:
2.3.1 the dermatoplasty blank BALB/c mouse cells in vivo toxicity of not going detects the streaming result
1) mouse 1. different time points cell mixing ratios change
As shown in table 1, (1) homogenic BALB/c splenocyte can be in the medium-term and long-term existence of BALB/c mouse peripheral blood, dropped to 1.8% from accounting for 3.2% of peripheral blood lymphocyte in six days behind the infusion, and decline rate there is the trend of slowing down, illustrates that simultaneously the CFSE cytotoxicity is less; The CFSE of higher concentration (6 μ M) dyeing is little to the activity influence of splenocyte.Homogenic hypocellular speed may be because not have repulsive interaction, cell substantially be naturally-aged death more slowly, and the beginning decline rate may be destroyed relevant through outer mechanical treatment with cell soon; (2) compare with the BALB/c splenocyte, heterogenic C57 splenocyte still showed at 2 hours does not have the specifically inactivating cell proportion still at 1: 1, and just disappearance fast later in a day, in the time of 1 day from 2 hours 3.3% drop to 1.8%, three days is to drop to 0.1% all to disappear basically, two kinds of cells except the MHC difference, obtain at cell in vitro, in the dyeing, body immune environment and handle when detecting all identical, quick inactivating can only the time specific antigen due in the body due to the specific killing.But C57 specific antibody or Specific CTL Cells owing to do not prestore in the BALB/c mouse body are so just significantly show later at 1 day the specific killing that kills and wounds the C57 splenocyte.(3) CFSE dyeing back spontaneity spills few: the splenocyte fluorescence intensity of CFSE dyeing in six days does not weaken, and is still the same in 103 zones with two hours streaming testing results, and it is few to illustrate that the CFSE spontaneity spills, and is a kind of fluorochrome of good Invivo Cellular tracking.(4) it is more simpler, directly perceived and can eliminate and pair dye errors than double-colored singly to dye the histogrammic result formats of design.
The flow cytometer testing result that table 1 mouse 1 different time points cell mixing ratio changes
| |
2 |
1 |
2 days | 3 days | 6 days |
| CFSE dyeing C57 cell | 3.3％ | 1.8％ | 0.6％ | 0.1％ | 0.03％ |
| CFSE dyeing BALB/c cell | 3.2％ | 2.8％ | 2.8％ | 1.7％ | 1.8％ |
2) it is as shown in table 2 that mouse 2 cells in vivo toxicity detect the streaming result, it is as shown in table 3 that mouse 3 cells in vivo toxicity detect the streaming result, its result and mouse 1 basically identical, the deactivation of allogene C57 cell speeded later at two days, and homogenic BALB/c splenocyte disappears in the medium-term and long-term existence of peripheral blood, losss slowly, still exists in a large number in the time of six days, and the CSFE fluorescence intensity is not seen in a week and is weakened, and illustrates that this method test findings is repeatable strong.
The flow cytometer testing result that table 2 mouse 1 different time points cell mixing ratio changes
| |
4 |
2 days | 3 days | 6 days |
| CFSE dyeing C57 cell | 3.1％ | 0.7％ | 0.1％ | 0.1％ |
| CFSE dyeing BALB/c cell | 3.6％ | 2.1％ | 2.2％ | 2.1％ |
The flow cytometer testing result that table 3 mouse 1 different time points cell mixing ratio changes
| |
10 |
2 days | 3 days | 6 days |
| CFSE dyeing C57 cell | 3.0％ | 1.0％ | 0.1％ | 0.1％ |
| CFSE dyeing BALB/c cell | 3.6％ | 2.7％ | 2.7％ | 2.6％ |
2.3.2 dermatoplasty model own control streaming result
Two kinds of cell proportion situations of change of different time points peripheral blood are as shown in table 4 behind same the dermatoplasty model mice injection mixed cell suspension: 1) model promptly only remains 0.3% at two hours allogene C57 cells, and homogenic contrast BALB/c splenocyte is up to 5.7%; Allogene C57 cell only remains 0.1% in the time of 1 day, and homogenic contrast BALB/c splenocyte is up to 3.4%, homogenic BALB/c splenocyte still has 2.5% in the time of 6 days, compare with not capable dermatoplastic normal BALB/c mouse result, the time unanimity that homogenic cell exists in peripheral blood, and transplantation model has powerful lethal effect to donor C57 splenocyte; 2) quick inactivating of allogene C57 splenocyte may have relation with transplanting sensitization, transplant in the body of back and have a large amount of donor specific antibody and special capable CTL cell, when with reagent of the present invention in fluorescent dye C57 cell it is initiated fierce immune attack when meeting, make it that molten cytological effect take place rapidly and disappear; 3) this test can be seen the experience of two kinds of cells in the model mice body intuitively, the allogene donorcells rapidly disappears, and homogenic cell energy long-term surviving, homogenic cell can find out that intuitively cell proportion changes by the streaming histogram as the confidential reference items photo cell, thereby understands the immune state of mouse.
Behind same dermatoplasty model mice injection of table 4 mixed cell suspension
Two kinds of cell proportion situations of change of different time points peripheral blood
| |
2 |
1 |
2 days | 6 days |
| CFSE dyeing C57 cell | 0.3％ | 0.1％ | 0.1％ | 0.1％ |
| CFSE dyeing BALB/c cell | 5.7％ | 3.4％ | 3.0％ | 2.5％ |
Description of drawings
Fig. 1 is model group and control group to fluorescent dye C57 cell killing rate change curve in time,
The expression control group,
The representation model group.
Fig. 2 is 1 hour model group mouse cells in vivo specific killing situation behind the injection reagent of the present invention, and M1 is the streaming peak area of fluorescent dye C57 cell, and M2 is the streaming peak area of fluorescent dye BALB/c cell.
Fig. 3 is 1 hour control group mice cells in vivo specific killing situation behind the injection reagent of the present invention, and M1 is the streaming peak area of fluorescent dye C57 cell, and M2 is the streaming peak area of fluorescent dye BALB/c cell.
Fig. 4 is 2 hours model group mouse cells in vivo specific killing situations behind the injection reagent of the present invention, and M1 is the streaming peak area of fluorescent dye C57 cell, and M2 is the streaming peak area of fluorescent dye BALB/c cell.
Fig. 5 is 2 hours control group mice cells in vivo specific killing situations behind the injection reagent of the present invention, and M1 is the streaming peak area of fluorescent dye C57 cell, and M2 is the streaming peak area of fluorescent dye BALB/c cell.
Fig. 6 is 4 hours model group mouse cells in vivo specific killing situations behind the injection reagent of the present invention, and M1 is the streaming peak area of fluorescent dye C57 cell, and M2 is the streaming peak area of fluorescent dye BALB/c cell.
Fig. 7 is 4 hours control group mice cells in vivo specific killing situations behind the injection reagent of the present invention, and M1 is the streaming peak area of fluorescent dye C57 cell, and M2 is the streaming peak area of fluorescent dye BALB/c cell.
Fig. 8 is 10 hours model group mouse cells in vivo specific killing situations behind the injection reagent of the present invention, and M1 is the streaming peak area of fluorescent dye C57 cell, and M2 is the streaming peak area of fluorescent dye BALB/c cell.
Fig. 9 is 10 hours control group mice cells in vivo specific killing situations behind the injection reagent of the present invention, and M1 is the streaming peak area of fluorescent dye C57 cell, and M2 is the streaming peak area of fluorescent dye BALB/c cell.
Figure 10 is 1 day model group mouse cells in vivo specific killing situation behind the injection reagent of the present invention, and M1 is the streaming peak area of fluorescent dye C57 cell, and M2 is the streaming peak area of fluorescent dye BALB/c cell.
Figure 11 is 1 day control group mice cells in vivo specific killing situation behind the injection reagent of the present invention, and M1 is the streaming peak area of fluorescent dye C57 cell, and M2 is the streaming peak area of fluorescent dye BALB/c cell.
Figure 12 is 2 days model group mouse cells in vivo specific killing situations behind the injection reagent of the present invention, and M1 is the streaming peak area of fluorescent dye C57 cell, and M2 is the streaming peak area of fluorescent dye BALB/c cell.
Figure 13 is 2 days control group mice cells in vivo specific killing situations behind the injection reagent of the present invention, and M1 is the streaming peak area of fluorescent dye C57 cell, and M2 is the streaming peak area of fluorescent dye BALB/c cell.
Figure 14 is 3 days model group mouse cells in vivo specific killing situations behind the injection reagent of the present invention, and M1 is the streaming peak area of fluorescent dye C57 cell, and M2 is the streaming peak area of fluorescent dye BALB/c cell.
Figure 15 is 3 days control group mice cells in vivo specific killing situations behind the injection reagent of the present invention, and M1 is the streaming peak area of fluorescent dye C57 cell, and M2 is the streaming peak area of fluorescent dye BALB/c cell.
Figure 16 is 6 days model group mouse cells in vivo specific killing situations behind the injection reagent of the present invention, and M1 is the streaming peak area of fluorescent dye C57 cell, and M2 is the streaming peak area of fluorescent dye BALB/c cell.
Figure 17 is 6 days control group mice cells in vivo specific killing situations behind the injection reagent of the present invention, and M1 is the streaming peak area of fluorescent dye C57 cell, and M2 is the streaming peak area of fluorescent dye BALB/c cell.
Embodiment
Example 1
1) take out splenocyte from C57 mouse and BALB/c mouse, suspending and adjust cell concentration with PBS respectively is 107/mL;
2) with concentration be the dimethyl sulfoxide solution of CFSE of 10mM after with 50 times PBS dilution 200 μ M CFSE working fluids;
3) adding the dyeing of CFSE working fluid in the C57 mouse boosting cell suspension, to make the final concentration of CFSE be 0.3 μ M, and adding the CFSE working fluid in the BALB/c mouse splenocyte suspension, to make the CFSE final concentration be 6 μ M, is 37 ℃ in temperature then, hatches and dyeed 15 minutes;
4) staining cell is resuspended in PBS with after 4 ℃ of PBS washing of 0.01M 2 times;
5) mix two kinds of fluorescent dye cells in 1: 1 ratio of cell number, and to adjust the cell total concentration be 5 * 107/mL, get final product.
Cell viability detects: after an amount of mixing with cells suspension dilution, get a dilution and one 0.5% trypan blue solution mixing, leave standstill 3min, add to cell counting count board, inverted microscope is observed down, and living cells is not painted, dead cell nuclear au bleu, living cell counting and dead cell quantity are calculated as follows number percent, and the living cells ratio reaches more than 95%:
Cell survival rate=0.5% trypan blue dyeing negative cells/100 cell/HP * 100.
Flow cytometry CFSE labeled cell: with the negative reference of PBS control treatment lymphocyte, with CFSE labelled lymphocyte sample up flow type cell instrument, set 488nm to excite/the 525nm filtration, measuring the positive cell ratio is greater than 95%.
Example 2
1) take out splenocyte from C57 mouse and BALB/c mouse, suspending and adjust cell concentration with PBS respectively is 107/mL;
2) with concentration be the dimethyl sulfoxide solution of CFSE of 10mM after with 50 times PBS dilution 200 μ M CFSE working fluids;
3) adding the dyeing of CFSE working fluid in the C57 mouse boosting cell suspension, to make the final concentration of CFSE be 0.3 μ M, and adding the CFSE working fluid in the BALB/c mouse splenocyte suspension, to make the CFSE final concentration be 6 μ M, is 37 ℃ in temperature then, hatches and dyeed 17 minutes;
4) staining cell is resuspended in PBS with after 4 ℃ of PBS washing of 0.01M 3 times;
5) mix two kinds of fluorescent dye cells in 1: 1 ratio of cell number, and to adjust the cell total concentration be 8 * 107/mL, get final product.
Example 3
1) take out splenocyte from C57 mouse and BALB/c mouse, suspending and adjust cell concentration with PBS respectively is 107/mL;
2) with concentration be the dimethyl sulfoxide solution of CFSE of 1 0mM after with 50 times PBS dilution 200 μ M CFSE working fluids;
3) adding the dyeing of CFSE working fluid in the C57 mouse boosting cell suspension, to make the final concentration of CFSE be 0.3 μ M, and adding the CFSE working fluid in the BALB/c mouse splenocyte suspension, to make the CFSE final concentration be 6 μ M, is 37 ℃ in temperature then, hatches and dyeed 20 minutes;
4) staining cell is resuspended in PBS with after 4 ℃ of PBS washing of 0.01M 3 times;
5) mix two kinds of fluorescent dye cells in 1: 1 ratio of cell number, and to adjust the cell total concentration be 1 * 108/mL, get final product.
Claims (2)
1. mouse immune state monitoring reagent, this reagent are that wherein the total concentration of two kinds of mice spleen cells is 0.5～1 * 10 through the PBS suspension of the mice spleen cell of two kinds of inbred strais of CFSE dyeing
8Individual/mL, the ratio of the two is 1: 1, and the fluorescent dye concentration difference is 1: 20.
2. the preparation method of the described immune state monitoring reagent of claim 1, this method is made up of following steps:
1) gets the mice spleen cell of two kinds of inbred strais respectively, suspend and adjust cell concentration, obtain two kinds of concentration and be 10 with PBS
7The cell suspension of individual/mL;
2) with concentration be the dimethyl sulfoxide solution of CFSE of 10mM after with 50 times PBS dilution 200 μ M CFSE working fluids;
3) add the CFSE working fluid of step 2 gained in a kind of cell suspension of step 1 gained, the final concentration that makes CFSE is 0.3 μ M, the CFSE working fluid that adds step 2 gained in the another kind of cell suspension of step 1 gained, making the CFSE final concentration is 6 μ M, is to hatch dyeing 15～20 minutes under 37 ℃ in temperature then;
4) collect staining cell in the cell suspension of step 3 gained respectively, be resuspended in PBS with after 4 ℃ of PBS washings of 0.01M 2～3 times;
5) in two kinds of staining cell suspensions of 1: 1 ratio blend step 4 gained of cell number, and to adjust the cell total concentration with PBS be 0.5～1 * 10
8Individual/mL, get final product.
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| CN88101256A (en) * | 1987-03-13 | 1988-09-21 | 科特电子有限公司 | Be used to strengthen biological respinse and carry out little volume stirring method and instrument |
| CN1427258A (en) * | 2001-12-18 | 2003-07-02 | 杜凤鸣 | Enzyme-linked immunosorbent assay (ELISA) reagent box for assaying low density lipoprotein content in human urine and its preparation method |
| CN1598586A (en) * | 2004-09-07 | 2005-03-23 | 江苏省农业科学院 | Immune antibody for detecting pesticide organic phosphorus residus and its application |
| WO2007037410A1 (en) * | 2005-09-30 | 2007-04-05 | Pulse-Immunotech Corporation | Method of assaying substance with affinity in sample including step of destroying blood-cell ingredient |
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| CN88101256A (en) * | 1987-03-13 | 1988-09-21 | 科特电子有限公司 | Be used to strengthen biological respinse and carry out little volume stirring method and instrument |
| CN1427258A (en) * | 2001-12-18 | 2003-07-02 | 杜凤鸣 | Enzyme-linked immunosorbent assay (ELISA) reagent box for assaying low density lipoprotein content in human urine and its preparation method |
| CN1598586A (en) * | 2004-09-07 | 2005-03-23 | 江苏省农业科学院 | Immune antibody for detecting pesticide organic phosphorus residus and its application |
| WO2007037410A1 (en) * | 2005-09-30 | 2007-04-05 | Pulse-Immunotech Corporation | Method of assaying substance with affinity in sample including step of destroying blood-cell ingredient |
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