CN108627641A - The check and evaluation method and kit of hepatopathy T cell function - Google Patents

The check and evaluation method and kit of hepatopathy T cell function Download PDF

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Publication number
CN108627641A
CN108627641A CN201810400121.3A CN201810400121A CN108627641A CN 108627641 A CN108627641 A CN 108627641A CN 201810400121 A CN201810400121 A CN 201810400121A CN 108627641 A CN108627641 A CN 108627641A
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cell
factor
antibody
pbs
function
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曾首杰
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Pu Xi (guangzhou) Biological Immune Technology Co Ltd
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Pu Xi (guangzhou) Biological Immune Technology Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label

Abstract

The invention discloses a kind of check and evaluation methods of hepatopathy related T-cell function, including step：1) it utilizes individual cells by flow cytometer, is combined with cell surface or intracellular specific markers using anti-cytokine antibodies, you can the secretion for detecting different cytokines obtains result according to different labels；It is selected simultaneously using special chemical and antibody, which can understand with the antibody that antibody is that specificity is combined with the cell factor of cell surface secretion, the coloured fluorescent labelling compound of antibody band and distinguish the immunocyte without this kind of cell factor of secretion；2) completely new big data biostatistics analytical technology is utilized, on each cell factor on causing contribution/effect/influence of whole immune function to carry out interpretation.The invention also discloses a kind of hepatopathy T cell Function detection kits.The kit of the present invention can effectively detect the general function of T cell.

Description

The check and evaluation method and kit of hepatopathy T cell function
Technical field
The invention belongs to biomedical and immunity inspection fields, are related to a kind of relevant overall T cell function of hepatitis B hepatopathy Detect and assess method, specifically, it is related to a kind of check and evaluation method of overall T cell function, and for this method Kit.
Background technology
Although hepatitis B vaccine, which is promoted, had for more than 30 years, still there are 2.4 hundred million hepatitis B infected persons at present, there are about 1,000,000 people are dead every year In hepatitis B hepatopathy, China occupies 93,000,000, accounts for the whole world 38.75%.Hepatitis B (HBV) is the Hepadna Virus of non-cell toxicity, The directly effect of liver cell inflammation damnification not instead of virus causes, and is mediated by host immune response.Meanwhile body removes virus Also immune response is relied primarily on, wherein T cell is immune plays an important role (see Fig. 1).T cell is divided into CD4+T cell and CD8+T is thin Born of the same parents, function are determined that activation sex factor mainly has by T cell activation sex factor and failure factor level：Interleukin-22 (IL-2), Interferon (IFN-γ), tumor necrosis factor α (TNF-α)；Failure molecule has：PD-1, CTLA-4, LAG3, TIM3,2B4 (figure 2)。
As HBV enters liver, a large amount of inflammatory cells also pour in liver, especially HBV specificity and non-specificity T therewith Cell is acted on by secrete cytokines or by direct cell cracking and further removes virus, but while removing virus Also cause infected hepatocellular apoptosis, necrosis, clinically show as transaminase and increase, HBV-DNA declines, remove.Acute HBV When infection, the inherent immunity and adaptive immunity of body can generate a large amount of antiviral agent and enough, full-featured T is thin Born of the same parents, so that fully erased virus.But strong inherent immunity cannot be generated in HBV chronic infection's bodies and adaptive immunity is anti- It answers, causes follow-up a large amount of HBV antigens to be accumulated in blood and hepatic tissue, further cause T cell afunction, failure and wither It dies, T cell failure is the main reason for body cannot remove virus, chronic sustained is caused to infect.Therefore, T cell functional assessment It is most important.
Lack the clinical detection reagent box for effectively assessing hepatitis B T cell general function at present.
With the development of life group, the relevant technologies of immune system are constantly progressive, the immune globulin since the seventies In vain, the measurement such as complement, can roughly and indirectly estimate body immune state, gradually by lymphocyte subgroup, cell because The detection of son substitutes, and representative detection means includes：
1) biological method based on DNA detections, such as PCR, RNA trace, in situ hybridization technology, such methods can be right Single cell immune factor is detected, and studies its regulatory pathway；
2) bioactivity detection method, such as ELISA, ELISPOT, such methods are only to single cell or single immune Cell-secretion factor is detected, rather than is detected to the mean cytokine levels of certain a group immunocyte；
3) Flow Cytometry and liquid chip detection：More accurately detection technique at present, can be to multiple cell factors It is detected, immune state is estimated according to different cytokines level；However, carrying stronger subjectivity.
Invention content
It is an object of the invention to be directed to the above technical problems to be solved, providing one kind, more accurately and efficiently T is thin The check and evaluation method of born of the same parents' function.
It is a further object to provide a kind of accurately and efficiently hepatopathy T cell Function detection kits.
The purpose of the present invention is what is realized using following technical scheme.
The present invention provides a kind of check and evaluation methods of hepatopathy T cell function comprising：
1) it utilizes individual cells by flow cytometer, uses anti-cytokine antibodies and cell surface or the specific mark of intracellular Note combination, detects the secretion of different cytokines, and result is obtained according to different labels；The anti-cytokine antibodies carry face The compound of the fluorescent marker of color can clearly distinguish the immunocyte that no Secretion answers cell factor；
2) big data biostatistics analytical technology is utilized, to each cell factor to causing the tribute of whole immune function Offer/acting on/influence carry out interpretation the interpretation to whole immune function is ultimately formed according to data model.
Preferably, the antibody is following a variety of：CD4+T cell and CD8+T cell expression IFN-γ, IL-2, TNF-α, CTLA-4、Tim3、2B4、PD-1、LAG3.It is highly preferred that the antibody be CD4_IFN- γ, CD4_IL-2, CD4_TNF- α, CD4_CTLA-4、CD4_2B4、CD4_PD-1、CD8_IFN-γ、CD8_IL-2、CD8_PD-1、CD8_Tim3、CD8_CTLA-4、 CD8_2B4、CD8_LAG3。
The T cell is the relevant T cell of liver diseases.
Preferably, step (1) specifically includes：PBMC is separately cultured；Flow cytometry T cell surface co-suppression point Son；The Flow cytometry T cell intracellular factor.
Include the following steps it is highly preferred that PBMC is separately cultured：
1) EDTA anticoagulations 20ml is adopted；Centrifugation, 500g, 8min rise 9, and drop 9, separated plasma marks, -80 DEG C of preservations of blood plasma For use, haemocyte layer is continued to employ；Dilution：Haemocyte is moved into 50ml centrifuge tubes, adds PBS to about 30ml, and is blown and beaten uniform；
2) 4 15ml centrifuge tubes are taken, often pipe plus 4.5ml lymphocyte separation mediums, then will add to above separating liquid；
3) without brake density gradient centrifugation：25 DEG C, 450g, 25min, 5 are risen, drop 0；
4) test tube is taken out, sees that it is divided into following four layers：Upper layer is PBS and some residual blood plasma, i.e. plasma layer, and lower layer is red Cell and granulocyte；Middle level is lymphocyte separation medium, the visible tunica albuginea layer between lymphocyte separation medium and plasma layer, is drawn Tunica albuginea confluent monolayer cells, with 1：5 volume ratios wash cell 2 times, 500g, 8min with PBS；
5) sedimentation cell is added culture medium and is resuspended, and is dyed with 2% trypan blu e, it was demonstrated that viable count is adjusted 95% or more Density is 1*e7/ml.
It is highly preferred that Flow cytometry T cell surface Co inhibitor includes the following steps：
1) usage amount of streaming antibody is 2-5 μ l, and every streaming pipe takes RPMI 1640+10%FBS complete medium weights 1*e5, outstanding cell；
2) 4 DEG C, 400g, 8min centrifugation abandon supernatant, are inverted in blotting paper and inhale, even using liquid shell remaining in pipe Cell；The antibody with respective fluorescent marker is added：CD4+T cell and CD8+T cell expression IFN-γ, IL-2, TNF-α, Each 2 μ l of CTLA-4, Tim3,2B4, PD-1, LAG3,4 DEG C are incubated 30min, even once every 15min bullets；
3) PBS 1ml are added to wash 1 time, 4 DEG C, 400g, 8min abandon supernatant, are inverted in blotting paper and inhale；120μlPBS+ It is resuspended at once on 40 μ l 4%PFA vortices, it is fixed；
4) machine testing on.
It is highly preferred that the Flow cytometry T cell intracellular factor includes the following steps：
1) 500 μ l of for use PBMC cells are taken, 10 μ l (50 ×) of lymphocyte stimulation agent are added, play even, 37 DEG C of cell culture Case is incubated 4h with the stimulation of leukocyte compound；
2) 2 μ l of surface antibody PECF-594 CD3 are added, 2 μ l of APC CD4,2 μ l of V450 CD8, play even, 4 DEG C of refrigerators It is incubated 30min, is shaken every 15min primary；
3) often pipe plus 1ml PBS, 450g 8min centrifugations, washing, abandons supernatant, nozzle back-off is utilized in the blotting paper last time The even and fine born of the same parents of remaining liq bullet；
4) often pipe adds 400 μ l of FIX＆PERM, and be vortexed mixing immediately, is incubated at room temperature 30min or 4 DEG C of incubation 45min；
5) often 1ml PBS centrifuge washings are added in pipe, and supernatant is abandoned in 750g, 10min centrifugation, and nozzle is buckled on blotting paper one It is secondary, utilize the even and fine born of the same parents of remaining liq bullet；
6) often pipe plus 500 μ l centrifuge washings of Perm buffer solutions, 4 DEG C, supernatant is abandoned in 750g, 10min centrifugation, nozzle back-off in Blotting paper is last, utilizes the even and fine born of the same parents of remaining liq bullet；
7) plus intracellular antibody IFN-γ, IL-2, TNF-α, CTLA-4, Tim3,2B4, PD-1, LAG3 bullet are even, 4 DEG C of incubations 45min, centre play even primary；
8) often pipe is added 500 μ l of Perm buffer solutions and washes 1 time, 750g, 10min；
9) 150 μ l PBS are resuspended, and upper machine carries out flow cytometer detection.
Preferably, in the step (2), method includes used by the big data biostatistics analysis：Using poly- T cell functional status is classified as that immune state is good and immune state is poor using immune indexes by alanysis method.
It is highly preferred that by cluster, the mark numerical value of each T cell factor is obtained, forms markization numerical model.
It is highly preferred that structure following equation carries out interpretation；
Z=c+a1X1+a2X2+...+apXp(formula 2)
Above equation meets distance (i.e. mahalanobis distance) maximum between 2 classes, i.e.,：
Wherein,The scoring score of two class crowds is indicated respectively,For variance；Find such one group of coefficient a1, a2,…apSo that D2Maximum, i.e. distance are maximum, then corresponding formula 2 is discriminant equation.
The T cell Function detection appraisal procedure of the present invention, 1) utilize individual cells by flow cytometer, use anti-cell Factor antibody is combined with cell surface or intracellular specific markers, you can the secretion for detecting different cytokines, according to different marks Note obtains result；It is selected simultaneously with antibody using special chemistry, it is ensured that the static minimum with the cell of acellular cytokine secretion Fluorescence background can effectively remove false positive results, contribute to the accuracy of result.The special chemical and antibody refer to special Property with the antibody that is combined of cell factor of cell surface secretion, the compound of the coloured fluorescent marker of antibody band, energy is clear Distinguish the immunocyte without this kind of cell factor of secretion in ground.2) completely new big data biostatistics analytical technology is utilized, to every One cell factor is right according to accurate data model on causing contribution/effect/influence of whole immune function to carry out interpretation Cell factor used forms finally to the whole immune function interpretation of T cell the effect of immune function.
The present invention also provides a kind of hepatopathy T cell Function detection kit, the kit includes comprising for detecting The reagent of a variety of T cell factor groups below：CD4+T and CD8+T cells expression IFN-γ, IL2, TNF-α, CTLA-4, Tim3, 2B4、PD-1、LAG3。
The hepatopathy T cell Function detection kit of the present invention, antibody are following a variety of：The expression of CD4+T and CD8+T cells IFN-γ、IL2、TNF-α、CTLA-4、Tim3、2B4、PD-1、LAG3。
The hepatopathy T cell Function detection kit of the present invention, the hepatopathy T cell are the relevant T cell of hepatitis B hepatopathy.
The invention has the advantages that and advantageous effect：
The present invention is by advanced Flow Cytometry to T cell activation factor group and T cell in hepatitis B large sample The failure molecular group of expression is detected, and obtains the concrete numerical value of each cellular immunity factor, and utilize the completely new big number of biology According to analysis, the comprehensive contribution of this serial T cell factor pair T cell immune function is obtained, the final accurate embodiment T cell of acquisition is immune The index of function.
In addition, the present invention also provides multi information advantages：Can provide representative 13 T cell activation factors and failure because The actual numerical value of son, represents these real-time quantity of cellular immunity factor and level；The level of each cell factor can be provided To forming the effect of T cell general function；Overall T cell function status can be provided.
Description of the drawings
Fig. 1 is the interaction schematic diagram of chronic hepatitis B T cell surface factor.
Its inhibition developed by molecule and cytokine secretion schematic diagram, show when Fig. 2 is chronic HBV infection T cell failure The mutual containing of T cell inflammatory factor and suppressive genes in liver.
Fig. 3 is the representative figure of each subgroup secreting function of FCM analysis T cell.
Fig. 4 is the dependency diagram of the T cell functional activation factor and the failure factor, has color filling to indicate correlation Property, color filling is deeper, and correlation is stronger；The packless expression non-correlation of blank.
Fig. 5 is that T cell function differentiates the Kmeans hierarchial-cluster analysis figures carried out.
Specific implementation mode
Below by way of specific embodiment, invention is further described in detail, it is not limited to following embodiment.
T cell immune function is divided into immune state by the present invention using clustering (K-means methods) using immune indexes Good and immune state is poor.
1, the Statistics of K-means methods clustering
The purpose of clustering is so that distance is gathered compared with the sample of nearly (property is similar) for different classifications.Common measurement The class statistic amount of distance is Euclidean distance.It is calculated following (being shown in Table 1)：
Table 1：The data structure of clustering
Wherein xijAnd xkjRespectively represent i and k sample of j-th of variable.Shadow caused by order to eliminate dimension difference It rings, before the computation, it should the standardization of advanced row data.
2, the specific calculating process of K-means methods is：
1. the seed of cluster is randomly choosed, with the center (oneself setting number, we set 2) that it is cluster；
2. calculating each sample to the distance at these centers, and sample is classified as apart from nearest center, the production of these results Temporary classification is given birth to；
3. based on above-mentioned temporary classification, algorithm calculates new center, and based on new center, sample is gathered again again Class；
4. algorithm iteration always, until the cluster result of sample does not change (Fig. 5).
We carry out the selection course of clustering, and clustering is carried out using 13 factors after markization；
Above-mentioned clustering method can obtain the classification (cluster) of each sample, we are named as immu_1 and immu_2.It is right Than the group difference of 13 immune factors (including immunofluorescence method etc.) between the immu_1 groups and immu_2 groups of each method.
3, discriminant analysis (is Fisher ' s Discriminant Analysis, also known as linear Discriminant Analysis)
The Statistics of K-means method clusterings：
We want that two crowds is made to separate, such as 2 classes of above-mentioned cluster result (immunity is uneven), it is assumed that this two class The explanatory variable of crowd is identical, then we can build so equation；
Z=c+a1X1+a2X2+...+apXp(formula 2)
It is maximum that this equation meets the distance (mahalanobis distance) between 2 classes
Wherein,The scoring score of two class crowds is indicated respectively,For variance.Our purpose is to find in this way One group
Coefficient (a1,a2,…ap) so that D2Maximum, i.e. distance are maximum.It is discriminant equation so to correspond to formula 2.
The present invention is by advanced Flow Cytometry to T cell activation factor group and T cell in hepatitis B large sample The failure molecular group of expression is detected, and obtains the concrete numerical value of each cellular immunity factor, and utilize the completely new big number of biology According to analysis, the comprehensive contribution of this serial T cell factor pair T cell immune function is obtained, the final accurate embodiment T cell of acquisition is immune The index (Fig. 3) of function.
In addition, the present invention also provides multi information advantages：Can provide representative 13 T cell activation factors and failure because The actual numerical value of son, represents these real-time quantity of cellular immunity factor and level；The level of each cell factor can be provided To forming the effect of T cell general function；Overall T cell function status (Fig. 4) can be provided.
The present invention is intended to provide a kind of T cell Function detection kit, including：1) detection reagent of T cell factor group, The T cell factor group has：CD4+T cell and CD8+T cell expression IFN-γ, IL-2, TNF-α, CTLA-4, Tim3,2B4, PD-1、LAG3；2) include the mathematical model for assessing T cell function.
Generally, the present invention includes for the method for check and evaluation T cell function：
1) it utilizes individual cells by flow cytometer, uses anti-cytokine antibodies and cell surface or the specific mark of intracellular Note combination, you can the secretion for detecting different cytokines obtains result according to different labels；Simultaneous selection it is special chemistry with Antibody, the special chemical and the antibody that antibody is that specificity is combined with the cell factor of cell surface secretion, carry color Fluorescent marker compound, can clearly distinguish without secretion this kind of cell factor immunocyte；
2) completely new big data biostatistics analytical technology is utilized, to work(is integrally immunized caused by each cell factor Contribution/effect of energy/influence carries out interpretation, according to accurate data model, evaluates work of the cell factor used to immune function With the simultaneously therefore formation finally interpretation to whole immune function.
The present invention is using Flow Cytometry to overall T cell group and T cell subgroup quantity and work(in hepatopathy large sample It can be identified, obtain the concrete numerical value of each cellular immunity factor, and using biological big data analysis, it is each to obtain this series The comprehensive contribution of general function is immunized in a T cell factor pair T cell, final to obtain the accurate index for embodying T cell immune function, To detect the general function of T cell.
The concrete operations of above-mentioned detection method are as follows：
(1) surrounding blood mononuclear cell (PBMC) detaches：
1) EDTA anticoagulations 20ml is adopted；Centrifugation, 500g, 8min rise 9, and drop 9, separated plasma marks, -80 DEG C of preservations of blood plasma For use, haemocyte layer is continued to employ；Dilution：Haemocyte is moved into 50ml centrifuge tubes, adds PBS to about 30ml, and is blown and beaten uniform.
2) 4 15ml centrifuge tubes are taken, often pipe plus 4.5ml lymphocyte separation mediums.Then centrifuge tube is tilted into 45 degree of angles, used Pasteur pipet pays attention to keeping above blood at away from lymphocyte separation medium interface 1cm is added slowly to separating liquid along test tube wall Two interfaces are clear, blood do not made to be mixed into separating liquid.
3) without brake density gradient centrifugation：25 DEG C, 450g, 25min, 5 are risen, drop 0.
4) test tube is taken out, sees that it is divided into following four layers：Upper layer is PBS and some residual blood plasma, and lower layer is red blood cell and grain Cell；Middle level is that lymph segments chaotropic, and the visible film layer between separating liquid and plasma layer, it is thin that suction pipe carefully draws tunica albuginea layer Born of the same parents, with 1:5 volume ratios wash cell 2 times (500g, 8min) with PBS.
5) sedimentation cell is added culture medium and is resuspended, and is dyed with 2% trypan blu e, it was demonstrated that viable count is 95% or more.It adjusts Density 1*e7/ml, for use.
(2) Flow cytometry T cell surface Co inhibitor
1) usage amount of streaming antibody is generally 2-5 μ l, and every streaming pipe is taken RPMI1640+10%FBS and cultivated completely 1*E5 outstanding, cell of base weight.
2) 4 DEG C, 400g, 8min centrifugation abandon supernatant, are inverted in blotting paper and inhale, play even and fine born of the same parents and (utilize remaining in pipe Liquid).The antibody with respective fluorescent marker is added：CD4+T cell and CD8+T cell expression IFN-γ, IL-2, TNF-α, CTLA-4, Tim3,2B4, PD-1, LAG3, each 2 μ l, 4 DEG C are incubated 30min, even once every 15min bullets.
3) PBS 1ml are added and wash 1 time (4 DEG C, 400g, 8min), abandon supernatant, be inverted in blotting paper and inhale；120μl PBS It is resuspended at once on+40 μ l 4%PFA vortices, it is fixed.
(4) machine testing on.
(3) the Flow cytometry T cell intracellular factor (obtains as shown in figure 3, detecting and analyzing for Flow Cytometry Obtain CD3+T cell and CD4+The level of T cell.)
1) for use 500 μ l of cell in (one) are taken, 10 μ l (50 ×) of lymphocyte stimulation agent are added, play even, 37 DEG C of cell trainings It supports case stimulation and is incubated 4h with Leukocyte Activation Cocktail (leukocyte compound).
2) surface antibody PECF-594 CD3 (2 μ l), APC CD4 (2 μ l), V450 CD8 (2 μ l) is added, plays even, 4 DEG C of ice Case is incubated 30min, is shaken every 15min primary.
3) often pipe plus 1ml PBS, 450g 8min centrifugations, washing, abandons supernatant, nozzle back-off is utilized in the blotting paper last time The even and fine born of the same parents of remaining liq bullet.
4) often pipe adds 400 μ l of FIX＆PERM (ebioscience, FIX＆PERM kit), and be vortexed mixing immediately, incubation at room temperature 30min or 4 DEG C of 45min.
5) often 1ml PBS centrifuge washings are added in pipe, and supernatant is abandoned in 750g, 10min centrifugation, and nozzle is buckled on blotting paper one It is secondary, utilize the even and fine born of the same parents of remaining liq bullet.
6) often pipe plus 500 μ l centrifuge washings of Perm buffer solutions, 4 DEG C, supernatant is abandoned in 750g, 10min centrifugation, nozzle back-off in Blotting paper is last, utilizes the even and fine born of the same parents of remaining liq bullet.
7) add intracellular antibody CD4+T cell and CD8+T cell expression IFN-γ, IL-2, TNF-α, CTLA-4, Tim3, 2B4, PD-1, LAG3, play even, 4 DEG C of incubation 45min, and centre bullet is even primary).
8) often pipe is added 500 μ l of Perm buffer solutions and washes 1 time, 750g, 10min.
9) 150 μ l PBS are resuspended, and upper machine carries out flow cytometer detection.
It is detected using 800 hepatopaths of the above Flow Cytometry pair and 250 Healthy Peoples, obtaining 13 altogether exempts from The mean (% total number of lymphocytes) of the data of epidemic disease index, as shown in table 2；
Table 2：Flow cytometer detection obtains the frequency disribution (mean ± standard deviation) of each cell factor
These data obtain contribution of each data to overall T cell immune function by K-means clusterings, from And obtain the T cell function mark value of each data.(as shown in Figure 4 and Figure 5, wherein Fig. 4 shows CD4+T cell and CD8+T is thin Correlation between the cell factor of intracrine,>0.5 is the positive, i.e., with the presence of correlation；In Fig. 4, there is color filling to indicate correlation Property, color filling is deeper, and correlation is stronger；The packless expression non-correlation of blank.Fig. 5 is the mathematical modulo that clustering method is established Type schematic diagram)
Table 3：The mark value of each cell factor is as shown in the following Table 3 in above-mentioned table 2：
Table 3：The mark value (mean) of each cell factor
Cell factor title/T cell functional class Immu_high Immu_low
CD4_PD-1 0.75 0.92
CD4_2B4 0.56 0.82
CD4_CTLA4 1.09 0.92
CD4_IFN-γ 1.11 0.83
CD4_IL-2 1.92 0.30
CD4_TNF-α 1.02 0.86
CD8_PD-1 0.82 0.87
CD8_TIM3 0.97 0.95
CD8_2B4 0.83 0.81
CD8_LAG3 0.60 0.52
CD8_CTLA4 1.03 0.85
CD8_IFN-γ 1.13 0.82
CD8_IL-2 1.70 0.38
(5) according to 3 each cell factor of table space bit inner in T cell function grouping (Immu_high and Immu_low) It sets, overall T cell function is evaluated：
(1) it is set to distance-1 with Immu_high distances, is set to distance-2 with Immu_low distances；
(2) function classification is carried out according to apart from length：Shorter with distance-1 distances is Immu_high, prompts T cell Immune function is higher；Shorter with distance-2 distances is Immu_low, prompts T cell immunologic hypofunction.
Such as：The mark space of the T cell factor of wherein one detection sample is as shown in the following Table 4：
Table 4：The mark space of the T cell factor of certain detection sample
distance-1 6.571491346
distance-2 5.332081736
Since the T cell factor mark value of the detection object is closer to " distance-2 ", its T cell function category is prompted In Immu_low, i.e. T cell hypofunction.
Example operation and analysis：
T lymphocyte function detections are carried out using 50ml peripheral bloods：
1) PBMC in people's lymph lymphocyte separating liquid separation fresh peripheral blood, is resuspended cell in complete serum free culture system liquid In, density 0.5-1*e7/ml is adjusted, for use.
2) usage amount of streaming antibody is generally 2-5 μ l, and every streaming pipe is taken RPMI1640+10%FBS and cultivated completely 1-2*E5 outstanding, cell of base weight.
3) 4 DEG C, 400g, 8min centrifugation abandon supernatant, are inverted in blotting paper and inhale, play even and fine born of the same parents and (utilize remaining in pipe Liquid).FITC LAG3, FITC CD8, PE-CY7CD4 is added, and the antibody with respective fluorescent marker is added：CD4+T is thin Born of the same parents and CD8+The IFN-γ of T cell expression, IL-2, TNF-α, CTLA-4, Tim3,2B4, PD-1, LAG3, each 2 μ l of surface antibody, 4 DEG C are incubated 30-45min, even once every 15min bullets.
4) it takes out, often pipe is added 500 μ l of Perm buffer solutions and washes 1 time, 750g, 10min.
5) 150 μ l PBS are resuspended, and upper machine carries out flow cytometer detection.
Experimental result：
As a result 1：As shown in table 5, the actually detected level of the T cell factor.
Table 5：The actually detected level of the various T cell factors：
T cell factor names Actual numerical value
CD4_IFN-γ 6.42
CD4_IL-2 0.5
CD4_TNF-α 68.7
CD8_IFN-γ 18
CD8_IL-2 0.21
CD4_PD-1 32.4
CD4_2B4 58
CD4_CTLA4 15.2
CD8_PD-1 38.9
CD8_TIM3 48.6
CD8_2B4 87.2
CD8_CTLA4 3.94
CD8_LAG3 0.4
As a result 2：As shown in table 6, it is the level of T cell factor mark：
Table 6：The level of T cell factor mark
T cell factor names Markization numerical value
CD4_IFN-γ -1.16815
CD4_IL-2 -0.75151
CD4_TNF-α 2.15087
CD8_IFN-γ -0.76154
CD8_IL-2 -0.69823
CD4_PD-1 0.541622
CD4_2B4 1.407876
CD4_CTLA4 -1.92606
CD8_PD-1 1.270279
CD8_TIM3 -1.04996
CD8_2B4 1.456711
CD8_CTLA4 -1.03625
CD8_LAG3 -0.60203
As a result 3：As shown in table 7, it is divided into standard T cell functional category：
Table 7：The mark space length of T cell function：
distance1 5.438312188
distance2 7.686902686
As a result 4：As a result judge, according to cluster tie, the overall T cell factor mark space length of the example with " distance1 " is closer to, therefore judges that the overall T cell function of the example belongs to " Immu-high ", i.e. T cell function It is active.
The whole immune functional state that can determine that T cell as a result, carries out it more accurate and effective detection.
It is concrete application case study on implementation below.
Application example 1:
T cell function differentiation is carried out according to following steps.
Step 1：Extract Patients with Fatty Liver peripheric venous blood 50ml；
Step 2：According to above-mentioned fluidic cell Examined effect method and steps, to 50ml vein blood specimens carry out T cell because Son (CD4+T cell and CD8+The IFN-γ of T cell expression, IL-2, TNF-α, CTLA-4, Tim3,2B4, PD-1, LAG3) detection；
Step 3：The actual numerical value of each cell factor is obtained by FCM analysis technology；The poly- of the present invention is utilized simultaneously Class technology, the contribution according to each cell factor to formation T cell function, obtains the mark value of each T cell factor, as a result such as Shown in the following table 8.
Table 8：The actual numerical value and markization value of each T cell factor
T cell factor names Actual numerical value Markization numerical value
CD4_IFN-γ 15.43 1.36
CD4_IL-2 4.50 1.73
CD4_TNF-α 5.70 -0.48
CD8_IFN-γ 2.80 2.48
CD8_IL-2 9.04 5.06
CD8_CTLA4 33.2 4.23
CD4_PD-1 52.4 3.52
CD4_2B4 14.3 1.40
CD4_CTLA4 5.20 -0.45
CD8_PD-1 18.32 0.44
CD8_TIM3 10.83 -4.04
CD8_2B4 31.20 1.43
CD8_LAG3 19.21 -1.0
Step 4：Differentiation data model according to the present invention obtains the space bit in a model of the example T cell factor It sets, as shown in table 9 below.
Table 9：The spatial position of the T cell factor in a model
distance1 4.4375188
distance2 6.8475297
Step 5：Finally judged according to mathematical model, the overall T cell factor mark space length of the example with " distance1 " is slightly close, therefore judges that the overall T cell function of the example belongs to " Immu_high ", i.e. T cell function It is active.
Application example 2
T cell function differentiation is carried out according to following steps.
Step 1：Extract hepatitis B patient peripheric venous blood 50ml；
Step 2：According to above-mentioned fluidic cell Examined effect method and steps, to 50ml vein blood specimens carry out T cell because Son (CD4+T cell and CD8+The IFN- of T cell expression is thin, IL-2, TNF- are thin, CTLA-4, Tim3,2B4, PD-1, LAG3) inspection It surveys；
Step 3：The actual numerical value of each cell factor is obtained by FCM analysis technology；The poly- of the present invention is utilized simultaneously Class technology, the contribution according to each cell factor to formation T cell function, obtains the mark value of each T cell factor, as a result such as Shown in the following table 10：
Table 10：The actual numerical value and markization value of each T cell factor
T cell factor names Actual numerical value Markization numerical value
CD4_IFN-γ 5.43 0.33
CD4_IL-2 5.80 2.73
CD4_TNF-α 1.70 -3.43
CD8_IFN-γ 0.80 -1.43
CD8_IL-2 5.04 1.39
CD4_PD-1 42.40 4.52
CD4_2B4 25.30 1.44
CD4_CTLA4 35.20 -5.55
CD8_PD-1 14.45 0.45
CD8_TIM3 14.83 -4.04
CD8_2B4 36.20 1.44
CD8_CTLA4 34.24 3.24
CD8_LAG3 21.21 -1.03
Step 4：Differentiation data model according to the present invention obtains the space bit in a model of the example T cell factor It sets, as shown in table 11 below：
Table 11：The spatial position of the T cell factor in a model
distance1 7.4375108
distance2 3.8455257
Step 5：Finally judged according to mathematical model, the overall T cell factor mark space length of the example with " distance2 " is close, therefore judges that the overall T cell function of the example belongs to " Immu_low ", i.e. T cell hypofunction.
It is worth noting that, the method for the present invention is only used for the function of assessment T cell, belong to indirect experimental data, and It cannot be directly used in diagnosis or assessment liver diseases, the diagnosis for disease itself, it is also necessary to refer in conjunction with various other clinics Mark side can determine.
The above described is only a preferred embodiment of the present invention, be not intended to limit the present invention in any form, therefore It is every without departing from technical solution of the present invention content, simply repaiied to any made by above example according to the technical essence of the invention Change, equivalent variations and modification, in the range of still falling within technical solution of the present invention.

Claims (10)

1. a kind of check and evaluation method of hepatopathy T cell function, which is characterized in that include the following steps：
1) it utilizes individual cells by flow cytometer, uses anti-cytokine antibodies and cell surface or intracellular specific markers group It closes, detects the secretion of different cytokines, result is obtained according to different labels；The anti-cytokine antibodies band is coloured The compound of fluorescent marker can clearly distinguish the immunocyte that no Secretion answers cell factor；
2) utilize big data biostatistics analytical technology, to each cell factor to cause the contribution of whole immune function/ Effect/influence carries out interpretation and ultimately forms the interpretation to whole immune function according to data model.
2. according to the method described in claim 1, it is characterized in that：The antibody is following a variety of：CD4+T cell and CD8+T is thin The IFN-γ of cellular expression, IL-2, TNF-α, CTLA-4, Tim3,2B4, PD-1, LAG3.
3. according to the method described in claim 1, it is characterized in that：The step 1) specifically includes：PBMC is separately cultured；Streaming Cell art detects T cell surface Co inhibitor；The Flow cytometry T cell intracellular factor.
4. according to the method described in claim 3, it is characterized in that：The PBMC, which is separately cultured, to be included the following steps：
1) EDTA anticoagulations 20ml is adopted；Centrifugation, 500g, 8min rise 9, and drop 9, separated plasma marks, and -80 DEG C of preservations of blood plasma wait for With haemocyte layer is continued to employ；Dilution：Haemocyte is moved into 50ml centrifuge tubes, adds PBS to about 30ml, and is blown and beaten uniform；
2) 4 15ml centrifuge tubes are taken, often pipe plus 4.5ml lymphocyte separation mediums, then add to blood above the separating liquid；
3) without brake density gradient centrifugation：25 DEG C, 450g, 25min, 5 are risen, drop 0；
4) test tube is taken out, sees that it is divided into following four layers：Upper layer is PBS and some residual blood plasma, i.e., plasma layer, lower layer are red blood cell And granulocyte；Middle level is lymphocyte separation medium, the visible tunica albuginea layer between lymphocyte separation medium and plasma layer, draws tunica albuginea Confluent monolayer cells, with 1：5 volume ratios wash cell 2 times, 500g, 8min with PBS；
5) sedimentation cell is added culture medium and is resuspended, and is dyed with 2% trypan blu e, it was demonstrated that viable count adjusts density 95% or more For 1*e7/ml.
5. according to the method described in claim 3, it is characterized in that：Flow cytometry T cell surface co-suppression point Son includes the following steps：
1) usage amount of streaming antibody is 2-5 μ l, and every streaming pipe takes the resuspension of RPMI 1640+10%FBS complete mediums 1*e5, cell；
2) 4 DEG C, 400g, 8min centrifugation abandon supernatant, are inverted water suction, utilize the even and fine born of the same parents of liquid shell remaining in pipe；It is added with each The antibody of autofluorescence label：CD4+T cell and CD8+T cell expression IFN-γ, IL-2, TNF-α, CTLA-4, Tim3,2B4, Each 2 μ l of PD-1, LAG3,4 DEG C are incubated 30min, even once every 15min bullets；
3) PBS 1ml are added to wash 1 time, 4 DEG C, 400g, 8min, abandons supernatant, is inverted water suction；120 whirlpools μ l PBS+40 μ l 4%PFA It is resuspended at once on spigot, it is fixed；
4) machine testing on.
6. according to the method described in claim 3, it is characterized in that：The Flow cytometry T cell intracellular factor includes Following steps：
1) take 500 μ l of for use PBMC cells, be added lymphocyte stimulation agent 10 μ l, 50 ×, play it is even, 37 DEG C of cell incubators with it is white The stimulation of cell activation object is incubated 4h；
2) 2 μ l of surface antibody PECF-594 CD3 are added, 2 μ l of APC CD4,2 μ l of V450 CD8, play even, 4 DEG C of refrigerators incubations 30min shakes primary every 15min；
3) often pipe plus 1ml PBS, 450g 8min centrifugations, washing, abandons supernatant, nozzle back-off utilizes residue in the blotting paper last time The even and fine born of the same parents of liquid shell；
4) often pipe adds 400 μ l of FIX＆PERM, and be vortexed mixing immediately, is incubated at room temperature 30min or 4 DEG C of incubation 45min；
5) often 1ml PBS centrifuge washings are added in pipe, and supernatant is abandoned in 750g, 10min centrifugation, and nozzle back-off is in blotting paper last time, profit With the even and fine born of the same parents of remaining liq bullet；
6) often pipe adds 500 μ l centrifuge washings of Perm buffer solutions, and 4 DEG C, supernatant is abandoned in 750g, 10min centrifugation, and nozzle is buckled in water suction Paper is last, utilizes the even and fine born of the same parents of remaining liq bullet；
7) plus intracellular antibody IFN-γ, IL-2, TNF-α, CTLA-4, Tim3,2B4, PD-1, LAG3 bullet are even, 4 DEG C of incubation 45min, Centre plays even primary；
8) often pipe is added 500 μ l of Perm buffer solutions and washes 1 time, 750g, 10min；
9) 150 μ l PBS are resuspended, and upper machine carries out flow cytometer detection.
7. according to the method described in claim 1, it is characterized in that：In the step 2), the big data biometrics credit Method includes used by analysis：
T cell functional status is classified as to using clustering methodology using immune indexes immune state is good and immune state is poor.
8. according to the method described in claim 7, it is characterized in that：By cluster, the mark number of each T cell factor is obtained Value forms markization numerical model.
9. according to the method described in claim 8, it is characterized in that：It builds following equation and carries out interpretation；
Z=c+a1X1+a2X2+...+apXp(formula 2)
Above equation meets the distance maximum between 2 classes, i.e.,：
Wherein,The scoring score of two class crowds is indicated respectively,For variance；Find such one group of coefficient a1,a2,… apSo that D2Maximum, i.e. distance are maximum, then corresponding formula 2 is discriminant equation.
10. a kind of hepatopathy T cell Function detection kit, which is characterized in that the kit includes for detecting following a variety of T The reagent of cell factor group：IFN-γ、IL-2、TNF-α、CTLA-4、Tim3、2B4、PD-1、LAG3.
CN201810400121.3A 2018-04-28 2018-04-28 The check and evaluation method and kit of hepatopathy T cell function Pending CN108627641A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110632217A (en) * 2019-11-05 2019-12-31 哈尔滨医科大学 Method for determining concentrations of four arsenic compounds in granulocyte by HPLC-ICP-MS method and application
CN113234159A (en) * 2021-05-12 2021-08-10 福州迈新生物技术开发有限公司 anti-LAG 3 protein monoclonal antibody, cell strain thereof, preparation method and application

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102590462A (en) * 2012-02-27 2012-07-18 贵州威门药业股份有限公司 Method for evaluating comprehensive quality of medicinal material Polygonum capitatum of Relinqing granules
CN103487558A (en) * 2013-07-30 2014-01-01 中国标准化研究院 Detection method for abnormal samples in mode identification and analysis of tea quality through intelligent sensory signals
CN105848680A (en) * 2013-10-25 2016-08-10 药品循环有限责任公司 Treatment using bruton's tyrosine kinase inhibitors and immunotherapy
CN107545135A (en) * 2017-08-15 2018-01-05 璞晞（广州）生物免疫技术有限公司 The construction method of hepatitis B liver diseases T lymphocyte immunity state application assessment models

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102590462A (en) * 2012-02-27 2012-07-18 贵州威门药业股份有限公司 Method for evaluating comprehensive quality of medicinal material Polygonum capitatum of Relinqing granules
CN103487558A (en) * 2013-07-30 2014-01-01 中国标准化研究院 Detection method for abnormal samples in mode identification and analysis of tea quality through intelligent sensory signals
CN105848680A (en) * 2013-10-25 2016-08-10 药品循环有限责任公司 Treatment using bruton's tyrosine kinase inhibitors and immunotherapy
CN107545135A (en) * 2017-08-15 2018-01-05 璞晞（广州）生物免疫技术有限公司 The construction method of hepatitis B liver diseases T lymphocyte immunity state application assessment models

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
周静芋 等: "常用费歇判别准则的比较", 《西北农林科技大学学报(自然科学版)》 *
姜钰 等: "多色流式细胞术检测CHB患者外周血多重细胞因子分泌的Th细胞亚群", 《中国肝脏病杂志（电子版）》 *
王济干 等: "《系统工程理论方法与应用》", 30 April 2016 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110632217A (en) * 2019-11-05 2019-12-31 哈尔滨医科大学 Method for determining concentrations of four arsenic compounds in granulocyte by HPLC-ICP-MS method and application
CN113234159A (en) * 2021-05-12 2021-08-10 福州迈新生物技术开发有限公司 anti-LAG 3 protein monoclonal antibody, cell strain thereof, preparation method and application
CN113234159B (en) * 2021-05-12 2022-04-08 福州迈新生物技术开发有限公司 anti-LAG 3 protein monoclonal antibody, cell strain thereof, preparation method and application

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