CN106198987A - A kind of detection label for evaluating tumor vaccine cells therapy and detection method thereof and application - Google Patents
A kind of detection label for evaluating tumor vaccine cells therapy and detection method thereof and application Download PDFInfo
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- CN106198987A CN106198987A CN201610784394.3A CN201610784394A CN106198987A CN 106198987 A CN106198987 A CN 106198987A CN 201610784394 A CN201610784394 A CN 201610784394A CN 106198987 A CN106198987 A CN 106198987A
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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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
Abstract
The present invention relates to the qualitative and quantitative analysis of cellular elements in human plasma, a kind of detection label for evaluating tumor vaccine cells therapy and detection method thereof and application.It is the combination of one or more in cytokine for evaluating the detection label of tumor vaccine cells therapy.The factor have highly sensitive, detection speed is fast, the advantages such as convenience, sample (blood plasma 20 DEG C is deposited) easy to store of drawing materials, this detection label and combinations thereof detection is one group of multiple relevant cell factor, by the low specificity overcoming single labelled thing to be brought because of individual variation and muting sensitivity, significantly improve accuracy.This detection label and detection method thereof can be used for judging the effect that tumor vaccine cells is treated in vitro, it is high that experiment shows that detection label that the present invention provides and combinations thereof has sensitivity, it is provided that the method detecting this label there is high flux, highly sensitive feature.
Description
Technical field
The present invention relates to the qualitative and quantitative analysis of cellular elements, specifically one in human plasma be used for evaluating tumor
The detection label of immune cell therapy and detection method thereof and application.
Background technology
Along with tumor is day by day serious to the threat that human survival is healthy, also there is day crescent in the Therapeutic mode of reply tumor
Different change, various novel drugs, new technique, new method emerge in an endless stream.In recent years, continuous along with what body immune system was recognized
Deeply and the developing rapidly of biotechnology, immunization therapy has become the important means of oncotherapy, and at combined therapy of tumour body
In occupation of the most important position in system, it it is the 4th class tumor therapeuticing method of development after operation, radiation and chemotherapy.Cancer
Being the cell disease of neoplastic cells escape immune surveillance system, the defect of body's immunity is that cancer occurs and the pass of development
Key.The antineoplastic immune of body is mainly T lymphocyte and is mediated.Cell biological treatment technology by autoimmune cell through external
Induce, break up, expand be fed back to again internal, walk around in-vivo tumour dysimmunity mechanism, by the immunoreation of excitating organism come
Resist, suppress and kill cancerous cell.
Along with immunotherapy of tumors basic research is increasing, increasing clinical trial occurs the most in succession.But when swollen
After tumor immunization therapy enters clinical experimental stage, with traditional appraisement system, such as WHO or solid tumor the standard of curative effect evaluation
(RECIST) go to evaluate its curative effect, but do not obtain expected results eventually, be partially into the project of III phase test, also grind at random controls
Count out in studying carefully.Immunotherapy of tumors meets with predicament in clinical trial so that Chinese scholars starts self-examination, applies mechanically tradition
The therapeutic efficacy assessment for the treatment of goes to evaluate immunization therapy, if having reasonability and feasibility.
For traditional treatment, the most i.e. clinical efficacy is evaluated.And with cell toxicity medicament not
Produce Graft Versus Tumor with, immune cell therapy usually through the immunoreation activating body, this process (as activating T cell,
Infiltration to tumor by local, produce clinical measurable Graft Versus Tumor) often need the most longer time several months.Pursue with chemotherapy
Occurring that complete incidence graph (CR) or part alleviate reaction differences such as (PR) in the recent period, patient benefits from the performance of immunization therapy the most for survival
Phase or SD time lengthening, quality of life improvement etc., this also complies with modern oncotherapy theory, is i.e. changed into from " one must be thorough in exterminating an evil "
" existence of band tumor ".In immunization therapy clinical trial, some patient be manifested initially by stable disease (SD), progression of disease (PD) or
, just there are the reactions such as tumor regression in hybrid reaction (MR) after a period of time.Therefore, immunization therapy therapeutic evaluation is totally different from carefully
Born of the same parents' poison class medicine, not only needs more evaluation index, must also observe the longer time, and this is accomplished by traditional therapeutic evaluation
System is modified or sets up new appraisement system.Outstanding problem in terms of immunization therapy reaction monitoring is at present, different real
Test testing result between room and have larger difference, especially in multicenter study, have impact on repeatability and the comparability of data.Lack
Can be as the method for quality control of goldstandard, this research also constraining immunization therapy and clinical practice.With traditional treatment (as changed
Treat) to directly act on tumor itself different, and immune cell therapy can direct killing tumor, it is often more important that, it is by acting on
In body immune system, improve immune response, finally affect the survival of patients phase.It plays a role main by 3 steps: 1. control
Activated immune cell and T cell propagation i.e. occur after treatment；2. the immunocyte activated mediates clinical measurable Graft Versus Tumor
(several weeks or several months produce the most after the treatment)；3. typically after treatment first, the several months just produces impact to the survival of patients phase.Just
Based on these features, tradition WHO or RECIST standard is not well suited for immunization therapy therapeutic evaluation, in addition to traditional index, exempts from
Epidemic disease reaction also should be included into the therapeutic efficacy assessment of immunization therapy.After evaluation immunization therapy, the immunoreation of body is to scientific evaluation
Its curative effect is significant.
To sum up, tumor vaccine cells treatment is carried out the evaluation of clinical efficacy, be this area research person must faced by one
Individual new problem, and set up the appraisement system of science, development and clinical practice to immunotherapy of tumors are significant.Now anxious
The detection mark that can be used for setting up tumour immunity Biotherapeutics clinical evaluation standard need to be found.
Summary of the invention
For filling up the immunization therapy clinical evaluation blank without unified standard, set up effectively evaluating standard directly perceived, it is provided that one
Plant the detection label for tumor vaccine cells treatment therapeutic evaluation and detection method thereof.
For achieving the above object, the present invention invent employing technical scheme be:
A kind of detection label for evaluating tumor vaccine cells therapy, for evaluating the detection mark of tumour immunotherapy
Note thing is the combination of one or more in cytokine.
Described cytokine be dipeptidyl peptidase Ⅳ (DPPIV), GDF-15 (GDF-15), growth hormone (GH),
Macrophage inflammatory protein 3α (MIP-3 α), Macrophage Cell albumen 3 β (MIP-3 β), growth stimulation expressing gene 2 albumen
(ST2), the receptor protein (Upar) of urokinase type plasminogen activator, AGER (RAGE), α swell
Tumor necrosis factor (TNF-α), one or more being situated between in-4 (IL-4), osteopontin (OPN), myeloperoxidase (MPO) (MPO) white
Combination.
CD26 is a kind of high conservative, is distributed in II type transmembrane glycoprotein of various kinds of cell.Its tertiary structure is the most clear at present
Chu.Initially because finding that it has proteolysis, therefore also known as dipeptidyl peptidase Ⅳ (DPPIV).The natural substrate of DPPIV is multiple many
Sample, performs multiple critical function in vivo.It or cell-membrane receptor and costimulatory molecules, thus participate in the immunity tune of body
Joint, cell migration, cell adhesion and natural death of cerebral cells process, thus relevant with the morbidity of multiple disease.Various CD26 inhibitor are to certainly
The treatment of body immune disease and cell proliferation disorders is significant.GDF-15 (growth
Differentiation factor-15, GDF-15) it is transforming growth factor-β (transforming growth factor-
β, TGF-β) remote branch of a clan of superfamily member is an important cardiovascular protection factor, as new label, GDF-15
Level has substantial connection with the diagnosis of multiple cardiovascular disease, risk stratification and Index for diagnosis.
Growth hormone (Growth Hormone, GH) is a kind of peptide hormone, and its structure is 191 strand peptides, contains
There are 191 amino acid moleculars, synthesized by the somatotroph in hypophysis, stored and secrete.Growth hormone major physiological effect
It is that tissue various to human body especially protein has promotion anabolic effect, bone articular cartilage and epiphyseal cartilage growth can be stimulated, because of
And can increase.Human body once lacks growth hormone and results in growth retardation.
Macrophage inflammatory protein 3α (Macrophage inflammatory protein 3-α, MIP-3 α), is chemotactic
The factor (Chemokine) superfamily member.The class CC race chemotactic factor mainly secreted by epidermis cell or mucomembranous epithelial cell,
Specificity chemotactic cell of Langerhan (Langerhans Cells, LC) migrates to epidermis or mucous epithelium.Known LC is in skin
Most important antigen presenting cell, it is resolved that the immunoreation feature of skin histology, is that immunity row occurs after heterogenous skin is transplanted
The main cause scolded；MIP-3 α also has chemotactic immature dendritic cell (DC) and the function of lymphocyte simultaneously, in inflammation
Play an important role in occurring with tumor.
Macrophage inflammatory protein 3 β (macrophage inflammatory protein, MIP-3 α), belongs to chemotactic factor
CC subfamily, its receptor is multiple CC-chemokine receptor (CCR).Granulocyte can be activated, regulate CD8+T cell and blood vessel endothelium
Cell adhesion, participates in hematopoietic regulation, induced NK cell proliferation and activation.
Growth stimulation expressing gene 2 albumen (ST2), is the member of interleukin I receptor family, in immunity and inflammatory reaction
Play the effect of key.ST2 has two kinds of forms: solubility ST2 and the ST2 of cross-film form, within 2002, discovery ST2 is on heart
Function, ST2 testing result not by renal function, ST2 albumen with sustain damage after fiberization have direct correlation.
UPAR is the receptor of urokinase type plasminogen activator (uPA), and this system is at mediation degrading matrix protein
During play an important role.UPAR is the polyglycosylated albumen of the strand rich in cysteine.UPAR is in many normal structures
Expression is all had with tumor tissues.UPAR can make uPA activate and be positioned cell surface, provides between cell and cell and cell
With the site that the uPAR of substrate junction is combined, provide uPA the having of proteolysis of mediation for expressing the tumor cell of uPAR
Profit environment.The uPA only effect of its activation fibrinolysin of specific combination competence exertion with the uPAR of surface of cell membrane.Therefore uPAR
It it is the key of the activating reaction of uPA mediation.UPAR is present on internal multiple human cell membrane, as ripe neutrophilic granulocyte, addicted to
The T cell etc. of eosinophil, mononuclear cell, vascular endothelial cell and activation.UPAR is epithelial in gastrointestinal cancer, bladder cancer etc.
High expressed in malignant tumor.UPAR, by being combined offer cell local fibrinolytic function, signal transduction in mediated cell with uPA, enters
And affect the propagation of cell, chemotactic, adhere to, migration etc..UPA, uPAR expression in the various malignant tumor tissue of the mankind
It is significantly larger than its corresponding normal structure.Research finds, uPA plays a significant role in tumor-infiltrated.With in situ hybridization and exempt from
The technique study such as epidemic disease group find, kinds of tumors tissue uPA and uPAR high expressed, and uPA and uPAR expresses the highest, patient's prognosis
The poorest, life cycle is the shortest.
AGER (receptor for advanced glycation end products, RAGE)
Newcomer for immunoglobulin superfamily.RAGE is distributed widely in vivo, can express on various kinds of cell surface.RAGE
Part have advanced glycosylation end products (advanced glycation end products, AGEs), amyloid beta
(amyloid-β protein, A β), Amphoterin, S100/ calgranulin and Transthyretin etc..Increasingly
Many biological evidences show, RAGE can be started by unique mechanism or accelerate atherosclerotic lesions.
Cachectin (TNF-α) is a kind of proinflammatory cytokine mainly produced by macrophage and mononuclear cell,
And participate in normal inflammation reaction and immunoreation.Cachectin produces under many pathological states and increases, including deteriorated blood
Disease, malignant tumor, heart failure and chronic inflammatory disease.
Bai Jie-4 (IL-4) is a kind of cytokine with various biological activity mainly secreted by T cell.Its energy
Promoting the growth of B cell, break up and breed, induction B cell produces IgE, IgM and IgG etc., and can strengthen MHCI class antigen and IL
The expression on resting B cells of one receptor；It also can promote propagation the generation of helper-inducer CTL toxicity of T cell；To monokaryon
The activity of macrophage antitumor and generation cytokine has a major impact.
Osteopontin (osteopontin, OPN) is a kind of electronegative non-Collagen bone matrix glycoprotein, and OPN is by multiple
Histiocyte synthesis and secretion, be distributed in Various Tissues and cell widely.1. cell adhesion OPN is by relying on RGD sequence
The combination of (α v β 1, α v β 3, α v β 5, α v β 1, α 8 β 1) and non-dependent RGD sequence (α 4 β 1, α 9 β 1) is present on cell surface many
Plant integrin receptor, play cell adhesion.OPN can adhere to JB6 cell and the HL60 cell (α v β 5 and α 4 β1receptor) converted,
And OPN combines the fibroblast converted with non-RGD form, the OPN of thrombin fracture can strengthen OPN Yu APP activation and Buddhist ripple
The platelet of ester activation and the adhesion of bone-marrow-derived lymphocyte and the adhesion of T cell.2. recruiting cells OPN in vitro is a kind of chemistry
Chemoattractant, exogenous OPN instructs fibroblastic migration with dosage-dependent manner (5.0～40mg/L)；It can stimulate greatly
Mus and the migration of bovine smooth muscle cells, can support to adhere to people and mouse T cell, in vivo after subcutaneous injection OPN, in injection part
Near Wei, OPN can directly inducing chemotactic effect and indirectly assist M move to other chemoattractant.In addition OPN can also promote brokenly
Osteocyte and the chemotaxis of B cell.3. cytokine-expressing OPN strengthens Th1 and suppresses the expression of Th2 cytokine, and it leads to
Crossing LPS stimulates directly induction to produce IL-12, the expression of suppression IL-10.Effect to IL-12 is to rely on OPN phosphorylation.OPN can
So that the polarization of earlier T h1 cytokine response.The also collaborative human T-cell of stimulation of OPN breeds and promotes that people's mononuclearcell expresses Th1
Cytokine.Intraarticular OPN is as a kind of intrinsic inhibitor producing IL-1, NO and PGE2.4. signal transduction osteopontin
As a kind of functional noncollagen protein of substrate, the main effect by two kinds of mechanisms play cell signaling molecules.One is to divide
In son, RGD primitive is combined with integral protein (integrin) family molecule；Two be with cell surface adhesion gC D44 with
Non-RGD relies on mode and combines.All by active cell internal specific signal transducting system, mediated cell glues two kinds of model of action
Attached, migrate and breed.After OPN is combined with integrin receptor, enabling signal transductory cascade reacts, and promotes the change of gene expression, and
Induction NF-К B activity, OPN can induce the focal adhesion kinase (FAK) of skeletin and the phosphorylation of paxillin (Paxillin) to change
Become, moreover it is possible to affect intracellular Ca2+Concentration.5. tumor occurs and the tumor in transfer medium source, generally expresses enhancing with OPN relevant,
The undue expression of OPN is relevant with people's gastric cancer progress.OPN promotes malignancy of tumor development by stimulating cell signalling and can strengthen
The existence of metastatic cell.The OPN paracrine as the advolution of carcinoma of prostate and the regulator of autocrine, recombinate OPN
By combining the H factor, these tumor cells can be made to survive.6. mineralization OPN contains the mineral with extracellular matrix
The acid domain of surface interaction.OPN can suppress the calcification of the vascular smooth muscle cell cultivated.7. cellular immunization
OPN knocks out rear mice and there is the immunodeficiency of Th1 mediation, and after corneal infection HSV-1, HSV can not be showed by the mice that OPN knocks out
, the most there is not keratitis adjoint for HSV, coding OPN gene and mice rickettsicidal gene in delayed hypersensitive reaction
(RicR) neighbouring, the RicR allele causing OPN early defect is relevant with rickettsial infection, and high level expression
The mice of OPN has resistance to rickettsia disease, and OPN plays an important role in the granulomatous formation that Th1 is cell-mediated.⑧
Other it mononuclear cell can be caused to break up；Accelerate angiogenesis；Participate in tissue reconstruction, such as bone resorption, angiogenesis and wound more
Close；The expression of induction urokinase type plasminogen activator (UPA)；The apoptosis of suppression endotheliocyte；By LPS and IFN-γ
The activity of the inducible NO-synthase (iNOS) of suppression renal cells；OPN is a kind of negative-feedback regu-lation of the INOS of mice M
Agent.It is also and atherosclerosis, autoimmune disease and other diseases associated with inflammation (pulmonary fibrosis) are relevant.
Myeloperoxidase (MPO) (myeloperoxidase, MPO) is by neutrophilic granulocyte, mononuclear cell and some tissue
The hemoprotein enzyme containing prosthetic heme group of macrophages secrete, is one of heme peroxidases superfamily member.MPO
Prompting patient can be raised as the predictor of coronary heart disease and acute coronary syndrome Index for diagnosis index: MPO and exist crown dynamic
Arteries and veins inflammation, speckle is unstable, but blood vessel is the most completely plugged, does not cause myocardial necrosis, so MPO can EARLY RECOGNITION unstable plaque
Block, is one of the Inflammation Marker of ACS early diagnosis.
A kind of detection method of the detection label for evaluating tumor vaccine cells therapy:
(1) by cellular elements detection labelling in the protein chip mensuration detected sample for detect plasma cell molecule
The change of thing；
(2) by the change of cellular elements detection label in above-mentioned acquisition detected sample, and then test sample to be checked is determined
The immune state of product.
Described protein chip detection method comprises the following steps:
(1) preparation of samples；
(2) the preparation of protein chip；
(3) protein chip is closed, hatches, is cleaned；
(4) hatch with biotinylation mixed antibody, clean；
(5) hatch with Streptavidin-HRP, clean；
(6) chip detection and data analysis.
Described label is dipeptidyl peptidase Ⅳ (DPPIV), GDF-15 (GDF-15), growth hormone (GH), huge
Phagocyte inflammatory protein 3 α (MIP-3 α), macrophage inflammatory protein 3 β (MIP-3 β), growth stimulation expressing gene 2 albumen
(ST2), the receptor protein (Upar) of urokinase type plasminogen activator, AGER (RAGE), α swell
Tumor necrosis factor (TNF-α), one or more being situated between in-4 (IL-4), osteopontin (OPN), myeloperoxidase (MPO) (MPO) white
Combination.
Concrete detection method:
The first step, take accept after immunotherapy of tumors effectively with the sample deteriorated
In the sample accepting immunotherapy of tumors in a large number, take the sample treating more than six courses for the treatment of continuously, take out wherein
Clinical feed back effective sample with accept immunotherapy of tumors after dead sample, be respectively effectively group and deterioration group.Select this
Blood plasma before a little each immunotherapy of tumors of sample.Simultaneously using normal human peripheral blood's separated plasma as comparison.
Second step, the preparation of protein chip
Within 20-30 minute in advance, from kits, take out chip to balance to room temperature.Chip is taken out from the plastic bag sealed,
Peel epiphragma, and allow its air be dried 1-2 hour.3rd step, protein chip are closed, hatch, are cleaned
100 μ L sample diluents are added in incubated at room 30 minutes in every hole, to close chip.Remove diluted sample completely
Liquid, 100 μ L sample, incubated at room 1-2 hour are added in every hole.Being removed from every hole by sample, 150 μ L/ holes are with 1 × washing buffer
Liquid washes 5 times (each 5 minutes), rocks the most gently.Lavation buffer solution is removed completely in each washing step.4th
Step is hatched with biotinylation mixed antibody, is cleaned
Add 1.4mL sample diluting liquid redissolution biotinylation mixed antibody, be sufficiently mixed dissolving.80 μ l mixing are added in every hole
Antibody-solutions, in incubated at room 1-2 hour.Being removed from every hole by sample, 150 μ L/ holes wash 5 times (often with 1 × lavation buffer solution
Secondary 5 minutes).Clean every time and need to rock the most gently, each washing step is removed lavation buffer solution completely.
5th step is hatched with Streptavidin-HRP, is cleaned
Add 1.4mL sample diluting liquid redissolution Streptavidin-HRP, be sufficiently mixed dissolving.80 μ l strepto-parents are added in every hole
With element-HRP, at room temperature lucifuge hatches 1 hour.Being removed from every hole by sample, 150 μ L/ holes wash 5 with 1 × lavation buffer solution
Secondary (each 5 minutes), rock the most gently, remove lavation buffer solution in each washing step completely.
6th step, chip detection, data analysis
Chip is pulled down carefully from pad.Chip is put in washer/exsiccator, adds enough lavation buffer solutions
To cover whole chip, rock 15 minutes the most gently.Lavation buffer solution is removed completely.It is dried chip.Use
Laser scanner scans chip, extracts data and is analyzed.
The application of a kind of cytokine, described cytokine is in the detection label for evaluating tumour immunotherapy
Application.
Described cytokine be dipeptidyl peptidase Ⅳ (DPPIV), GDF-15 (GDF-15), growth hormone (GH),
Macrophage inflammatory protein 3α (MIP-3 α), macrophage inflammatory protein 3 β (MIP-3 β), growth stimulation expressing gene 2 albumen
(ST2), the receptor protein (Upar) of urokinase type plasminogen activator, AGER (RAGE), α swell
Tumor necrosis factor (TNF-α), one or more being situated between in-4 (IL-4), osteopontin (OPN), myeloperoxidase (MPO) (MPO) white
Combination.
The present invention has the advantage that and good effect is: the present invention utilizes human cell factor protein chip to filter out 12 kinds of spies
Different stable existence in human plasma and the combination of detectable human cell factor are as evaluating tumor vaccine cells treatment curative effect
Detection label, these 12 kinds of factors can more sensitive reflect to tumor vaccine cells treatment be benefited degree degree, have
Highly sensitive, detection speed is fast, the advantages such as convenience, sample (blood plasma-20 DEG C is deposited) easy to store of drawing materials, this detection label
And combinations thereof detection be one group of multiple relevant cell factor, by overcome single labelled thing because of individual variation brought low special
Property and muting sensitivity, significantly improve clinical immunization cell therapy evaluate accuracy.This detection label and detection method thereof can
For judging the effect that tumor vaccine cells is treated in vitro, it was predicted that and evaluate the curative effect that tumor vaccine cells is treated, analyze swollen
The benefited intensity of tumor immune cell therapy, can as setting up the detection mark of tumor vaccine cells Therapy evaluation criterion,
Set up the new standard of immune cell therapy clinical evaluation.
Accompanying drawing explanation
Fig. 1 is Cytokine protein chip detection scanning figure in healthy group blood plasma 1；
Fig. 2 is Cytokine protein chip detection scanning figure in healthy group blood plasma 2；
Fig. 3 is Cytokine protein chip detection scanning figure in healthy group blood plasma 3
Fig. 4 is Cytokine protein chip detection scanning figure before effectively group sample A Levels of tumor immunization therapy；
Cytokine protein chip detection scanning figure when Fig. 5 is four courses for the treatment of of effectively group sample A Levels of tumor immunization therapy；
Cytokine protein chip detection scanning figure when Fig. 6 is eight courses for the treatment of of effectively group sample A Levels of tumor immunization therapy；
Fig. 7 is Cytokine protein chip detection scanning figure before deterioration group sample a Levels of tumor immunization therapy；
Cytokine protein chip detection scanning figure when Fig. 8 is four courses for the treatment of of deterioration group sample a Levels of tumor immunization therapy；
Cytokine protein chip detection scanning figure when Fig. 9 is eight courses for the treatment of of deterioration group sample a Levels of tumor immunization therapy.
Detailed description of the invention
Traditional biological chemistry and Protocols in Molecular Biology that disease carries out clinical diagnosis at present are the most comparatively laborious and coarse.
The new technique that development in recent years is got up has gene chip and protein (antibody) chip technology etc..In human body particularly in blood plasma
Containing ten hundreds of albumen and polypeptide fragments, the distribution of their concentration is wide, and the albumen clearly reported is little, quantification the most less
?.Cellular elements is the molecule that at present research is relatively more, these clearly with Ia cellular elements in look for
Immunotherapy of tumors has a specific cellular elements of close association, and to understand its change during immunotherapy of tumors be ten
Dividing significant work, Cytokine protein detection technique can analyze cellular elements in plasma sample the most with high throughput
Composition and content, clinical applicability is extremely strong.Owing to the physiological status change of organ-tissue can cause cellular elements content in blood plasma
Change, protein chip can detect in biological sample to the containing of the tumor vaccine cells treatment relevant all protein of curative effect
Amount situation, i.e. phenotype fingerprint (phenomic fingerprint).The phenotype fingerprint process to monitoring immunotherapy of tumors, or in advance
Survey and judge that the effect for the treatment of is significant.
The present invention can be by collecting blood in routine physical examination, owing to blood can be recycled to whole body institute in a organized way, and to carefully
Born of the same parents carry nutrition and remove refuse, and therefore blood can reflect the physiological and pathological situation of whole body, and its testing result is to people
Body health has directive significance.There is multiple protein in known blood plasma, including hundreds of kind of cellular elements, it is to a certain extent
The immune change of reaction body.Detect hundreds of cellular elements, compare screening and can react tumor vaccine cells treatment curative effect
Specific cells molecule, and then as detection label.
Below in conjunction with accompanying drawing, the invention will be further described, and particular implementation described here is by way of example
Representing, it is not intended as limitation of the present invention.In the case of not deviating from the scope of the invention, the principal character of the present invention
May be used for various embodiment.
Embodiment 1:
The protein chip test experience of cytokine in blood plasma, the present embodiment uses R&D Systems company Proteome
ProfilerTMHuman XL Cytokine Array Kit, concrete operation step is as follows:
Step one. sample and chip pretreatment
1. the plasma sample after treating through tumor vaccine cells takes the sample treating more than six courses for the treatment of continuously, at it
20, the sample that middle taking-up clinic is fed back effectively or focus reduces is as effective group, and taking-up simultaneously accepts tumor vaccine cells treatment
20, the sample of rear death is as deterioration group.And prepare freezing before above-mentioned two groups of different samples accept tumor vaccine cells treatment
Deposit plasma sample, accept tumor vaccine cells treatment four courses for the treatment of time frozen plasma sample with accept tumor vaccine cells treatment
Frozen plasma sample during eight courses for the treatment of.Simultaneously using the frozen blood plasma of 20 healthy plasma samples as comparison.To be the most frozen
Blood plasma is in 37 DEG C of fast melt, and 2100 × g is centrifuged 10min and goes oil removal, each blood plasma to be testing sample.
2. within 20-30 minute in advance, from test kit, take out (chip) Human XL Cytokine Array
Nitrocellulose membranes balances to room temperature.Human XL Cytokine is taken out from the plastic bag sealed
Array nitrocellulose membranes, peels epiphragma, and allows its air be dried 1-2 hour.Step 2. protein chip
Closing, hatch, clean
1. 100 μ L Array Buffer are added in incubated at room 30 minutes in every hole, to close chip.
2. removing Array Buffer, the detected sample prepared in 100 μ L step one the 1st steps is added in every hole, and room temperature is incubated
Educate 1-2 hour.
3. with sterilized water, 25 × Wash Buffer Concentrate is diluted 25 times, be 1 × lavation buffer solution, standby
With.4. detection sample added in upper step is removed from every hole, wash 5 times (often with the amount in 150 μ L/ holes with 1 × lavation buffer solution
Secondary 5 minutes), rock the most gently.Lavation buffer solution is removed completely in each washing step.
Step 3. hatch with biotinylation mixed antibody, clean
1. with 1.4mL Array Buffer redissolution Human XL Cytokine Array Detection Antibody
Cocktail, is sufficiently mixed dissolving.
2. mixed solution prepared by 80 μ l step 3 the 1st steps is added, in incubated at room 1-2 hour in every hole.
3. sample is removed from every hole, wash 5 times (each 5 minutes) with 1 × lavation buffer solution 150 μ L/ hole.Clean every time
Need to rock the most gently, each washing step is removed lavation buffer solution completely.
Step 4. hatch with Streptavidin-HRP, clean
1., with 1.4mL Array Buffer redissolution Streptavidin-HRP, it is sufficiently mixed dissolving.
2. mixed solution prepared by 80 μ l step 4 the 1st steps is added in every hole, and at room temperature lucifuge hatches 1 hour.
3. sample is removed from every hole, wash 5 times (each 5 minutes) with 1 × lavation buffer solution 150 μ L/ hole, at room temperature
Rock gently, each washing step is removed lavation buffer solution completely.
Step 5. chip detection
Chip is pulled down the most carefully from pad.
2. chip is put into and washes in device/exsiccator, add enough lavation buffer solutions with cover whole chip (about 30mL),
Rock 15 minutes the most gently.Lavation buffer solution is removed completely.Remove liquid completely, be dried chip.Use
LuxScan 10K/A twin-channel laser scanner scanning detects the chip of the front plasma sample of effective patient tumors treatment, is somebody's turn to do
Chip scanning figure (see Fig. 4).
Step 6. data acquisition
1. use LuxScan3.0 image analysis software that the chip image of Fig. 4 is analyzed, picture signal is turned
Turn to digital signal (factor content of detection), i.e. can directly display each factor by each point analysis software on scanning chip
Content (is shown in Table 1).Other each detection sample operations herewith.
Table 1 (unit: gray scale)
The most last statistical analysis selects the cytokine of differential expression
2.1. healthy group histone analysis
Data to health group, carry out one factor analysis of variance, and result display Sig, less than 0.05, illustrates between healthy group
The obvious albumen of the property of there are differences, needs to carry out further statistical analysis.Each sample of health group is carried out twice experiment,
Twice experiment is carried out paired-samples T-test, draws variant protein 14, by these 14 variant eggs in normal group
White reject after, health group data are carried out one factor analysis of variance again, found that Sig is 0.052 more than 0.05, it are described
Remaining data do not have notable difference the most as broad as long.
2.2. histone analysis is deteriorated
For deterioration group, detect respectively each sample accept tumor vaccine cells treatment before, four courses for the treatment of of treatment time with eight
The sample of 3 treatment time points of frozen plasma sample during the individual course for the treatment of, it is considered to the diversity between them utilizes repeated measure
Variance analysis, result shows that the Sig that each sample time is corresponding is both less than 0.05, and 3 treatment time points of each sample are described
Sample be distinguishing.
2.2.1 3 treatment time points of deterioration group do differential protein analysis respectively
Data to deterioration group, according to treatment time, the difference of point does paired-samples T-test respectively, finds out differential protein, deletes
The differential protein data that in deterioration group data, 3 treatment time point detections are corresponding, the differential protein 30 deleted altogether.
2.2.2 the analysis after deterioration group rejects differential protein
The deterioration group of remaining 80 protein data is carried out one factor analysis of variance, result display P value both greater than 0.05, says
After bright deterioration group carries out the deletion of data, remaining data, do not have diversity the most as broad as long between the detection of each sample homogeneous.Enter
One step discusses the diversity of 3 treatment time point detections of each sample, does the one factor analysis of variance of repetition, and result shows often
Sig between individual sample is measured every time is both less than 0.05, illustrates that the diversity of 3 treatment time point detections of each sample is significantly i.e. every
The data of 3 treatment time point detections of individual sample are distinguishing.
2.3 effective histone analyses
For effective group of group, detect respectively each sample accept tumor vaccine cells treatment before, treatment four courses for the treatment of time with
The sample of 3 treatment time points of frozen plasma sample during eight courses for the treatment of, it is considered to the diversity between them utilizes to have and repeats to survey
The variance analysis of amount, result shows that the Sig that each sample time is corresponding is less than 0.05,3 treatment time points of each sample is described
Sample be distinguishing.
The most effectively 3 treatment time point differential protein analyses respectively of group
Data to effectively group, according to treatment time, the difference of point does paired-samples T-test respectively, finds out differential protein, deletes
The differential protein data that effectively in group group data, 3 treatment time point detections are corresponding, the differential protein 13 deleted altogether.
2.3.2 effectively group rejects differential protein post analysis
Effectively group data after rejecting have done repeated measure one factor analysis of variance, result at different point of care time points
The Sig of display time is less than 0.05, illustrates effective group there were significant differences between treatment time point and treatment time put i.e. effectively
The data that the sample difference treatment time point of group records are distinguishing.
2.4. deterioration group and effective group compare
Data after data after deterioration group is deleted differential protein delete differential protein with effective group have compared weight
Multiple two-way analysis of variance, the Sig of deterioration group and effectively group, less than 0.05, illustrates between effective group and deterioration group it is to have difference
Be i.e. distinguishing between effective group and the data of deterioration group.
2.5. relative analysis further
2.5.1 normal group and deterioration histone analysis
Further the common significant albumen in healthy group, deterioration group and effective group is analyzed, normal group is common
The meansigma methods of significant Protein Detection carries out independent sample T with each protein data of the 3rd treatment time point detection of deterioration group
Inspection, finds the protein 15 of the property of there are differences, and illustrates that these albumen are normal group and the common distinguishing mark of deterioration group
Property albumen.
2.5.2 deterioration group and effective histone analysis
The protein data of the 3rd treatment time point detection of deterioration group is organized first treatment time spot check with effective respectively
Protein data, the protein data of effectively the 3rd treatment time point detection of group surveyed carry out independent sample T inspection, analyze and find to dislike
The protein data of the 3rd treatment time point detection of change group is carried out with the protein data of effectively first treatment time point detection of group
Relatively 48, finding differences property albumen；The protein data of the 3rd treatment time point detection of deterioration group and effectively the 3rd treatment of group
The protein data of time point detection compares finding differences property protein 32.When Integrated comparative finds deterioration group the 3rd treatment
Between put the protein data of the protein data of detection and effectively first, the 3rd treatment time point detection of group and find common difference
Property protein 28, these albumen are deterioration groups and effectively organize common distinguishing significant albumen, deterioration group and effective group
There is notable difference.
When Integrated comparative 15 deteriorates, differentially expressed protein and 28 treatments are effective, expressing protein finds, dipeptidyl peptidase Ⅳ
(DPPIV), GDF-15 (GDF-15), growth hormone (GH), macrophage inflammatory protein 3α (MIP-3 α), huge bite
Cellular inflammatory albumen 3 β (MIP-3 β), growth stimulation expressing gene 2 albumen (ST2), urokinase type plasminogen activator
Receptor protein (Upar), AGER (RAGE), cachectin (TNF-α), white-4 (IL-4) that are situated between,
Osteopontin (OPN), myeloperoxidase (MPO) (MPO) totally 12 Cytokine protein are that the two has, and this 12 kinds of cells are described
The factor has extremely close relationship with the generation development of tumor.Except dipeptidyl peptidase Ⅳ after being treated by tumor vaccine cells
(DPPIV) with fail to respond to any medical treatment in negative correlation outside, the deterioration that other 11 kinds of cytokines and tumor vaccine cells are treated is proportionate,
To the good sample of tumor vaccine cells therapeutic response then without this rule.Effectively in group pattern detection data these 12 kinds of cells because of
The content of son can be along with the number increase course for the treatment of close to the average in healthy blood plasma.It may be said that by these 12 kinds of cytokines at blood
In slurry, changes of contents can reflect and the clinical effectiveness of tumor vaccine cells treatment.
Embodiment 2:
Filter out 12 kinds of cytokine-expressing amount changes for checking further and can react tumor vaccine cells treatment curative effect, make
With h company Proteome ProfilerTMHuman XL Cytokine Array Kit, verifies.Concrete operation step is such as
Under:
Step one. sample and chip pretreatment
1. before after preparing tumor vaccine cells treatment, dead sample and each tumor vaccine cells of this sample are treated
Frozen plasma sample.By 37 DEG C of fast melt of frozen blood plasma, 2100 × g is centrifuged 10min and goes oil removal, each plasma sample to be
Testing sample.
2. within 20-30 minute in advance, from box, take out Human XL Cytokine Array nitrocellulose
Membranes balances to room temperature.Human XL Cytokine Array is taken out from the plastic bag sealed
Nitrocellulose membranes, peels epiphragma, and allows its air be dried 1-2 hour.
Step 2. the closing of protein chip, hatch, clean
1. 100 μ L Array Buffer are added in incubated at room 30 minutes in every hole, to close chip.
2. removing Array Buffer, the detected sample prepared in 100 μ L step one the 1st steps is added in every hole, and room temperature is incubated
Educate 1-2 hour.
3. with sterilized water, 25 × Wash Buffer is diluted 25 times, be 1 × lavation buffer solution, standby.
4. detection sample added in upper step is removed, with the amount 1 × lavation buffer solution with 150 μ L/ holes from every hole
Wash 5 times (each 5 minutes), rock the most gently.Lavation buffer solution is removed completely in each washing step.
Step 3. hatch with biotinylation mixed antibody, clean
1. with 1.4mL Array Buffer redissolution Human XL Cytokine Array Detection Antibody
Cocktail, is sufficiently mixed dissolving.
2. the mixed solution of preparation in 80 μ l step 3 the 1st steps is added in every hole, in incubated at room 1-2 hour.
3. sample is removed from every hole, wash 5 times (each 5 minutes) with 1 × lavation buffer solution 150 μ L/ hole, clean every time
Need to rock the most gently, each washing step is removed lavation buffer solution completely.
Step 4. hatch with Streptavidin-HRP, clean
1., with 1.4mL Array Buffer redissolution Streptavidin-HRP, it is sufficiently mixed dissolving.
2. the mixed solution of preparation in 80 μ l step 4 the 1st steps is added in every hole, and at room temperature lucifuge hatches 1 hour.
3. sample is removed from every hole, wash 5 times (each 5 minutes) with 1 × lavation buffer solution 150 μ L/ hole, at room temperature
Rock gently, each washing step is removed lavation buffer solution completely.
Step 5. chip detection and data acquisition
Chip is pulled down the most carefully from pad.
2. chip is put into and washes in device/exsiccator, add enough lavation buffer solutions with cover whole chip (about 30mL),
Rock 15 minutes the most gently.Lavation buffer solution Ι is removed completely.Remove liquid completely, be dried chip.Use
LuxScan 10K/A twin-channel laser scanner scanning detects this deterioration group sample when accepting the 8th course for the treatment of of immunotherapy of tumors
The detection chip of plasma sample, obtains this chip scanning figure (see Fig. 9).
3. use LuxScan3.0 image analysis software that the chip image of Fig. 9 is analyzed, picture signal is turned
Turn to digital signal, i.e. can directly display each factor content (being shown in Table 2) by each point analysis software on scanning chip.
Table 2 (unit: gray scale)
During other each courses for the treatment of, plasma sample detection chip operates herewith.
Step 6. data analysis
The data that when relatively this sample accepts tumor vaccine cells treatment different course, plasma sample detection chip obtains, choose
Selecting 12 kinds of cellular elementss filtered out in each chip, analyze these data lateral comparisons, result proves that its change becomes
Gesture meets present invention, i.e. except dipeptidyl peptidase Ⅳ (DPPIV) with fail to respond to any medical treatment in negative correlation in addition to, other 11 kinds of cytokines with
The deterioration of immunotherapy of tumors is proportionate.Prove that the combination of one or more in these 12 kinds of cytokines can be made further
For the detection label for tumor vaccine cells treatment therapeutic evaluation.
Fig. 1-3 is the testing result of cytokine in healthy group blood plasma, and Fig. 4-6 is effectively to organize the inspection of cytokine in blood plasma
Surveying result, Fig. 7-9 is the testing result of cytokine in deterioration group blood plasma, and in figure, every bit represents a kind of cellular elements, its face
The depth of color represents the number of this factor content.Can be seen that from scanning figure and not all cytokine has Gao Feng in the sample
Degree is expressed, and some cytokine is very trace in the sample, even can not normally detect.The result display health of Fig. 1-3
In blood plasma some cytokine be stable existence and content close.Fig. 4-6 and Fig. 7-9 result show at improvement and deteriorating course
In the content of some factor really occur significantly to change.Filter out these according to statistical analysis and significantly reflect swollen
12 cytokines of tumor immune cell therapy therapeutic effect.It can thus be seen that the present invention provide detection label and
Combination there is higher content and more sensitive reaction tumor vaccine cells therapy for treating effects capability, it is provided that this labelling of detection
The method of thing has high flux, highly sensitive feature.
Claims (7)
1. the detection label being used for evaluating tumor vaccine cells therapy, it is characterised in that: it is used for evaluating tumour immunity thin
The detection label of born of the same parents' therapy is the combination of one or more in cytokine.
2. as described in claim 1 for evaluating the detection label of tumor vaccine cells therapy, it is characterised in that: described carefully
Intracellular cytokine is dipeptidyl peptidase Ⅳ (DPPIV), GDF-15 (GDF-15), growth hormone (GH), Macrophage Inflamatory egg
White 3 α (MIP-3 α), macrophage inflammatory protein 3 β (MIP-3 β), growth stimulation expressing gene 2 albumen (ST2), urokinase type are fine
The receptor protein (Upar) of fibrillarin zymoexcitator, AGER (RAGE), cachectin (TNF-
α), the white combination of one or more being situated between in-4 (IL-4), osteopontin (OPN), myeloperoxidase (MPO) (MPO).
3. a detection method for the detection label for evaluating tumor vaccine cells therapy described in claim 1, it is special
Levy and be:
(1) by cellular elements detection label in the protein chip mensuration detected sample for detect plasma cell molecule
Change；
(2) by the change of cellular elements detection label in above-mentioned acquisition detected sample, and then detected sample is determined
Immune state.
4. the detection method of the detection label for evaluating tumor vaccine cells therapy as described in claim 3, its feature
It is: described protein chip detection method comprises the following steps:
(1) preparation of samples；
(2) the preparation of protein chip；
(3) protein chip is closed, hatches, is cleaned；
(4) hatch with biotinylation mixed antibody, clean；
(5) hatch with Streptavidin-HRP, clean；
(6) chip detection and data analysis.
5. the detection method of the detection label for evaluating tumor vaccine cells therapy as described in claim 3, its feature
It is: described label is dipeptidyl peptidase Ⅳ (DPPIV), GDF-15 (GDF-15), growth hormone (GH), huge bites
Cellular inflammation albumen 3 α (MIP-3 α), macrophage inflammatory protein 3 β (MIP-3 β), growth stimulation expressing gene 2 albumen (ST2),
The receptor protein (Upar) of urokinase type plasminogen activator, AGER (RAGE), α neoplasm necrosis
The factor (TNF-α), the white combination of one or more being situated between in-4 (IL-4), osteopontin (OPN), myeloperoxidase (MPO) (MPO).
6. the application of a cytokine, it is characterised in that: described cytokine is for evaluating tumor vaccine cells therapy
Application in detection label.
7. the application of the cytokine as described in claim 6, it is characterised in that: described cytokine is dipeptidyl peptidase Ⅳ
(DPPIV), GDF-15 (GDF-15), growth hormone (GH), macrophage inflammatory protein 3α (MIP-3 α), huge bite
Cellular inflammatory albumen 3 β (MIP-3 β), growth stimulation expressing gene 2 albumen (ST2), urokinase type plasminogen activator
Receptor protein (Upar), AGER (RAGE), cachectin (TNF-α), white-4 (IL-4) that are situated between,
The combination of one or more in osteopontin (OPN), myeloperoxidase (MPO) (MPO).
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| CN113109565A (en) * | 2021-04-12 | 2021-07-13 | 中国人民解放军空军军医大学 | Detection kit for evaluating sensitivity of patient to PD-1 monoclonal antibody product and application thereof |
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| CN1335505A (en) * | 2001-07-11 | 2002-02-13 | 上海晶泰生物技术有限公司 | Protein chip based on labeling streptavidin-biotin technology |
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