CN103235135A - Detection method of characteristic cell mass for representing inhibitory activity of peripheral regulatory T cell, and application thereof - Google Patents
Detection method of characteristic cell mass for representing inhibitory activity of peripheral regulatory T cell, and application thereof Download PDFInfo
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Abstract
The invention relates to the field of medicine, particularly relates to a detection method of a characteristic cell mass for representing inhibitory activity of a peripheral regulatory T cell and application thereof, and more particularly relates to the detection method of the characteristic cell mass for representing the inhibitory activity of the peripheral regulatory T cell, application of the detection method in preparation of a kit for testing the inhibitory activity of the regulatory T cell and application in preparation of a kit for screening early pancreatic lesions targets. The industrial universal theory that the contradiction exists between a peripheral immune regulatory cell mass and the required adjusting function, and the quantity of the immune regulatory cells is independent from the played role is negated; and a group of cell masses having characterization value on the inhibitory activity of the regulatory T cell are found out. The invention further discloses a corresponding cell mass detection method. Thus, an important foundation is established for representing the inhibitory activity of the regulatory T cell and screening the early pancreatic lesions targets.
Description
Technical field
The present invention relates to field of medicaments, particularly relate to a kind of for characterizing detection method and the application thereof that the periphery regulatory T cells suppresses the feature cell mass of activity, it is a kind of for characterizing regulatory T cells inhibition activity change and target tissue immune response mechanism and using to say so especially, be to find a kind ofly for characterizing that regulatory T cells has the state variation of the cell mass that suppresses active and the mechanism of mediated targeted histogenic immunity between replying, and be used for the application that the dynamic monitoring regulatory T cells suppresses the active agent box, and for the preparation of the application in the kit of screening pancreas early lesion target.
Background technology
Discover that the autoimmune diabetes are a kind of spontaneous immunity diseases and present gradual evolution, because immunoregulatory activity descends, reduce it to endogenous effect T cell inhibiting effect, the specificity attack beta Cell of islet that is mediated by effect T cell-be the unit of unique synthesis secretion insulin in the body, therefore, the result that pancreas islet destroys directly causes insulin secretion quantity to reduce, and the function of insulin is transported to the glucose in the blood in the corresponding cell, needs for the cell normal physiological activity.Insulin quantity reduces the glucose metabolism utilization that directly has influence on again in the blood.When the pancreas islet of 80 ≈ 90% is destroyed when making that insulin secretion quantity is down to critical value, the endogenous insulin that remains can't be kept the required glycometabolism of physiology, and the glucose in the blood raises unusually, develops into the 1 type diabetes of clinical phase this moment.Disease progression so far, patient table reveals corresponding clinical symptoms and sign, laboratory examination finds that the dextrose equivalent in blood and the urine raises, and other some auxiliary examinations can assisted diagnosis, but mainly is the rising degree diagnosing diabetes according to blood sugar.
At present to having limitation in the pathogenesis of 1 type diabetes and the diagnosis and treatment; At first, though academia is unbalance to the periphery immune tolerance to be to cause the pathogenetic origin cause of formation of autoimmunity that common recognition has been arranged, the key that namely tolerates balance adjustment depends on immunity regulatory cell group's expression, but find in some scientific experiments to have contradiction between regulatory function that immunity regulatory cell group height and it should possess, the effect irrelevant (Anne Cooke univ cambridge uk is published in international Journal of Immunology) that the quantity height of discovering immunity regulatory cell should be brought into play with it, have " corpse cell " among the immunity regulatory cell group, therefore how defining the target position with definite regulatory function activity becomes key; Secondly, because the unusual rising that blood sugar continues is subjected to the influence of several factors, fluctuation has error more greatly and easily, and only pancreas islet is destroyed reduce to critical value after, inherent pathological development to clinical after date just shows.Therefore, can not be comprehensively and system embody generation and the progress of disease, the early lesion that especially can't reflect the immune attack islet cells, in addition, though the pancreatic tissue biopsy can reflect the pathological change of pathology part, the restriction that it is subjected to complication such as operation wound that biopsy brings and can't be accepted by doctor and patient.
Summary of the invention
The present invention breaks through the quagmire in the research of present autoimmune disease pathogenesis, solved the height of the simple dependence periphery immunity regulatory cell expression contradiction between can't the regulatory function of its actual performance of objective embodiment, find the specificity target position can scientifically reflect the regulatory T cells immunosuppressive activity, and corresponding cell mass detection method further disclosed, thereby realize that the dynamic monitoring regulatory T cells suppresses activated state, and the target of the target organ pancreas early lesion of conduct screening autoimmunity diabetes, established important basis.
Particularly, the invention discloses a kind of detection method for the targeted cells group who characterizes periphery regulatory T cells inhibition activity, may further comprise the steps:
Step 1) is handled sample to be tested, forms single cell suspension; Specifically, peripheral blood to be measured or animal spleen are handled according to conventional method, gone to prepare the lymphocyte suspension after the red blood cell filtration;
Step 2) antibody staining of feature cell mass:
With above-mentioned single cell suspension respectively with monoclonal antibody body CD3, CD4, the CD69 mixing of the different fluorescence of mark, hatched on ice 30 minutes;
Step 3) mixed liquor PBS washing lotion is mixed well, and adds cell rupture of membranes lysate, and mixing was hatched 30 minutes on ice, and is with the auxilliary liquid of cell rupture of membranes, fully centrifugal behind the mixing again, removes supernatant;
The fresh preparation deoxyribonuclease of step 4) liquid joins in the step 4) in the gained precipitation, hatches behind the mixing 60 minutes, and is with the auxilliary liquid of cell rupture of membranes, fully centrifugal behind the mixing again, removes supernatant,
Step 5) adds the antibody Fc blocking agent, fully acts on 20 minutes;
Step 6) adds certification mark respectively and regulates the transcription factor (FoxP3) of cell epitope and the antibody that DNA (deoxyribonucleic acid) (BrdU) copies; Described BrdU, FoxP3 adopt different fluorescence labelings respectively, divide to get 1.5 μ l antibody and hatched on ice 30 minutes, and again with the auxilliary liquid 200 μ l of cell rupture of membranes, the washed cell suspension, fully centrifugal behind the mixing, remove supernatant;
The step 7) flow cytometer detects:
Centrifugal behind the abundant mixing of adding fluidic cell washing lotion, remove supernatant, add 500ul fluidic cell washing lotion, flow cytometer detects the accounting of the active regulatory T cells that obtains dyeing.
Further, the invention also discloses described step 2) in, single cell suspension with mark the blending ratio of monoclonal antibody body of different fluorescence be respectively: single cell suspension and CD3 by volume calculate for 1:400, single cell suspension and CD4 by volume calculate for 1:200, single cell suspension be 1:150 with the calculation of CD69 by volume; Single cell suspension and BrdU with the volume mass ratio be calculated as 1.5ul:1 ug, single cell suspension is calculated as 1.5ul:1ug with FoxP3 with the volume mass ratio.
As further optimization, the final concentration that the invention also discloses single cell suspension described in the step 1) is 1 * 10
7/ ml, the cell suspension of getting 100ul behind the mixing is experimental subjects.
Simultaneously, the invention also discloses described step 2) in, cell suspension and CD3, CD4, CD69 in conjunction with hatching 30 minutes, wash 2 times with PBS under 4 ℃ condition again.
Moreover, the invention also discloses in the described step 4), the gained precipitation with the auxilliary liquid repeated washing of cell rupture of membranes once, and is fully centrifugal behind the mixing, removes supernatant.
The present invention finds the cell mass with practical adjustments function by the discovery target position in overall adjustment T cell, and by antibody staining method disclosed by the invention, characterizing this feature cell mass also simultaneously, quantitative statistics comes out, reach the purpose that scientific quantification has the regulatory T cells that suppresses active, solved at present ubiquitously because paying close attention to the regulatory T cells total quantity merely in the industry, and caused the problem to the misjudgement of immunoregulation functional state; Also scientific explarnation regulation mechanism autoimmune disease particularly 1 type diabetes take place, effect in the pathology process of development.
Simultaneously, feature cell mass detection method disclosed in this invention, be generalized to the ill and potential morbidity crowd of type 1 diabetes, with peripheric venous blood as detecting sample, thereby suppress active and have wound ground screening pancreas early lesion target and lay a good foundation for detecting regulatory T cells.
Simultaneously, the present invention further discloses described detection method for the feature cell mass that characterizes regulatory T cells inhibition activity and be used for the application that regulatory T cells suppresses the active agent box.Particularly be used for the application that target detects regulatory T cells inhibition active agent box.
And it is described for characterizing the active method of regulatory T cells inhibition in the application for the preparation of the kit that screens pancreas early lesion target.Particularly target detects the periphery regulatory T cells and suppresses active method for the preparation of the application in the kit of screening pancreas early lesion target.
Can't reflect objective and comprehensively that autoimmunity attacks the limitation of islet cells different phase pathology thereby overcome conventional blood sugar monitoring, and the difficult problem of the complication such as operation wound brought of pancreatic tissue biopsy.
FoxP3 is finger transcription factor herein; BrdU refers to 5-bromodeoxyuridine nucleosides.
Description of drawings
Fig. 1-1 is for being in the lymphocyte flow cytometer testing result of female mouse around the model the;
Fig. 1-2 is the lymphocyte flow cytometer testing result that is in model the tenth all female mouse;
Fig. 1-3 is for being in the lymphocyte drain cell instrument testing result of model the 16 all female mouse;
Fig. 2 is for being in the curve synoptic diagram of female mouse characteristic of correspondence cell mass accounting in the regulatory T cells total amount of disease process different phase among the embodiment 10;
Fig. 3-1 is the lymphocyte flow cytometer testing result of A group mouse among the embodiment 11;
Fig. 3-2 is the lymphocyte drain cell instrument testing result of B group mouse among the embodiment 11;
Fig. 4-1 is for being in the pancreas stained preparation of the female mouse around the model the;
Fig. 4-2 is the pancreas stained preparation of A group mouse;
Fig. 4-3 is the pancreas stained preparation that is in the female mouse in the tenth week of model;
Fig. 4-4 is the pancreas stained preparation of B group mouse;
Fig. 4-5 is the pancreas stained preparation that is in the female mouse in the 16 week of model;
Fig. 5 is the curve synoptic diagram that is in female mouse characteristic of correspondence cell mass accounting in the regulatory T cells total amount of pancreatic disorders different phase;
Fig. 6 is the blood sugar synoptic diagram of the female mouse of pancreatic disorders different phase among the embodiment 13;
Fig. 7 is the end-state synoptic diagram of feature cell mass accounting in the regulatory T cells total amount for the treatment of group and the female mouse of control group among the embodiment 14;
Fig. 8 is the blood sugar synoptic diagram for the treatment of group and the female mouse of control group among the embodiment 14.
Embodiment
Further specify the present invention below in conjunction with embodiment and accompanying drawing, in following examples, unless stated otherwise, otherwise the reagent of using, instrument, equipment are the conventional commercially available prod that meets the corresponding function requirement.
Embodiment 1
Gather person under inspection's peripheric venous blood, peripheric venous blood to be measured is handled also therefrom separated the T cell, concrete separate mode can be with reference to prior art.After adopting nylon membrane net filtration partition method to obtain monocyte, preparation forms single cell suspension, and for the ease of follow-up operations such as dyeing, we become 1 * 10 with the single cell suspension prepared at concentrations usually
7/ ml, getting 100ul behind the mixing is experimental subjects.
We carry out antibody staining to the feature cell mass in the single cell suspension then, and concrete operation steps is as follows:
Above-mentioned single cell suspension is mixed with fluorescence antibody CD3, CD4, CD69 and BrdU, FoxP3 respectively;
The method of operating of mixed mark is according to the operation instruction operation of associated antibodies.
Mix well with the fluidic cell washing lotion through the mixed liquor of mark, add cell rupture of membranes lysate, behind the mixing, on ice or with identical on ice temperature conditions under hatched 30 minutes, wash with the auxilliary liquid of cell rupture of membranes again, centrifugal behind the abundant mixing, remove supernatant;
With fresh preparation deoxyribonuclease liquid, join in the abovementioned steps in the gained precipitation, hatched behind the mixing 60 minutes, with the auxilliary liquid of cell rupture of membranes, fully centrifugal behind the mixing again, remove supernatant,
Add the antibody Fc blocking agent in the precipitation that obtains, fully act on 20 minutes;
Add certification mark respectively and regulate the transcription factor (FoxP3) of cell epitope) and the antibody of DNA replication (BrdU), the fluorescence labeling that described BrdU, FoxP3 difference is different, divide and to get 1.5 μ l antibody and hatched on ice 30 minutes, again with the auxilliary liquid 200 μ l of cell rupture of membranes, the washed cell suspension, fully centrifugal behind the mixing, remove supernatant;
To precipitate by flow cytometer and detect:
Centrifugal behind the abundant mixing of adding fluidic cell washing lotion, remove supernatant, add 500ul fluidic cell washing lotion, flow cytometer detects the accounting of the active regulatory T cells that obtains dyeing.
At our goal of the invention, above-mentioned colouring method selects these to represent the molecular marker of lymphocyte particular attribute effectively, and consider to remove the interference of non-specific staining, both utilized the exclusive sign of each molecule, itself and other molecular marker can be united to define new function target again.
Embodiment 2
Gather person under inspection's peripheric venous blood, peripheric venous blood to be measured is handled also therefrom separated the T cell, concrete separate mode can be with reference to prior art.After adopting nylon membrane net filtration partition method to obtain monocyte, preparation forms single cell suspension, and for the ease of follow-up operations such as dyeing, we become 1 * 10 with the single cell suspension prepared at concentrations usually
7/ ml, getting 100ul behind the mixing is experimental subjects.
We carry out antibody staining to the feature cell mass in the single cell suspension then, and concrete operation steps is as follows:
Above-mentioned single cell suspension is mixed with fluorescence antibody CD3, CD4, CD69 and BrdU, FoxP3 respectively;
The dilution ratio of preferred single cell suspension and fluorescence antibody is respectively in the present embodiment: it is 1:150 that single cell suspension and the calculation of CD3 by volume are calculated for 1:200, single cell suspension and CD69 by volume for 1:400, single cell suspension and the calculation of CD4 by volume, and reaching envelope-bulk to weight ratio after the different proportion dilution is 1ul:1 ug; And single cell suspension and BrdU with the volume mass ratio be calculated as 1.5ul:1 ug, single cell suspension is calculated as 1.5ul:1ug with FoxP3 with the volume mass ratio.
Mix well with the fluidic cell washing lotion through the mixed liquor of mark, add cell rupture of membranes lysate, behind the mixing, on ice or with identical on ice temperature conditions under hatched 30 minutes, wash with the auxilliary liquid of cell rupture of membranes again, centrifugal behind the abundant mixing, remove supernatant;
With fresh preparation deoxyribonuclease liquid, join in the abovementioned steps in the gained precipitation, hatched behind the mixing 60 minutes, with the auxilliary liquid of cell rupture of membranes, fully centrifugal behind the mixing again, remove supernatant;
Add the antibody Fc blocking agent in the precipitation that obtains, fully act on 20 minutes;
To regulate the transcription factor of cell epitope and the antibody of DNA replication (different fluorescence labeling BrdU, FoxP3 respectively) through adding certification mark in the solution after the effect of antibody Fc blocking agent respectively, divide and to get 1.5 μ l antibody and hatched on ice 30 minutes, again with the auxilliary liquid 200 μ l of cell rupture of membranes, the washed cell suspension, fully centrifugal behind the mixing, remove supernatant;
Centrifugal behind the abundant mixing of adding fluidic cell washing lotion, remove supernatant, add 500ul fluidic cell washing lotion, flow cytometer detects the accounting of the active regulatory T cells that obtains dyeing.
Embodied in experiment is implemented, optimized antibody concentration and dosage, ensured that the stable and antibody of statistics efficiently acts on.
Embodiment 3
Gather person under inspection's peripheric venous blood, peripheric venous blood to be measured is handled also therefrom separated the T cell, concrete separate mode can be with reference to prior art.After adopting nylon membrane net filtration partition method to obtain monocyte, preparation forms single cell suspension, and for the ease of follow-up operations such as dyeing, we become 1 * 10 with the single cell suspension prepared at concentrations usually
7/ ml, getting 100ul behind the mixing is experimental subjects.
We carry out antibody staining to the feature cell mass in the single cell suspension then, and concrete operation steps is as follows:
Above-mentioned single cell suspension is mixed with fluorescence antibody CD3, CD4, CD69 and BrdU, FoxP3 respectively; Cell suspension and CD3, CD4, CD69 in conjunction with hatching 30 minutes, wash 2 times with PBS under 4 ℃ condition again.
Mix well with the fluidic cell washing lotion through the mixed liquor of mark, add cell rupture of membranes lysate, behind the mixing, on ice or with identical on ice temperature conditions under hatched 30 minutes, wash with the auxilliary liquid of cell rupture of membranes again, centrifugal after the washing, get precipitation;
With fresh preparation deoxyribonuclease liquid, join in the abovementioned steps in the gained precipitation, hatched behind the mixing 60 minutes, again with the auxilliary liquid of cell rupture of membranes, washing, centrifugal, get precipitation,
Add the antibody Fc blocking agent in the precipitation that obtains, fully act on 20 minutes;
To regulate the transcription factor (FoxP3) of cell epitope through adding certification mark in the solution after the effect of antibody Fc blocking agent respectively) and the antibody of DNA replication (BrdU), the fluorescence labeling that described BrdU, FoxP3 difference is different, divide and to get 1.5 μ l antibody and hatched on ice 30 minutes, again with the auxilliary liquid 200 μ l of cell rupture of membranes, the washed cell suspension, fully centrifugal behind the mixing, remove supernatant;
Centrifugal behind the abundant mixing of adding fluidic cell washing lotion, remove supernatant, add 500ul fluidic cell washing lotion, flow cytometer detects the accounting of the active regulatory T cells that obtains dyeing.
We optimize conditions such as temperature that experiment needs, brooding time, thereby detect the functional status of two kinds of different film inner cell organs in same cell sample, have improved traditional colouring method.
Gather person under inspection's peripheric venous blood, peripheric venous blood to be measured is handled also therefrom separated the T cell, concrete separate mode can be with reference to prior art.After adopting nylon membrane net filtration partition method to obtain monocyte, preparation forms single cell suspension, and for the ease of follow-up operations such as dyeing, we become 1 * 10 with the single cell suspension prepared at concentrations usually
7/ ml, getting 100ul behind the mixing is experimental subjects.
We carry out antibody staining to the feature cell mass in the single cell suspension then, and concrete operation steps is as follows:
Above-mentioned single cell suspension is mixed with fluorescence antibody CD3, CD4, CD69 and BrdU, FoxP3 respectively;
Mix well with the fluidic cell washing lotion through the mixed liquor of mark, add cell rupture of membranes lysate, behind the mixing, on ice or with identical on ice temperature conditions under hatched 30 minutes, wash with the auxilliary liquid of cell rupture of membranes again, centrifugal after the washing, get precipitation;
With fresh preparation deoxyribonuclease liquid, join in the abovementioned steps in the gained precipitation, hatched behind the mixing 60 minutes, again with the auxilliary liquid of cell rupture of membranes, washing, centrifugal, get precipitation, the gained precipitation is with the auxilliary liquid repeated washing of cell rupture of membranes once, and is centrifugal, gets precipitation;
Add the antibody Fc blocking agent in the precipitation that obtains, fully act on 20 minutes;
To regulate the transcription factor (FoxP3) of cell epitope through adding certification mark in the solution after the effect of antibody Fc blocking agent respectively) and the antibody of DNA replication (BrdU), the fluorescence labeling that described BrdU, FoxP3 difference is different, divide and to get 1.5 μ l antibody and hatched on ice 30 minutes, again with the auxilliary liquid 200 μ l of cell rupture of membranes, the washed cell suspension, fully centrifugal behind the mixing, remove supernatant;
Centrifugal behind the abundant mixing of adding fluidic cell washing lotion, remove supernatant, add 500ul fluidic cell washing lotion, flow cytometer detects the accounting of the active regulatory T cells that obtains dyeing.
Thereby antibody titer and the dosage of two kinds of film inner cell organs have been optimized.
Gather person under inspection's peripheric venous blood, peripheric venous blood to be measured is handled also therefrom separated the T cell, concrete separate mode can be with reference to prior art.After adopting nylon membrane net filtration partition method to obtain monocyte, preparation forms single cell suspension, and for the ease of follow-up operations such as dyeing, we become 1 * 10 with the single cell suspension prepared at concentrations usually
7/ ml, getting 100ul behind the mixing is experimental subjects.
We carry out antibody staining to the feature cell mass in the single cell suspension then, and concrete operation steps is as follows:
Above-mentioned single cell suspension is mixed with fluorescence antibody CD3, CD4, CD69 and BrdU, FoxP3 respectively;
The dilution ratio of preferred single cell suspension and fluorescence antibody is respectively in the present embodiment: it is 1:150 that single cell suspension and the calculation of CD3 by volume are calculated for 1:200, single cell suspension and CD69 by volume for 1:400, single cell suspension and the calculation of CD4 by volume, and reaching envelope-bulk to weight ratio after the different proportion dilution is 1ul:1 ug; And single cell suspension and BrdU with the volume mass ratio be calculated as 1.5ul:1 ug, single cell suspension is calculated as 1.5ul:1ug with FoxP3 with the volume mass ratio.
Cell suspension and CD3, CD4, CD69 in conjunction with hatching 30 minutes, wash 2 times with PBS under 4 ℃ condition again.
Mix well with the fluidic cell washing lotion through the mixed liquor of mark, add cell rupture of membranes lysate, behind the mixing, on ice or with identical on ice temperature conditions under hatched 30 minutes, wash with the auxilliary liquid of cell rupture of membranes again, centrifugal after the washing, get precipitation;
With fresh preparation deoxyribonuclease liquid, join in the abovementioned steps in the gained precipitation, hatched behind the mixing 60 minutes, again with the auxilliary liquid of cell rupture of membranes, washing, centrifugal, get precipitation,
Add the antibody Fc blocking agent in the precipitation that obtains, fully act on 20 minutes;
To regulate the transcription factor (FoxP3) of cell epitope through adding certification mark in the solution after the effect of antibody Fc blocking agent respectively) and the antibody of DNA replication (BrdU), the fluorescence labeling that described BrdU, FoxP3 difference is different, divide and to get 1.5 μ l antibody and hatched on ice 30 minutes, again with the auxilliary liquid 200 μ l of cell rupture of membranes, the washed cell suspension, fully centrifugal behind the mixing, remove supernatant;
Centrifugal behind the abundant mixing of adding fluidic cell washing lotion, remove supernatant, add 500ul fluidic cell washing lotion, flow cytometer detects the accounting of the active regulatory T cells that obtains dyeing.
Embodiment 6
Gather person under inspection's peripheric venous blood, peripheric venous blood to be measured is handled also therefrom separated the T cell, concrete separate mode can be with reference to prior art.After adopting nylon membrane net filtration partition method to obtain monocyte, preparation forms single cell suspension, and for the ease of follow-up operations such as dyeing, we become 1 * 10 with the single cell suspension prepared at concentrations usually
7/ ml, getting 100ul behind the mixing is experimental subjects.
We carry out antibody staining to the feature cell mass in the single cell suspension then, and concrete operation steps is as follows:
Above-mentioned single cell suspension is mixed with fluorescence antibody CD3, CD4, CD69 and BrdU, FoxP3 respectively;
Mixed liquor through mark is mixed well with fluidic cell washing lotion 100 μ l, adds cell rupture of membranes lysate 100 μ l, behind the mixing, on ice or with identical on ice temperature conditions under hatched 30 minutes, wash with the auxilliary liquid of cell rupture of membranes, the washing back is centrifugal, gets precipitation again;
With fresh preparation deoxyribonuclease liquid 100 μ l, join in the abovementioned steps in the gained precipitation, hatched behind the mixing 60 minutes, again with the auxilliary liquid of cell rupture of membranes, washing, centrifugal, get precipitation,
Add the antibody Fc blocking agent in the precipitation that obtains, fully act on 20 minutes;
To regulate the transcription factor of cell epitope and the antibody of DNA replication (different fluorescence labeling BrdU, FoxP3 respectively) through adding certification mark in the solution after the effect of antibody Fc blocking agent respectively, divide and to get 1.5 μ l antibody and hatched on ice 30 minutes, again with the auxilliary liquid 200 μ l of cell rupture of membranes, the washed cell suspension, fully centrifugal behind the mixing, remove supernatant;
Centrifugal behind the abundant mixing of adding fluidic cell washing lotion, remove supernatant, add 500ul fluidic cell washing lotion, flow cytometer detects the accounting of the active regulatory T cells that obtains dyeing.
Gather person under inspection's peripheric venous blood, peripheric venous blood to be measured is handled also therefrom separated the T cell, concrete separate mode can be with reference to prior art.After adopting nylon membrane net filtration partition method to obtain monocyte, preparation forms single cell suspension, and for the ease of follow-up operations such as dyeing, we become 1 * 10 with the single cell suspension prepared at concentrations usually
7/ ml, getting 100ul behind the mixing is experimental subjects.
We carry out antibody staining to the feature cell mass in the single cell suspension then, and concrete operation steps is as follows:
Above-mentioned single cell suspension is mixed with fluorescence antibody CD3, CD4, CD69 and BrdU, FoxP3 respectively;
Mixed liquor through mark is mixed well with fluidic cell washing lotion 100 μ l, adds cell rupture of membranes lysate 100 μ l, behind the mixing, on ice or with identical on ice temperature conditions under hatched 30 minutes, wash with the auxilliary liquid of cell rupture of membranes, the washing back is centrifugal, gets precipitation again;
With fresh preparation deoxyribonuclease liquid 100 μ l, join in the abovementioned steps in the gained precipitation, hatched behind the mixing 60 minutes, again with the auxilliary liquid 200 μ l of cell rupture of membranes, washing, centrifugal, get precipitation,
Add antibody Fc blocking agent 10 μ l in the precipitation that obtains, fully act on 20 minutes;
To regulate the transcription factor (FoxP3) of cell epitope through adding certification mark in the solution after the effect of antibody Fc blocking agent respectively) and the antibody of DNA replication (BrdU), the fluorescence labeling that described BrdU, FoxP3 difference is different, divide and to get 1.5 μ l antibody and hatched on ice 30 minutes, again with the auxilliary liquid 200 μ l of cell rupture of membranes, the washed cell suspension, fully centrifugal behind the mixing, remove supernatant;
To precipitate by flow cytometer and detect:
Centrifugal behind the abundant mixing of adding fluidic cell washing lotion, remove supernatant, add 500ul fluidic cell washing lotion, flow cytometer detects the accounting of the active regulatory T cells that obtains dyeing.
Embodiment 8
Gather person under inspection's peripheric venous blood, peripheric venous blood to be measured is handled also therefrom separated the T cell, concrete separate mode can be with reference to prior art.After adopting nylon membrane net filtration partition method to obtain monocyte, preparation forms single cell suspension, and for the ease of follow-up operations such as dyeing, we become 1 * 10 with the single cell suspension prepared at concentrations usually
7/ ml, getting 100ul behind the mixing is experimental subjects.
We carry out antibody staining to the feature cell mass in the single cell suspension then, and concrete operation steps is as follows:
Above-mentioned single cell suspension is mixed with fluorescence antibody CD3, CD4, CD69 and BrdU, FoxP3 respectively;
The dilution ratio of preferred single cell suspension and fluorescence antibody is respectively in the present embodiment: it is 1:150 that single cell suspension and the calculation of CD3 by volume are calculated for 1:200, single cell suspension and CD69 by volume for 1:400, single cell suspension and the calculation of CD4 by volume, and reaching envelope-bulk to weight ratio after the different proportion dilution is 1ul:1 ug; And single cell suspension and BrdU with the volume mass ratio be calculated as 1.5ul:1 ug, single cell suspension is calculated as 1.5ul:1ug with FoxP3 with the volume mass ratio.
Cell suspension and CD3, CD4, CD69 in conjunction with hatching 30 minutes, wash 2 times with PBS under 4 ℃ condition again.
Mixed liquor through mark is mixed well with fluidic cell washing lotion 100 μ l, adds cell rupture of membranes lysate 100 μ l, behind the mixing, on ice or with identical on ice temperature conditions under hatched 30 minutes, wash with the auxilliary liquid of cell rupture of membranes, the washing back is centrifugal, gets precipitation again;
With fresh preparation deoxyribonuclease liquid 100 μ l, join in the abovementioned steps in the gained precipitation, hatched behind the mixing 60 minutes, again with the auxilliary liquid 200 μ l of cell rupture of membranes, washing, centrifugal, get precipitation,
Add antibody Fc blocking agent 10 μ l in the precipitation that obtains, fully act on 20 minutes;
To regulate the transcription factor (FoxP3) of cell epitope through adding certification mark in the solution after the effect of antibody Fc blocking agent respectively) and the antibody of DNA replication (BrdU), the fluorescence labeling that described BrdU, FoxP3 difference is different, divide and to get 1.5 μ l antibody and hatched on ice 30 minutes, again with the auxilliary liquid 200 μ l of cell rupture of membranes, the washed cell suspension, fully centrifugal behind the mixing, remove supernatant;
To precipitate by flow cytometer and detect:
Centrifugal behind the abundant mixing of adding fluidic cell washing lotion, remove supernatant, add 500ul fluidic cell washing lotion, flow cytometer detects the accounting of the active regulatory T cells that obtains dyeing.
The mentality of designing of present embodiment, be by the modeling technology, manual construction is because far-end organ immune response directtissima islet cells generation type 1 diabetes model, utilize this model to construct predictable regulatory T cells and suppress active respective change model, and the dynamic change state of the mouse feature cell mass of technological means tracking and monitoring model different phase disclosed according to the present invention, expectation forms one and has regular relation.
In order to obtain relevant relation more accurately, we are a time point with every half cycle, have obtained correlation curve according to following mode.
In the present embodiment for convenience of explanation, we have only chosen three wherein representative time points and have carried out related description, but other time point is all in mean value that this curve allows, standard value deviation range.
Mode of operation:
The first step, the animal model of type 1 diabetes are set up the NOD mouse and are divided and to get three groups as experimental subjects:
A.
1 group 4 the week age female mouse randomly draw 8;
2 group 10 age in week female mouse and continuous 2 days tail vein sugar be lower than 200mg/dl and randomly draw 8;
3 group 16 age in week female mouse and continuous 2 days tail vein sugar be higher than 350mg/dl and randomly draw 8 as experimental subjects.
B.
Each group experiment NOD mouse takes respectively that to contain DNA precursor-5-bromo-2-deoxyuridine(commercially available) sterilized water, concentration is that 1mg/ml fully dissolves, drink was supported in continuous 5 days, observing general state during this time comprises: activity, diet, drinking-water, ight soil Non Apparent Abnormality, and monitoring record tail vein sugar value.Attention is with fresh preparation 5-bromo-2-deoxyuridine every day and lucifuge operation.
C.
Spleen, the pancreas peripheral lymph node of each experimental group animal are prepared single cell suspension, and final concentration is 1 * 107/ml.
D.
To prepare spleen and lymphocyte single cell suspension and be used for antibody staining, the dilution ratio of single cell suspension and fluorescence antibody is respectively: single cell suspension and CD3 are that 1:400, single cell suspension and CD4 are that 1:200, single cell suspension and CD69 are calculated as 1:150 with dilution with the volume dilution with the volume dilution, and reaching the antibody envelope-bulk to weight ratio after the different proportion dilution is 1ul:1 ug; Single cell suspension and BrdU antibody are calculated as 1.5ul:1 ug, single cell suspension and antibody FoxP3 with envelope-bulk to weight ratio and are calculated as 1.5ul:1ug with envelope-bulk to weight ratio.
Cell suspension and CD3, CD4, CD69 in conjunction with hatching 30 minutes, wash 2 times with PBS under 4 ℃ condition again.
E.
Cell suspension is mixed well with fluidic cell washing lotion 100ul, adds cell rupture of membranes lysate 100ul, and mixing was hatched 30 minutes on ice, again with the auxilliary liquid 200ul washed cell suspension of cell rupture of membranes, outwells supernatant after centrifugal.
F.
Fresh preparation DNase liquid, 100ul adds cell, hatches behind the mixing 60 minutes, again with the auxilliary liquid 200ul washed cell suspension of cell rupture of membranes, outwells supernatant after centrifugal, and repeated washing is once.
G.
Add the antibody Fc blocking agent, 10ul fully acts on 20 minutes.
H.
Add nucleus transcription factor and differentiation activation detection antibody, divide and to get the 1.5ul fluorescent-labeled antibody and hatch on ice, again with the auxilliary liquid 200ul washed cell suspension of cell rupture of membranes, outwell supernatant after centrifugal, last fluidic cell washing lotion 200ul is washed cell again, outwell supernatant after centrifugal, finally add 500ul fluidic cell washing lotion, the up flow type cell instrument detects.
Testing result as Fig. 1-1 to shown in Fig. 1-3, sick time prolongation along with mouse in the model, the inhibition activity of its regulatory T cells should reduce gradually, we distinguish the cell quantity in the feature cell mass after the dyeing among the statistical graph 1-1 to Fig. 1-3, and pass through statistical analysis, the accounting of calculated characteristics cell group in the regulatory T cells total amount, obtain curve map as shown in Figure 2, find As time goes on, the accounting of feature cell mass cell in the regulatory T cells total amount descends gradually, be corresponding with the inhibition activity of regulatory T cells, and present positive correlation.
We with the curve shown in Fig. 2 and related data thereof as the active standard reference data of the inhibition of regulatory T cells; And the accounting with feature cell group is weighed parameter as activity index.The corresponding regulatory T cells of point that is positioned at the curve upper end suppresses active high, moves to the curve lower end, and is more low the closer to its corresponding regulatory T cells inhibition activity of point of lower end.
Embodiment 11 regulatory T cells suppress application and the checking of activity curve figure
First step: set up the mode modeling according to the animal model of the type 1 diabetes among the embodiment 10.
Second step: the mouse of picked at random different phase experimentizes.
Third step:
3-1. gather the peripheric venous blood of mouse A group, B group respectively, and handle, analyze according to the mode among the embodiment 8, obtain the regulatory T cells quantity of corresponding dyeing, shown in Fig. 3-1 and Fig. 3-2.By statistical analysis, the accounting of calculated characteristics cell group in the regulatory T cells total amount, wherein the accounting data of mouse A group are 29, the accounting data of mouse B group are 16.
Win, dye 3-2. mouse A group, the B group of having gathered peripheric venous blood among the 3-1 are carried out pancreas, the regulatory T cells that is used for presenting under the corresponding pancreas state of check suppress active whether with 3-1 by typical curve obtain data corresponding.Concrete operation can be with reference to following steps,
1. under aseptic condition, conventional anesthesia and operation technique separate pancreatic tissue.
2. pancreatic tissue is with formalin fixed, pancreatic tissue paraffin enclosed mass, and routine is prepared into the section of 4um.
3. section is finished through dewaxing, aquation, dyeing and mounting.The pancreatic tissue of each mouse is equipped with 10 of sheets at least continuously as sample.Singly dye a slice hematoxylin eosin staining, microexamination every 3.
4. the white sheet between haematine-Yihong dyeing carries out monolithic with dying with the insulin antibody of DAPI and Texas red marker respectively, insulin antibody is that 1:50 doubly dilutes, and hatches back PBS in 2 hours and develops a film, and adds and incubates DAPI dyeing, PBS cleans, with fluorescence microscopy.
5. the state the when dyeing of haematine-Yihong manifests pancreas islet structure under the health status or lymphocyte infiltration on can form and destroys pancreas islet.
6. and DAPI and Texas red colouring can show pancreas islet form and structure on year-on-year basis, whether periphery has lymphocyte infiltration, invades that pancreas islet structure after the profit is first not to be destroyed, and whether the quantity of islet secretion insulin is normal
Or reduce, can diagnose immune attack beta Cell of islet state and lesion degree and rank of living in by pathologic finding
Section.
The section preparation coloration result of pancreas is referring to Fig. 4-2(mouse A group) and Fig. 4-4(mouse B group), for the ease of observing us with among the embodiment 10, around, the pancreas in the tenth week, the 16 week handles according to the preparation of specimen's mode among the present embodiment 3-2, respectively shown in Fig. 4-1,4-3,4-5.
The pancreas pathological manifestations of mouse A goes out one peripheral lymphoid cellular infiltration, form change and insulin secretion minimizing as can be seen, and its order of severity is between the pathological manifestations of Fig. 4-1 and Fig. 4-3 demonstration; That is to say around the and between the tenth week, corresponding with the 16 corresponding mouse residing pathology times of accounting data.
Simultaneously we as can be seen the pancreas pathological manifestations of mouse B go out even more serious lymphocyte and invade pancreas, the state that causes insulin dyeing obviously to reduce.Observe its coloration result, its pathological manifestations that is to say that this mouse B is positioned at the model stage in 16 weeks of the tenth week to the between the pathological manifestations of Fig. 4-3 and 4-5 demonstration, corresponding with the 29 corresponding mouse residing pathology times of accounting data.
Embodiment 12 screening pancreas early lesion targets
The mentality of designing of present embodiment, be by the modeling technology, manual construction is because far-end organ immune response directtissima islet cells generation type 1 diabetes model, and the dynamic change state of the mouse feature cell mass of technological means tracking and monitoring model different phase disclosed according to the present invention, and expectation forms a relation with regularity.
In order to obtain relevant relation more accurately, we are a time point with every half cycle, have obtained correlation curve according to following mode.
In the present embodiment for convenience of explanation, we have only chosen three wherein representative time points and have carried out related description, but other time point is all in mean value that this curve allows, standard value deviation range.
First step: set up the mode modeling according to the animal model of the type 1 diabetes among the embodiment 10.
Second step: divide and get three groups as experimental subjects,
1 group 4 the week age female mouse randomly draw 8;
2 group 10 age in week female mouse and continuous 2 days tail vein sugar be lower than 200mg/dl and randomly draw 8;
3 group 16 age in week female mouse and continuous 2 days tail vein sugar be higher than 350mg/dl and randomly draw 8 as experimental subjects.
Third step:
3-1. gather 1 group of mouse, 2 groups, 3 groups peripheric venous blood respectively, and handle, analyze according to the mode among the embodiment 8, obtain the regulatory T cells quantity of corresponding dyeing, shown in Fig. 1-1, Fig. 1-2 and Fig. 1-3.By statistical analysis, the accounting of calculated characteristics cell group in the regulatory T cells total amount, wherein the accounting data of 1 group of mouse are 32, and the accounting data that mouse is 2 groups are 18.5, and the accounting data that mouse is 3 groups are 13.
Win, dye 3-2. the mouse of having gathered peripheric venous blood among the 3-1 is carried out pancreas for 1 group, 2 groups, 3 groups, concrete operation can be with reference to following steps,
1. under aseptic condition, conventional anesthesia and operation technique separate pancreatic tissue.
2. pancreatic tissue is with formalin fixed, pancreatic tissue paraffin enclosed mass, and routine is prepared into the section of 4um.
3. section is finished through dewaxing, aquation, dyeing and mounting.The pancreatic tissue of each mouse is equipped with 10 of sheets at least continuously as sample.Singly dye a slice hematoxylin eosin staining, microexamination every 3.
4. the white sheet between haematine-Yihong dyeing carries out monolithic with dying with the insulin antibody of DAPI and Texas red marker respectively, insulin antibody is that 1:50 doubly dilutes, and hatches back PBS in 2 hours and develops a film, and adds and incubates DAPI dyeing, PBS cleans, with fluorescence microscopy.
5. the state the when dyeing of haematine-Yihong manifests pancreas islet structure under the health status or lymphocyte infiltration on can form and destroys pancreas islet.
6. and DAPI and Texas red colouring can show pancreas islet form and structure on year-on-year basis, whether periphery has lymphocyte infiltration, invades that pancreas islet structure after the profit is first not to be destroyed, and whether the quantity of islet secretion insulin is normal
Or reduce, can diagnose immune attack beta Cell of islet state and lesion degree and rank of living in by pathologic finding
Section.
Testing result as Fig. 1-1 to shown in Fig. 1-3, sick time prolongation along with mouse in the model, the inhibition activity of its regulatory T cells should reduce gradually, we distinguish the cell quantity in the feature cell mass after the dyeing among the statistical graph 1-1 to Fig. 1-3, and pass through statistical analysis, the accounting of calculated characteristics cell group in the regulatory T cells total amount, obtain curve map as shown in Figure 2, find that As time goes on the accounting of feature cell mass cell in the regulatory T cells total amount descends gradually.
We are the pancreas state that reflects among Fig. 4-1, Fig. 4-3 and Fig. 4-5 reference basis as horizontal ordinate, and with model stage at its place as horizontal ordinate, obtain the curve shown in Fig. 5, and with the normative reference of this curve as the pancreatic disorders state.The corresponding pancreas of point that is positioned at curve upper end is in the pathology initial stage, moves to the curve lower end, is in pathology the closer to its corresponding pancreas of point of lower end and gets over late period.
The contrast test of embodiment 13 different modes screening pancreas early lesion target
First step |: set up the mode modeling according to the animal model of the type 1 diabetes among the embodiment 10.
Second step: divide and get three groups as experimental subjects,
1 group 4 the week age female mouse randomly draw 8;
2 group 10 age in week female mouse and continuous 2 days tail vein sugar be lower than 200mg/dl and randomly draw 8;
3 group 16 age in week female mouse and continuous 2 days tail vein sugar be higher than 350mg/dl and randomly draw 8 as experimental subjects.
Third step: gather 1 group of mouse, 2 groups, 3 groups peripheric venous blood respectively, carry out following experiment then respectively,
3-1. handle, analyze according to the mode among the embodiment 8, obtain the regulatory T cells quantity of corresponding dyeing, shown in Fig. 1-1, Fig. 1-2 and Fig. 1-3.By statistical analysis, the accounting of calculated characteristics cell group in the regulatory T cells total amount, its curve map is as shown in Figure 5.
3-2. according to the detection method of blood sugar, detect the blood-sugar content of 1 group, 2 groups, 3 groups correspondence respectively, its curve map correspondence as shown in Figure 6.Horizontal line place ordinate value is normal critical value among the figure, be positioned at critical value following for blood sugar is normal, be positioned at critical value above be pathoglycemia.
Carry out pancreas for 1 group, 2 groups, 3 groups and win, dye 3-3. will gather the mouse of peripheric venous blood, concrete operation can be with reference to following steps,
1. under aseptic condition, conventional anesthesia and operation technique separate pancreatic tissue.
2. pancreatic tissue is with formalin fixed, pancreatic tissue paraffin enclosed mass, and routine is prepared into the section of 4um.
3. section is finished through dewaxing, aquation, dyeing and mounting.The pancreatic tissue of each mouse is equipped with 10 of sheets at least continuously as sample.Singly dye a slice hematoxylin eosin staining, microexamination every 3.
4. the white sheet between haematine-Yihong dyeing carries out monolithic with dying with the insulin antibody of DAPI and Texas red marker respectively, insulin antibody is that 1:50 doubly dilutes, and hatches back PBS in 2 hours and develops a film, and adds and incubates DAPI dyeing, PBS cleans, with fluorescence microscopy.
5. the state the when dyeing of haematine-Yihong manifests pancreas islet structure under the health status or lymphocyte infiltration on can form and destroys pancreas islet.
6. and DAPI and Texas red colouring can show pancreas islet form and structure on year-on-year basis, whether periphery has lymphocyte infiltration, invades that pancreas islet structure after the profit is first not to be destroyed, and whether the quantity of islet secretion insulin is normal
Or reduce, can diagnose immune attack beta Cell of islet state and lesion degree and rank of living in by pathologic finding
Section.
The sample of dyeing the results are shown in Figure 4-1, Fig. 4-3 and Fig. 4-5.
By above experimental result as can be seen, when model was in for 10 weeks, pathology has taken place in the pancreas of mouse, but in the blood sugar test, still be in the normal envelope, therefore by blood sugar as type 1 diabetes take place, the monitoring means of development has defective, can not in time filter out the target of early lesion, delay treatment opportunity.
And the accounting data of feature cell mass in the regulatory T cells that obtains by colouring method disclosed in this invention can in time reflect the pathological change process of pancreas, particularly before 12 weeks, just in the following blind spot scope of critical blood glucose value.
Though if directly can accurately obtain the judgement of pancreatic disorders by the pancreatic tissue test alive, this mode needs the live body sampling, and the patient is caused operation wound and operation risk.
Based on the principle of accuracy, we will utilize feature cell mass disclosed by the invention to detect to the method on basis and traditional biopsy mode to have carried out the comparison of using value, result such as table 2.
Table 2
Embodiment 14 treatment back demonstration tests
First step |: set up the mode modeling according to the animal model of the type 1 diabetes among the embodiment 10.
Second step: the female mouse that 8 of picked at random were in after 16 weeks is tested.
Wherein 4 are organized in contrast, carry out the blank test with dummy;
Other 4 as the treatment group, through immunologic intervention treatment.
Third step: every two hours, respectively the mouse of control group and treatment group is taken a blood sample, sampling is from its peripheric venous blood.
Handle in accordance with the following methods respectively then, analyze: handle, analyze according to blood sugar detecting method, obtain experimental result as shown in Figure 8.
When observing blood sugar recovery to normal value, according to the mode among the embodiment 8, mouse is handled, analyzes, obtain the regulatory T cells quantity accounting of corresponding dyeing, test figure is as shown in Figure 7.
As can be seen, the accounting of the active regulatory T cells quantity of untreated control group remains on reduced levels from the result, and the accounting of its active regulatory T cells quantity of mouse by treatment has returned in the normal data scope.
Claims (7)
1. one kind is used for characterizing the detection method that the periphery regulatory T cells suppresses the feature cell mass of activity, it is characterized in that this method may further comprise the steps:
Step 1) is handled sample to be tested, forms single cell suspension;
Step 2) antibody staining of feature cell mass:
Above-mentioned single cell suspension is mixed with fluorescence antibody CD3, CD4, CD69 respectively;
The step 3) mixed liquor is mixed well with the fluidic cell washing lotion, adds cell rupture of membranes lysate, and mixing was hatched 30 minutes on ice, and is with the auxilliary liquid of cell rupture of membranes, fully centrifugal behind the mixing again, removes supernatant;
The fresh preparation deoxyribonuclease of step 4) liquid joins in the step 4) in the gained precipitation, hatches behind the mixing 60 minutes, and is with the auxilliary liquid of cell rupture of membranes, fully centrifugal behind the mixing again, removes supernatant,
Step 5) adds the antibody Fc blocking agent, fully acts on 20 minutes;
Step 6) adds certification mark respectively and regulates the transcription factor (FoxP3) of cell epitope and the antibody of DNA replication (BrdU); Described BrdU, FoxP3 adopt different fluorescence labelings respectively, divide to get 1.5 μ l antibody and hatched on ice 30 minutes, and again with the auxilliary liquid 200 μ l of cell rupture of membranes, the washed cell suspension, fully centrifugal behind the mixing, remove supernatant;
The step 7) flow cytometer detects:
Add fluidic cell washing lotion 500ml, fully centrifugal behind the mixing, remove supernatant, add the fluidic cell washing lotion, machine testing on the flow cytometer.
2. according to claim 1 a kind of for characterizing the dyeing detection method that the periphery regulatory T cells suppresses the feature cell mass of activity, it is characterized in that: described step 2), the dilution ratio of single cell suspension and fluorescence antibody is respectively: single cell suspension and CD3 by volume are calculated to 1:400, single cell suspension and CD4 by volume and are calculated to 1:200, single cell suspension and the calculation of CD69 by volume are 1:150, and reaching envelope-bulk to weight ratio after the different proportion dilution is 1ul:1 ug; And single cell suspension and BrdU with the volume mass ratio be calculated as 1.5ul:1 ug, single cell suspension is calculated as 1.5ul:1ug with FoxP3 with the volume mass ratio.
3. according to claim 1 a kind of for characterizing the detection method that the periphery regulatory T cells suppresses the feature cell mass of activity, it is characterized in that: the final concentration of single cell suspension described in the step 1) is 1 * 10
7/ ml.
4. according to claim 1 a kind of for characterizing the detection method that the periphery regulatory T cells suppresses the feature cell mass of activity, it is characterized in that: described step 2), cell suspension and CD3, CD4, CD69 in conjunction with hatching 30 minutes, wash 2 times with PBS under 4 ℃ condition again.
5. according to claim 1 for characterizing the detection method that the periphery regulatory T cells suppresses the feature cell mass of activity, it is characterized in that: in the described step 4), the gained precipitation with the auxilliary liquid repeated washing of cell rupture of membranes once, and is centrifugal behind the mixing, removes supernatant.
In the claim 1 to 5 any described detection method in the application that suppresses for the preparation of regulatory T cells in the active test kit.
In the claim 1 to 5 any described detection method for the preparation of the application in the kit of screening pancreas early lesion target.
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| CN201310150133.2A CN103235135B (en) | 2013-04-26 | 2013-04-26 | Detection method of characteristic cell mass for representing inhibitory activity of peripheral regulatory T cell, and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108508196A (en) * | 2018-04-13 | 2018-09-07 | 沈阳汇敏源生物科技有限责任公司 | A kind of kit and detection method of detection people's regulatory T cells hypotype |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1364093A (en) * | 1999-04-15 | 2002-08-14 | 莫纳希大学 | Improvement of T cell mediated immunity |
| CN1981031A (en) * | 2004-03-05 | 2007-06-13 | 宾久法尼亚大学理事会 | Regulatory T cells and their use in immunotherapy and suppression of autoimmune responses |
| CN101329230A (en) * | 2008-07-14 | 2008-12-24 | 中国人民解放军第三军医大学 | Improved method for dyeing immunofluorescence cell |
| CN101392294A (en) * | 2008-11-03 | 2009-03-25 | 复旦大学附属中山医院 | Drug screening method for regulating regulatory T cell and Th17cell mutual transformation |
| CN101631851A (en) * | 2006-11-29 | 2010-01-20 | 贝斯以色列护理医疗中心 | Novel regulatory T cells and uses thereof |
| CN101883844A (en) * | 2007-10-12 | 2010-11-10 | 分子医学马克斯德尔布吕克中心 | The method of rapid isolation of human foxp 3+treg cells and test kit |
| US20100318749A1 (en) * | 2009-06-15 | 2010-12-16 | Broadcom Corporation | Scalable multi-bank memory architecture |
| WO2012041968A1 (en) * | 2010-09-29 | 2012-04-05 | Uti Limited Partnership | Methods for treating autoimmune disease using biocompatible bioabsorbable nanospheres |
| CN102460161A (en) * | 2009-04-09 | 2012-05-16 | 洛菲厄斯生物科学有限责任公司 | Method for polypeptide transfer into cells |
-
2013
- 2013-04-26 CN CN201310150133.2A patent/CN103235135B/en not_active Expired - Fee Related
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1364093A (en) * | 1999-04-15 | 2002-08-14 | 莫纳希大学 | Improvement of T cell mediated immunity |
| CN1981031A (en) * | 2004-03-05 | 2007-06-13 | 宾久法尼亚大学理事会 | Regulatory T cells and their use in immunotherapy and suppression of autoimmune responses |
| CN101631851A (en) * | 2006-11-29 | 2010-01-20 | 贝斯以色列护理医疗中心 | Novel regulatory T cells and uses thereof |
| CN102766596A (en) * | 2006-11-29 | 2012-11-07 | 贝斯以色列护理医疗中心 | Novel regulatory T cells and uses thereof |
| CN101883844A (en) * | 2007-10-12 | 2010-11-10 | 分子医学马克斯德尔布吕克中心 | The method of rapid isolation of human foxp 3+treg cells and test kit |
| CN101329230A (en) * | 2008-07-14 | 2008-12-24 | 中国人民解放军第三军医大学 | Improved method for dyeing immunofluorescence cell |
| CN101392294A (en) * | 2008-11-03 | 2009-03-25 | 复旦大学附属中山医院 | Drug screening method for regulating regulatory T cell and Th17cell mutual transformation |
| CN102460161A (en) * | 2009-04-09 | 2012-05-16 | 洛菲厄斯生物科学有限责任公司 | Method for polypeptide transfer into cells |
| US20100318749A1 (en) * | 2009-06-15 | 2010-12-16 | Broadcom Corporation | Scalable multi-bank memory architecture |
| WO2012041968A1 (en) * | 2010-09-29 | 2012-04-05 | Uti Limited Partnership | Methods for treating autoimmune disease using biocompatible bioabsorbable nanospheres |
Non-Patent Citations (5)
| Title |
|---|
| QIXIN SHI ET AL.: "Abrogation of recurrent autoimmunity in the NOD mouse: a critical role for host interleukin 4", 《SURGERY》 * |
| SHARVAN SEHRAWAT ET AL.: "In Vitro-Generated Antigen-Specific CD4+ CD25+Foxp3+ Regulatory T Cells Control the Severity of Herpes Simplex Virus-Induced Ocular Immunoinflammatory Lesions", 《JOURNAL OF VIROLOGY》 * |
| 姚军霞 等: "恶性肿瘤患者CD4+CD25+/CD4+CD25high调节性T细胞的研究", 《中国免疫学杂志》 * |
| 杨庆华 等: "流式细胞仪BrdU/DNA双参数分析的方法与利用", 《上海第二医科大学学报》 * |
| 谭政 等: "T细胞功能亚群", 《生命科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108508196A (en) * | 2018-04-13 | 2018-09-07 | 沈阳汇敏源生物科技有限责任公司 | A kind of kit and detection method of detection people's regulatory T cells hypotype |
| CN108508196B (en) * | 2018-04-13 | 2020-10-27 | 何韶衡 | Kit and method for detecting human regulatory T cell subtype |
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