CN107050427A - Applications of the MBL in preparing prevention or treating using Tregs as the disease medicament of target spot - Google Patents
Applications of the MBL in preparing prevention or treating using Tregs as the disease medicament of target spot Download PDFInfo
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Abstract
The invention discloses applications of the MBL in preparing prevention or treating using Tregs as the disease medicament of target spot, belong to the new application technical field of mannan-binding lectin.Technical scheme main points are：Applications of the MBL in infectious diseases, autoimmune disease or/and the allergic disease medicine of prevention or treatment using Tregs as target spot is prepared, the MBL realizes effect of medicine by promoting CD4+CD25 T cells to break up to Tregs cell inductions.The present invention has been obtained adjusting the new factor that Tregs cell inductions break up by exploring, i.e. MBL directly facilitates CD4+CD25 T cells to be broken up to Tregs cell inductions；Highlight a kind of MBL New function, important anti-infectious function not only played in inherent immunity, and in the intervention broken up for Tregs cell inductions be probably treat various autoimmune disease and diseases associated with inflammation be directed to target spot.
Description
Technical field
The present invention relates to the new application technical field of mannan-binding lectin, and in particular to MBL prepare prevention or
Application in treating using Tregs as the disease medicament of target spot.
Background technology
Mannose binding lectin (mannan-binding lectin, MBL) is that the main height by hepatocytes secrete is protected
The plasma protein kept, is collectin (collectins) family member in c-type agglutinin superfamily.MBL is also a kind of acute stage
Reactive protein, stress (such as pathogenic infection, surgical operation) when, its plasma concentration can raise 2-3 times.With preceding antibody
(ante-antibody) MBL of title passes through its carbohydrate recognition domain (carbohydrate recongnition domain, CRD)
Identification is distributed in the sugared structure on the multiple pathogens such as surface such as bacterium, virus, parasite, fungi, including D-MANNOSE, L- extensively
Fucose, N-Acetyl-D-glucosamine, ManNAc etc..Its collagen-like region (collagen-like region, CLR) is tied
Close mannosan associated serine protease (MBL-associated serine protease, MBL-MASP), activating complement
Lectin pathway and play molten broken and indirect conditioning functions；Or combined with phagocyte collectin acceptor, with independent of complement
Mode start opsonophagocytosis.Science once published the article (Thompson C.Protein proves to be a key link
in innate immunity[J].Science,1995,269(5222):301-302.) it is called " in natural immune system
Key molecule ".Available data is it has been shown that MBL is most important anti-infective innate immune molecule in not immune host.
Have now been found that 3 point mutation (CGT52TGG, GGC54GAC and GGA57GAA) of MBL structural genes and start
Opsonophagocytosis defect caused by blood plasma MBL caused by son and 5 ' non-translational regions are mutated is low is the most common something lost that is found so far
Transmissibility immunodeficiency disease.MBL defect persons among the infection risk in height, can suffer from various infection or even threaten life at any time throughout one's life
The infection of life.Recent researches show that MBL disease resistance mechanisms are related to its serum level, and low-level MBL is easily caused
The defense function of body substantially weakens, easily infect Crohn disease, Autoimmune neuropathies obstacle, primary immunodeficiency and
Invasive infections with fungi；On the contrary, recent studies have shown that high-caliber MBL can improve the survival of Patients with Chronic Obstructive Pulmonary Disease
Rate.
With deepening continuously for studying MBL, it is found that it, except can recognize that and remove pathogen, is also equipped with many endogenous
Function (endongenous functions), such as combines autoimmunity globulin；Recognize ischemical reperfusion injury in exposure from
The autoantigen modified under body antigen or morbid state；Participate in the phagocytosis to apoptotic cell；Can be thin with monocytic series THP1
Born of the same parents interact, and suppress the cytokine secretion of C.albicans inductions, while to bone-marrow-derived lymphocyte system Raji cells, and T cell
It is that Jurkat cell is respectively provided with immunoregulation effect.Therefore, MBL is used as pattern-recognition having multi-functions in the innate immunity point
Son, is not only only involved in the innate immune defence of body, and its effect is also played between the innate immunity and acquired immunity.Gu research
MBL and acquired immunity relation, with important scientific meaning and potential medical practice meaning.
T lymphocytes are the Primary Actors of acquired immunity, wherein regulatory T cells (regulatory T cell,
Tregs the regulation of body immunity) is participated in.As the important defence line of human autoimmune, Tregs cells are exempted from maintenance body
There is considerable effect in terms of epidemic disease tolerance.On the one hand, the missing of Tregs cells can cause x linked recessive genetic disease
Generation.This is a kind of serious multiple autoimmune disease, causes the enteron aisle of Juvenile onset and the inflammation of multiple incretory
Disease, significantly shortens patient's life-span.On the other hand, in the patient and animal model of various autoimmune disease and diseases associated with inflammation
In, find the reduction for there are Tregs cell numbers, including multiple sclerosis, systemic loupus erythematosus, type 1 diabetes, class wind
Wet arthritis, inflammatory bowel disease, psoriasis and atherosclerosis etc..These achievements in research are all pointed out to Tregs cell numbers
Intervention be probably treat various autoimmune disease and diseases associated with inflammation be directed to target spot.
Recent study finds that Tregs cells are as the CD4+T cell subsets in vivo with immunoloregulation function, and it can
To be divided into the adaptability regulation that nature regulatory T cells (natural regulatory T cell, nTreg) and induction are produced
Property T cell (induced regulatory T cell, iTreg), nTreg cells develop in thymus gland, are maintaining immunity of organism
Played an important role in terms of tolerance, trnasplantion immunity and autoimmunity disease；ITreg cells are in some spies by CD4+T cells
Determine to induce in periphery under physiological condition and differentiate, the same functional characteristics similar with nTreg cells.Therefore, find newly
Tregs cell inductions differentiation factor is adjusted, maintains immune tolerance, treatment various autoimmune disease to have for body important
Scientific meaning and clinical application.Existing document report TGF-β, IL-1 β, IL-2 are in the induction differentiation of Tregs cells
Most important, use in conjunction CD3/CD28 monoclonal antibodies and TGF-β 1 can improve the expression of Tregs cells.But, if it is also unknown
Promotion Tregs cells induction differentiation factor urgently further deeply develop research.
This seminar always works on the work of MBL correlative studys in recent years.In terms of MBL participates in immunological regulation, MBL is found
Energy induced monocyte is to differentiation of dendritic cells and promotes its function maturation, high concentration MBL to suppress LPS in a dose-dependent manner
The THP1/CD14 cells that the maturing dendritic cell and LPS of induction are stimulated produce TNF-α and IL-12, MBL can also be with B lymphs
Cell line Raji cells and the Jurkat cell interaction of T cell system, adjust immune response.Illustrate that MBL has above a variety of
Immunoregulation effect.
The content of the invention
The purpose of the present invention is to propose to MBL answering in preparing prevention or treating using Tregs as the disease medicament of target spot
With mainly having inquired into the influence that MBL breaks up to CD4+CD25-T cells to Tregs cell inductions, as a result shown that MBL can be direct
Promote CD4+CD25-T cells to Tregs cell inductions break up, this be not only in theory have conspicuousness break through (before this from
Relevant MBL does not induce the document report of Tregs cells), and with potential clinical medicine meaning, for treat using Tregs as
Infectious diseases, autoimmune disease and allergic disease of target spot etc. provide a kind of new therapy approach.
The present invention adopts the following technical scheme that MBL is preparing prevention or treated using Tregs as target spot to achieve the above object
Infectious diseases, autoimmune disease or/and allergic disease medicine in application, the MBL is by promoting CD4+
CD25-T cells break up the effect for realizing medicine to Tregs cell inductions.
Further preferably, the infectious diseases include infections with leishmaniasis, Infected With Plasmodium, Listeria infection,
Throttle pylori (Hp) infection, herpes simplex infections, HIV infection, hepatitis b virus infected and the third type liver
Scorching virus infection.
Further preferably, the autoimmune disease includes systemic loupus erythematosus, non-obese diabetes, rheumatoid
Arthritis, type 1 diabetes, atopic dermatitis, Graves diseases and alopecia areata.
Further preferably, the allergic disease includes allergic rhinitis and bronchial astehma.
Further preferably, the MBL is use in conjunction part and the natural MBL of monoclonal antibody affinity chromatography Purified human plasma
Albumen.
Further preferably, the concentration of the MBL is higher than its physiological concentration in vivo.
The present invention can improve the number phase of Tregs cells with prior art use in conjunction CD3/CD28 monoclonal antibodies and TGF-β 1
Than, the invention has the advantages that：
1st, the present invention has obtained adjusting the new factor that Tregs cell inductions break up by exploring, i.e. MBL directly facilitates CD4+
CD25-T cells break up to Tregs cell inductions；
2nd, the present invention highlights a kind of MBL New function, and important anti-infectious function is not only played in inherent immunity, and
And be probably the pin for treating various autoimmune disease and diseases associated with inflammation in the intervention broken up for Tregs cell inductions
To target spot；
3rd, the immunocyte used in the present invention comes from human cord blood, is different from the peripheral blood in existing research, in view of
The advantages of umbilical cord abundance, the candidate stem cell rich in high-quality and immune precursor and strong power of regeneration, therefore, Cord blood
First choice as experimentation Specimen origin of the present invention.
Brief description of the drawings
Fig. 1 is the expression figure of Foxp3 in the fresh CBMC of Flow cytometry；
Fig. 2 is Flow cytometry CBMC transcription factor Foxp3 at different conditions expression figure；
Fig. 3 is the expression figure that RT-PCR methods detect Foxp3mRNAs of the fresh CBMC and CBMC in different condition culture；
Fig. 4 is proliferative effect figures of the CCk-8 methods detection iTreg to PBMCs；
Fig. 5 is the purity figure of immunological magnetic bead sorting CD4+CD25-T, CD4+CD25+T cell；
Fig. 6 is that Flow cytometry MBL induces differentiation figure to CD4+CD25-T cells to iTreg；
Fig. 7 is that RT-PCR analysis MBL induce differentiation figure to CD4+CD25-T cells to iTreg；
Fig. 8 is the influence figure that CCK-8 methods detection iTreg breeds to CD4+CD25-T cells.
Embodiment
The above to the present invention is described in further details by the following examples, but this should not be interpreted as to this
The scope for inventing above-mentioned theme is only limitted to following embodiment, and all technologies realized based on the above of the present invention belong to this hair
Bright scope.
Embodiment 1
Experiment material
1st, cell derived used is tested
Umbilical cord blood picks up from Third Affiliated Hospital of Xinxiang Medical Coll and gynemetrics of First People's Hospital of Xinxiang City is mature
After healthy newborn, delivery of baby immediately from the blood sampling of placenta umbilical vein puncture is put into the blood taking bag for preserving liquid containing 28mL.Institute
Have in blood sample after sampling 4h and be sent to laboratory.
2nd, main agents
Culture medium RPMI1640, hyclone (Fetal bovine serum, FBS), 10 × PBS solution are purchased from
Thermo Fisher Scientific companies.
Lymphocyte separation medium (Ficoll-Paque PLUS), is the general of FicollTMPM400 and density 1.077g/mL
Sterile endotoxin (＜ 0.12EU/mL) the test solution of shadow acid sodium, purchased from GE Healthcare Life Sciences companies.
Anti-human CD3 monoclonal antibodies (CD3mAb), anti-human CD28 monoclonal antibodies (CD28mAb), PE-Cyanine7 marks
CD3mAb, FITC mark CD4mAb, PE mark ROR γ tmAb and Cell Stimulation Cocktail (plus
Protein transport inhibitors) it is purchased from eBioscience companies.
CD4+CD25+Tregs sortings kit, LD sortings post, MS sortings post, MACS sorting framves are purchased from Miltenyi
Biotec companies.
Recombined human MBL is purchased from R＆D System companies.
Cell Counting Kit-8 (CCK-8) are purchased from colleague's chemistry institute.
3rd, key instrument and equipment
Superclean bench, SW-CJ-2FD types, Purifying Equipment Co., Ltd., Suzhou.
Water bath, SSW-420-2S types, Shanghai Bo Xun companies.
CO2Incubator, Thermo, Thermo companies.
Low speed desk centrifuge, TDL-5 types, Town in Shanghai enjoys scientific instrument factory.
Flow cytometer, FACS Calibur types, U.S. company BD.
Inverted microscope, TS100-F types, Japanese NIKON companies.
Refrigerated centrifuge, Eppendorf 5810R types, German Eppendorf companies.
Nucleic acid-protein analyzer, SmartSpecTM3000 types, BIO-RAD companies of the U.S..
PCR instrument, TGradient types, Biometra companies.
Electrophoresis apparatus, DYC-31C types, Machines Plant of Beijing 61.
GIS gel imaging systems, Tanon GIS-3500 types, Shanghai Tian Neng Science and Technology Ltd.s.
Pure water meter, NW types, Heal Forece companies.
4th, common agents are prepared
(1) 1 × PBS solution
The parts by volume distilled water of 1 10 × PBS solution of parts by volume+9；
(2) 50 × TAE electrophoresis liquids
Mended above with ddH2O to 1000mL, room temperature preservation, it is necessary to when, be diluted to 1 × TAE.For example：Need
Using 200mL 1 × TAE, then 4mL 50 × TAE, 196mL ddH2O, mixing are measured；
(3) 1 × Fix/Perm solution
1 parts by volume Fix/Perm Concentrate+3 parts by volume Fix/Perm Diluent
For example：4mL 1 × Fix/Perm solution is needed to use, then measures 1mL Fix/Perm Concentrate, 3mL
Fix/Perm Diluent, mixing；
(4) 1 × Perm Buffer solution
1 parts by volume 10 × Perm Buffer+9 parts by volume Deionized Water
For example：10mL 1 × Perm Buffer solution is needed, then measures 1mL 10 × Perm Buffer, 9mL's
Deionized Water, mixing.
Experimental method
1st, the preparation of human umbilical cord blood mononuclear cell (Cord blood mononuclear cell, CBMC)
The Cord blood of ACD anti-freezings, is diluted with the precooling PBS solution of equivalent, is placed on lymphocyte separation medium, conventional density
Gradient centrifugation (400 × g, 30min) separation obtains mononuclearcell (CBMC), and three (each 1500r/ are washed with sterile PBS
Min, 10min), cell is resuspended with the culture mediums of RPMI 1640 containing 10wt%FBS, and count.
2nd, it is coated with anti-CD3mAb
1mg/mL anti-CD3mAb is diluted to 2 μ g/mL and 5 μ g/mL with sterile 1 × PBS respectively, 96 orifice plates are added,
50 μ L/ holes, 4 DEG C overnight.
3rd, inductions of the various concentrations MBL to Tregs cells is broken up
Above-mentioned CBMC suspensions every 106The implantation of/mL cell numbers is coated with anti-CD3mAb (2 μ g/mL) 96 hole cells in advance
In culture plate, per the μ L of hole 200, each hole adds anti-CD28mAb (1 μ g/mL), TGF-β 1 (5ng/mL), and experimental group adds respectively
Enter various concentrations (1-10ng/mL) MBL, control group is not added with MBL, and every kind of condition of culture is respectively provided with 5 multiple holes.Cell is placed in
37 DEG C, volume fraction is 5% CO2Cultivated in incubator.It is used for related experiment in the cell of collecting for the 3rd day of culture.
4th, Flow cytometry fresh CBMC and CBMC transcription factor Foxp3 at different conditions expression
(1) collect cell suspensions of the fresh CBMC and CBMC after different condition culture and add 1.5mL EP pipes, 106It is individual thin
Born of the same parents/pipe (about 100 μ L)；
(2) in 4 DEG C of 400 × g of centrifuge, 5min is centrifuged, supernatant is abandoned；
(3) 400 1 × PBS of μ L are added and cell is resuspended, in 4 DEG C of 400 × g of centrifuge, centrifuged 5min, abandon supernatant；
(4) 200 1 × PBS of μ L are separately added into cell is resuspended, be separately added into PEcyanine7-CD3 (1 μ L), FITC-CD4
(2 μ L), APC-CD25 (2 μ L), the 30min of lucifuge incubation on ice；
(5) after the completion of being incubated, in 4 DEG C of 400 × g of centrifuge, 5min is centrifuged, supernatant is abandoned；
(6) 400 μ 1 × PBS of L are added, in 4 DEG C of 400 × g of centrifuge, 5min is centrifuged, abandons supernatant；
(7) 200 1 × Fix/Perm of μ L are separately added into cell is resuspended, lucifuge is incubated 30min on ice；
(8) after the completion of being incubated, 200 μ L 1 × Perm Buffer are directly added into, in 4 DEG C of 500 × g of centrifuge, centrifugation
6min, abandons supernatant；
(9) 400 μ L1 × Perm Buffer are separately added into cell is resuspended, in 4 DEG C of 500 × g of centrifuge, centrifuged 6min, abandon
Supernatant；
(10) 100 μ L1 × Perm Buffer and 2 μ L PE-Foxp3 are separately added into, 30min is incubated in lucifuge on ice, it
After 4 DEG C of 500 × g of centrifuge, 6min is centrifuged, supernatant is abandoned；
(11) 400 μ L1 × Perm Buffer are separately added into cell is resuspended, in 4 DEG C of 500 × g of centrifuge, centrifuged 6min, abandon
Supernatant；
(12) 400 1 × PBS of μ L are separately added into cell is resuspended, treat machine testing, if upper machine testing can not add in time
Enter 500 μ L 4wt% paraformaldehydes and cell is resuspended, in upper machine in 24-48h.
As a result：It will be seen from figure 1 that fresh CMBC low expressions CD4+CD25+Foxp3+T cells, i.e. CBMC low expressions
Tregs cells；Figure it is seen that MBL improves the ratio of CD4+CD25+Foxp3+T cells in CBMC, it is indicated above being higher than
The MBL of physiological concentration can significantly improve the ratio of CD4+CD25+Foxp3+T cells in CBMC.
5th, RT-PCR methods detect Foxp3mRNAs of the fresh CBMC and CBMC in different condition culture expression
(1) fresh CBMC and culture after CBMC total serum IgEs extraction
The cracking of cell：Collect fresh CBMC and CBMC and cultivate the cell of 3 days at different conditions in 1.5mL EP pipes,
4 DEG C of centrifugation 2min of 8000g, abandon supernatant.Often pipe adds 1mL RNAiso Plus, with liquid-transfering gun repeatedly pressure-vaccum until lysate
It is middle without obvious sediment, be stored at room temperature 5min.
RNA separation：Then chloroform (200 μ L) is added into homogenate lysate, covers tightly centrifuge tube, acutely shaken with hand
15s (chloroform low boiling point, volatile, should carefully centrifuge lid during concussion and flick suddenly).Treating solution, fully emulsified (no phase separation is existing
As) after, then 5min is stored at room temperature, 4 DEG C of 12000g centrifuge 15min.It is careful from centrifuge to take out centrifuge tube after centrifugation, now
Homogenate is divided into three layers, i.e., colourless supernatant, middle white egg white and with coloured lower floor's organic phase.
Precipitation：Aspirate supernatant is transferred in another new centrifuge tube (never suction out white intermediate layer), reset and add upwards into
Isometric isopropanol, after the centrifuge tube that turns upside down fully is mixed, 10min, 4 DEG C of centrifugations of 12000g are stood at 15-30 DEG C
10min.Typically after centrifugation, precipitation occurs in test tube bottom.
RNA washing：Careful supernatant discarding, slowly adds volume fraction along centrifugation tube wall and (is cut for 75% ethanol 1mL
Do not touch precipitation), gently turned upside down washing centrifuge tube wall, and carefully ethanol is discarded (in order to more after 4 DEG C of centrifugation 5min of 12000g
The salt ion content in RNA is controlled well, should try one's best cleared ethanol).
RNA dissolving：Drying at room temperature precipitation 2-5min (cannot be centrifuged or heat drying, otherwise RNA will be difficult molten
Solution), plus the appropriate RNase-free water dissolving precipitation of people, precipitation can be gently blown and beaten with liquid-transfering gun if necessary, treats that RNA precipitate is complete
Dissolving is after -80 DEG C of preservations.
(2) reverse transcription (RT) reacts
The PrimeScriptTMRT regent Kit with provided according to precious bioengineering (Dalian) Co., Ltd
GDNA Eraser specifications are operated.
Remove genomic DNA reaction：
By following composition in reaction mixture processed on ice, in order to ensure the accuracy of reaction solution preparation, every reaction is carried out
When, Master Mix first should be prepared by the amount of stoichiometric number+2, then be dispensed into again in each reaction tube, be eventually adding RNA sample.
Synthesize cDNA reactions：
Reaction solution is prepared and please carried out on ice.In order to ensure the accuracy of reaction solution preparation, when carrying out every reaction, Ying Xian
Master Mix are prepared by the amount of stoichiometric number+2,10 μ L are then dispensed again into each reaction tube.Soft mix is carried out instead immediately
Responsive transcription.
When the cDNA of synthesis needs long-term preserve, please preserved in -20 DEG C or lower temperature；
(3) the design synthesis and dilution of PCR primer
Foxp3, β-actin primers are designed according to design of primers principle according to people β-actin, Foxp3 DNA sequence dna, and
Its specificity is verified by the NCBI blast programs provided, above primer is synthesized by Shanghai Sheng Gong bio-engineering corporations.Plus it is suitable
Autoclaving ultra-pure water is measured, primer storage liquid is prepared, concentration is 10 μm of ol/L, -20 DEG C of storages after packing.Primer sequence and reaction
Condition see the table below：
The size of primer sequence and RT-PCR products
PCR reaction systems：
| Reagent | Usage amount |
| cDNA | 0.8μL |
| Forward Primer | 0.8μL |
| Reverse Primer | 0.8μL |
| 2×Taq plus PCR Mix | 10μL |
| ddH2O | 7.6μL |
Reaction condition：
It is simple to mix after low-speed centrifugal, expanded on grads PCR instrument.95 DEG C of pre-degenerations 3min, subsequent 95 DEG C of 30s, 60 DEG C are moved back
Fiery 30s, 72 DEG C of extension 1min, totally 35 circulations.
PCR primer is identified：
8 each amplified productions of μ L electrophoresis on 1wt% agaroses (containing EB) gel is taken, as a result in GIS gel images processing systems
Shot in system and record and preserve, and each net OD value of band is determined with Image J softwares, calculate purpose band and β-actin
The ratio between net OD value.
As a result：As can be drawn from Figure 3, MBL promotes CBMC height expression Foxp3mRNA.
6th, proliferative effects of the iTreg to PBMCs is detected with CCK-8 methods
By the CD4+CD25+T cells sub-elected from experimental group (i.e. iTreg) and the single core of freshly extd human peripheral
Cell (Peripheral blood mononuclear cells, PBMCs) is with 1:8、1:4、1:2、1:The mixing of 1 ratio is as real
Group is tested, PBMCs is control group, be separately added into coating AntiCD3 McAb (2 μ g/mL) and CD28 (1 μ g/mL) monoclonal antibody, at 37 DEG C,
Volume fraction is 5% CO2Cultivated 4 days in incubator.4h before culture is terminated, 20 μ L CCK-8 reagents are added per hole, continue to train
Optical density (OD) value of Detection wavelength 450nm on 4h, ELISA instrument is supported, iTreg Carbazole alkaloids are calculated by following equation
Rate.
Suppress propagation (%)=[(CD4+ being stimulated in the hole not containing iTreg cells of CD4+CD25-T cells
The CD4+CD25-T cells being stimulated in the hole of CD25-T cells-addition iTreg cells)/(in the hole not containing iTreg cells
The blank well of CD4+CD25-T cells being stimulated-comprise only culture medium)] × 100%.
As a result：As can be drawn from Figure 4, iTreg cells suppress PBMCs propagation with concentrationdependent manner, and as a result prompting is lured
The iTreg cells led have obvious Cellular immunity suppression function, and concentration dependent (as shown in Figure 4 B) is presented in inhibiting rate.
Embodiment 2
Experiment material
1st, cell derived used is tested
Reference example 1
2nd, main agents
Reference example 1
3rd, key instrument and equipment
Reference example 1
4th, common agents are prepared
Reference example 1
Experimental method
1st, the preparation of human umbilical cord blood mononuclear cell
Reference example 1
2nd, it is coated with anti-CD3mAb
Reference example 1
3rd, CD4+CD25-T cells and CD4+CD25+T cell magnetic sortings
Preparation of samples
Peripheral blood mononuclear cells are separated using density gradient centrifugation, to remove blood platelet, cell and 200 is resuspended with buffer
× g centrifuges 10-15min, carefully draws supernatant, repeated washing.
The non-CD4+T cells of magnetic mark
Whole process will be rapidly completed, and keep cell to be in pre-cold state, prevent capping phenomena from occurring.
It is 2-8 DEG C to recommend the temperature being incubated.
MS posts magnetic mark≤107Cell, LD posts magnetic mark≤108Cell.
Using the filter process cell suspension of the blood nylon filter with 30 μm, filter is using preceding using buffer
Moisten.
(1) cell is counted；
(2) cell suspension centrifuges 10min in 300 × g, and supernatant is removed completely；
(3) every 107Add 90 μ L buffer and cell is resuspended；
(4) every 107Add 10 μ L CD4+T Cell Biotin-Antibody Cocktail；
(5) mix and in (2-8 DEG C) incubation 5min of refrigerator；
(6) every 107Add 20 μ L Anti-Biotin MicroBeads；
(7) mix and in (2-8 DEG C) incubation 10min of refrigerator；
(8) enter next step magnetic sorting (required cell suspension at least 500 μ L such as can not enough add buffer)；
The negative sorting of the magnetic of CD4+T cells
The sorting post suitable according to the selection of total cell number.
Always will be until the liquid in splitter flow to end, then perform next step.
Sorted with LD splitters
LD splitters are positioned in MACS sorters.
Add 2 × 1mL buffer rinse splitters.
Cell suspension is placed in LD splitters.
The liquid of outflow is collected, and splitter is rinsed 2 times with 1mL buffer.The liquid being collected into is unlabelled
CD4+T cells.
Magnetic mark CD4+CD25+ regulatory T cells
(1) cell suspension collected centrifuges 10min in 300 × g, and supernatant is abandoned completely；
(2) 90 μ L buffer are added and cell is resuspended；
(3) 10 μ L CD25MicroBeads are added；
(4) mix and be incubated 15min in refrigerator (2-8 DEG C) lucifuge；
(5) (option) adds staining antibodies.Such as, 10 μ L CD25-PE, 10 μ L CD4-FITC or CD4-APC, in ice
Case (2-8 DEG C) lucifuge is incubated 5min；
(6) 1-2mL buffer washing cells are added, 300 × g centrifugation 10min abandon supernatant completely；
(7) 500 μ L buffer are added and cell is resuspended.Cell number should be less than 108, more than 108Buffer should accordingly be increased
Volume；
(8) next step magnetic sorting is entered.
MS splitters positive sorting CD4+CD25+ regulatory T cells
Highest is reached, it is necessary to using two MS splitters for the cell purity that makes sorting.
(1) MS splitters are placed in MACS sorters；
(2) 500 μ L buffer rinse splitters are added；
(3) 500 μ L cell suspensions are placed in MS splitters, collect the liquid (CD4+CD25-T cells) of outflow；
(4) 500 μ L buffer are added and rinses MS splitters 3 times, (CD4+CD25-T is thin for the liquid of collection outflow completely
Born of the same parents)；
(5) MS splitters are removed into MACS sorters, placed on suitable collecting pipe；
(6) 1mL buffer are added to be placed in MS splitters, promotes piston that the cell of mark is placed in collecting pipe immediately；
(7) it is the purity of increase CD4+CD25+ cells, the CD4+CD25+ cells that previous step is collected must pass through second
MS splitters, repeat 1-6；
(8) CD4+CD25-T cells 1000rpm centrifuges 5min；
(9) supernatant discarding, CD4+CD25-T cells add 5mL 10wt%FBS RPMI-1640 complete culture solutions and thin
Born of the same parents count.
4th, inductions of the various concentrations MBL to Tregs cells is broken up
Above-mentioned CBMC suspensions every 106The implantation of/mL cell numbers is coated with anti-CD3mAb (2 μ g/mL) 96 hole cells in advance
In culture plate, per the μ L of hole 200, each hole adds anti-CD28mAb (1 μ g/mL), TGF-β 1 (5ng/mL), and experimental group adds respectively
Enter various concentrations (1-10ng/mL) MBL, control group is not added with MBL, and every kind of condition of culture is respectively provided with 5 multiple holes.Cell is placed in
37 DEG C, volume fraction is 5% CO2Cultivated in incubator.It is used for related experiment in the cell of collecting for the 3rd day of culture.
5th, fresh CD4+CD25-T, CD4+CD25+T and CD4+CD25-T cell of Flow cytometry is at different conditions
Transcription factor Foxp3 expression, method reference example 1.
As a result：From fig. 5, it can be seen that by immunological magnetic bead sorting can draw high-purity CD4+CD25-T cells and
CD4+CD25+T cells；As can be drawn from Figure 6, MBL promotes CD4+CD25-T cells to break up to iTreg cell inductions, thus table
It is bright, higher than the induction differentiation that physiological concentration MBL can increase iTreg cells.
6th, RT-PCR methods detect Foxp3mRNAs of the fresh CD4+CD25-T and CD4+CD25-T in different condition culture table
Reach
Fresh CD4+CD25-T and culture after CD4+CD25-T total serum IgEs extraction, method reference example 1.
As a result：As can be drawn from Figure 7, MBL promotes CD4+CD25-T height expression Foxp3mRNA.
7th, proliferative effects of the iTreg to CD4+CD25-T is detected with CCK-8 methods
Method reference example 1, as a result：As can be drawn from Figure 8, iTreg cells suppress CD4+CD25- with concentrationdependent manner
T propagation, as a result showing the iTreg cells of induction has obvious Cellular immunity suppression function, and concentration is presented in inhibiting rate
Dependence.
Embodiment above describes general principle, principal character and the advantage of the present invention, the technical staff of the industry should
Understand, the present invention is not limited to the above embodiments, the original for simply illustrating the present invention described in above-described embodiment and specification
Reason, under the scope for not departing from the principle of the invention, various changes and modifications of the present invention are possible, and these changes and improvements are each fallen within
In the scope of protection of the invention.
Claims (6)
1.MBL is preparing infectious diseases, autoimmune disease or/and the metamorphosis of prevention or treatment using Tregs as target spot instead
Application in answering property disease medicament, the MBL realizes medicine by promoting CD4+CD25-T cells to break up to Tregs cell inductions
Effect.
2. MBL according to claim 1 is preparing prevention or is treating infectious diseases using Tregs as target spot, itself exempts from
Application in epidemic disease disease or/and allergic disease medicine, it is characterised in that：It is former that the infectious diseases includes Li Shiman
Insect infection, Infected With Plasmodium, Listeria infection, throttle pylori (Hp) infection, herpes simplex infections, human immunodeficiency
Poison infection, hepatitis b virus infected and infection with hepatitis C virus.
3. MBL according to claim 1 is preparing prevention or is treating infectious diseases using Tregs as target spot, itself exempts from
Application in epidemic disease disease or/and allergic disease medicine, it is characterised in that：The autoimmune disease includes system
Property lupus erythematosus, non-obese diabetes, rheumatoid arthritis, type 1 diabetes, atopic dermatitis, Graves disease and alopecia areata.
4. MBL according to claim 1 is preparing prevention or is treating infectious diseases using Tregs as target spot, itself exempts from
Application in epidemic disease disease or/and allergic disease medicine, it is characterised in that：The allergic disease includes allergy
Property rhinitis and bronchial astehma.
5. MBL according to claim 1 is preparing prevention or is treating infectious diseases using Tregs as target spot, itself exempts from
Application in epidemic disease disease or/and allergic disease medicine, it is characterised in that：The MBL is use in conjunction part and list
The natural MBL albumen of clonal antibody affinitive layer purification human plasma.
6. MBL according to claim 1 is preparing prevention or is treating infectious diseases using Tregs as target spot, itself exempts from
Application in epidemic disease disease or/and allergic disease medicine, it is characterised in that：The concentration of the MBL is higher than it in vivo
Physiological concentration.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110051824A (en) * | 2019-04-30 | 2019-07-26 | 新乡医学院 | The method of the drug of application of the MBL in preparation prevention or in terms for the treatment of fat drug, screening prevention or treatment obesity |
| CN113645983A (en) * | 2018-10-17 | 2021-11-12 | 基础科学研究院 | Structural and functional characterization of yeast-derived polysaccharides to induce Treg cells |
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| CN1359298A (en) * | 1999-05-14 | 2002-07-17 | 斯蒂芬·蒂尔 | Novel indications of mannan-bindie lectin (MBL) in the treatment of immanocompromised individuals |
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2017
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| CN1359298A (en) * | 1999-05-14 | 2002-07-17 | 斯蒂芬·蒂尔 | Novel indications of mannan-bindie lectin (MBL) in the treatment of immanocompromised individuals |
| CN1359420A (en) * | 1999-05-14 | 2002-07-17 | 斯蒂芬·蒂尔 | Novel indications of mannan-binding lectin (MBL) in the treatment of immunocompromised individuals |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113645983A (en) * | 2018-10-17 | 2021-11-12 | 基础科学研究院 | Structural and functional characterization of yeast-derived polysaccharides to induce Treg cells |
| CN110051824A (en) * | 2019-04-30 | 2019-07-26 | 新乡医学院 | The method of the drug of application of the MBL in preparation prevention or in terms for the treatment of fat drug, screening prevention or treatment obesity |
| CN110051824B (en) * | 2019-04-30 | 2022-09-30 | 新乡医学院 | Application of MBL in preparation of medicament for preventing or treating obesity, and method for screening medicament for preventing or treating obesity |
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