CN117683724A - Tolerogenic dendritic cell, and preparation method and application thereof - Google Patents
Tolerogenic dendritic cell, and preparation method and application thereof Download PDFInfo
- Publication number
- CN117683724A CN117683724A CN202311700521.3A CN202311700521A CN117683724A CN 117683724 A CN117683724 A CN 117683724A CN 202311700521 A CN202311700521 A CN 202311700521A CN 117683724 A CN117683724 A CN 117683724A
- Authority
- CN
- China
- Prior art keywords
- aim2
- cell
- dendritic cells
- preparation
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000004443 dendritic cell Anatomy 0.000 title claims abstract description 82
- 230000003614 tolerogenic effect Effects 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 35
- 108091034117 Oligonucleotide Proteins 0.000 claims abstract description 30
- 108060000255 AIM2 Proteins 0.000 claims abstract description 27
- 102100024064 Interferon-inducible protein AIM2 Human genes 0.000 claims abstract description 23
- 230000001105 regulatory effect Effects 0.000 claims abstract description 14
- 230000005764 inhibitory process Effects 0.000 claims abstract description 7
- 206010052779 Transplant rejections Diseases 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 43
- 230000002401 inhibitory effect Effects 0.000 claims description 26
- 108090000623 proteins and genes Proteins 0.000 claims description 25
- 239000004055 small Interfering RNA Substances 0.000 claims description 24
- 102000004169 proteins and genes Human genes 0.000 claims description 20
- 230000006698 induction Effects 0.000 claims description 19
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 17
- 102000055501 telomere Human genes 0.000 claims description 17
- 108091035539 telomere Proteins 0.000 claims description 17
- 101150051235 AIM2 gene Proteins 0.000 claims description 15
- 238000002054 transplantation Methods 0.000 claims description 12
- 238000000338 in vitro Methods 0.000 claims description 6
- 230000004853 protein function Effects 0.000 claims description 6
- 210000003289 regulatory T cell Anatomy 0.000 claims description 6
- 230000001939 inductive effect Effects 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 238000011282 treatment Methods 0.000 claims description 5
- 230000001154 acute effect Effects 0.000 claims description 3
- 230000004069 differentiation Effects 0.000 claims description 3
- 230000006058 immune tolerance Effects 0.000 claims description 3
- 239000003112 inhibitor Substances 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims description 2
- 210000002540 macrophage Anatomy 0.000 claims description 2
- 210000000822 natural killer cell Anatomy 0.000 claims description 2
- 210000002707 regulatory b cell Anatomy 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 238000011321 prophylaxis Methods 0.000 claims 1
- 241000701161 unidentified adenovirus Species 0.000 abstract description 17
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 abstract description 15
- 230000000694 effects Effects 0.000 abstract description 11
- 210000005259 peripheral blood Anatomy 0.000 abstract description 10
- 239000011886 peripheral blood Substances 0.000 abstract description 10
- 210000001185 bone marrow Anatomy 0.000 abstract description 9
- 238000005516 engineering process Methods 0.000 abstract description 6
- 210000000056 organ Anatomy 0.000 abstract description 5
- 230000001276 controlling effect Effects 0.000 abstract description 3
- 230000003832 immune regulation Effects 0.000 abstract description 3
- 239000006228 supernatant Substances 0.000 description 24
- 229940124276 oligodeoxyribonucleotide Drugs 0.000 description 19
- 238000001514 detection method Methods 0.000 description 17
- 238000002474 experimental method Methods 0.000 description 17
- 241000699666 Mus <mouse, genus> Species 0.000 description 15
- 238000000684 flow cytometry Methods 0.000 description 14
- 239000006285 cell suspension Substances 0.000 description 12
- 238000001890 transfection Methods 0.000 description 12
- 230000000638 stimulation Effects 0.000 description 10
- 238000005406 washing Methods 0.000 description 8
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 7
- 241000699670 Mus sp. Species 0.000 description 7
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 7
- 230000035755 proliferation Effects 0.000 description 7
- 239000012980 RPMI-1640 medium Substances 0.000 description 6
- 241000700605 Viruses Species 0.000 description 6
- 230000006907 apoptotic process Effects 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 230000006870 function Effects 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- 238000001356 surgical procedure Methods 0.000 description 5
- 102100022297 Integrin alpha-X Human genes 0.000 description 4
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 239000003018 immunosuppressive agent Substances 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 230000035800 maturation Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000007799 mixed lymphocyte reaction assay Methods 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 3
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 3
- 102000004388 Interleukin-4 Human genes 0.000 description 3
- 108090000978 Interleukin-4 Proteins 0.000 description 3
- 210000000662 T-lymphocyte subset Anatomy 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 3
- 229940124589 immunosuppressive drug Drugs 0.000 description 3
- 229940028885 interleukin-4 Drugs 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 229940121649 protein inhibitor Drugs 0.000 description 3
- 239000012268 protein inhibitor Substances 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 210000004989 spleen cell Anatomy 0.000 description 3
- 210000002303 tibia Anatomy 0.000 description 3
- 210000000689 upper leg Anatomy 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 2
- 210000000447 Th1 cell Anatomy 0.000 description 2
- 210000000068 Th17 cell Anatomy 0.000 description 2
- 230000000735 allogeneic effect Effects 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 238000003197 gene knockdown Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 229960004857 mitomycin Drugs 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 1
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 description 1
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
- -1 CD86 Proteins 0.000 description 1
- SPJOZZSIXXJYBT-UHFFFAOYSA-N Fenson Chemical compound C1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 SPJOZZSIXXJYBT-UHFFFAOYSA-N 0.000 description 1
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 1
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 1
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 1
- 206010067268 Post procedural infection Diseases 0.000 description 1
- 230000033540 T cell apoptotic process Effects 0.000 description 1
- 230000006052 T cell proliferation Effects 0.000 description 1
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 239000000370 acceptor Substances 0.000 description 1
- 230000004721 adaptive immunity Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000005427 lymphocyte apoptotic process Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000007425 progressive decline Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000024664 tolerance induction Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a tolerogenic dendritic cell, a preparation method and application thereof. The invention provides a target point for regulating and controlling the tolerance of dendritic cells, namely AIM2, and can induce the tolerant dendritic cells by regulating and controlling the AIM2 target point, and provides a method for preparing the tolerant dendritic cells by an adenovirus vector technology and an oligonucleotide targeted inhibition technology. The tolerogenic dendritic cells obtained by the preparation method have obvious negative immune regulation effect, and can effectively prevent or treat organ transplant rejection. In addition, the method for preparing the tolerogenic dendritic cells is efficient and stable, has low cost, can take bone marrow-derived BMDC or peripheral blood PBMC as a source of the dendritic cells, has good expansibility, can realize the supply of a large number of tolerogenic dendritic cells, and has good application value.
Description
Technical Field
The invention belongs to the technical field of tolerogenic dendritic cells. More particularly, to a tolerogenic dendritic cell, a preparation method and application thereof.
Background
Organ transplantation is currently the most effective means of treating numerous end-stage diseases, but long-term, effective control of rejection remains a major challenge in the field of transplantation immunization. Although the development of immunosuppressants has significantly promoted the control of rejection after transplantation in recent years, chronic rejection is still difficult to avoid and long-term survival of the transplant recipients is still not optimal. Meanwhile, the use of immunosuppressive drugs also has a plurality of side effects, and long-term postoperative application of immunosuppressive schemes has the risks of increasing postoperative infection, damaging graft function and promoting malignant tumor generation.
Therefore, it is important to seek new therapeutic strategies to increase survival of patients after transplantation and to reduce the use of immunosuppressive drugs. In this context, studies of Dendritic Cells (DCs) have attracted a great deal of attention. As a bridge between innate and adaptive immunity, the antigen presenting function of DCs plays a key role in the development of a variety of immune-related diseases, and DCs play a key role in tolerance induction and maintenance of immune balance. Basic research in the direction of DC has driven the breakthrough of many therapeutic approaches, including tolerogenic dendritic cells modified and induced by various methods (tolerogenic dendritic cells, tolDC). toldcs are a hotspot and focus in the current field of immunomodulation research. In cell therapy following organ transplantation, tolDC can reduce the use of immunosuppressive drugs and have the opportunity to create specific tolerance to allografts. tolDC provides a new direction with broad prospect for treatment after transplantation.
The prior art methods for preparing tolerogenic dendritic cells (toldcs) mainly convert DCs to toldcs by means of induction, including by Oligodeoxyribonucleotide (ODN) or adenovirus induction, as in patent US6936468B2. However, the induction target point, sample source and the like can influence the induction effect and activity of the tolerogenic dendritic cells, so that the research of the technology is less at present, and the research of tolDC induction production technology is significant.
Disclosure of Invention
The invention AIMs at exploring a preparation technology of a tolerogenic dendritic cell (tolDC), and provides a method for inducing the tolerogenic dendritic cell by regulating an AIM2 target. The tolDC induced by taking AIM2 as a target point has high IL-10 secretion level, has obvious negative immunoregulation effect, and the imDC can be derived from bone marrow and peripheral blood PBMC, thereby having good expansibility; the induction method is efficient and stable, has high cell purity and lower cost, and can realize the supply of a large number of tolerating dendritic cells.
It is a first object of the present invention to provide a dendritic cell tolerance regulatory target.
It is a second object of the present invention to provide a method for preparing tolerogenic dendritic cells.
It is a third object of the present invention to provide a tolerogenic dendritic cell.
It is a fourth object of the present invention to provide the use of the tolerogenic dendritic cells described above.
The above object of the present invention is achieved by the following technical scheme:
the invention provides a dendritic cell tolerance regulation target, namely AIM2 gene or protein. Studies of the present invention show that inhibition of AIM2 expression by immature dendritic cells (imDCs) or inhibition of AIM2 protein function can induce production of tolDCs. Accordingly, the present invention claims:
use of AIM2 gene/protein as a target for modulating dendritic cell tolerance in vitro.
Use of AIM2 gene/protein as a target for the induction of the preparation of tolerogenic dendritic cells.
Use of an AIM2 gene/protein inhibitor for modulating dendritic cell tolerance in vitro.
Use of an AIM2 gene/protein inhibitor for the preparation of a tolerogenic dendritic cell.
Preferably, the inhibitor of the AIM2 gene/protein is an AIM2-shRNA capable of inhibiting expression of the AIM2 gene/protein, or an inhibitory oligonucleotide capable of inhibiting function of the AIM2 gene/protein.
As an alternative embodiment, the AIM2 gene/protein inhibitor may be: aim2-shRNA capable of inhibiting AIM2 gene/protein expression realizes AIM2 gene/protein expression inhibition in an adenovirus transfection mode; or an inhibitory Oligonucleotide (ODN) capable of competitively binding to the AIM2 protein, inhibiting the function of the AIM2 protein, such as the commercially available ODN TTAGGG.
The invention also provides a preparation for inducing the preparation of tolerogenic dendritic cells, the preparation comprising an Aim2-shRNA capable of inhibiting the expression of AIM2 genes/proteins, or an inhibitory oligonucleotide capable of inhibiting the function of AIM2 genes/proteins.
As one of the preferable embodiments, the target sequence of the Aim2-shRNA is 5'-GCTGACAGGATCCTGATATAG-3'.
As one of the preferred embodiments, the inhibitory oligonucleotide is ODN TTAGGG.
The invention also provides a method for preparing the tolerogenic dendritic cells, which is to prepare the tolerogenic dendritic cells by inhibiting the expression of AIM2 of the immature dendritic cells or the protein function of AIM 2. The above-described formulations may be used in particular to inhibit the expression of the immature dendritic cells AIM2 or its protein function.
Tolerogenic dendritic cells produced by the above-described methods are also within the scope of the present invention.
In addition, in the method for producing a tolerogenic dendritic cell provided by the present invention, the ODN TTAGGG is preferably used at a concentration of not less than 3. Mu.M (more preferably at a concentration of 3 to 10. Mu.M). Considering cost and other factors in combination, it is recommended to use a concentration of 3 μm.
As an alternative embodiment, the method for preparing the tolerogenic dendritic cells by using adenovirus transfection technology to inhibit AIM2 expression is to transfect the prepared Aim2-shRNA adenovirus (Aim 2-shRNA-ADV) with imDC (preferably with MOI of 50) and half-transferring 24-36 hours (preferably 24 hours) after transfection to maintain cell activity and continue culture; resistant dendritic cells (toldcs) were obtained 48-72 hours (preferably 48 hours) after transfection.
As an alternative embodiment, the method for preparing the tolerogenic dendritic cells by inhibiting AIM2 protein function by using the inhibitory oligonucleotide is to add the inhibitory oligonucleotide (such as ODN TTAGGG) on the 3 rd day in the imDC induction process and continue to culture until the 6 th day, thus obtaining the tolerogenic dendritic cells (tolDC).
The imDC may be derived from bone marrow or peripheral blood PBMC.
The tolerogenic dendritic cells prepared by the invention are verified to have the effects of inhibiting inflammation and negatively regulating immune response in vivo, remarkably improve the capability of secreting IL-10, can stimulate and weaken lymphocyte proliferation capability, induce lymphocyte apoptosis, and have the potential of further inducing allograft immune tolerance. The invention therefore also provides the use of the tolerogenic dendritic cells obtained above for the preparation of a medicament for the prevention and/or treatment of transplant rejection (including cell transplantation, organ transplantation or tissue transplantation), for the inhibition of acute rejection or for the induction of differentiation of naive T cells into tregs.
The invention also provides the use of the tolerogenic dendritic cells described above for the preparation or induction of transplantation immune tolerance-modulating cells, including regulatory T cells, regulatory B cells, regulatory NK cells or regulatory macrophages.
The invention has the following beneficial effects:
the invention creatively prepares the tolerogenic dendritic cells by regulating and controlling AIM2 targets, the tolerogenic dendritic cells prepared by the preparation method can secrete higher level of IL-10, have obvious negative immune regulation effect, and can effectively prevent or treat organ transplant rejection by inducing CD4+ Treg cell subsets and inhibiting Th1 and Th17 cell subsets in a receptor body.
In addition, the preparation method of the tolerogenic dendritic cells provided by the invention can induce the initial cells imDC to be derived from bone marrow and peripheral blood PBMC, and has good expansibility.
In addition, the method for preparing the tolerogenic dendritic cells is efficient and stable, the cell purity is more than 85%, the cell purity after purification can reach more than 95%, the cost is low, and the supply of a large number of tolerogenic dendritic cells can be realized.
Drawings
FIG. 1 is a graph of the results of flow cytometer measurements processed in example 2 and a graph of the morphology of imDC and mDC under a light microscope.
FIG. 2 is a graph showing the expression of AIM2 in DC using Aim2-shRNA adenovirus.
FIG. 3 is a graph of the results of flow cytometry detection of DC-Aim2-shRNA after LPS stimulation and IL-10 detection of DC-Aim 2-shRNA.
FIG. 4 is a graph showing the results of flow cytometry induced ODN-tolDC by different ODN TTAGGG concentration protocols.
FIG. 5 is a flow chart of a preferred preparation scheme of ODN-tolDC.
FIG. 6 is a graph showing the results of detection by a flow cytometer of the proliferation of the ODN-tolDC-inhibited T cells.
FIG. 7 is a graph showing the results of flow cytometry detection of the promotion of T cell apoptosis by ODN-tolDC.
FIG. 8 is a schematic flow chart of embodiment 9, embodiment 10 and embodiment 11
FIG. 9 is a graph showing the results of flow cytometry using ODN-tolDC to induce differentiation of Treg subpopulations in example 9.
FIG. 10 is a graph showing the results of flow cytometry using ODN-tolDC to inhibit Th1, th17 subpopulations in example 10.
FIG. 11 is a graph showing the results of flow cytometry detection using PBMC as a source for induction of ODN-tolDC.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
C57BL/6 mice, balb/C mice, purchased from Si Bei Fu (Beijing) Biotechnology Co., ltd.
IL-10ELISA kits were purchased from Hangzhou Union Biotechnology Co.
Aim2-shRNA adenovirus (Aim 2-shRNA-ADV) was produced by Shanghai Ji Kai Gene technologies Co.
The inhibitory oligonucleotide ODN TTAGGG (sequence 5'-TTAGGGTTAGGGTTAGGGTT AGGG-3') is commercially available.
EXAMPLE 1 preparation of mouse bone marrow derived dendritic cells (bone marrow derived dendritic cell, BMDC)
The specific preparation method of the mouse BMDC is as follows:
1. balb/c mice at 6 weeks of age were sacrificed using a cervical removal procedure, sterilized in 75% ethanol, the femur and tibia were separated using a sterile surgical instrument at a super clean bench, the separated femur and tibia were placed in 4℃PBS solution (Gibco, USA), and surface excess muscle tissue was carefully separated;
2. the femur and tibia after treatment are moved into RPMI-1640 solution with the temperature of 4 ℃, and the RPMI-1640 liquid with the temperature of 4 ℃ is repeatedly extracted to flush the marrow cavity by using a sterile insulin syringe or a 1mL syringe;
3. filtering the flushing liquid by using a 200-mesh screen, centrifuging at the temperature of 4 ℃ and at the speed of 1200rpm for 5 minutes, and discarding the supernatant to obtain cells;
4. the cells were resuspended in 5mL of 1 Xerythrocyte lysate, incubated at 4℃for 5 minutes, then 20mL of 4℃RPMI-1640 solution was added, centrifuged at 1200rpm at 4℃for 5 minutes, and the supernatant was discarded;
5. resuspension of cells in 5mL RPMI-1640, one wash, centrifugation and removal of supernatant, followed by 2X 10 cells 6 The concentration of/mL was inoculated into a flat bottom 6-well plate containing 2mL of complete medium; the complete culture medium is based on RPMI-1640 culture solution containing 10% of fetal bovine serum FBS, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) are added, wherein the GM-CSF concentration is 20ng/mL, and the IL-4 concentration is 10ng/mL;
6. the plates were placed at 37℃with 5% CO 2 Is a cell of (a)Culturing in an incubator for 3 days, performing half-volume liquid exchange on the 3 rd day, removing 1mL of supernatant from each well, and adding 1mL of newly prepared complete culture medium containing cytokines with the same concentration;
7. all cells were collected on day 6 to obtain mouse bone marrow-derived dendritic cells.
Example 2 mouse BMDC cell function and phenotypic characterization
1. Mouse BMDCs were prepared as in example 1, and immature dendritic cells (imDCs) on day 6 of culture were collected and divided into two groups: immature group (imDC, no LPS added), mature stimulated group (mDC, 1. Mu.g/mL LPS added); 4 compound holes are arranged in each group, and the culture is continued for two days in a 6-hole culture plate;
2. two days later, collecting cell suspensions of one compound well of the immature group and the mature group of cells, adding 2mL of PBS into each sample tube, centrifuging at 4 ℃ and 1200rpm for 5 minutes, discarding supernatant, and observing cell morphology change by using a laser confocal microscope (FV 3000, OLYMPUS) after the cells are resuspended in 500 mu LPBS;
3. meanwhile, respectively collecting the cells of the immature group and the mature group of the other three compound holes, adding 2mL of flow Cell Staining Buffer (CSB), then cleaning, centrifuging at 4 ℃ for 5 minutes at 1200rpm, and then discarding the supernatant;
4. the cell suspension was again washed with 2mL of CSB, and then centrifuged at 1200rpm for 5 minutes at 4 ℃, and the supernatant was discarded;
5. mouse monoclonal antibodies including FITC CD11c, PE-Cy7 CD86, PE MHC-II, and BV421 CD80 (5. Mu.L each) were added to each sample tube, and after thoroughly mixing, incubated at 4℃for 30 minutes in the absence of light;
6.2 mL of CSB was added to terminate the staining, and the mixture was centrifuged at 1200rpm at 4℃for 5 minutes, and the supernatant was discarded;
7. the cell suspension was again washed with 2mL of CSB, and then centrifuged at 1200rpm for 5 minutes at 4 ℃, and the supernatant was discarded;
8. the cells were resuspended using 500 μl CSB, the cell suspension transferred into a flow tube, and dendritic cell surface markers were detected using a flow cytometer (BD LSRFortessa, usa).
The result of detecting CD11c mark by flow cytometry is shown in A diagram in FIG. 1, and the imDC purity obtained by the method for preparing mouse bone marrow-derived dendritic cells is more than 85% and accords with the purity standard of DC obtained by classical cytokine culture method.
The morphological characteristics of imDC and mDC were observed using a laser confocal microscope, and it was found that the imDC surface was smooth, no protrusions had been formed (panel B in FIG. 1), and that a typical dendritic structure existed on the mDC surface (panel C in FIG. 1).
Further, according to the detection result of the flow cytometer (D diagram in fig. 1): the imDC obtained by the method provided by the implementation has low expression of CD80, CD86 and MHC-II (23.97% +/-2.71%, 22.01+/-3.26% and 26.22% +/-4.65%) before being stimulated by LPS, and accords with the phenotype characteristics of the standard imDC; the significantly increased expression levels of CD80, CD86 and MHC-II on the cell surface (94.73% + -2.78%, 96.28% + -1.91%, 93.36% + -3.67%) of the imDC of this example after 48h of LPS stimulation, indicated that the DCs obtained in this example had perfect dendritic cell function.
Example 3 expression of AIM2 in specific knock-down DCs using Aim2-shRNA adenoviruses
The experimental method comprises the following steps:
for the gene sequence of Aim2, a vector virus, namely an Aim2-shRNA adenovirus (Aim 2-shRNA-ADV), is designed, and the target sequence of the Aim2-shRNA is 5'-GCTGACAGGATCCTGATATAG-3'.
The negative control virus was empty vector (GFP-ADV) containing only the selectable marker EGFP.
The adenovirus prepared as described above was used to transfect mouse BMDC, and the transfection method was performed with reference to the virus operation manual provided by Shanghai Ji Kai Gene technologies Co. The specific flow is as follows:
1. mouse BMDC prepared as in example 1, immature dendritic cells (imDC) cultured until day 6 were collected, and the cell concentration was adjusted to 1X 10 with the complete medium as in example 1 6 1mL system by inoculating cells into 24-well plate; cells were divided into three groups, each group comprising 6 duplicate wells: blank (imDC, no adenovirus added), GFP negative (DC-GFP, GFP-ADV used), aim2-shRNA (DC-Aim 2-shRNA, aim 2-shRNA-ADV);
2. GFP-ADV and Aim2-shRNA-ADV ice-bath were dissolved, and the virus concentration was adjusted to 1X 10 with RPMI1640 solution 10 TU/mL, MOI was set to 50 (5. Mu.L of the diluted virus added per well);
3. half-dose liquid exchange is carried out 24 hours after the mouse BMDC is transfected with virus, so that the cell activity is maintained;
4. detecting the intensity of EGFP fluorescence of an adenovirus transfection group by a flow cytometer after 48 hours of transfection, and verifying the transfection efficiency; the expression changes of AIM2 after air 2-shRNA adenovirus transfection were simultaneously verified from the transcription level (qPCR) and protein level (Western Blot).
(II) experimental results:
the expression result of the fluorescent marker EGFP detected by the flow cytometry is shown as a graph A in FIG. 2, and the transfection efficiency of adenovirus to DC can reach more than 80% as shown in the graph A in FIG. 2. The results of AIM2 qPCR are shown in figure 2, panel B, showing that: the relative expression level of AIM2 mRNA after DC transfection of Aim2-shRNA adenovirus was significantly lower than that of the blank and negative control groups. The results of further protein expression levels of AIM2 in each group of DCs by Western Blots are shown in FIG. 2, which shows that the protein expression levels of AIM2 after transfection of AIM2-shRNA adenovirus in DCs were significantly lower than in the blank and negative control groups. In summary, experiments demonstrated that adenovirus constructed using this example was able to effectively knock down expression of the AIM2 gene in DCs.
Example 4 validation of functional and phenotypic Change of DC-Aim2-shRNA
Experimental methods
1. DC cells with reduced Aim2 expression (i.e., DC-Aim 2-shRNA), as well as blank control cells (imDC) and negative control cells (DC-GFP) were obtained by the same procedure as in step 1-step 3 of example 3;
2. at 48 hours post-transfection, 3 duplicate wells within each group were not added with LPS, and the other 3 duplicate wells were added with 1 μg/mL LPS;
on day 2 after LPS stimulation, culture supernatants were collected and assayed for IL-10 levels using an IL-10ELISA kit; all groups of cells were harvested simultaneously and flow-antibody stained with the same staining protocol as in example 2;
4. dendritic cell surface markers were detected using a flow cytometer (BD LSRFortessa, usa).
(II) results of experiments
The flow cytometry detection results are shown in A diagram in FIG. 3, and after LPS stimulation, the DC-Aim2-shRNA still has low expression of CD80, CD86 and MHC-II, and has the capacity of resisting LPS stimulation maturation compared with imDC.
The results of IL-10 detection are shown in panel B of FIG. 3, and the results show that the level of IL-10 secretion by DC-Aim2-shRNA (217.95 + -36.45 pg/mL) is significantly improved compared with that of imDC and DC-GFP (109.05 + -21.53, 102.91 + -19.27 pg/mL).
The experiment in the embodiment proves that Aim2 is an important target point for regulating DC tolerance, and inhibition of Aim2 can improve the capacity of DC to resist LPS stimulation maturation and induce generation of tolerizing dendritic cells.
Example 5 phenotypic identification and preparation protocol optimization of inhibitory oligonucleotide-tolerant dendritic cells (ODN-tolDCs)
Experimental methods
1. BMDCs were obtained using the method of example 1 and cultured to day 3 using the same induction conditions;
2. the BMDC half-volume liquid change is carried out on the 3 rd day in the example 1, meanwhile, ODN TTAGGG with different concentrations is respectively added into a culture system to continue culture, and the concentrations of the ODN TTAGGG are respectively set to be 0 mu M, 1 mu M, 3 mu M, 5 mu M and 10 mu M; the DC cells induced by ODN TTAGGG treatment are marked as ODN-tolDC;
3. continuing to culture cells until day 6, and adding 1 μg/mL LPS for stimulation to each ODN TTAGGG concentration group on day 6;
4. all groups of cells were harvested by culture to day 8 (day 2 after LPS stimulation) and stained with flow-through antibodies, staining protocol was as in example 2;
5. dendritic cell surface markers were detected using a flow cytometer (BD LSRFortessa, usa).
(II) results of experiments
The flow cytometer detection results are shown in fig. 4, and as can be seen from graph a in fig. 4, imDC not treated with ODN TTAGGG in this example was completely mature after LPS stimulation; whereas ODN TTAGGG-treated imdcs had the ability to resist LPS-stimulated maturation, and with increasing concentrations of ODN TTAGGG (1 μm, 3 μm, 5 μm, 10 μm, respectively), an increase in the ability of ODN TTAGGG-stem-prognosis imdcs to resist LPS-stimulated, i.e. progressive decrease in surface co-stimulatory molecules CD80, CD86 and MHC II expression, was observed; at a concentration of 3. Mu.M, the trend of the change in the DC surface co-stimulatory molecules was stable.
According to the detection result, the optimized ODN TTAGGG intervention scheme can induce to generate stable ODN-tolDC by using 3 mu M of ODN TTAGGG. Under this condition, CD11c expression of ODN-tolDC was stable with purity >85% (panel B in FIG. 4), while surface co-stimulatory molecules CD80, CD86, MHC II (32.46% + -6.71%; 24.56% + -4.23%; 19.64% + -2.82%) were expressed at low levels.
EXAMPLE 6 IL-10 level detection of ODN-tolDC
imDC in this example was prepared according to the method in example 1.
The preparation method of the ODN-tolDC in the embodiment is a preferable preparation scheme, and the preparation flow is shown in FIG. 5, and the specific method is as follows:
s1, BMDC is obtained by using the method of the example 1, and is cultured until the 3 rd day under the same induction condition;
s2, BMDC half-volume liquid change is carried out on the 3 rd day in the same embodiment 1, and 3 mu M of ODN TTAGGG is added into a culture system respectively.
The induced ODN-tolDC and control imDC were cultured in complete medium for 48 hours, and culture supernatant was collected and IL-10 level was detected using IL-10ELISA kit. Comparing the level of IL-10 in the supernatant of ODN-tolDC with that of the control group (imDC), the result shows that the ability of ODN-tolDC to secrete IL-10 is significantly improved (212.6+ -45.68 vs.139.9+ -57.32) pg/mL compared with the control group imDC.
Example 7 ability of ODN-tolDC to stimulate lymphocyte proliferation was decreased
Experimental methods
In this example, the ODN-tolDC provided by the present invention was confirmed to have an ability to modulate immune negative direction by constructing an in vitro mixed lymphocyte reaction (mixed lymphocyte reaction, MLR).
ODN-tolDC was prepared according to the preferred preparation scheme of example 6. ODN-tolDC was treated with mitomycin C.
Cd3+ T cells were sorted using immunomagnetic beads according to the instructions (meiteni, germany) and stained using a cell proliferation detection dye (sameifei, usa). Cd3+ T cells were divided into three groups and then as DC: T cell=1: 5 ratio ODN-tolDC, imDC, mDC were mixed with CD3+ T cells for 72h, respectively. After 72h, each set of cell suspensions was collected and the T cell surface was stained and examined as follows:
1. 2mL of flow cytometry buffer (CSB) was added and washed, and centrifuged at 2400rpm at 4℃for 5 minutes to discard the supernatant;
2. the cell suspension was again washed with 2mL of CSB, and then centrifuged at 2400rpm at 4 ℃ for 5 minutes, and the supernatant was discarded;
3. mouse monoclonal antibodies, including PerCP-Cy5.5-CD3, BV711-CD4, BV510-CD8 (BD Co., U.S.A., 5. Mu.L each), were added to each sample tube;
4. after fully mixing, shading and incubating for 30 minutes at the temperature of 4 ℃;
5. 2mL of CSB was added to terminate the staining, and centrifugation was performed at 2400rpm at 4℃for 5 minutes, and the supernatant was discarded;
6. the cell suspension was again washed with 2mL of CSB, and then centrifuged at 2400rpm at 4 ℃ for 5 minutes, and the supernatant was discarded;
7. the cells were resuspended using 500 μl CSB and the cell suspension transferred into a flow tube;
8. the change in fluorescence intensity of the proliferation dye on the cell surface of CD3+ T cells was detected using a flow cytometer, and the proliferation ratio of CD4+ to CD8+ T cell subsets in CD3+ T cells was further analyzed.
(II) results of experiments
The results of flow cytometry are shown in FIG. 6, which shows that the proliferation ratio of CD4+ to CD8+ T cell subsets is significantly lower (CD 4:11.34% + -3.29% vs.48.75% + -3.80%; CD8:18.62% + -3.74% vs.82.76% + -6.52%) in the imDC group compared to the mDC group, indicating that the technical method used in this example was correct and the results were reliable.
Comparing the T cell proliferation ratio of ODN-tolDC group with that of mDC group, the proliferation ratio of CD4+ and CD8+ T cell subgroup after ODN-tolDC stimulation was lower (CD 4:22.44% + -2.64%vs. 48.75% + -3.80%; CD8:29.58% + -3.58%vs. 82.76% + -6.52%), indicating that its immunostimulatory capacity was reduced.
Example 8ODN-tolDC induces apoptosis of lymphocytes
Experimental methods
In this example, the ODN-tolDC provided by the present invention was confirmed to have an ability to modulate immune negative direction by constructing an in vitro mixed lymphocyte reaction (mixed lymphocyte reaction, MLR).
ODN-tolDC was prepared according to the preferred preparation scheme of example 6. ODN-tolDC was treated with mitomycin C and cd3+ T cells were sorted using immunomagnetic beads according to the instructions (meiteni, germany). Cd3+ T cells were divided into three groups, ODN-tolDC, imDC, mDC was combined with cd3+ T cells according to dc:t cell=1: 5, and culturing for 72h. The surface marker staining method for T cells was the same as in example 7, and apoptosis of cd4+ and cd8+ T cells was detected using apoptosis detection kit staining (sameiaway, usa).
(II) results of experiments
The results of the detection are shown in FIG. 7, which shows that the imDC group has an increased apoptosis ratio of CD4+ to CD8+ T cell subsets (CD 4:38.78% + -3.16% vs.5.05% + -1.82%; CD8:20.94% + -4.17% vs.5.39% + -1.90%) compared to the mDC group, indicating that the results obtained in this example are reliable.
After 3 days of co-culture of ODN-tolDC and T cells, the apoptosis rate of each subgroup is obviously increased compared with that of control mDC (CD 4:21.09% + -1.71%vs.5.05% + -1.82%; CD8:17.54% + -2.68%vs.5.39% + -1.90%), and the apoptosis of CD4+ and CD8+ T cells is obviously promoted by ODN-tolDC (P < 0.01).
The test results of comprehensive examples 6, 7 and 8 show that the ODN-tolDC prepared by the invention has obvious negative immune regulation effect.
A brief flow of examples 9, 10 and 11 is shown in fig. 8.
Example 9 in vivo induction of ODN-tolDC to generate CD4+ Treg cell subsets
Experimental methods
ODN-tolDC was prepared as in the preferred preparation scheme of example 6, and further purified using CD11c magnetic beads (Meitian and Geneva, germany) to obtain cells with a purity of more than 95%.
A model of allogeneic abdominal heart transplantation of the mice is constructed by taking Balb/C mice as donors and C57 mice as acceptors. Recipient mice were injected 2X 10 by tail vein injection 1 day prior to surgery 6 ODN-tolDC for each cell number (two control groups were simultaneously set, and equal volumes of PBS solution and equal cell numbers of imdcs were injected respectively). The recipients were sacrificed on day 7 post-surgery and spleens were obtained. Spleen was prepared as single cell suspension, and 1X 10 samples were added to each tube 6 Cells were pooled, 2mL of CSB was added, centrifuged, and the supernatant was removed. The same procedure was followed for 1 more washing. After 100 μl CSB was resuspended, mouse monoclonal flow antibody was added: each of CD45, CD3, CD4, and CD25 was 5. Mu.L (Bidi, USA), and incubated at 4℃for 30min under light protection. 2mL of CSB was added, centrifuged, and the supernatant was removed. The same procedure was followed for 1 more washing. Cell rupture reagent (Sieimer, USA) was added to each tube and incubated at 4℃for 45min at 1mL in the absence of light. 2mL of membrane-disrupting buffer (Siemens, USA) was added, and the supernatant was removed by centrifugation. The same procedure was followed for 1 more washing. After 100. Mu.L of membrane-disrupting buffer was resuspended, the mouse monoclonal flow antibody Foxp3 5. Mu.L (Bidi, USA) was added and incubated at 4℃for 60min in the absence of light. 2mL of CSB was added, centrifuged, and the supernatant was removed. The same procedure was followed for 1 more washing. The proportion of Treg subpopulations (cd4+, cd25+, foxp3+) in the spleen cell suspension was measured using a flow cytometer, resuspended at 500 μl CSB.
(II) results of experiments
The flow cytometer detection results are shown in fig. 9, and the results show that ODN-tolDC significantly up-regulates the proportion of cd4+cd25+ Treg (9.31% ± 0.67% vs.5.13% ± 0.91%) sub-populations in the recipient at day 7 after surgery compared to the control group (PBS group); it follows that ODN-tolDC has the ability to induce CD4+ Treg in vivo, and has the potential to further induce allograft immune tolerance.
Example 10ODN-tolDC effectively inhibits Th1 and Th17 cell subsets
Experimental methods
A mouse allogeneic heart transplant acute rejection model was constructed as in example 9, and a recipient spleen single cell suspension was obtained on day 7 post-surgery as in example 9. 1X 10 of each sample tube was added 6 Spleen cells were added with 2mL of CSB, centrifuged, and the supernatant was removed. The same procedure was followed for 1 more washing. After 100 μl CSB was resuspended, mouse monoclonal flow antibody was added: CD45, CD3, CD4 were incubated at 4℃for 30min at 5. Mu.L each. 2mL of CSB was added, centrifuged, and the supernatant was removed. The same procedure was followed for 1 more washing. 1mL of a membrane breaker (Sieimer, america) is added into each sample tube, the mixture is blown and evenly mixed, and the mixture is incubated for 45min at 4 ℃ in the dark. 2mL of membrane-disrupting buffer was added, and the supernatant was removed by centrifugation. The same procedure was followed for 1 more washing. After 100 μl membrane-breaking buffer was resuspended, mouse monoclonal flow antibody was added: IFN-gamma, IL-17. Mu.L, 4℃and incubation in the dark for 60min. 2mL of CSB was added, centrifuged, and the supernatant was removed. The same procedure was followed for 1 more washing. The spleen cell suspension was resuspended in 500. Mu.L of CSB and the ratio of the Th1 subset (CD4+ IFN-. Gamma. +) to the Th17 subset (CD4+ IL-17+) was examined using a flow cytometer.
(II) results of experiments
The results of flow cytometry are shown in FIG. 10, and show that ODN-tolDC significantly inhibited the proportion of Th1 and Th17 sub-populations (Th 1:8.04% + -1.69% vs.19.69% + -3.37%; th17:0.38% + -0.15% vs.1.05% + -0.22%) in the recipients at day 7 post-surgery compared to the control (PBS). The detection result shows that ODN-tolDC has the effects of inhibiting inflammation and negatively regulating immune response in vivo.
EXAMPLE 11 preparation of peripheral blood PBMC-derived ODN-tolDC
Experimental methods
PBMC-derived ODN-toldcs were prepared using mouse peripheral blood PBMC. The preparation method comprises the following steps: mice peripheral blood 3mL was collected at 1:1 was diluted with PBS and peripheral blood PBMCs were obtained by density gradient centrifugation using peripheral blood lymphocyte separation medium (solebao, china) according to the instructions. 2mL of PBS was added, centrifuged, and the supernatant was removed and washed once. Adding 4mL of erythrocyte lysate, incubating at 4 ℃ for 5min in a dark place, adding 10mL of RPMI-1640 medium, centrifuging, removing the supernatant, and cleaning for 2 times; imDC cell induction protocol was the same as in example 1, and PBMC-derived imDC was obtained on day 6 after culture, and the same ODN-tolDC preparation protocol as in example 6 was used since day 6. The phenotype was examined by flow cytometry using the induced ODN-tolDC, and the specific technical method was the same as in example 2.
(II) results of experiments
The detection results of the flow cytometry are shown in FIG. 11, and the results show that the ODN-tolDC derived from peripheral blood PBMC low express CD80, CD86 and MHC-II; compared to imDC, PBMC-derived ODN-tolDC has the ability to resist LPS-stimulated maturation. The expression level of IL-10 in the PBMC-derived ODN-tolDC culture broth was examined in the same manner as in example 6, and it was revealed that the PBMC-derived ODN-tolDC had a high IL-10 secretion capacity (195.87.+ -. 38.73vs. 133.42.+ -. 62.18) pg/mL as compared with imDC. Therefore, the ODN-tolDC induction scheme provided by the invention can induce the initial cell imDC to be derived from bone marrow and peripheral blood PBMC, and has good expansibility.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
- Use of an aim2 gene/protein as a target for modulating dendritic cell tolerance or inducing the preparation of tolerogenic dendritic cells in vitro.
- Use of an inhibitor of the aim2 gene/protein for modulating dendritic cell tolerance in vitro or for the preparation of tolerogenic dendritic cells.
- 3. The use according to claim 2, wherein the inhibitor of AIM2 gene/protein is an AIM2-shRNA capable of inhibiting expression of AIM2 gene/protein or an inhibitory oligonucleotide capable of inhibiting function of AIM2 gene/protein.
- 4. A preparation for the induction of the preparation of tolerogenic dendritic cells, characterized in that it comprises an AIM2-shRNA capable of inhibiting the expression of AIM2 genes/proteins or an inhibitory oligonucleotide capable of inhibiting the function of AIM2 genes/proteins.
- 5. The use according to claim 3 or the formulation according to claim 4, wherein the Aim2-shRNA has a target sequence of 5'-GCTGACAGGATCCTGATATAG-3'; the inhibitory oligonucleotide is ODN TTAGGG.
- 6. A method for preparing a tolerogenic dendritic cell, characterized in that the expression of the AIM2 of the immature dendritic cell or the protein function thereof is inhibited, whereby the tolerogenic dendritic cell is prepared.
- 7. The method of claim 6, wherein the agent of claim 4 or 5 is used to inhibit expression of immature dendritic cells AIM2 or protein function thereof.
- 8. A AIM2 targeted inhibited tolerogenic dendritic cell prepared by the method of claim 6 or 7.
- 9. Use of a tolerogenic dendritic cell according to claim 8 for the preparation of a medicament for the prophylaxis and/or treatment of transplant rejection, for the inhibition of acute rejection or for the induction of differentiation of naive T cells into tregs.
- 10. Use of a tolerogenic dendritic cell according to claim 8 for the preparation or induction of a transplantation immune tolerance regulatory cell, wherein said regulatory cell comprises a regulatory T cell, a regulatory B cell, a regulatory NK cell or a regulatory macrophage.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311700521.3A CN117683724A (en) | 2023-12-11 | 2023-12-11 | Tolerogenic dendritic cell, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311700521.3A CN117683724A (en) | 2023-12-11 | 2023-12-11 | Tolerogenic dendritic cell, and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117683724A true CN117683724A (en) | 2024-03-12 |
Family
ID=90131397
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311700521.3A Pending CN117683724A (en) | 2023-12-11 | 2023-12-11 | Tolerogenic dendritic cell, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117683724A (en) |
-
2023
- 2023-12-11 CN CN202311700521.3A patent/CN117683724A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11339372B2 (en) | Serum-free medium inducing differentiation of umbilical cord mesenchymal stem cell into insulin-secretion-like cell and preparation method and use thereof | |
| CN107164332B (en) | Leukemia cell exosome with TGF-beta 1 mutism modified by interference sequence and preparation method and application thereof | |
| EP1501918A1 (en) | Antigen-presenting cell populations and their use as reagents for enhancing or reducing immune tolerance | |
| US20130323832A1 (en) | Antigen-presenting cell populations and their use as reagents for enhancing or reducing immune tolerance | |
| CN106754664B (en) | Culture medium for inducing adipogenic differentiation of skeletal muscle myogenic stem cells, application of culture medium and adipogenic differentiation method | |
| CN113881707B (en) | Product, method and application for regulating and controlling immune suppression effect of umbilical cord mesenchymal stem cells | |
| CN115011561A (en) | Chimeric antigen receptor macrophage and preparation method and application thereof | |
| Gosavi et al. | Optimization of ex vivo murine bone marrow derived immature dendritic cells: A comparative analysis of flask culture method and mouse cd11c positive selection kit method | |
| CN115044553B (en) | mTOR-targeted tolerant dendritic cell and preparation method and application thereof | |
| CN111057719B (en) | Efficient transfection method and application of mouse bone marrow-derived macrophages | |
| CN111944755A (en) | Method for enhancing antigen presenting capability of dendritic cells | |
| CN117683724A (en) | Tolerogenic dendritic cell, and preparation method and application thereof | |
| CN115094034B (en) | Human NKT cell line and application thereof | |
| CN116286638A (en) | Promotion of CD34 + cell-to-CD 16 + Method for differentiating macrophages and polarizing macrophages towards M2 | |
| CN107574149B (en) | Maturation promoting method of dendritic cells and application thereof | |
| Xu et al. | Immunoregulatory effect of neuronal-like cells in inducting differentiation of bone marrow mesenchymal stem cells. | |
| CN110643575A (en) | Culture reagent and culture method for mature dendritic cells | |
| CN117701500B (en) | Culture method and application of mesenchymal stem cells | |
| CN110564692A (en) | Stem cells with enhanced immunoregulatory capacity, and preparation method, composition and application thereof | |
| CN111748581B (en) | Method for transfecting dendritic cells by using hypoxia inducible factor recombinant expression plasmid | |
| CN109954139B (en) | Application of matrine as immunoadjuvant in preparation of gastric cancer dendritic cell vaccine | |
| CN107988157B (en) | Dendritic cell induction culture medium and application thereof | |
| CN116179483B (en) | Method for rapidly amplifying stem cell-like memory cervical cancer tumor infiltrating lymphocytes in vitro | |
| WO2021227573A1 (en) | Xeno-free culture medium and method for expansion of mesenchymal stem cells by means of using same | |
| CN109897824A (en) | Application using TIPE2 gene as the tumour MDSC of target spot in the drug of preparation prevention or treatment tumour |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |