CN108531479A - A method of inducing regulatory T cells by using synthetic dsdna - Google Patents
A method of inducing regulatory T cells by using synthetic dsdna Download PDFInfo
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- CN108531479A CN108531479A CN201810287362.1A CN201810287362A CN108531479A CN 108531479 A CN108531479 A CN 108531479A CN 201810287362 A CN201810287362 A CN 201810287362A CN 108531479 A CN108531479 A CN 108531479A
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- 238000000034 method Methods 0.000 title claims description 18
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Abstract
The invention discloses a kind of double-stranded DNAs, wherein the base sequence of a chain is：5 ' ctcgaggg 3 ', and second C base methylates.Meanwhile the application the invention also discloses the double-stranded DNA in inducing regulatory T cells.Double-stranded DNA provided by the invention in vitro no immunogenicity, leucocyte will not be damaged, the regulatory T cells of the FoxP3 positives can be induced, it can be effectively used for diseases associated with inflammation after non-infectious inflammation, infection, the treatment of the immune correlated diseases such as respiratory anaphylactic disease, gastrointestinal allergies disease, skin allergic disease and autoimmune disease.
Description
Technical field
The invention belongs to field of biomedicine technology, and in particular to a kind of to be adjusted by using synthetic dsdna to induce
The method of property T cell.
Background technology
Regulatory T cells (Regulatory cell, Treg) are the T cells of autoimmune response in a kind of control volume
Subgroup can be divided into the adaptability regulatory T cells of naturally-produced Natural regulation T cell (nTreg) and induction generation
(iTreg), be body maintain self tolerance important component, by thymus gland or by periphery activation cell development from.Its
In, transcription factor Foxp3 is the characteristic marker of Treg, and Foxp3 is a variety of by direct regulation and control as a transcription regulatory factor
Gene adjusts the activity of Treg, plays a key role in Treg cell developments process and function.
Treg has close relationship, research to have confirmed with autoimmune disease and the immunological rejection etc. of organ transplant
Treg can be used for the treatment of above-mentioned immune correlated disease, and to feed back to patient's body again by amplification in vitro Treg can improve disease
The progress of disease.However existing Treg quantity is seldom in vivo, only accounts for normal human peripheral blood CD4+5%~10% in T cell.Cause
This, establishes a kind of method that external a large amount of inductions generate Treg, to obtain sufficient amount of Treg for immune correlated disease
Treatment, have very great clinical meaning.
Invention content
Based on this, a kind of double-stranded DNA is provided it is an object of the invention to overcome above-mentioned the deficiencies in the prior art place,
In vitro no immunogenicity, leucocyte will not be damaged, regulatory T cells can be induced, have immune suppression function.
To achieve the above object, the technical solution that the present invention takes is：A kind of double-stranded DNA, wherein the base sequence of a chain
It is classified as：5 '-ctcgaggg-3 ', and second C base methylates.
Preferably, the double-stranded DNA constitutes the biological double-stranded DNA oligomers that molecular weight is 10~50000MW, described
Biological double-stranded DNA oligomers are named as Metvax.
The double-stranded DNA in vitro no immunogenicity, leucocyte will not be damaged；And antigen, silk can be inhibited in vitro
The T cell hyperplasia of rimocidin and autoantigen induction, and this inhibiting effect is at dose dependent.
It is another object of the present invention to provide application of the double-stranded DNA in inducing regulatory T cells.
It is still a further object of the present invention is to provide a kind of methods of induction regulatory T cells.
Regulatory T cells of the present invention refer to CD4+FoxP3+The regulatory T cells of immunophenotype.
To achieve the above object, the technical solution adopted by the present invention is, a method of induction regulatory T cells, including
Following steps：
(1) the initial CD4 of people is obtained+T cell；
(2) the initial CD4 of people for obtaining step (1)+T cell and people's AntiCD3 McAb/CD28 antibody, TGF-β, IL-2 and described
Double-stranded DNA co-cultured；
(3) in step (2) after cultivating system culture 3 days, the culture solution containing the double-stranded DNA is updated；
(4) cultivating system in step (3) continues culture at least 5 days, that is, obtains the regulatory T cells that induction generates.
Preferably, it is obtained from human peripheral using the method that magnetic bead sorting or flow cytometry sort in the step (1)
Obtain the initial CD4 of people+T cell.
Preferably, a concentration of 1-100ug/mLs of step (2) the double center chain DNA in cultivating system.
Present inventor has found, in the presence of IL-2 and TGF-β, can reinforce double-stranded DNA to regulatory T cells
Inducing action.
The regulatory T cells being prepared according to the method for induction regulatory T cells provided by the invention can be used for preparing
Diseases associated with inflammation, respiratory anaphylactic disease, gastrointestinal allergies disease, skin allergy after treatment non-infectious inflammation, infection
Property disease, autoimmune disease, graft-versus-host reaction or the drug of graft-rejection.
The regulatory T cells being prepared according to the method for induction regulatory T cells provided by the invention can be fed back into edge
It is reduced by regulatory T cells number or afunction and pathogenic patient's body corrects the regulatory T cells of patient's body
Number and function it is unbalance.
It is still a further object of the present invention is to provide the double-stranded DNAs to prepare treatment non-infectious inflammation, inflammation after infection
Property disease, respiratory anaphylactic disease, gastrointestinal allergies disease, skin allergic disease, autoimmune disease, graft it is anti-
Purposes in host response or the drug of graft-rejection.
Preferably, can also include vitamin A acid, rapamycin or sodium butyrate aliphatic acid in the drug.
Preferably, the disease include systemic loupus erythematosus, it is dermatomyositis, rheumatoid arthritis, vasculitis, multiple
Dull-witted, amyloidosis caused by sclerosis, Parkinson disease, Alzheimer disease, wound and CAIDs- childhoods, the Gulf War
Disease, ICIS and IRAES etc. caused by disease, silicon or vaccine.
Preferably, the double-stranded DNA can be sucked by respiratory tract, and the approach such as oral or intravascular, intramuscular injection make
With, and no antigen is shown as in vitro.
Preferably, the immune suppression function of the double-stranded DNA can pass through anti-IL-6 antibody, bromocryptine, vitamin
D and its separate excitation usually improve.
It is still a further object of the present invention is to provide a kind of drug, the drug, which contains the double-stranded DNA and materia medica, to be connect
The carrier received.
Compared with the existing technology, beneficial effects of the present invention are：Double-stranded DNA provided by the invention is in vitro without immunogene
Property will not damage leucocyte, can induce regulatory T cells, to play immune suppression function, can be effectively used for non-infectious
Diseases associated with inflammation after inflammation and infection, the immune phases such as respiratory tract, the anaphylactia of gastrointestinal tract and skin and autoimmune disease
The treatment of related disorders.
Description of the drawings
Fig. 1 is the sequence chart of double-stranded DNA of the present invention.
Fig. 2 is the comparative result figure that double-stranded DNA of the present invention induces regulatory T cells with reference material, wherein Foxp3+/CD4+
The cell of FoxP3 and CD4 are represented while expressing, DNA-1 represents double-stranded DNA of the present invention, and DNA-4 represents control group double-stranded DNA.
Fig. 3 is the result figure that regulatory T cells are induced using various concentration double-stranded DNA of the present invention, wherein Foxp3+/CD4+
Represent while expressing the cell of FoxP3 and CD4.
Specific implementation mode
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment to the present invention
It is described further.
Embodiment 1
Double chain DNA sequence of the present invention is as shown in Figure 1, base with adjacent base is keyed by di-phosphate ester, base
To being connected by hydrogen bond, and second C base methylates.
Embodiment 2
In order to study double-stranded DNA of the present invention to CD4+FoxP3+The induced efficiency of T regulatory T cells, the present embodiment is to this hair
Bright double-stranded DNA generates CD4 with double-stranded DNA induction is compareed+FoxP3+The effect of regulatory T cells is into comparing, the control double-strand
Difference lies in its second C bases not to methylate with double-stranded DNA of the present invention by DNA.
(1) experimental design
The effect of regulatory T cells can be induced in order to study double-stranded DNA of the present invention, experimental group and control are set
Group, it is specific as shown in table 1：
1 experimental design of table
(2) experimental method
A method of induction regulatory T cells include the following steps：
(1) normal human peripheral blood is taken, after detaching mononuclearcell, utilizes magnetic bead sorting (MASC) technology or flow cytometry
Sorting obtains the initial CD4 of people+T cell；
(2) the initial CD4 of people for obtaining step (1)+T cell is trained altogether with people's AntiCD3 McAb/CD28 antibody, TGF-β and IL-2
It supports, experimental group and control group are separately added into the inducer in table 1 in the culture starting stage；
(3) in step (2) after cultivating system culture 3 days, culture solution (the culture solution experimental group containing same concentrations respectively is replaced
With control group inducer)；
(4) cultivating system in step (3) continued culture to the 5th day, collects cell, utilizes Flow cytometry culture
Regulatory T cells ratio in system.
(3) experimental result
Experimental result after changing liquid as shown in Fig. 2, as shown in Figure 2, continue culture to the 5th day, in experimental group cultivating system
The CD4 of (containing double-stranded DNA of the present invention)+Foxp3+The ratio of regulatory T cells is apparently higher than control group (double-stranded DNA containing control group),
Difference has statistical significance.Illustrate that second C base provided by the invention is that the double-stranded DNA induction to methylate generates CD4+
Foxp3+The effect of regulatory T cells is more preferable.
A kind of method of induction regulatory T cells of embodiment 3
A kind of method of induction regulatory T cells provided by the invention, includes the following steps：
(1) normal human peripheral blood is taken, after detaching mononuclearcell, utilizes magnetic bead sorting (MASC) technology or flow cytometry
Sorting obtains the initial CD4 of people+T cell；
(2) the initial CD4 of people for obtaining step (1)+T cell is trained altogether with people's AntiCD3 McAb/CD28 antibody, TGF-β and IL-2
Support, culture the starting stage i.e. double-stranded DNA of the present invention is added, make its concentration in cultivating system be respectively 0ug/mL,
10ug/mL, 50ug/mL and 100ug/mL；
(3) in step (2) after cultivating system culture 3 days, the culture solution of the double-stranded DNA containing same concentrations is replaced；
(4) cultivating system in step (3) continued culture to the 5th day, that is, obtained the regulatory T cells that induction generates.
The method of the present invention induces regulatory T cells, and the results are shown in Figure 3, from the figure 3, it may be seen that double-strand provided by the invention
DNA usage amounts can successfully induce regulatory T cells when being 1~100ug/mL, and there are dose-dependants for this inducing action
Property.When double-stranded DNA usage amount is 100ug/mL, up to 55% CD4 is can get using invention abductive approach+FoxP3+It adjusts
Property T cell.
Present invention applicant is using above-mentioned abductive approach to the initial CD4 in Peripheral Blood in Patients with Systemic Lupus Erythematosus+T is thin
Born of the same parents induce, and equally can successfully induce CD4+FoxP3+The formation of regulatory T cells, as a result in normal human peripheral blood
Initial CD4+The induction result of T cell is similar, and specific data are omitted.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than is protected to the present invention
The limitation of range is protected, although being explained in detail to the present invention with reference to preferred embodiment, those skilled in the art should
Understand, technical scheme of the present invention can be modified or replaced equivalently, without departing from the essence of technical solution of the present invention
And range.
Claims (10)
1. a kind of double-stranded DNA, which is characterized in that the base sequence of wherein one chain is：5 '-ctcgaggg-3 ', and second C
Base methylates.
2. a kind of double-stranded DNA oligomers, which is characterized in that include double-stranded DNA described in claim 1.
3. double-stranded DNA oligomers according to claim 2, which is characterized in that the molecular weight of the double-stranded DNA oligomers is
10~50000MW.
4. application of the double-stranded DNA according to claim 1 in inducing regulatory T cells.
5. a kind of method of induction regulatory T cells, which is characterized in that include the following steps：
(1) the initial CD4 of people is obtained+T cell；
(2) the initial CD4 of people for obtaining step (1)+T cell and people's AntiCD3 McAb/CD28 antibody, TGF-β, IL-2 and claim 1
The double-stranded DNA is co-cultured；
(3) in step (2) after cultivating system culture 3 days, the culture solution containing double-stranded DNA described in claim 1 is updated；
(4) cultivating system in step (3) continues culture at least 5 days, that is, obtains the regulatory T cells that induction generates.
6. the method for induction regulatory T cells according to claim 5, which is characterized in that utilize magnetic in the step (1)
Pearl sorts or the method for flow cytometry sorting obtains the initial CD4 of people from human peripheral+T cell.
7. the method for induction regulatory T cells according to claim 5, which is characterized in that step (2) double center chain
A concentration of 1-100ug/mLs of the DNA in cultivating system.
8. double-stranded DNA according to claim 1 diseases associated with inflammation, breathing after preparing treatment non-infectious inflammation, infection
Road anaphylactia, gastrointestinal allergies disease, skin allergic disease, autoimmune disease, graft-versus-host reaction or
Purposes in the drug of graft-rejection.
9. purposes according to claim 8, which is characterized in that can also include vitamin A acid, rapamycin in the drug
Or sodium butyrate aliphatic acid.
10. a kind of drug, which is characterized in that it is acceptable that the drug contains double-stranded DNA and materia medica as described in claim 1
Carrier.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104740648A (en) * | 2013-12-27 | 2015-07-01 | 江苏命码生物科技有限公司 | Application of miRNA-214 inhibitor for inhibition of regulatory T cells |
-
2018
- 2018-03-30 CN CN201810287362.1A patent/CN108531479A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104740648A (en) * | 2013-12-27 | 2015-07-01 | 江苏命码生物科技有限公司 | Application of miRNA-214 inhibitor for inhibition of regulatory T cells |
Non-Patent Citations (5)
| Title |
|---|
| JAU-LING SUEN等: "Altered homeostasis of CD4+FoxP3+regulatory T-cell subpopulations in systemic lupus erythematosus", 《IMMUNOLOGY》 * |
| OLIVER J LAWLESS等: "In vitro induction of T regulatory cells by a methylated CpG DNA sequence in humans: Potential therapeutic applications in allergic and autoimmune diseases", 《ALLERGY AND ASTHMA PROCEEDINGS》 * |
| SHIO KOBAYASHI等: "TGF-β induces the differentiation of human CXCL13-producing CD4+ T cells", 《EUR. J. IMMUNOL.》 * |
| WHEATON JD等: "登录号：NM_001199348", 《GENBANK》 * |
| 张帆 等: "CD4+Foxp3+调节性T细胞在恶性血液病中的变化及机制研究", 《中国免疫学杂志》 * |
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