CN113209124A - Application of DNA tetrahedron in preparation of medicines for preventing and treating type 1 diabetes - Google Patents
Application of DNA tetrahedron in preparation of medicines for preventing and treating type 1 diabetes Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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Abstract
The invention discloses an application of a DNA tetrahedron in preparing a medicament for preventing and treating type 1 diabetes, and belongs to the field of medicaments for treating diabetes. The invention utilizes the regulation effect of the DNA tetrahedron on immune cells to inhibit autoimmunity and block the generation and development process of type 1 diabetes from the source; in addition, the DNA tetrahedron and metabolites thereof have no toxicity and good biological affinity, and the DNA tetrahedron has good application prospect in preparing medicines for preventing and treating type 1 diabetes.
Description
Technical Field
The invention belongs to the field of diabetes related medicines.
Background
At present, the main treatment means of type 1 diabetes is subcutaneous insulin injection, but the method often causes large blood sugar fluctuation of patients and easily causes hypoglycemia syncope; and the condition can be only temporarily controlled by injecting insulin, and the type 1 diabetes cannot be radically treated.
Islet transplantation can help patients produce insulin themselves, but clinical practice shows that patients can only do not rely on exogenous insulin within a few months after operation, and need to inject exogenous insulin at a later stage, so that the sustainable effect is lacked. In addition, islet transplantation suffers from difficulty in obtaining a homologous islet, low survival rate of the transplanted islets, side effects of immunosuppressive agents (used to reduce rejection of the transplanted islets by autoimmunity), and high cost.
The stem cell therapy can solve the problem of difficulty in islet sourcing by directionally inducing stem cells into insulin-producing stem cells, and further solve the problem of side effects of immunosuppressive agents if stem cells are autologous stem cells and do not cause autoimmune rejection during transplantation. The gene therapy method is to introduce insulin gene into body to raise the insulin producing level and avoid most of the problems of insulin transplantation. Stem cell therapy and gene therapy are also far from clinical practice.
The aforementioned methods are started from the direction of increasing the insulin source, and the problem of attack of beta cells by T cells cannot be solved from the source. Accordingly, it is thought that regulatory T cells are expanded by means of various drugs (e.g., tellurium-based small molecules, cyclosporine a, etc.), thereby avoiding excessive activation of T cells, preventing attack of T cells on β cells, and fundamentally preventing the occurrence and development of type 1 diabetes.
DNA Tetrahedrons (TDNs), also called tetrahedral framework nucleic acids (tFNAs), are nucleic acid molecules synthesized from several single strands of DNA (typically 4) by base-complementary pairing. The original single DNA strand is changed into a helical double strand in two-dimensional structure, and a tetrahedral structure is formed in three-dimensional structure.
the tFNAs has better stability in vivo, can be used as a carrier of certain medicines and also can be used as a skeleton structure of certain detection probes; meanwhile, tFNAs can promote proliferation, differentiation and migration of neural stem cells and have the activity of promoting neural repair.
There are no reports of tFNAs about the treatment or prevention of type 1 diabetes.
Disclosure of Invention
The invention aims to solve the problems that: provides the application of the DNA tetrahedron in preparing the medicine for preventing and treating type 1 diabetes.
The technical scheme of the invention is as follows:
use of DNA tetrahedron in the preparation of a medicament for the prevention and treatment of type 1 diabetes.
The term "drug for preventing and treating type 1 diabetes" as used herein means a drug that can be used for both preventing diabetes and treating diabetes.
Further, the type 1 diabetes is autoimmune-induced type 1 diabetes.
Further, the DNA tetrahedron is synthesized by base complementary pairing of 4 DNA single strands;
the sequence of the DNA single strand is shown as SEQ ID NO. 1-4.
Further, the preparation method of the DNA tetrahedron comprises the following steps:
1) heating the DNA single strand with equal concentration to 95 ℃ to open the hydrogen bond in the single strand;
2) the temperature is rapidly reduced to 4 ℃ to carry out base complementary pairing between the single strands.
Further, the DNA single strand is in a buffer solution with a pH value of 8 during the preparation process.
A medicament for preventing and treating type 1 diabetes is prepared by taking DNA tetrahedron as an active ingredient and adding pharmaceutically acceptable auxiliary ingredients.
Further, the type 1 diabetes is autoimmune-induced type 1 diabetes.
Further, the DNA tetrahedron is synthesized by base complementary pairing of 4 DNA single strands;
the sequence of the DNA single strand is shown as SEQ ID NO. 1-4.
Further, the preparation method of the DNA tetrahedron comprises the following steps:
1) heating the DNA single strand with equal concentration to 95 ℃ to open the hydrogen bond in the single strand;
2) the temperature is rapidly reduced to 4 ℃ to carry out base complementary pairing between the single strands.
Further, the DNA single strand is in a buffer solution with a pH value of 8 during the preparation process.
The invention has the following beneficial effects:
experiments show that the DNA tetrahedron can avoid over-activation of T cells and reduce CD4 in spleen and pancreas+、 CD8+T cells (including NRP-V7+CD8+T cells), the proportion of regulatory T cells (tregs) in the spleen and pancreas is increased, and the protective effect on pancreatic islets is achieved, so that the problem of insufficient insulin secretion caused by damaged pancreatic beta cells is solved from the source, and the obvious prevention and treatment effect on type 1 diabetes is achieved.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1: schematic synthesis and identification of tFNAs. A, a schematic synthesis diagram of tFNAs; b, capillary electrophoresis detection results; c, detecting results of PAGE gel; d, detecting results by an atomic force microscope; and E, detecting a result by using a transmission electron microscope.
FIG. 2: results related to blood glucose and insulin in mice. A, a mouse treatment time flow; b, blood glucose level; c, proportion of mice protected from diabetes; d, survival rate of the mouse; e, insulin level; f, performing immunofluorescence staining on insulin-positive cells; g, insulin positive beta cell number.
FIG. 3: results of T cell detection in pancreas. Immunohistochemical detection of CD4 positive T cells; b, CD4 positive T cells account for visual field; immunohistochemical detection of C, CD8 positive T cells; d, CD8 positive T cells account for visual field; e, CD4 positive T cells and CD8 positive T cells account for the proportion of lymphocytes respectively; proportion of F, type I diabetes specific cells NRP-V7 and CD8 double positive cells in CD8 positive T cells.
FIG. 4: t cell assay results in spleen. A, CD4+And CD8+Specific gravity of T cells in lymphocytes; b, detecting the positive proportion of a plurality of immune markers in the lymphocytes; c, Th1, Th2, Th17 and Treg in CD4+Proportion in T cells.
Detailed Description
Example 1 Synthesis of tFNAs
1. Synthesis method
Four DNA single strands (S1, S2, S3, S4) were dissolved in TM Buffer (10mM Tris-HCl, 50mM MgCl2pH 8.0) so that the final concentration of the four DNA single strands is 1000nM, thoroughly mixing, rapidly heating to 95 ℃ for 10 minutes, and then rapidly cooling to 4 ℃ for 20 minutes or more to obtain tFNAs. The synthetic scheme is shown in FIG. 1A.
The four single-stranded sequences (5 '→ 3') are as follows:
S1：
ATTTATCACCCGCCATAGTAGACGTATCACCAGGCAGTTGAGACGA ACATTCCTAAGTCTGAA(SEQ ID NO.1)
S2：
ACATGCGAGGGTCCAATACCGACGATTACAGCTTGCTACACGATTC AGACTTAGGAATGTTCG(SEQ ID NO.2)
S3：
ACTACTATGGCGGGTGATAAAACGTGTAGCAAGCTGTAATCGACGG GAAGAGCATGCCCATCC(SEQ ID NO.3)
S4：
ACGGTATTGGACCCTCGCATGACTCAACTGCCTGGTGATACGAGGA TGGGCATGCTCTTCCCG(SEQ ID NO.4)
2. identification
From the results of PAGE and capillary electrophoresis, tFNAs of about 200bp in size was observed (FIG. 1B, C); the scattered point-like objects can be seen by transmission electron microscope and atomic force microscope (fig. 1D), and it can be observed that some point-like objects are in tetrahedral shape (fig. 1E). From the foregoing identification results, it can be considered that tFNAs were successfully synthesized.
The invention will be further illustrated in the form of experimental examples in which the tFNAs used were prepared by the method of example 1.
Experimental example 1 in vivo experiments for prevention of diabetes by tFNAs
NOD/ShiLtJ mice are a typical animal model that mimics type I diabetes, with lymphocytes appearing around the islets at weeks 4-5, followed by massive leukocyte entry into the entire islets, and finally, beta-cell failure, insulin deficiency, and the onset of diabetic symptoms. The decrease of pancreatic insulin is obvious in about 12 weeks (males are later than females for several weeks), and the blood sugar level rises to more than 2.50 mg/mL; at 24-30 weeks 90% of the islet beta cells are destroyed, the probability of a female developing diabetes is 90% and the probability of a male developing diabetes is 50% -60%.
In this example, female NOD/ShiLtJ mice were treated with tFNAs, and the effect of tFNAs in preventing and inhibiting the progression of diabetes was evaluated.
1. Method of producing a composite material
(1) Female NOD/ShiLtJ mice at 8 weeks of age were purchased and acclimatized for one week.
(2) 9 weeks old female NOD/ShiLtJ mice were used as pre-stage animal models of type I diabetes.
(3) The experimental group was injected with 100. mu.L of 250nM or 500nM tFNAs, and the saline group was injected with equal volumes of saline once every other day for four weeks. One batch of mice was sacrificed 6 weeks, 12 weeks, and 18 weeks after the injection of the drug, and blood, spleen, and pancreas were collected and examined. Mice were monitored for blood glucose levels, morbidity and mortality throughout the course of experimental dosing and observation.
(4) The collected blood was subjected to ELISA detection to detect the insulin concentration in the blood.
(5) Collecting pancreas and spleen, and performing immunofluorescence staining to observe the functions of pancreatic islets; flow cytometry assays were performed to explore CD4+T cell, CD8+T cell, antigen specific T cell changes; intoImmunohistochemical detection exploration of CD4+T cells and CD8+T cell changes.
2. Results
(1) Detection of blood sugar and insulin
FIG. 2A is a flow chart of treatment time for mice. Fig. 2B-D show the results of blood glucose, diabetes-free ratio and survival rate tests, respectively, showing that tFNAs significantly inhibit the increase of blood glucose and prevent the onset and progression of type 1 diabetes. FIG. 1E shows the measurement of insulin level, and it can be seen that the insulin level in the experimental group is significantly higher than that in the normal saline group. Fig. 2F and G are the detection of insulin positive cells specific for NRP-V7, showing that the number of insulin positive β cells in the experimental group is significantly higher than that in the saline group. In FIGS. 2B-G, the experimental mice injected with 250nM tFNAs showed comparable assay results to healthy mice.
The results show that tFNAs can protect beta cells of islet tissues, maintain the level of insulin in blood, further stabilize blood sugar and prevent the occurrence and development of type 1 diabetes to a great extent.
(2) Detection of T cells in pancreas
CD8 positive T cells (CD 8)+T cell) is a cytotoxic T cell which can kill target cells specifically; CD4 positive T cells (CD 4)+T cells) are primarily responsible for assisting CD8+T cells exert a killing effect. CD4+、CD8+After the T cells simultaneously enter the islet beta cells in a large amount, the islet beta cells are attacked, and the functions of the islet beta cells are influenced. FIGS. 3A-E show CD4 in pancreatic islets+、CD8+Number of T cells, compared to saline group, Experimental group CD4+、CD8+T cell numbers were significantly reduced and the experimental group injected with 250nM tFNAs was comparable to healthy mice.
NRP-V7+CD8+T cells are antigen-specific immune cells of type 1 diabetes and are markers of type 1 diabetes. FIG. 2F shows NRP-V7+CD8+T cells in CD8+The ratio in T cells was found to be significantly lower in the experimental group than in the saline group, and the experimental group injected with 250nM tFNAs was comparable to healthy mice.
The above results indicate that tFNAs can prevent T cells from entering pancreas, attack pancreas islet, and prevent the occurrence and development of type 1 diabetes.
(3) T cell detection in spleen
The spleen is the largest lymphatic organ in the human body and is close to the pancreatic islets, and the T cell groups in the spleen have important influence on the pancreatic islets.
As shown in FIG. 4A, CD4 from tFNAs treatment group+And CD8+The specific gravity of T cells in lymphocytes was significantly reduced compared to saline group, consistent with the results in pancreas, suggesting that tFNAs may delay the progression of type 1 diabetes by modulating T cell populations in spleen.
IFN-gamma in lymphocytes+CD4+The T cells are Th1 cells, IL-4+CD4+The T cell is a Th2 cell, the Th1 cell can secrete related cytokines to inhibit the differentiation of the Th2 cell, and the secreted cytokines can destroy and kill insulin-secreting islet beta cells, so that the Th1 plays an important role in the development of type I diabetes. Th2 has effect in inhibiting Th1 differentiation. IL-17+ CD4+ T cells are Th17 cells, Th17 can secrete related cytokines to destroy islet beta cells, and the secreted cytokines can promote other immune cells to differentiate to further destroy the islet beta cells. The Th1 and Th17 ratios were significantly increased in type I diabetes, and after treatment with tFNAs, the Th1 and Th17 ratios were significantly decreased, returning to normal levels (fig. 4B, C). CD4+CD25+Foxp3+T cells are Treg cells, the Treg cells have an important immune regulation function, and the proportion of Tregs in type I diabetes is obviously reduced. Treg rates were significantly increased following treatment with tFNAs, returning to normal levels (figure 4B, C).
The above results indicate that tFNAs can reduce intra-splenic CD4+And CD8+T cell ratio, decreased Thl, Th17 in CD4+The proportion of T cells improves the proportion of Tregs, so that the related autoimmunity of the T cells is maintained at a lower level, and the attack of the T cells on islet B cells is reduced from the source.
In conclusion, the DNA tetrahedron can prevent attack of T cells on the pancreatic islets, protect the pancreatic islet cells (especially pancreatic islet beta cells), and maintain the insulin secretion level, thereby playing a role in preventing the occurrence and development of diabetes. By means of the principle, the DNA tetrahedron is used for preparing the medicine for preventing and treating type 1 diabetes, and is favorable for fundamentally preventing the occurrence and the development of type 1 diabetes.
SEQUENCE LISTING
<110> Sichuan university
Application of <120> DNA tetrahedron in preparation of medicines for preventing and treating type 1 diabetes
<130> GYKH1118-2020P0112148CC
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<170> PatentIn version 3.5
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<213> Artificial sequence (Artificial sequence)
<400> 2
acatgcgagg gtccaatacc gacgattaca gcttgctaca cgattcagac ttaggaatgt 60
tcg 63
<210> 3
<211> 63
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
actactatgg cgggtgataa aacgtgtagc aagctgtaat cgacgggaag agcatgccca 60
tcc 63
<210> 4
<211> 63
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
acggtattgg accctcgcat gactcaactg cctggtgata cgaggatggg catgctcttc 60
ccg 63
Claims (10)
- Use of DNA tetrahedrons for the preparation of a medicament for the prevention and treatment of type 1 diabetes.
- 2. Use according to claim 1, characterized in that: the type 1 diabetes is type 1 diabetes caused by autoimmunity.
- 3. Use according to claim 1, characterized in that: the DNA tetrahedron is synthesized by 4 DNA single-strands through base complementary pairing;the sequence of the DNA single strand is shown as SEQ ID NO. 1-4.
- 4. Use according to claim 3, characterized in that: the preparation method of the DNA tetrahedron comprises the following steps:1) heating the DNA single strand with equal concentration to 95 ℃ to open the hydrogen bond in the single strand;2) the temperature is rapidly reduced to 4 ℃ to carry out base complementary pairing between the single strands.
- 5. Use according to claim 4, characterized in that: the DNA single strand is in a buffer at pH 8 during the preparation.
- 6. A medicament for preventing and treating type 1 diabetes, which is characterized in that: the medicine is prepared by taking DNA tetrahedron as an active ingredient and adding pharmaceutically acceptable auxiliary ingredients.
- 7. The medicament of claim 6, wherein: the type 1 diabetes is type 1 diabetes caused by autoimmunity.
- 8. The medicament of claim 6, wherein: the DNA tetrahedron is synthesized by 4 DNA single-strands through base complementary pairing;the sequence of the DNA single strand is shown as SEQ ID NO. 1-4.
- 9. The medicament of claim 8, wherein: the preparation method of the DNA tetrahedron comprises the following steps:1) heating the DNA single strand with equal concentration to 95 ℃ to open the hydrogen bond in the single strand;2) the temperature is rapidly reduced to 4 ℃ to carry out base complementary pairing between the single strands.
- 10. The medicament of claim 9, wherein: the DNA single strand is in a buffer at pH 8 during the preparation.
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| CN113209124B (en) | 2022-04-15 |
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Effective date of registration: 20240621 Address after: Room 1-17, 15th Floor, Building 2, No. 89, North Section 4, Second Ring Road, Jinniu District, Chengdu City, Sichuan Province, 610000 Patentee after: Chengdu Yunhai Tetrahedral Biotechnology Co.,Ltd. Country or region after: China Address before: 610000 No. 24 south part of Wuhou District first ring road, Chengdu, Sichuan. Patentee before: SICHUAN University Country or region before: China |