阅读说明：本技术 Dna四面体在制备预防和治疗舍格伦综合征的药物中的用途 (Application of DNA tetrahedron in preparation of medicine for preventing and treating Sjogren syndrome ) 是由 林云锋 高邵静雅 蔡潇潇 于 2021-04-30 设计创作，主要内容包括：本发明公开了DNA四面体在制备预防和治疗舍格伦综合征的药物中的用途,属于核酸药物领域。实验证明,DNA四面体可特异性地上调调节性B细胞,抑制自身反应B细胞,减少生发中心形成,阻碍舍格伦综合征病发生发展。另一方面,DNA四面体可以重建唾液腺唾液分泌功能,以及维持下颌下腺正常状态,并维持下颌下腺细胞增殖,减少下颌下腺细胞凋亡。DNA四面体预防和治疗舍格伦综合征的效果十分优异,应用前景良好。(The invention discloses an application of a DNA tetrahedron in preparation of a medicine for preventing and treating Sjogren syndrome, and belongs to the field of nucleic acid medicines. Experiments prove that the DNA tetrahedron can specifically up-regulate regulatory B cells, inhibit autoreactive B cells, reduce the formation of a generation center and block the generation and development of Sjogren syndrome. On the other hand, the DNA tetrahedron can rebuild salivary gland salivary secretion function, maintain the normal state of the submandibular gland, maintain the proliferation of submandibular gland cells and reduce the apoptosis of the submandibular gland cells. The DNA tetrahedron has excellent effect of preventing and treating Sjogren syndrome and good application prospect.)

Use of DNA tetrahedra for the preparation of a medicament for the prevention and treatment of Sjogren's syndrome.

2. Use according to claim 1, characterized in that: the medicine is a medicine for promoting and recovering salivary gland salivary secretion function.

3. Use according to claim 1, characterized in that: the medicine is used for protecting submandibular glands;

and/or, the drug is a drug that inhibits B cell activation.

4. Use according to any one of claims 1 to 3, wherein: the DNA tetrahedron is formed by complementary pairing of DNA single strands with sequences shown as SEQ ID NO. 1-4 or DNA single strands with homology of more than 95% with the DNA single strands in a molar ratio of 1: 1.

5. Use according to claim 4, characterized in that: the medicine contains 7.9 mu mol of DNA tetrahedron per unit preparation.

6. A medicament for preventing and treating sjogren's syndrome, which is characterized in that: the medicine is prepared by taking a DNA tetrahedron as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

7. The medicament of claim 6, wherein: the medicine is used for reconstructing salivary gland salivary secretion function.

8. The medicament of claim 6, wherein: the medicine is used for protecting submandibular glands;

and/or, the drug modulates B cell;

the regulatory B cells are up-regulatory B cells and inhibit autoreactive B cells.

9. The medicament according to any one of claims 6 to 8, wherein: the DNA tetrahedron is formed by complementary pairing of DNA single strands with sequences shown as SEQ ID NO. 1-4 or DNA single strands with homology of more than 95% with the DNA single strands in a molar ratio of 1: 1.

10. The medicament of claim 9, wherein: the medicine contains 7.9 mu mol of DNA tetrahedron per unit preparation.

Technical Field

The invention belongs to the field of nucleic acid medicines.

Background

Sjogren's syndrome: (syndrome, SS) is a systemic autoimmune disease, mainly involving salivary, lacrimal, submandibular glands, etcExocrine glands and local tissues have lymphocyte infiltration, normal functions of glands are affected, systemic damage can occur simultaneously, and clinical characteristics comprise mouth stem, eye stem, submandibular gland pathological changes, reduction of the number of regulatory B cells, abnormal increase of autoreactive B cells and the like.

There is currently no ideal method for the treatment and prevention of sjogren's syndrome. For the reduction of saliva, high fluoride toothpastes are usually used for tooth brushing, chlorhexidine gargling, saliva substitutes and chewing sugar-free chewing gums to relieve dry mouth symptoms; or the use of systemic stimulating drugs, such as muscarinic agonists (pilocarpine), to stimulate salivation of the salivary glands by selectively acting directly at the M choline receptor. However, the aforementioned methods only alleviate mild symptoms and are not effective for treatment. More potent immunomodulatory drugs, such as corticosteroids, hydroxychlorozine, methotrexate, azathioprine, cyclosporine a, cyclophosphamide, cause an overall suppression of the immune system, leading to the susceptibility of the user to side effects and reduced resistance to disease.

DNA tetrahedrons, also called tetrahedral framework nucleic acids (tFNAs), are made from single DNA strands (usually 4 strands) by base complementary pairing, wherein the original single DNA strands are changed into helical double strands in two-dimensional structure and tetrahedral structure is formed in three-dimensional structure. DNA tetrahedra are more stable than single stranded DNA or ordinary linear double stranded DNA and are therefore useful for synthesizing in vivo detection probes or as carriers for certain nucleic acid drugs.

There is currently no report of the use of DNA tetrahedron for the treatment of Sjogren's syndrome.

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 Sjogren syndrome.

The technical scheme of the invention is as follows:

use of DNA tetrahedra for the preparation of a medicament for the prevention and treatment of Sjogren's syndrome.

Further, the drug is a drug for promoting recovery of salivary gland salivary secretion function.

Further, the medicament is a medicament for protecting submandibular glands;

and/or, the drug is a drug that inhibits B cell activation;

it inhibits B cell activation by up-regulating regulatory B cells, inhibiting autoreactive B cells, and reducing the formation of hair centers.

Further, the DNA tetrahedron is formed by complementary pairing of DNA single strands with sequences shown as SEQ ID NO. 1-4 or DNA single strands with homology of more than 95% with the DNA single strands in a molar ratio of 1: 1.

"homology" in the present invention refers to the similarity of sequence alignment.

Further, the drug contains 7.9. mu. mol of DNA tetrahedron per unit preparation.

The medicine for preventing and treating sjogren syndrome is prepared by taking DNA tetrahedron as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

Further, the drug is a drug for reconstructing salivary gland salivary secretion function.

Further, the medicament is a medicament for protecting submandibular glands;

and/or, the drug modulates B cell;

the regulatory B cells are up-regulatory B cells and inhibit autoreactive B cells.

Further, the DNA tetrahedron is formed by complementary pairing of DNA single strands with sequences shown as SEQ ID NO. 1-4 or DNA single strands with homology of more than 95% with the DNA single strands in a molar ratio of 1: 1.

Further, the drug contains 7.9. mu. mol of DNA tetrahedron per unit preparation.

Experiments show that after the DNA tetrahedron is applied to the Sjogren syndrome mouse, the saliva secretion function of a salivary gland can be promoted to be recovered, a submandibular gland is protected, B cells (up-regulating regulatory B cells and self-reaction B cells are inhibited) are regulated, a better treatment effect is achieved on the Sjogren syndrome, particularly, 250nM concentration is injected for 1 time every 1 day, and the Sjogren syndrome mouse can be recovered to be close to a normal state after 4 weeks of continuous injection. Therefore, the DNA tetrahedron can be used for preparing the medicine for treating the Sjogren syndrome.

For mice, the optimal specification for a drug is 25nmol of DNA tetrahedra per unit formulation. According to the body surface area coefficient of a 70kg adult relative to a mouse, it is presumed that the DNA tetrahedral content in the human unit preparation is: 25nmol × 387.9 (body surface area coefficient of 70kg adult to mouse) 7.9 μmol.

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 diagram and identification of DNA tetrahedron. a, a schematic diagram of a synthetic principle; b, capillary electrophoresis results; c, PAGE gel results; and d, transmission electron microscope results.

FIG. 2: saliva flow and H & E staining assay results. a, an experimental flow chart; b-c, H & E dyeing detection results; d, saliva flow test results.

FIG. 3: and (3) performing immunofluorescence detection on structural proteins in submandibular glands. a to e are respectively the detection of CK5, c-kit, alpha-SMA, AQP4 and AQP 5.

FIG. 4: immunofluorescence and flow cytometry detection of B cells infiltrating in the submandibular gland. a, immunofluorescence against CD 19; b, immunofluorescence against BAFF; c, quantitative statistical plots of immunofluorescence signals for CD19 and BAFF; d to e, flow cytometry detection results.

Detailed Description

Example 1 Synthesis and characterization of DNA tetrahedrons (tFNAs)

1. Synthesis method

Four single-stranded DNAs (S1, S2, S3, S4) were dissolved in TM Buffer (10mM Tris-HCl, 50mM MgCl)₂pH 8.0) to a final concentration of 1000nM, mixing well, rapidly heating to 95 ℃ for 10 minutes, rapidly cooling to 4 ℃ for 20 minutes or more, and obtaining tFNAs.

The four single-stranded sequences (5 '→ 3') are as follows:

S1：

ATTTATCACCCGCCATAGTAGACGTATCACCAGGCAGTTGAGACGAACATTCCTAAGTCTGAA(SEQ ID NO.1)

S2：

ACATGCGAGGGTCCAATACCGACGATTACAGCTTGCTACACGATTCAGACTTAGGAATGTTCG(SEQ ID NO.2)

S3：

ACTACTATGGCGGGTGATAAAACGTGTAGCAAGCTGTAATCGACGGGAAGAGCATGCCCATCC(SEQ ID NO.3)

S4：

ACGGTATTGGACCCTCGCATGACTCAACTGCCTGGTGATACGAGGATGGGCATGCTCTTCCCG(SEQ ID NO.4)

2. identification

As a result of capillary electrophoresis, the tFNAs was found to have a size of about 164bp (FIG. 1 b); from the PAGE results, tFNAs of about 200bp size (FIG. 1 c); the scattering point-like objects can be seen by transmission electron microscopy, and some point-like objects can be observed to be in a tetrahedral form (fig. 1 d).

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 therapeutic Effect of tFNAs on Sjogren's syndrome mice

1. Model mouse

Female NOD/ShiLtJ mice are a common model of sjogren's syndrome (jannere et al, based on NOD/Ltj model mice to explore the regulation of vitamin C on the pathological factors related to sjogren's syndrome, 2019 national oral biomedical academic annual meeting, acacia etc., Toll-like receptor 9-dependent mechanism of action of p38MAPK signaling pathway in primary sjogren's syndrome, oromaxillofacial surgery journal 2015 4 month, volume 25, phase 2). In this example, 11-week-old female NOD/ShiLtJ mice were acclimatized for one week. Female NOD/ShiLtJ mice, 12 weeks old, were designated as Sjogren's syndrome mice.

2. Packet processing

The experimental group was injected with 250nM or 500nM tFNAs in a volume of 100. mu.L, and the model group was injected with an equal volume of physiological saline once every other day for four weeks.

3. Detection of

Mouse salivary flow rate was monitored throughout the experimental dosing and observation.

The mice were sacrificed 5 weeks, 10 weeks, and 15 weeks after the injection of the drug, and the submaxillary glands were harvested for detection.

(1) Flow cytometry. Collecting submaxillary gland for flow cytometry detection, exploring CD4⁺T and B cell subsets.

(2) And (6) dyeing. Collecting submaxillary gland, performing H & E staining, and observing lymphocyte infiltration condition; specific proteins associated with salivary secretory functions and normal structures of the submandibular gland were observed by immunofluorescence staining.

4. Results

FIG. 2a is a flow chart of the experiment. FIGS. 2b-c show that inflammatory cells of submandibular glands of mice with Sjogren's syndrome infiltrated clumps and areas of infiltration significantly decreased following treatment with tFNAs. Figure 2d shows that after treatment with tFNAs, salivary secretion function was restored in sjogren's syndrome mice and dry mouth was relieved. Wherein, after 250nM tFNAs treatment, the salivation function of Sjogren's syndrome mice is substantially restored to normal mouse level. This indicates that treatment with tFNAs restored salivary secretory function in the sjogren's syndrome mice, reducing inflammatory cell infiltration in the salivary glands.

Proteins in the submandibular gland that are critical to the normal structure of the submandibular gland, such as CK5, c-Kit and α -SMA, all returned to normal levels after tFNAs treatment (FIGS. 3 a-c). Similarly, AQP-4 and AQP-5 are important proteins secreted by saliva and were also restored to normal levels following tFNAs treatment (FIG. 3b, d).

B cells infiltrating in the submandibular gland were also explored by immunofluorescence and flow cytometry. After tFNAs treatment, B cells and the important cytokine BAFF secreted by B cells were reduced (FIGS. 4 a-c)). CD5 inhibiting disease progression⁺CD1D⁺CD19⁺B220⁺B cells (Breg) and IL-10⁺CD5⁺CD1D⁺CD19⁺B220⁺B cells (IL-10)⁺Breg) had an increased proportion following treatment with tFNAs. CD19 promoting disease progression⁺CD138⁺B cells and CD19⁺CD95⁺GL-7⁺B cells, and at the same time, these cells are also important cells for the formation of germinal centers (germinal centers). The odds decreased after treatment with tFNAs. Similarly, the submandibular gland-infiltrated B cell subpopulation after 250nM tFNAs treatment all returned to normal mouse levels (fig. 4d, e).

Sjogren's syndrome is an autoimmune disease caused by excessive B cell activation and production of large amounts of autoantibodies. The above results indicate that tFNAs specifically up-regulate regulatory B cells, inhibit autoreactive B cells, reduce the formation of hair centers, further inhibit over-activation of B cells, reduce the production of autoantibodies, and thus treat Sjogren's syndrome.

In conclusion, tFNA can rebuild salivary gland salivary secretion function, protect submandibular gland, regulate B cells (up-regulating regulatory B cells and inhibiting autoreactive B cells), has a better treatment effect on Sjogren syndrome, and the Sjogren syndrome mouse can be recovered to be close to a normal state under a specific dosage. Therefore, the tFNA has good application prospect in preparing the medicine for treating Sjogren syndrome.

SEQUENCE LISTING

<110> Sichuan university

Application of <120> DNA tetrahedron in preparation of medicine for preventing and treating Sjogren syndrome

<130> GYKH1118-2021P0112784CC

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<170> PatentIn version 3.5

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