MiR-29 sponge, nucleic acid construct containing same and applications of miR-29 sponge

A technology of nucleic acid constructs and sponges, applied in the field of biomedicine, can solve problems such as application limitations, inability to meet the needs of metabolism, and individual differences in patients, so as to achieve the effects of improving survival rate, inhibiting secretion, and reducing the incidence of disease

Active Publication Date: 2021-01-15
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the traditional treatment of T1DM is still insulin injection, but long-term use can not meet the metabolic demands, and can lead to various complications, such as hypoglycemia, retinopathy, cardiovascular disease, etc.
In addition, islet / pancreas transplantation is also unable to fundamentally solve the problem due to the existence of autoimmune rejection and the limited availability of donors.
Stem cell therapy can treat diabetes at the cell and gene levels, but most of them are in the research and trial stage, and there are individual differences and application limitations for patients

Method used

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  • MiR-29 sponge, nucleic acid construct containing same and applications of miR-29 sponge
  • MiR-29 sponge, nucleic acid construct containing same and applications of miR-29 sponge
  • MiR-29 sponge, nucleic acid construct containing same and applications of miR-29 sponge

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Effect of miR-29sponge on the onset of type 1 diabetes in NOD mice.

[0036] Experimental animals: 8-9-week-old female NOD mice and feed, provided by Beijing Huafukang Biotechnology Co., Ltd., license number: SCXK (Beijing) 2019-0008. Animals were housed in random cages, with free access to food and water.

[0037] Experimental drug: HBLV-mmu-miR-29 sponge was prepared and purified by Hanheng Technology (Shanghai) Co., Ltd. The negative control was HBLV-mmu-GFP NC, which was purchased from Hanheng Technology Company. Among them, HBLV-mmu-miR-29 sponge The lentiviral expression vector used was pHBLV-U6-MCS-CMV-ZsGreen; the competent cell was Escherichia coli strain DH5α; resistance: Amp.

[0038] Experimental steps:

[0039] 1) 8-9 week-old female NOD mice entered the Experimental Animal Center of Nanjing Medical University, raised in a clean environment, temperature (21±2)℃, humidity (35±2)%, 12h:12h day and night lighting, free to eat Drinking water, drink...

Embodiment 2

[0056] Example 2 The effect of miR-29 sponge on the levels of pro-inflammatory and anti-inflammatory cytokines in the serum of NOD mice.

[0057] Take 50ul of the collected serum sample and add it to the flow tube, add 50ul of the inflammatory factor magnetic bead mixture in the CBA kit (BD company, USA), incubate at room temperature for 1 hour, then add 50ul of the PE detection kit (BD company in the United States) mixture, and keep at room temperature Incubate for 1 hour. After the incubation, add 1ml of cleaning solution and centrifuge at room temperature 200g for 5min, discard the supernatant, resuspend with 300ul cleaning solution, and detect on the flow cytometer.

[0058] The results are shown in Figure 8. It was found that compared with the GFP control group, the miR-29 sponge treatment group could significantly reduce the levels of inflammatory cytokines TNF-α, IL-6 and MCP-1 in the serum of NOD mice. Figure A The content distribution of TNF-α, IL-6, IL-1β and MCP-1 ...

Embodiment 3

[0059] Example 3 Effect of miR-29 sponge on circulating monocytes of NOD mice.

[0060] The number of monocytes in the circulation of the mice in Example 1 was analyzed by flow cytometry, and the difference between the GFP group and the miR-29 sponge experimental group was compared. Specific steps are as follows:

[0061] Add 20ul of 6250U / ml heparin sodium to 80ul of peripheral blood of the mouse, mix well, let stand at 37°C for 1h, and absorb plasma and buffy coat. Plasma is used for CBA and other detections. Add 3 times the volume of erythrocyte lysate to the middle white blood cell layer, room temperature for 5 minutes, mix well, after lysing, wash with PBS and mark the corresponding flow cytometry antibody F4 / 80, MHCII (both purchased from eBioscience, Inc., USA) ). Incubate at room temperature in the dark for 15 minutes, wash with PBS once, and discard the supernatant. Finally, resuspend in 300ul PBS to make mononuclear cell suspension. Take 1×10 6 The cells were co...

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Abstract

The invention discloses a miR-29 sponge, and further provides a nucleic acid construct containing the same. The invention finds that the miR-29 sponge can adsorb miR-29a-3p and miR-29b-3p in the body,close the functions of the miR-29a-3p and the miR-29b-3p and inhibit secretion of pro-inflammatory cytokines, so that insulitis of NOD mice is relieved, the serum insulin level is increased, the blood glucose tends to be normal, and finally, the onset of Type 1 diabetes mellitus (T1DM) of NOD (non-obese diabetic) mice is remarkably reduced, and the survival rate of the NOD mice is increased.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a miR-29 sponge inhibiting miR-29, a nucleic acid construct comprising the miR-29 sponge and its application in preparing medicines for treating or preventing type 1 diabetes. Background technique [0002] Diabetes mellitus (DM) has become a common and frequently-occurring disease that seriously affects human health in modern society. According to the latest IDF report, as of 2019, approximately 463 million adults aged 20-79 worldwide suffer from diabetes. Although type 2 diabetes accounts for 90-95%, the prevalence of type 1 diabetes is also increasing at a rate of 2% to 5%, among which the incidence rate of children and adolescents under the age of 15 is the fastest growing. Although the incidence of type 1 diabetes is not as high as that of type 2 diabetes, it has a rapid onset, tends to be younger, and causes serious complications such as ketoacidosis and hyp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867A61K31/7105A61P3/10
CPCA61K31/7105A61P3/10C12N15/113C12N15/86C12N2310/113C12N2310/14C12N2320/32C12N2740/15043C12N2800/107
Inventor 朱云霞韩晓石颖孙艺周云财姚正建唐伟常晓嫒李雅婷
Owner NANJING MEDICAL UNIV
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