Application of MiR-503 cluster sponge in preparation of medicine for treating type 2 diabetes
A type 2 diabetes, mir-503 technology, applied in the field of biomedicine, can solve the problems of lack of treatment methods and high treatment costs, and achieve the effects of peripheral tissue insulin sensitivity recovery, function improvement, and glucose tolerance improvement.
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Embodiment 1
[0028] Example 1 Expression of miR-503 in chronic inflammation model mouse tissues.
[0029] Experimental animals: Male C57BL / 6J mice were purchased from the Institute of Model Animals, Nanjing University. All animal experiments were approved by the Experimental Animal Management Committee of Nanjing Medical University, and all experimental animals were kept in a barrier facility in the Experimental Animal Center of Nanjing Medical University in a clean environment with a temperature of (21±2)°C and a humidity of (35±2) )%, 12h: 12h day and night without lighting, free to eat and drink, the drinking water is the sterile water prepared by the experimental animal center.
[0030] Experimental results: if figure 1 As shown, in order to screen miRNAs that change and have significant effects in cells under chronic inflammatory conditions, we treated INS-1 cells with 10 ng / ml IL-1β, and collected four groups of samples at different time points to prepare miRNA chip, the results ar...
Embodiment 2
[0031] Example 2 The role of miR-503 cluster in vivo during the development of type 2 diabetes.
[0032] Experimental animals: In order to explore the role of miR-503 cluster in the development of diabetes, we used miR-503 cluster knockout (KO) mice.
[0033] experimental method:
[0034] 1) Construct miR-503 cluster whole-body knockout mice, 10 WT and KO mice, fed normal diet (NCD) and high-fat diet (HFD), and monitored fasting and postprandial blood glucose levels once a month. The mice were fasted for 12-16 hours in advance during the fasting blood glucose test; the postprandial blood glucose test was fasted and then fed, and the blood glucose level was detected 2 hours after eating.
[0035] 2) When differences in fasting or postprandial blood glucose levels were detected, glucose tolerance (GTT), in vitro islet perfusion and insulin tolerance (ITT) experiments were performed on the mice. The GTT detection method is to measure the blood glucose value at 0, 5, 15, 30, 60,...
Embodiment 3
[0038] Example 3 Blocking the effect of β-cell-derived miR-503 clusters can reverse the diabetic phenotype in aged mice.
[0039] Experimental drugs: MIP1-mmu-miR-503-424-Sponge, negative control MIP1-NC-GFP, all commissioned to Weizhen Biotechnology Co., Ltd., all using type 8 adeno-associated virus vector pAAV-MCS to construct recombinant plasmids; auxiliary plasmids Select pAAV-RC; select pHelper for adenovirus plasmid; select AAV-293 cells for competent cells; resistance: Amp, insulin promoter for the promoter, specifically use the mouse insulin 1 promoter (mouse insulin1 promoter, MIP1).
[0040] experimental method:
[0041] 1) Select 13 60-week-old mice, and use 10-week-old mice as controls to detect their body weight, fasting blood glucose level, glucose tolerance and insulin tolerance, and then divide the mice into two batches and inject the recombinant virus. The 6 rats in the batch were divided into two groups and injected with the control virus and the miR-503 spo...
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