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Application of miR-148 to proliferation of pancreatic beta cells

1. The technology of mir-148 and β cells is applied in the direction of drug combinations, preparations for in vivo experiments, medical preparations containing active ingredients, etc., which can solve the problems of activity increase and decrease, and achieve the effect of promoting proliferation

Active Publication Date: 2013-12-04
RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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Problems solved by technology

The results showed that the expression of insulin, sugar transporter, glucokinase and other mRNA levels in islet β cells of patients with type 2 diabetes was significantly reduced, while the activities of caspase-3 and caspase-8 related to apoptosis were significantly increased. These results also verified Islet β cells in type 2 diabetes do have increased apoptosis and defects in secretory function of the cells

Method used

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  • Application of miR-148 to proliferation of pancreatic beta cells
  • Application of miR-148 to proliferation of pancreatic beta cells
  • Application of miR-148 to proliferation of pancreatic beta cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Inhibitor control and Anti-miR-148 were transiently transfected in the mouse islet β cell line MIN6, and the inhibitory efficiency was verified by Real-time PCR. It was found that the inhibitor can very effectively inhibit endogenous miR-148 in cells expression, and the degree of inhibition was more than 80%.

[0034] The 24-well plate cells and the 8-well plate slide cells were transiently transfected with blank vector inhibitor control and Anti-miR-148. 48 hours after transfection, half of the fresh culture medium was replaced, and 10mM EDU was added at a ratio of 1:1000. Incorporate cell DNA, fix the cells after 4 hours, slide the cells for EDU-Alexa647 staining, and perform flow cytometric analysis on the cells in the 24-well plate. The results showed that compared with the control group, the proportion of EDU-positive cells in the Anti-miR-148 group was significantly reduced. At the same time, the results of flow cytometry analysis also showed that the proportion o...

Embodiment 2

[0040] The isolated mouse primary islets were cultured overnight, digested and broken up, and transiently transfected with inhibitor control and Anti-miR-148. After 48 hours of transfection, the nuclei were fixed and stained. The transfection efficiency of the cells was observed under a fluorescent microscope, which was about 90%. Real-time PCR was used to verify its inhibition efficiency, and it was found that its inhibitor can very effectively inhibit the expression of endogenous miR-148 in cells, with an inhibition degree of more than 90%.

[0041] 48 hours after transfection, the results of immunofluorescence chemistry after fixation and permeabilization showed that both groups of cells had a small amount of Ki67-positive cells expressed in the nuclei. Photographs were taken under a fluorescent microscope, and statistical analysis after counting the positive cells showed that Ki67-positive cells accounted for insulin. The proportion of positive cells decreased from 9‰ to ab...

Embodiment 3

[0044] The cell line MIN6 was transiently transfected with the control blank vector and Anti-miR-148, and 48 hours after transfection, the protein kinase C inhibitor staurosprine was added at a ratio of 1:1000 to induce cell apoptosis. The cells were stained with Annexin-FITC / 7-AAD, and then analyzed by flow cytometry, the results were as follows figure 2 .

[0045]The results showed that compared with the control group, the proportion of early apoptotic cells (Annexin-FITC positive / 7-AAD negative cells) in the Anti-MiR-148 group was significantly increased, and the proportion of surviving cells (Annexin-FITC / 7-AAD negative cells ) was significantly reduced, and the proportion of dead cells or late apoptotic cells (both Annexin-FITC / 7-AAD positive cells) was not significantly changed.

[0046] Therefore, Anti-miR-148 can promote the apoptosis of the islet β-cell line MIN6, and at the same time, it indicates that the pathway of the reduction of surviving cells after inhibitin...

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Abstract

The invention relates to the field of biology and medical drugs, and discloses the function of miR-148 on proliferation and apoptosis of pancreatic beta cells, which provides a new target spot for diabetes treatment as well as preparation or screening of drugs for curing 2-type diabetes. According to the invention, miR-148 can be applied to the preparation or screening of drugs for controlling pancreatic beta cells, and preparation or screening of drugs for controlling apoptosis of pancreatic beta cells, or preparation or screening of drugs for curing 2-type diabetes.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular to the application of miRNA. Background technique [0002] Diabetes mellitus is a chronic systemic metabolic disease characterized by persistent high blood sugar as its basic biochemical feature. It is a comprehensive disease that leads to carbohydrate, protein and fat metabolism disorders mainly caused by glucose metabolism disorders. According to the cause and mechanism of the disease, it can be divided into two types: type 1 diabetes (T1D) and type 2 diabetes (T2D). Of these, type 2 diabetes characterized by defective insulin secretion or / and insulin resistance of surrounding tissues accounts for 90–95%. Relevant statistics show that the number of people suffering from diabetes has reached 220 million, and its incidence has reached the scale of epidemic diseases in the world. By 2030, the number of diabetes cases worldwide will reach 360 million. In the past two dec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K31/7105A61K49/00A61P3/10
Inventor 宁光山爱景曹亚南姜秀丽顾卫琼
Owner RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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