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Application of miRNA-382 capable of regulating and controlling AKT3 gene expression

A lv-mirna-382, gene expression technology, applied in the field of small molecule non-coding RNA miRNA-382, can solve the problems of no heroin and low addiction, and achieve the effect of making up for the gap of gene drugs

Active Publication Date: 2016-05-25
宁波市微循环与莨菪类药研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In my country, heroin addicts account for nearly 50% of drug addicts. For the treatment of heroin addiction, methadone oral liquid is mainly used as a therapeutic drug, and long-term maintenance treatment is carried out for heroin addicts. The purpose is to reduce their dependence on heroin, and methadone itself has an Addictive, but less addictive than heroin, so there is currently no effective drug for heroin addiction

Method used

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  • Application of miRNA-382 capable of regulating and controlling AKT3 gene expression
  • Application of miRNA-382 capable of regulating and controlling AKT3 gene expression
  • Application of miRNA-382 capable of regulating and controlling AKT3 gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Establishment of heroin self-administration model in rats and cue-induced heroin relapse test after withdrawal:

[0048] see figure 1 , to create a line graph for the rat heroin addiction model. Adult male rats were reared in single cages, weighing 280-320g, anesthetized by intraperitoneal injection of pentobarbital sodium (50mg / kg) plus atropine sulfate (1mL / kg), followed by cannulation of the right jugular vein, and intravenous administration Hose, jugular vein ligated, skin sutured. Postoperative intramuscular injection of penicillin (15000U) for 3 days to prevent infection. After one week of recovery, self-administration training of heroin (50 mg / kg) was performed in the self-administration training box, 4 hours a day for 12 consecutive days, and the last 3 days were required. The daily effective nasal contact number does not change more than 10%, and it is determined that the heroin addiction model in rats has been successfully established.

Embodiment 2

[0050] Analysis of miRNA expression profiling in rat nucleus accumbens:

[0051] After cue-induced relapse in each group of rats, total-RNA was extracted from the nucleus accumbens of rats for miRNA expression profile analysis. The specific methods are as follows:

[0052] 2.1Total-RNA purification steps:

[0053] (1) Put the excised rat brain nucleus accumbens tissue into a 2.5mL RNase-free EP tube, add 1mL Trizol reagent, fully homogenate, fully lyse the tissue, place it on ice for 5min, and then put it in -70℃ Refrigerator, long-term storage;

[0054] (2) Take out the experimental sample from the -70°C refrigerator, place it on ice to dissolve it, add 200 μL of chloroform, shake it up and down, and place it on the ice surface for 15 minutes, layering can be seen;

[0055] (3) Put the experimental sample into a pre-opened high-speed centrifuge at 4°C with a rotation speed of 12000rpm and fully centrifuge for 15min;

[0056] (4) Remove the centrifuged sample to the ice sur...

Embodiment 3

[0065] Applying RT-PCR technology to verify in rat heroin addiction model:

[0066] In order to verify the changes of miRNA-382 in the rat heroin addiction model, RT-PCR was used for verification. The specific verification method is as follows:

[0067] 3.1 Refer to 2.1 Total-RNA purification steps to extract the total-RNA of rat nucleus accumbens.

[0068] 3.2 Total-RNA was generated by reverse transcription reaction to cDNA, which was completed using Qiagen reverse transcription kit and purchased from Qiagen Enterprise Management (Shanghai) Co., Ltd.

[0069] (1) Reverse transcription reaction 20μL system:

[0070] ①The Total-RNA concentration was measured by NanoDrop2000, and the volume of 1 μg Total-RNA template in the reaction system was calculated.

[0071] ②Reaction system:

[0072]

[0073]

[0074] (2) Reverse transcription reaction program

[0075] ① Place the mixed reaction solution in a PCR reaction tube, adjust the incubation temperature to 37°C, and inc...

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Abstract

The invention discloses application of miRNA-382 capable of regulating and controlling AKT3 gene expression. The invention provides application of miRNA-382 in preparation of drugs used for inhibiting relapse of heroin addiction and application of a target gene, i.e., an AKT3 gene, in preparation of drugs used for inhibiting relapse of heroin addiction. In the invention, miRNA-382 is overexpressed in mice; and overexpression results show that overexpression of miRNA-382 can decrease expression of the target gene AKT3 and effectively inhibits relapse of heroin addiction. The miRNA-382 related to relapse of heroin addiction in the invention provides a novel therapeutic target for heroin addiction.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to the use of small molecule non-coding RNA miRNA-382 capable of regulating AKT3 gene expression. Background technique [0002] According to statistics, in 2015, there were 2.955 million registered drug users nationwide, of which 1.458 million were opioid abusers and 1.459 million were synthetic drug abusers, accounting for 49.3% and 49.4% respectively. [0003] In 1874, Wright, a chemist at St. Mary's Hospital in London, England, added acetic anhydride and other substances to morphine, and for the first time extracted a semi-synthetic derivative with better analgesic effect, morphine diacetate, which was the earliest synthetic heroin. Heroin addiction refers to the abuse of heroin and other opioids for non-medical purposes caused by any reason. In the process of compulsive repeated and continuous drug use that is difficult for self-control, the abuser forms a pharmacologi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61P25/36
Inventor 周文华刘惠芬马宏陈为升朱华强赖苗军庄丁丁
Owner 宁波市微循环与莨菪类药研究所
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