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Application of GABRD methylation as heroin relapse resisting target

A technology of methylation and heroin, applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems such as addiction, and achieve the effect of reducing heroin relapse behavior

Pending Publication Date: 2022-03-22
宁波市康宁医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Long-term activation of opioid receptors by heroin leads to compensatory adaptation of opioid receptors and non-opioid receptor systems, leading to addiction

Method used

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  • Application of GABRD methylation as heroin relapse resisting target
  • Application of GABRD methylation as heroin relapse resisting target
  • Application of GABRD methylation as heroin relapse resisting target

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Establishment of heroin intravenous self-administration model

[0032] Intravenous Drug Self-Administration (Intravenous Drug Self-Administration) is one of the most classic animal models for studying drug addiction. Drug motivation and active compulsive drug use.

[0033] The classic rat heroin self-administration model can well simulate the heroin smoking behavior of clinical drug addicts. Rats can produce heroin self-administration after training. At the same time, the behavioral response rate and The dosage can reflect the compulsive drug-seeking behavior of drug addicts from the initial exposure to heroin to the addiction to heroin.

[0034]We used the following method to establish a self-administration model in rats: first, male SD rats underwent jugular vein cannulation, inserted a section of PE tube into the right jugular vein, and passed it out of the body through the back, antibacterial and recovery for more than a week after the operation . Subse...

Embodiment 2

[0035] Example 2 Illumina HiSeq sequencing

[0036] 12 hours after the heroin self-administration model was established stably, the rats were intraperitoneally administered with sodium pentobarbital, and sacrificed after deep anesthesia to isolate the NAc brain region. A heroin self-administration group (Heroin self-administration), a heroin passive administration group (Yoke heroin group) and a passive saline control group (Control) were established (n=3 for each group). The genomic DNA of the tissue samples was extracted with a PerkinElmer automatic nucleic acid extraction instrument (PerkinElmer Medical Diagnostic Products Shanghai Co., Ltd.), and the concentration of the obtained DNA was detected by a Qubit4.0 nucleic acid and protein analyzer. After DNA fragmentation and purification, end-repair and sequencing adapters were added, followed by Bisulfite treatment, library construction and quality inspection after amplification, and then Illumina HiSeq genome-wide methylati...

Embodiment 4

[0057] Example 4 Changes in the expression level of GABRD gene mRNA in the NAc brain region

[0058]The expression of GBRD gene mRNA was detected by reverse transcription real-time quantitative PCR (RT-qPCR) on Roche LightCycler 480 instrument. Extract heroin self-administration group, passive administration group and control group rat NAc brain tissue RNA, total RNA used (Invitrogen; Thermo Fisher Scientific, Inc.) kit extraction. Total RNA was reverse transcribed into cDNA using the miScript IIRT kit (Qiagen gmbH). The nucleotide sequences of qPCR primers are shown in Table II, and the GAPDH gene was used as an internal reference. The reaction system contained 5 μl SYBR Green premix, 2 μl primer (10 μM), 1.5 μl cDNA and 1.5 μl water. The amplification conditions are: 95°C, 10min, 40 cycles: 95°C, 10S, 55°C, 15S and 72°C, 15S. use 2 -ΔΔCq Methods Analysis of RT-qPCR data to analyze the relative change of GABRD mRNA expression level. The analysis of the results showed t...

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Abstract

The invention relates to an application of GABRD methylation as an anti-heroin relapse target spot, and compared with the prior art, the application has the advantages that the heroin relapse behavior can be effectively reduced by reducing the GABRD gene methylation level through research; therefore, the medicine for treating heroin relapse by taking the GABRD gene methylation as the target spot has important research and development value and development significance.

Description

technical field [0001] The invention relates to a GABRD methylation target, in particular to an application of GABRD methylation as an anti-heroin relapse target. Background technique [0002] Opioid addiction, especially heroin addiction, causes enormous medical and economic harm. Survey data from the United Nations Office on Drugs and Crime (UNODC) shows that globally, in 2018, 109,600 drug-related crimes were cracked, 137,400 criminal suspects were arrested, and 67.9 tons of various drugs were seized. Taking a relatively large proportion, the situation of heroin abuse is severe. Long-term activation of opioid receptors by heroin leads to compensatory adaptation of opioid receptors and non-opioid receptor systems, leading to addiction. Long-term chronic use of heroin leads to strong physical and mental dependence, which leads to a strong desire for drug use again, which is the main reason for the high relapse rate. The dopaminergic system in the brain region from the ve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12Q1/6883C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/136C12Q2600/154C12Q2537/164C12Q2531/113
Inventor 刘惠芬洪青晓徐文锦刘婧陈为升朱华强赖苗军周文华
Owner 宁波市康宁医院
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