SiRNA of targeted CRH gene and application of siRNA in treatment of neuropathic pain

A neurological and genetic technology, applied in the fields of basic medicine and biomedicine, can solve problems such as high doses and long-term use restrictions, achieve good therapeutic effects, relieve mechanical hyperalgesia, and have significant application and promotion value

Pending Publication Date: 2021-07-30
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Clinically, salicylic acid drugs have many side effects, and non-steroidal anti-inflammatory drugs are not sensitive to them. Although opioids can relieve them

Method used

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  • SiRNA of targeted CRH gene and application of siRNA in treatment of neuropathic pain
  • SiRNA of targeted CRH gene and application of siRNA in treatment of neuropathic pain
  • SiRNA of targeted CRH gene and application of siRNA in treatment of neuropathic pain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1, the design and synthesis of siRNA

[0049] Regarding the siRNA sequence design, use software such as RNAi Designer, and set the parameters as follows: GC content is 35%-55%; the target region is the gene coding region and the evolutionarily conserved region. According to the human (NM_000756.4) and mouse (NM_205769.3) CRH mRNA sequences, the following three groups of double-stranded siRNA were designed and synthesized:

[0050]

[0051] The three groups of siRNA designed for CRH in the present invention can simultaneously target the CRH genes of humans and mice, and the target sequence is located in the coding region of the CRH gene. For specific targeting positions, see Figure 1-3 .

Embodiment 2

[0052] Example 2. Detection of CRH mRNA expression changes in DRG of neuropathic pain model mice

[0053] The results of real-time quantitative PCR showed that after SNI, the mRNA expression of CRH in DRG increased from the first day, which could last for more than 21 days, and the expression reached the peak on the 10th day. The specific experimental process is as follows:

[0054] 2.1 Preparation of neuropathic pain model mice

[0055] Adult healthy mice were properly deprived of water and food before the operation, pre-anesthetized in a transparent anesthesia box, shaved the leg hair with an animal shaver, and then placed on an animal heating blanket, and kept the mice in an anesthetized state through a mask anesthesia machine. After disinfecting the leg surgery site with alcohol and iodophor, use a scalpel to make a longitudinal incision near the knee of the mouse, separate the muscle layer with pointed and curved forceps, expose the sciatic nerve through the biceps femor...

Embodiment 3

[0083] Example 3. Detection of CRH protein expression in model mouse DRG

[0084] The results of immunofluorescence showed that compared with the Sham 3d group, the expression of CRH protein and the number of positive cells in the DRG of SNI 3d were significantly higher than that of the Sham 3d group. The specific experimental process was as follows:

[0085] 3.1 Tissue section preparation

[0086] After the mice were deeply anesthetized with isoflurane, they were perfused with normal saline and paraformaldehyde and placed on ice to take DRG. Place the DRG in a 1.5ml tube with 4% paraformaldehyde and place it at 4°C until the DRG sank to the bottom. Change to 20% sucrose and place at 4°C. After the DRG sinks to the bottom, change to 30% sucrose and place at 4°C for dehydration. After the DRG sinks to the bottom again. Take out the DRG, spread a shallow layer of embedding agent in the model groove, place the trimmed DRG in the model groove in order, put the model groove into t...

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Abstract

The invention belongs to the technical field of biological medicine, and discloses three groups of siRNAs targeting human and mouse CRH genes and application thereof in treatment of neuropathic pain. The three groups of siRNAs targeting the CRH genes (adrenocorticotropic hormone-releasing hormone) are designed and synthesized, the three groups of siRNAs are intrathecally injected into a neuropathic pain model mouse during in-vivo experiments. Molecular biology experiments show that the two groups of siRNAs can block CRH expression increase induced by nerve injury in mouse dorsal root ganglion (DRG) in vivo, and show that the two groups of siRNAs can effectively reduce CRH gene expression, but the effect of the second group is more obvious. Pain behavioral detection shows that the neuropathic pain can be effectively relieved through intrathecal injection of the second group of CRH siRNA. The invention provides non-chemical modification and chemical modification nucleic acid molecules of the targeted CRH gene, has a better treatment effect on neuropathic pain, provides a new thought and experimental basis for clinical research and development of new analgesic drugs, and has important clinical application value.

Description

technical field [0001] The invention belongs to the fields of basic medicine and biomedicine, and is mainly aimed at regulating the expression of corticotropin-releasing hormone (CRH, also known as corticotropin-releasing factor, CRF) gene expression and / or regulating the traits of CRH gene expression pathway , compounds, compositions and methods for diseases, specifically relate to small interfering RNA (Small interfering RNA, siRNA) targeting human and mouse CRH genes and its application in the treatment of neuropathic pain. Background technique [0002] Acute pain acts as an alarm system for tissue damage and has a protective function for the body. But if it still persists after completing the alarm task, this kind of pain becomes pathological pain (or chronic pain) that endangers health, and neuropathic pain is one of them. Neuropathic pain refers to a group of pain syndromes caused by the disturbance of somatosensory transmission in the central nervous system and perip...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K31/713A61P25/04
CPCA61K31/713A61P25/04C12N15/1136C12N2310/141
Inventor 张志军符元元马灵杰
Owner NANTONG UNIVERSITY
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