A small RNA interfering with TLR4 receptor and its application

A technology of receptors and carriers, which is applied in the field of preparation of drugs for the treatment of chronic pain, can solve problems such as interference, and achieve good effects and convenient administration routes

Inactive Publication Date: 2015-10-28
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, effective small RNAs that interfere with human TLR4 receptors are still needed for human therapy
[0006] There are currently no literature reports on small RNAs that interfere with human TLR4 receptors and their use in the treatment of chronic pain

Method used

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  • A small RNA interfering with TLR4 receptor and its application
  • A small RNA interfering with TLR4 receptor and its application
  • A small RNA interfering with TLR4 receptor and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Screening process for small RNA sequences that interfere with TLR4

[0033] 1. Construction and identification of pEGFPC1 reporter plasmid containing human TLR4 receptor gene

[0034] According to the human TLR4 receptor sequence, design primers:

[0035] Upstream primer: 5'-CGGAGCGTTTCAGACTCCGGAGCCTCAGCTGTT-3' (SEQ ID NO: 7);

[0036] Downstream primer: 5'-CGCGTCTTGCCCAGCTGGGTCCAATAAATT-3' (SEQ ID NO: 8).

[0037] Both ends of the primers contain SacI and SalI restriction sites respectively. RT-PCR is used to obtain the partial sequence in the expression frame of the human TLR4 sequence containing the interference target sequence.

[0038] This fragment and pEGFPC1 plasmid (purchased from Invitrogen, USA) were digested with SacI and SalI respectively, and then ligated with T4 DNA ligase overnight at 16°C to form pEGFPC1-hNMDA. Transform E.coli.DH5α competent cells, pick clones to extract plasmid DNA, perform PCR amplification to obtain the correct band, ...

Embodiment 2

[0088] Example 2: Inhibition of small RNA of the present invention on NMDA receptor NR2B in human neuroglial cells

[0089]Conventionally cultured human microglial cells (Human Microglia, BH-HP1900, purchased from Shanghai Biotechnology Co., Ltd.), were divided into four groups: siRNA group (siRNA-TLR4Ⅱ), mismatched RNA group (Mismathch RNA , MM group), normal saline group (NS group) and Control group.

[0090] Using the liposome method (see: Lipofection-mediated DNA transfection. J. Sambrook, translated by Huang Peitang. Molecular Cloning Experiment Guide. 3rd Edition. Published by Science Press. 2002, 1276-1288.) will wrong 5 μg (20 μl) of the prepared RNA and siRNA-TLR4Ⅱ were transfected into human microglial cells, and an equal amount of normal saline was added to the normal saline group. 12 hours after transfection, endotoxin was added to the culture medium of siRNA-TLR4 group, mismatched RNA group (Mismathch RNA, MM group) and normal saline group (NS group) to a final c...

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Abstract

The invention belongs to the technical field of biological medicals. The chronic pain is one of manifestations of the plasticity variation of nerve cells; the physiological characteristics thereof are increased reactivity of algesia, troublesome treatment and unclear mechanism; the treatment thereof mainly adopts an opiate receptor retardant which has more side effects. The invention provides a small RNA (Ribonucleic Acid) for disturbing a TLR4 receptor; the sequence thereof is shown as SEQ ID NO: 5. The invention further provides application of the small RNA in medicines for treating the chronic pain. The small RNA can adopt a direct injecting manner, or can adopt a carrier expression manner for restraining the expression of TLR4 in vitro and vivo to act the effect of treating the chronic pain; meanwhile, the small RNA has the advantages of convenient administration route, no existence of habituation and tolerance and long-time use.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a small RNA that inhibits TLR4 receptors and its application in the preparation of medicines for treating chronic pain. Background technique [0002] Chronic pain is one of the manifestations of neuronal plasticity changes, and its physiological characteristics are increased pain responsiveness, mainly manifested as hyperalgesia and allodynia. The treatment of this type of pain is difficult, mainly because its mechanism is unknown. At present, in treatment, opioid receptor blockers are mainly represented by morphine, but there are many side effects, addiction, tolerance and other reactions, which limit its long-term use. Therefore, there is an urgent need for a new method with good therapeutic effect, long acting time and light side effects, so as to achieve the purpose of long-term and effective control of chronic pain. [0003] Toll like receptor 4 (TLR4), one...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A61K48/00A61P25/04
Inventor 吴飞翔俞卫锋张金旻
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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