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Small RNA for human and rat-homologous NMDA receptor NR2B and application thereof

A homology and receptor technology, applied in DNA/RNA fragments, recombinant DNA technology, liquid delivery, etc., can solve the problems of difficulty in obtaining small RNA, loss of effectiveness, and high interference efficiency, and achieve good effect and route of administration. Convenience, efficacy in the treatment of chronic pain

Inactive Publication Date: 2011-01-12
俞卫锋 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is difficult to obtain small RNA with high interference efficiency and strong specificity, and the difference of different sequences can lead to the loss of effectiveness, thus affecting its pharmacological value

Method used

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  • Small RNA for human and rat-homologous NMDA receptor NR2B and application thereof
  • Small RNA for human and rat-homologous NMDA receptor NR2B and application thereof
  • Small RNA for human and rat-homologous NMDA receptor NR2B and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: the screening process of the small RNA sequence of the homologous NMDA of human and mouse

[0030] 1. Construction and identification of pEGFPC1 reporter plasmid containing human NMDA receptor NR2B gene According to the sequence of human NMDA receptor NR2B, primers were designed:

[0031] 5'-CGGAGCGAAGCCCAGAGCGGAGTGCTGTT-3'; (as shown in SEQ ID NO: 2)

[0032] 5'-CGCGTCGAC CAGGATCCCATAAATAAATT-3'. (as shown in SEQ ID NO: 3)

[0033] Both ends of the primers contain Sac I and Sal I restriction sites respectively. RT-PCR is used to obtain the partial sequence in the expression frame of the human NMDA receptor NR2B sequence containing the interference target sequence. This fragment and the pEGFPC1 plasmid (purchased from Invitrogen, USA) were digested with Sac I and Sal I respectively, and then T 4 DNA ligase was ligated into pEGFPC1-hNMDA overnight at 16°C. Transform E.coli.DH5α competent cells, pick clones to extract plasmid DNA, perform PCR amplificat...

Embodiment 2

[0136] Embodiment 2: The inhibitory effect of small RNA of the present invention on NMDA receptor NR2B in the human nerve cell HN

[0137] Human nerve cells HN (purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences) were routinely cultured and divided into four groups: siRNA group (siRNA-NMDA), mismatched RNA group (Mismathch RNA, MM group), saline group ( NS group) and Control group. The siRNA group used the No. 2 small RNA obtained in Example 1 (siRNA2-NMDA: CAACUCCGUACCUGUGCAG). Adopt the liposome method (referring to: DNA transfection mediated by lipid staining. J. Shumbrook, 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 NMDA small interfering RNA were transfected into human nerve cells HN, and an equal amount of normal saline was added to the normal saline group. 12h after transfection, glutamic acid was added to the cult...

Embodiment 3

[0139] Example 3: Effect of subarachnoid injection of NMDA receptor NR2B small RNA of the present invention on thermal pain threshold and mechanical pain threshold of rats

[0140] The neuropathic pain model was constructed by ligation of the right sciatic nerve in rats, and the effect of subarachnoid injection of NMDA receptor NR2B small RNA on the thermal pain threshold of the rats was observed. Rats were intubated with PE-10 catheter through the subarachnoid space, and the rats were divided into sham operation group (Sham group), sciatic nerve ligation group (CCI group), small RNA group and MM group. Intra-injection, 20μg / d, for 7 consecutive days. The MM group gave mismatched small RNAs. In the sham operation group, the sciatic nerve was not ligated.

[0141] 1. Construction of CCI model

[0142]The rats were rested in the animal room for 3 days before the model operation. Anesthetized by intraperitoneal injection of pentobarbital (40ml / kg), increased by about 40mg if ...

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Abstract

The invention belongs to the field of biological genes. Chronic pain is one of forms of expression for neuronal plasticity change, and has the physiological characteristics of increased reactivity of pain sense, treatment difficulty, unclear mechanism, and more side effects by the use of opiate receptor antagonist in treatment. Researches prove that the NMDA regulation is related to the chronic pain. Therefore, the invention provides a small RNA for a human and rat-homologous NMDA receptor NR2B, which has a sequence shown as SEQ ID NO:1. The invention also provides application of the small RNA to medicaments for treating the chronic pain. The small RNA can be directly injected, can inhibit the expression of the NMDA receptor NR2B for human and a rat in vitro and in vivo to achieve the effect of treating the chronic pain, and has the advantages of convenient administration route, no addiction, tolerance and other reactions, and long-term use.

Description

technical field [0001] The invention belongs to the field of biological genes and relates to small RNA interference and its application, in particular to a small RNA inhibiting the expression of NMDA receptor NR2B in humans and rats 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 an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61K9/10A61P25/04
Inventor 俞卫锋吴飞翔
Owner 俞卫锋
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