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Therapeutic agent for acceleration of spinal nerve repair comprising ghrelin, derivative thereof or substance capable of acting on GHS-R1a as active ingredient

A technology of spinal nerves and therapeutic agents, applied in the field of proliferation of spinal nerve precursor cells

Inactive Publication Date: 2009-08-26
UNIVERSITY OF MIYAZAKI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no description of the effect of substances acting on GHS-R1a (such as ghrelin) on fetal spinal cord neural precursor cells that are transplanted into the central nervous system and are expected to treat spinal cord injuries through the growth and development of nerve cells. Further research progress is expected in technology targeting regeneration and transplantation

Method used

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  • Therapeutic agent for acceleration of spinal nerve repair comprising ghrelin, derivative thereof or substance capable of acting on GHS-R1a as active ingredient
  • Therapeutic agent for acceleration of spinal nerve repair comprising ghrelin, derivative thereof or substance capable of acting on GHS-R1a as active ingredient
  • Therapeutic agent for acceleration of spinal nerve repair comprising ghrelin, derivative thereof or substance capable of acting on GHS-R1a as active ingredient

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0230] Example 1 Expression of GHS-R1a mRNA in Rat Fetal Spinal Cord

[0231] Spinal cord tissues were excised from Wistar rat fetuses on days 13 to 19 of pregnancy and rat fetuses just after birth, using Trizol reagent (Life Technologies, Inc., Gaithersburg, MD USA), according to Nakahara et al.: Biochem.Biophys.Res. Total RNA was extracted by the method described in Commun. 303: 751-755 (2003). 1 μg of total RNA was reverse-transcribed with random primers, and single-stranded cDNA was synthesized using Superscript 3 preamplification reagent (Life Technologies, Inc.). Using the sense and antisense primers specific for GHS-R1a, the PCR method (using BDAdvantage TM 2 PCR Enzyme System, BD Science, CA USA) After amplifying the obtained cDNA, electrophoresis was performed using 2% agarose gel. In addition, as a control mRNA, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) stably expressed in cells was used.

[0232] As PCR primers specific to GHS-R1a, 5'-GATACCTCTTTTTCCAAG...

Embodiment 2

[0235] Example 2 Existence of GHS-R1a in spinal cord cells

[0236] Fetal spinal cords were collected from 17-day pregnant Wistar rats, and frozen sections with a thickness of 14 μm were prepared. The sections were fixed with 4% paraformaldehyde / 0.1 M phosphate buffer for 30 minutes, washed with 0.1 M phosphate buffer, and incubated with 2% normal goat serum in PBS for 30 minutes at room temperature. Then, the sections were washed 3 times with PBS, incubated overnight at 4°C with rabbit anti-GHS-R antibody, washed with PBS, incubated with goat anti-rabbit IgG conjugated to Alexa Fluoro 488, and immunostained. After washing the residual antibody, the sections were embedded and observed with a fluorescence microscope.

[0237] The result is as figure 2 shown. According to immunostaining using an anti-GHS-R antibody, it was shown that in the gray matter where the cell bodies of neurons are present, GHS-R1a ( figure 2 A). After pretreatment with anti-GHS-R antibody, the i...

Embodiment 3

[0238] During the cell proliferation of embodiment 3, nestin in spinal cord nerve cells and spinal cord neural precursor cells, Coexistence of Map2 and GHS-R 1a

[0239] Double immunostaining was performed to confirm the coexistence of Nestin, a marker of neuron precursor cells, and Map2, and GHS-R, a marker of neuron cells, in proliferating cells (cells incorporating BrdU).

[0240] Fetus were removed by laparotomy under anesthesia from gestational 17-day Wistar rats. The spinal cord was collected from the fetus, digested with papain in cold Hanks solution, and mechanically separated by a pipette to obtain a fetal spinal cord cell dispersion. After the dispersed cells were filtered and centrifuged, they were suspended in NaHCO-containing 3 , 5% fetal calf serum, penicillin (100 U / mL) and streptomycin (100 μg / mL) in DMEM medium, on a laminin-coated 96-well multi-well plate in 10 5 Cells / well Seed cells.

[0241] 5-Bromo-2'-deoxyuridine (BrdU) (10 µM) was added thereto a...

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Abstract

The invention provides a spinal neuron damage treating agent for use in the treatment of spinal neuron damage, or an agent for promoting the proliferation of spinal neuronal precursor cells in the culture of spinal neuronal precursor cells, or an agent for promoting the regeneration of spinal nerves after transplantation of cultured spinal neuronal precursor cells, and the like. The invention provides an agent that contains a substance (e.g., ghrelin) that acts on the growth hormone secretagogue-receptor as an active ingredient, the agent being a spinal neuron damage treating agent for use in the treatment of spinal neuron damage, or an agent for promoting the proliferation of cultured spinal neuronal precursor cells in the culture of spinal neuronal precursor cells, or an agent for promoting the regeneration of spinal nerves after transplantation of cultured spinal neuronal precursor cells, and the like.

Description

technical field [0001] The present invention relates to the proliferative effect of a substance acting on growth hormone secretagogue receptors in spinal cord neural precursor cells. More specifically, it relates to a therapeutic agent for spinal nerve injury for treating spinal nerve injury, an agent for promoting proliferation of spinal nerve precursor cells when culturing spinal nerve precursor cells, and an agent for transplanting cultured spinal nerve precursor cells, which contain the substance as an active ingredient. Spinal nerve regeneration promoters, etc. Background technique [0002] Due to traffic accidents, accidents in sports, and industrial accidents, more than 6,000 people suffer from spinal cord injuries every year, and the total number reaches about 110,000 people nationwide. According to statistics, its social losses are as high as 300 billion yen per year, but there is no treatment (non-patent document 1). So far, spinal nerves have not been able to be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/00A61K38/22A61P19/08A61P25/00A61P43/00
CPCA61K38/00C07K14/60A61P19/08A61P25/00A61P43/00A61K38/16A61K47/40A61K9/08
Inventor 村上昇中原桂子桥本美穗林友二郎寒川贤治
Owner UNIVERSITY OF MIYAZAKI
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