Pharmaceutical composition for treating and repairing spinal cord injury and application thereof

A technique for spinal cord injury and composition, applied in the field of pharmaceutical composition for treating and repairing spinal cord injury

Active Publication Date: 2022-05-24
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the technical problems existing in the treatment process of spinal cord injury repair in the prior art, thereby providing a new treatment target for spinal cord injury repair, and investigating the function and m

Method used

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  • Pharmaceutical composition for treating and repairing spinal cord injury and application thereof
  • Pharmaceutical composition for treating and repairing spinal cord injury and application thereof
  • Pharmaceutical composition for treating and repairing spinal cord injury and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Preparation of plasmids and constructs

[0071] (1) Rab3A and spastin sequences were obtained for preparation of cDNA, which were then cloned into pEGFP-C1 (Clontech, CA, USA), pGEX-5x-3 (Amer-sham Pharmacia Biotech, NJ, USA) and pCMV-Tag2 (Stratagene, CA, USA) vector, and the constructed construct was confirmed by DNA sequencing method;

[0072] (2) Pull-down analysis using glutathione S-transferase (GST): Spinal cord tissue sections were ground and lysed, then GST-agarose beads (Invitrogen, CA, USA) were rinsed and mixed with lysis buffer at 4 Incubate for 1 h at °C, then centrifuge at 1000 g for 10 min. 4°C. The supernatant was then collected and these steps were repeated one more time. Appropriate bead fusion proteins were then added to the spinal cord tissue, and the samples were incubated overnight at 4°C. After spinning at 1000 g for 5 min at 4°C, unbound protein was washed with 1 mL of wash buffer, and then the samples were spun at 1000 g for 1 min...

Embodiment 2

[0076] Example 2 Construction of rat SCI model

[0077] The Sprague-Dawley (SD) rats used in the experiment were purchased from the Laboratory Animal Center of Sun Yat-Sen University. The rats were housed individually in a facility at 25±3°C and provided with regular food and water. Specifically, the following steps were included:

[0078] (1) SD rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.35 mL / 100 g body weight), after which a 2.5 cm longitudinal dorsal incision was made to expose the T9-11 spinous process and lamina;

[0079] (2) The entire T10 lamina is removed, and the exposed area of ​​the spine is about 2.5mm × 3mm;

[0080] (3) Use a stabilizer to fix the T10 section bilaterally, and set the nitrogen tank to control the impact head to 18psi or 124kPa. Load the U-shaped stabilizer with the rat onto the platform of the Louisville Injury System Equipment (LISA) and adjust the dura / spinal height directly below the impactor while monitorin...

Embodiment 3

[0085] Example 3 Rab3A regulates neuron growth and development

[0086] Firstly, the research on the growth and development of neurons by Rab3A overexpression includes the following steps:

[0087] (1) GFP-Rab3A and GFP (blank control group) were transfected into primary cultured hippocampal neurons for 72 hours, respectively;

[0088] (2) After 48 hours, the hippocampal neurons were fixed and photographed, and the scale was 100 μm.

[0089] The result is as Figure 1-3 shown, where figure 1 Schematic diagram of the immunofluorescence staining results of hippocampal neurons, figure 2 Schematic diagram of the quantitative statistics of the length of axons and neurites in hippocampal neurons, image 3 Schematic diagram of the quantitative statistical results of the number of axons and processes in hippocampal neurons, n=20 / group, the results are represented by Mean±SD, and * represents p <0.05. The scale bar is 100 μm. According to the results, overexpression of Rab3A in...

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Abstract

The invention relates to a pharmaceutical composition for treating and repairing spinal cord injury and application of the pharmaceutical composition. According to the invention, research finds that Rab3A plays a key role in SCI and interacts with Spastatin to regulate and control outward growth of neurite. The identification of differentially expressed proteins in SCI proves that the expression level of Rab3A is down-regulated in the SCI process. In addition, the invention also finds that Rab3A can generate physical interaction with the Spastatin, and the degradation of the Spastatin is regulated and controlled through a lysosome way, so that the function of the Spastatin is influenced. These findings collectively emphasize the signal transduction pathway, where Rab3A mediates spastin degradation to modulate the formation and growth of neurite branches. As SCI is a major cause of disability, repair of spinal cord structure defects caused by injury or degeneration is crucial in the field of modern regenerative medicine. Various drug intervention measures can be designed to treat SCI and assist in related tissue repair, and other cell intervention measures can be combined.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular, the present invention relates to a pharmaceutical composition for treating and repairing spinal cord injury and its application. Background technique [0002] Spinal cord injury (SCI) can lead to permanent spinal cord dysfunction, seriously affecting the quality of life of patients. The main cause of dysfunction after SCI is direct mechanical injury leading to neuronal damage, and subsequent secondary damage, including inflammatory response, oxidative stress response and excitatory injury, etc., resulting in neuronal axonal damage and glial Plasma cells proliferate, eventually leading to neuronal cell death. Neurological dysfunction caused by SCI is permanent because after spinal cord tissue damage, the ability of axons to regenerate is inhibited and it is difficult to pass through the damaged area, thereby irreversibly impairing the transmission of motor and sensory information...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61K31/713A61K38/17A61K49/00A61P25/00G01N33/68G01N33/53
CPCA61K45/00A61K31/713A61K38/1709A61K49/0008A61P25/00G01N33/6893G01N33/53G01N2800/28
Inventor 张国威陈云杨裕豪林宏生
Owner JINAN UNIVERSITY
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